Caveolin-1 reduces HIV-1 infectivity by restoration of HIV Nef mediated impairment of cholesterol efflux by apoA-I

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Caveolin-1 reduces HIV-1 infectivity by restoration of HIV Nef mediated impairment of cholesterol efflux by apoA-I
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Lin, Shanshan
Nadeau, Peter E.
Wang , Xiaomei
Mergia, Ayalew
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BioMed Central
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Abstract:
Background: HIV infection results in inhibited cholesterol efflux by apolipoprotein A-I (apoA-I) in macrophages, and this impairment involves Nef mediated down-regulation and redistribution of ATP-binding cassette transporter A1 (ABCA-1). We investigated the effect of caveolin-1 (Cav-1) on the cholesterol efflux by apoA-I in HIV infected primary and THP-1 cell-differentiated macrophages as well as astrocyte derived glioblastoma U87 cells. Results: Our results reveal that Cav-1 restores the Nef -mediated impairment of cholesterol efflux by apoA-I in both cell types. Co-immunoprecipitation studies indicate a physical association of Cav-1 and Nef. The level of ABCA-1 expression remains the same whether Cav-1 is over-expressed or not. In addition, we examined the cholesterol composition of HIV particles released from Cav-1 treated cells and identified that the cholesterol content is dramatically reduced. The infectivity level of these virus particles is also significantly decreased. Conclusions: These observations suggest that the interplay of Cav-1 with Nef and cholesterol subsequently counters Nef induced impairment of cholesterol efflux by apoA-l. The findings provide a cellular mechanism by which Cav-1 has an ability to restore HIV mediated impairment of cholesterol efflux in macrophages. This subsequently influences the cholesterol content incorporated into virus particles thereby inhibiting HIV infectivity and contributing to HIV’s persistent infection of macrophages. Keywords: HIV, Caveolin-1, Cholesterol efflux, Nef, Apolipoprotein A-I
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Lin et al. Retrovirology 2012, 9:85 http://www.retrovirology.com/content/9/1/85; Pgs.1-15
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doi:10.1186/1742-4690-9-85 Cite this article as: Lin et al.: Caveolin-1 reduces HIV-1 infectivity by restoration of HIV Nef mediated impairment of cholesterol efflux by apoA-I. Retrovirology 2012 9:85.

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Lin et al. Retrovirology 2012, 9:85
http://www.retrovirology.com/content/9/1/85


RETROVIROLOGY


Caveolin-1 reduces HIV-1 infectivity by restoration

of HIV Nef mediated impairment of cholesterol

efflux by apoA-I


Shanshan Lin, Peter E Nadeau, Xiaomei Wang and Ayalew Mergia


Background
Caveolin 1 (Cav-1), a 21~24-kDa scaffolding protein, is
an important structural component of caveolae [1], small
invaginations of the plasma membrane, which are en-
riched in cholesterol, phospholipids, and sphingolipids.
This protein is highly expressed in terminally differen-
tiated cells including endothelial cells, macrophages, den-
dritic cells and adipocytes [2,3]. Functional studies have
shown that Cav-1 is involved in a wide range of cellular
processes, including cell cycle regulation, signal transduc-
tion, endocytosis, cholesterol trafficking and efflux [3-9].
Multiple lines of evidence indicate that Cav-1 acts as a
scaffolding protein capable of directly interacting with and
modulating the activity of caveolin-bound signaling mole-
cules. The Cav-1 scaffolding domain (CSD), residues 82 to

SCorrespondence mergiaa@ufl edu
Department of Infectious Disease and Pathology, University of Florida,
Gainesville, Florida 32611, USA


101, is essential for both Cav-1 oligomerization and the
interaction of caveolin with other proteins [10]. Associa-
tions with other proteins through the CSD help provide
coordinated and efficient signal transduction [11,12]. The
CSD serves as a receptor for binding proteins containing
the sequence (XbXXXXX, bXXXXbXXb, or bXbXXX
X4XX4 (4 representing any aromatic amino acid and X
any other amino acid)[10]. HIV Env has been shown
to interact with Cav-1 via a motif (WNNMTWMQW)
localized within the ectodomain (the C-terminal heptad
repeats) of HIV-1 gp41 [13-15]. Our group has shown the
binding of Cav-1 with HIV Env in the lipid rafts which
subsequently blocks cell fusion and innocent bystander
killing mediated by HIV envelope [16]. We have also
demonstrated that HIV infection in primary human
monocyte derived macrophages (MDMs) results in a
dramatic up-regulation of Cav-1 expression mediated
by HIV Tat [17]. Furthermore, over-expression of Cav-1


S 2012 Lin et al., licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Bioliled Central Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted ue, distribution, and
reproduction in any medium, provided the original work is properly cited.


Abstract
Background: HIV infection results in inhibited cholesterol efflux by apolipoprotein A-I (apoA-I) in macrophages, and
this impairment involves Nef mediated down-regulation and redistribution of ATP-binding cassette transporter Al
(ABCA-1). We investigated the effect of caveolin-1 (Cav-1) on the cholesterol efflux by apoA-I in HIV infected
primary and THP-1 cell-differentiated macrophages as well as astrocyte derived glioblastoma U87 cells.
Results: Our results reveal that Cav-1 restores the Nef mediated impairment of cholesterol efflux by apoA-I in both
cell types. Co-immunoprecipitation studies indicate a physical association of Cav-1 and Nef The level of ABCA-1
expression remains the same whether Cav-1 is over-expressed or not. In addition, we examined the cholesterol
composition of HIV particles released from Cav-1 treated cells and identified that the cholesterol content is
dramatically reduced. The infectivity level of these virus particles is also significantly decreased.
Conclusions: These observations suggest that the interplay of Cav-1 with Nef and cholesterol subsequently
counters Nef induced impairment of cholesterol efflux by apoA-I. The findings provide a cellular mechanism by
which Cav-1 has an ability to restore HIV mediated impairment of cholesterol efflux in macrophages. This
subsequently influences the cholesterol content incorporated into virus particles thereby inhibiting HIV infectivity
and contributing to HIV's persistent infection of macrophages.
Keywords: HIV, Caveolin-1, Cholesterol efflux, Nef, Apolipoprotein A-I






Lin et al. Retrovirology 2012, 9:85
http://www.retrovirology.com/content/9/1/85


causes significant reduction in HIV replication in macro-
phages. Cav-1 inhibits HIV replication through transcrip-
tional repression of viral gene expression by modulating
the NF-xB pathway [18]. The up-regulation of Cav-1 by
HIV infection and subsequent inhibition of HIV replica-
tion suggest a role for Cav-1 in macrophage persistent
infection.
Cav-1 plays an important role in cellular cholesterol
homeostasis, a process that controls intracellular lipid
composition and prevents cholesterol accumulation.
Cav-1 has been implicated in modulating the expres-
sion of lipoprotein receptors and interacts with many
lipid transporter molecules [11,19-21]. Furthermore, it
is involved in the transport of newly synthesized cho-
lesterol from the endoplasmic reticulum (ER) to the
plasma membrane [11,22,23] and promotes cholesterol
efflux in hepatic cells [9,24]. HIV appears to manipulate
cellular cholesterol metabolism to ensure that there is a
sufficient ..l'.1 of cholesterol and that it is located in
the appropriate compartments such as lipid rafts for ef-
ficient virus release and subsequent infectivity [25-28].
Cholesterol is an important component that influences
HIV production and efficient virus infectivity. Choles-
terol depletion significantly reduces HIV-1 particle
production [29-34]. Virus infectivity is also negatively
affected when HIV is produced from cholesterol depleted
cells -. .J.
The HIV accessory protein Nef has an ability to exploit
cholesterol metabolism, r. .. 1 mechanisms for this
i. ai. include 1.....;1. to cholesterol and aiding the
transport of newly synthesized cholesterol into lipid
rafts and viral particles as well as enhancing cholesterol
synthesis [36,37]. Nef has also been shown to impair
ATP 1,|.1;..l cassette transporter protein 1 (ABCA-1)-
dependent cholesterol efflux from human macrophages
by down-regulation and redistribution of ABCA-1 [38].
This suggests that Nef is involved in HIV mediated
cholesterol accumulation. Since Cav-1 has a high af-
finity for cholesterol and aids in the transport of
newly synthesized cholesterol from the ER to the plasma
membrane and indirectly promoting the transfer to extra-
cellular acceptors such as lipid free apolipoprotein A-I
(apoA-I) we hypothesize it would influence the level of
cholesterol accumulation as well as virus production
and infectivity. Macrophages are major targets for
HIV infection and also play an important role in its
pathogenesis. The up-regulation of Cav-1 by HIV in-
fection and the role of Cav-1 in cholesterol trafficking
':I-. I a mechanism for a Cav-1/cholesterol mediated
impact on HIV replication in macrophages. In this report,
we establish evidence for a Cav-1/cholesterol mediated
mechanism of inhibition of HIV replication for the first
time providing a new angle in :,,.:.. :... .-L- HIV's per-
sistent infection of macrophages.


Results
Cav-1 restores HIV Nef mediated impairment of cholesterol
efflux by apoA-I in U87 cells and macrophages
HIV infection impairs ATP-binding cassette transporter
Al (ABCA-1) dependent cholesterol efflux by apoA-l.
The Nef protein is identified as the key molecule respon-
sible for this effect [38]. Since Cav-1 is an important
regulator of cholesterol I, .::., i .1 : and is involved in the
transport of newly synthesized cholesterol from the ER
to the plasma membrane, it is likely to influence Nef
mediated ABCA-1 dependent down modulation of chol-
esterol efflux. To determine whether Cav-1 counters the
influence of Nef on cholesterol t :.ff;.1 .. !-. first, we tes-
ted the impairment of cholesterol efflux in HIV infected
THP-1 cell-differentiated macrophages. HIV AD8 infec-
ted THP-1 cells were exposed to lipid-free apoA-I or
HDL treatment to induce cholesterol efflux. Cholesterol
efflux was measured as the fraction of total radiolabeled
cholesterol appearing in the i, ....i in the presence
of apoA-I after subtraction of values for apoA-I-free
medium [39]. ApoA-I stimulated cholesterol efflux from
HIV infected THP-1 ., ii 1,-::- ...., .(. ,I macrophages was
markedly decreased in a dose dependent manner with
the reduction reaching 71.6% as compared to uninfected
cells T" ..... 1A). No significant i-11. .- was observed
between HIV infected and uninfected cells in HDL
mediated cholesterol efflux T'i,.-,- 1B). The decrease in
cholesterol efflux to apoA-I by HIV infection was not
present in the presence of AZT, an inhibitor of the HIV
replication IF-. ..- 1C). These results suggest that HIV
infection decreased the apoA-1 i., .. i ,. I cholesterol ef-
flux substantially and are in accordance with previous
findings [38]. To further examine impairment of choles-
terol efflux due to HIV infection the level of cholesterol
accumulation was tested by oil red O staining of infected
macrophages. As shown in Figure ID, accumulated chol-
esterol was markedly increased in AD8 or Bal infected
macrophages as compared to uninfected cells which is
similar to previous findings [38].
To address the influence of Cav-1 on cholesterol efflux
of HIV infected cells, we examined whether Cav-1 can res-
tore Nef mediated impairment of cholesterol efflux. First,
U87-CD4-CXCR4 cells were transfected with a Cav-1 ex-
pression construct (pCZ-Cav-1) in the presence or ab-
sence of a Nef expression plasmid (pcDNA3.1SF2Nef).
ApoA-l or HDL mediated cholesterol efflux was measured
by harvesting the culture media as well as cell lysate sam-
ples. As a control U87-CD4-CXCR4 cells were also trans-
fected with expression vector lacking Cav-1 (pCZ-vector)
or Nef (pcDNA3.1). The expression of Cav-1 and Nef in
transfected cells was determined by Western blot analy-
sis (Figure 2A). As expected apoA-l mediated choleste-
rol efflux from cells transfected with the Nef expression
construct alone was reduced by 77% compared to cells


Page 2 of 16







Lin et al. Retrovirology 2012, 9:85
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P<0.05
00P<.05
_ 0.06 P<0.05

a 0.06



0.02
.4^1^

1-1 |


0 1-
(AD8 Inf MOI)


C.


Mock 0.001


0.14
0.12
0.10
0.08
0.06
0.04
0.02


0.01


Mock AD8


120%
100%
80%
60%
40%
20%
0%


P<0.05
i I


*


Mock AD8 AD8+AZT
HIVRTActivity(cpm/Al) Blank 2834 418


HIV RT Activity(cpm/pl)


Figure 1 HIV-1 impairs apoA-l mediated cholesterol efflux from THP-1 cell-differentiated macrophages. (A) HIV AD8 infected and
uninfected THP-1 cell-differentiated macrophages were cultured for 14 days. MOI represents multiplicity of infection. Cells were labeled with 3H]
cholesterol, and subsequently incubated with media in the presence and absence of apoA- (50 pg/ml). ApoA-1-induced cholesterol efflux was
measured and analyzed as described in the Materials and Methods. (B) Cells were treated the same as above but incubated with medium in the


presence or in absence of
determined in the present
shown are mean + SD wit
macrophages cultured for
accumulation was determ
bottom panel.


HDL (50 pg/ml) and HDL mediated cho
ce and absence of the HIV replication in
th P values. (D) Uninfected or infected w
10 days then incubated with AcLDL (50
ined by Oil red O staining by light micr


lesterol efflux was measured. (C) ApoA-I mediated cholesterol efflux was
hibitor AZT (5uM). All experiments were performed in triplicate, and results
ith HIV AD8 (moi 0.01) and HIV Bal (moi 0.001) THP-1 cell-differentiated
pg/ml) for 48 h followed by 30pg/ml apoA-I stimulation for 18 hours. Lipid
oscopy. HIV reverse transcriptase activity in culture medium is shown in the


transfected with the plasmid construct devoid of nef
or cells that received pCZ-Cav-1 (Figure 2B). Interes-
tingly, apoA-1 mediated cholesterol efflux from cells co-
transfected with pCZ-Cav-1 and pcDNA3.1SF2Nef was
comparable, and even slightly higher, to that of mock
which did not receive Nef treatment, suggesting that
Cav-1 can restore the impairment of cholesterol efflux
caused by Nef. Neither Nef nor Cav-1 had significant ef-
fect on HDL mediated cholesterol efflux (data not shown).
We confirmed our findings by conducting these studies
in physiologically relevant primary monocyte derived
macrophages (MDMs). We infected MDMs with vesicular
stomatitis virus glycoprotein (VSV-G) Env pseudotyped
pSG3aenv HIV provirus carrying wild type nef (psHIVwt-
Nef) or defective nef (psHIVANef) for one round of re-
plication. Infection of MDMs with psHIVwtNef showed
a significant reduction (70%) in apoA-1 mediated choles-
terol efflux as compared to uninfected cells (Figure 2C)
similar to what is observed in the U87-CD4-CXCR4 cells


(Figure 2B). The reduction in cholesterol efflux was 75%
when compared to Nef defective HIV. There was no sig-
nificant difference in cholesterol efflux between Nef de-
fective HIV infected and uninfected MDMs. Introduction
of exogenous Cav-1 into psHIVwtNef MDMs using
adenovirus expressing Cav-1 (Ad-Cav-1) increased choles-
terol efflux by 43% compared to cells only infected with
psHIVwtNef. The control adenovirus carrying GFP (Ad-
GFP) was not able to counter cholesterol efflux impair-
ment induced by psHIVwtNef infection of the MDMs
(Figure 2C). As expected, Western blot analysis of MDMs
transduced with Ad-GFP revealed endogenous Cav-1
expression, with increased amounts of Cav-1 in MDMs
treated with Ad-Cav-1 (Figure 2D). Furthermore, intro-
duction of exogenous Cav-1 or GFP using adenovirus did
not alter cholesterol efflux from MDMs infected with the
Nef defective HIV. In addition, we compared apoA-I
mediated cholesterol efflux in MDMs infected with wild
type AD8 and replication competent nef deleted AD8


Page 3 of 16







Lin et al. Retrovirology 2012, 9:85
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A.
Nef
Mock Nef Cav-1Cav-1

Cav-1 60
Nef f *27KD

B-acin




B.


P<0.05 P0.05
150% -
0 120% -
a 90%-
60%
! e 30%
U 0%


Mock


psHIVwtNef Ad-Cav-1+psHIVwtNef


Mock Nef Cav-1 Nef+Cav-1 .




0.06 ---


0.0-

0
Mock AD8 ADnefmut -
Figure 2 Cav-1 restores HIV -1 Nef mediated impairment of cholesterol efflux to apoA-I. U87-CD4-CXCR4 cells were transfected with Nef
expression plasmid (pcDNA3.1SF2Nef) along with pCZ-cav-1 or pCZ vector. Cells were labeled with [H] cholesterol for 36 h. (A) The level of Nef
and Cav-1 expression was determined by Western blot analysis. (B) ApoA-I induced cholesterol efflux is shown. All experiments were performed
in triplicate, and results shown are mean + SD with P values. (C) Primary monocyte derived macrophages (MDMs) were infected with Ad-Cav-1 c
Ad-GFP, followed by infection with VSV-G pseudotyped HIV, either carrying Nef (psHIVwtNef) or defective Nef (psHIVANef at an moi of 5. ApoA-
mediated cholesterol efflux was performed and analyzed by incubating cells in medium in the presence or absence of 50 g/ml apoA-. The
results are presented as a percentage of cholesterol efflux to apoA-I from control (set as 100%), and are the mean+ SD of triplicate
determinations. The expression levels of Cav-1 and GFP are shown in (D). (E) Primary macrophages were infected with ADS or nef defective ADS
(ADnefmut) at an moi of 0.01. ApoA-I mediated cholesterol efflux was measured 15 days post infection. (F) THP-1 cell-differentiated macrophage
were infected with psHIVwtNef or psHIVANef at an moi 3 with or without Ad-Cav-1. On day 5 after infection, Oil red 0 staining was performed
and a representative area in each well is shown.


(ADnefmut) viruses. Cholesterol efflux was decreased by
56.3% in wild type infected macrophages compared to un-
infected cells while ADnefmut infection had no significant
effect (Figure 2E). These results taken together suggest
that Cav-1 is capable of restoring HIV induced impair-
ment of apoA-1 mediated cholesterol efflux. To confirm
our findings, we further examined the affect of Cav-1
on cholesterol accumulation by oil red O staining in
HIV infected THP-1 cell-differentiated macrophage
cells. As shown in Figure 2F, psHIVwtNef infected cells
had significantly increased cholesterol accumulation com-
pared to uninfected cells (mock) which is similar to HIV
AD8 infected THP-1 cell-differentiated macrophages
(Figure 1D). The co-infection of macrophages with Ad-


Cav-1 and psHIVwtNef, on the other hand, showed a dra-
matic reduction of intracellular cholesterol inclusions
when compared to psHIVwtNef only infected cells. As
expected, no significant influence on intracellular choles-
terol accumulation was observed when THP-1 cell-diffe-
rentiated macrophages were infected with Nef defective
HIV (psHIVANef). In addition, over-expression of Cav-1
by Ad-Cav-1 infection did not alter intracellular choles-
terol inclusions in cells infected with psHIVANef.
We further determined whether endogenous Cav-1
has an effect on Nef mediated suppression of cholesterol
efflux to apoA-1 in U87 cells and THP-1 cell-differentiated
macrophages. The expression of endogenous Cav-1 was
knocked down using specific siRNA, and the expression of


Page 4 of 16


U87.CD4.CXCR4


Ad-GFP Ad-Cav-1
Cav-1 0
actinn


s







Lin et al. Retrovirology 2012, 9:85
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Nef was accomplished by transfecting U87 cells with
pCDNA3.1SF2Nef or pseudotyped HIV (psHIVwtNef) in-
fection of THP-1 cell-differentiated macrophages. The
siRNA treatment reduced the expression of Cav-1 by 76%
in U87 cells and 38% in THP-1 cell-differentiated macro-
phages (Figure 3A and 3B, respectively). Cholesterol efflux
to apoAl was reduced by 61% in Nef expressing U87 cells
in the absence of siRNA targeting Cav-1 (Figure 3A). The
level of cholesterol was further reduced (96%) when Cav-1
expression was knocked-down with Cav-1 specific siRNA.
Similar results were observed in THP-1 cell-differentiated
macrophages showing a decrease in cholesterol efflux by


[A.


120%
100%
80%
60%
40%
20%
0%


P<0.05 P<0.05
-- P<0.05



ii


+

+


+
+
4-


U87 cells *

Cay-1 MO

B-actin


50% in Nef expressing cells and by 79% in Cav-1 siRNA
treated Nef expressing cells (Figure 3B). Furthermore, the
levels of cholesterol efflux correlate with the efficiency of
siRNA knock-down in U87 and THP-1 cells (Figure 3A
and 3B).
In addition, to determine whether Cav-1 specifically
restores Nef mediated impairment of cholesterol efflux
to apoA-1, U87 cells were co-transfected with a Nef
mutant (NefG2A) and Cav-1 expressing plasmids. The
NefG2A is a Nef mutant that cannot undergo myristoy-
lation [40]. Its association with the plasma membrane is
impaired [26,35], and it lacks the ability to decrease


B.


140%
o 120%
* 100% -
o 80%-
- 60%

0 20%
U 0% -

CTL-siRNA
siRNA-Cav-1
pSHIVwtNef


,
+

+


P<0.05 P<0.05








+ +
+ +
+ +


THP-1 c or

Cav-1

B-actin

Cav-1 ratio 1.0 0.62


Cav-1 ratio 1.0


0.24 C.


0.1
0.08
0.06
0.04
0.02
0


P<0.05
I I


7L FL


Mock


Nef NefG2A NefG2A+Cav-1


Mock Nef NefG2A
Nef a -

B-actin

Figure 3 siRNA knockdown of Cav-1 and its effect on cholesterol of cells expressing wild type Nef or NefG2A. (A). U87-CD4-CXCR4 cells
were transfected with Cav-1 siRNA or control siRNA (CTL-siRNA) followed by transfection with Nef expressing plasmid (pSHIVwtNef) or pCDNA3.1
vector. The level of Cav-1 expression in siRNA treated cells was detected by Western blotting analysis. Cells were labelled with [ H] cholesterol for
36 hours and apoA-I induced cholesterol efflux was measured. Results are shown as the percentage of cholesterol efflux relative to control.
(B) THP-1 cell-differentiated macrophages were first transfected with Cav-1 siRNA or CTL-siRNA which was followed by infection with VSV-G
pseudotyped HIV (psHIVwtNef) at an moi of 3. Cells were again treated with siRNA the day after infection. Cholesterol efflux was measured as
described above four days post-infection. (C) U87-CD4-CXCR4 cells were transfected with pCI (Mock), pCI NL4-3 Nef-HA-WT (Nef), pCI NL4-3
NefG2A-HA (NefG2A), or pCI NL4-3 NefG2A-HA along with pCZ-cav-1 (Nef plus Cav-1). Cholesterol efflux to apoA- was determined. All experiments
were performed in triplicate and results shown are mean + SD with P values.


Page 5 of 16


a0-


uc
0-
U


CTL-siRNA
siRNA-Cav-1
pCDNA3.1
pCDNA3.1SF2Nef







Lin et al. Retrovirology 2012, 9:85
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apoA-1 stimulated cholesterol efflux [36,41]. As shown
in Figure 3C, cells expressing Nef experienced 62% less
cholesterol efflux to apoA-I compared to Mock. In con-
trast, NefG2A had no effect on apoA-1 mediated choles-
terol efflux in the presence of either endogenous or over-
expressing Cav-1 cells. These studies, therefore, clearly es-
tablish that Cav-1 counters Nef mediated impairment of
cholesterol efflux by apoA-1.

Cav-1 over-expression has no effect on ABCA-1
expression
Nef has been shown to impair ABCAl-dependent cho-
lesterol efflux from human macrophages, and the ex-
pression of ABCA-1 is shown to be down regulated by
HIV infection or Nef expression [38,42]. Cav-1 is im-
plicated in positive regulation of ABCA-1 expression
and ABCA-1 expression is down regulated in Cav-1


knockout mice [43]. In order to understand the mechan-
ism responsible for Cav-1 mediated restoration of chol-
esterol efflux upon HIV infection we examined the
expression of ABCA-1 in Cav-1 over-expressing cells.
U87-CD4-CXCR4 cells were transfected with pCZ-Cav-1
in a dose-dependent manner, and the level of ABCA-1
expression was monitored by Western blot analysis. As
shown in Figure 4A, Cav-1 over-expression did not alter
the level of ABCA-1 expression. To further confirm our
findings, we co-transfected U87 cells with pCZ-Cav-1
and pcDNA3.1SF2Nef, and then analyzed ABCA-1 ex-
pression by Western blot in the presence and absence of
Cav-1 or Nef. Although Nef down-regulated ABCA-1
(by 69%) the level of ABCA-1 expression remained the
same in Nef treated cells whether Cav-1 is over-expressed
or not (Figure 4B), suggesting that Cav-1 mediated resto-
ration of cholesterol efflux is not related to the regulation


Cav-1(Ig) Mock 0.5 1.0

Cayv-

ABCA1 r0

B-actin I1

ABCA1 ratio 1.0 0.99 0.98


- + + -


Nef
Mock Nef Cav-1
Cav-1
Cav-1

Nef


Alld ilNO
B-actin
ABCAlratio 1.0 0.31 0.29 0.98


psHIVwtNef
Ad-Cav-1


+ +
+ +


ABCAl l -*

Nef

Cav-1 w Goi

R-actin mo

ABCA1 ratio 1.0 0.26 0.98 0.25


+ -


- + + +
+ +
+ + -


CTL-siRNA siRNA-Cav-1


CTL-siRNA siRNA-Cav-1


ABCA1 MO lwi W w w

Nef m0

Cav-1 m l

B-actin -.. .


Cav-1 -- Cav-1

ABCA-1 ABCA-1

R-actin B-actin

ABCA1 ratio 1.0 1.12 ABCA1 ratio 1.0 1.02
U87 cells THP-1


ABCA1 ratio 1.0 0.51 0.96 0.42 0.94 0.36 0.97
Figure 4 Cav-1 over-expression has no influence on ABCA-1 expression. (A) U87-CD4-CXCR4 cells were transiently transfected either with
pCZ-vector (mock) or with different doses of Cav-1 (pCZ-Cav-1). Expression of ABCA-1 protein was examined by Western blot analysis. (B) Cells
were transfected with pCZ-vector (mock), Nef expressing construct (pcDNA3.1SF2Nef) and pCZ-Cav-1 or pCZ-Cav-1 only. The expression levels of
ABCA-1 in the presence or absence of Nef were determined in the cells with endogenous or over-expressing Cav-1. (C) MDMs were infected with
VSV-G pseudotyped HIV (psHIVwtNef), Ad-Cav-1, or both. The level of Cav-1 and ABCA-1 expression was examined by Western blot analysis in the
presence and absence of Cav-1 and/or Nef Representative results from three experiments are shown in A, B and C. (D) MDMs were co-infected
with wild type ADS or nef defective ADS (ADnefmut) and Ad-Cav-1 or Ad-GFP. The level of expression of ABCA-1, Nef, and Cav-1 was examined
by Western blot analysis. (E) To determine whether siRNA knock-down of Cav-1 affects the expression of ABCA-1 samples used in experiment 3A
and B were used to measure the level of ABCA-1 expression. Note that the siRNA Cav-1 knock out and 3-actin are the same bands shown in
Figure 3 because the same samples were used to demonstrate the level of ABCA-1 expression. The densities of bands corresponding to each
protein were quantified using image densitometer analysis. The numbers at the bottom of each blot are the relative values of ABCA1 expression
in transfected or transduced cells compared to those in control cells (mock).


Page 6 of 16


AD8
ADnefmut
Ad-Cav-1
Ad-GFP






Lin et al. Retrovirology 2012, 9:85
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of ABCA-1 expression. Likewise, VSV-G pseudotyped
HIV (psHIVwtNef) infection down-regulated ABCA-1 ex-
pression in MDMs, and co-infection of MDMs with
psHIVwtNef and Ad-Cav-1 did not restore the reduced
ABCA-1 levels (Figure 4C). MDMs were also infected
with AD8 or ADnefmut virus along with infection of Ad-
Cav-1 or Ad-GFP to determine the level of ABCA-1 ex-
pression. ADnefmut HIV infection did not affect the ex-
pression of ABCA-1 with either Ad-Cav-1 or Ad-GFP co-
infection (Figure 4D). ABCA-1 expression, however, was
decreased when MDMs were co-infected with AD8 and
Ad-Cav-1 or Ad-GFP confirming the role of Nef in the re-
duction of ABCA-1 expression while over expression of
Cav-1 has no impact on ABCA-1 expression. Further-
more, we examined the level of ABCA-1 expression in
U87 or THP-1 cells where the expression of endogenous
Cav-1 was knocked down with siRNA using samples
described for results in Figure 3A and 3B. As shown
in Figure 4E reduced endogenous Cav-1 expression by
siRNA treatment did not alter the level of ABCA-1
expression.


Interaction of Nef and Cav-1
Since over-expression of Cav-1 does not alter ABCA-1
expression, the mechanism of restoration of cholesterol
efflux by Cav-1 that is impaired by Nef may not involve
the level of ABCA-1 expression. Nef has been shown to
bind to ABCA-1 [38,42], and it is not known whether
Cav-1 interacts with Nef. Cav-1 may associate, either
directly or indirectly, with Nef thereby countering the
impairment of cholesterol efflux. To determine whether
there is a physical association between Cav-1 and Nef,
we performed co-immunoprecipitation and immuno-
blotting experiments. U87-CD4-CXCR4 cells were co-
transfected with pCZ-Cav-1 and HA-tagged wild type
Nef (pCI NL4-3 Nef-HA-WT) or the NefG2A mutant
(pCI NL4-3 NefG2A-HA). Transfected cells were then
cultured in medium containing cholesterol (30tg/ml) for
48 hours followed by treatment with apoA-I (20tg/ml)
for 30min. Cell lysates were then subjected to co-immu-
noprecipitation and analyzed for Cav-1 and Nef interac-
tions by immunoblotting. As shown in Figures 5A and
5B the association of Cav-1 and Nef is evident in U87
cells. Interestingly, there was no NefG2A mutant inter-
action with Cav-1 implicating the association of Nef and
Cav-1 is at the cell membrane. We examined the en-
dogenous interaction of Cav-1 and ABCA-1 in U87 cells
and were unable to show ABCA-1 association with Cav-
1 by co-immunopreciptation and immunoblotting ana-
lysis (data not shown). The interaction of Cav-1 with
Nef suggests that Cav-1 by associating with Nef blocks
the activity of Nef and subsequently helps restore cho-
lesterol efflux impaired by Nef.


A.
ak $
d^ ~ > ^


IP : HA-tag
WB: Cav-1






B.



IP: Cav-1
IB: Nef


IB: Cav-1


a- Cav-1


Nef










S avNef-

~~-Cay-1


Figure 5 Interaction of Cav-1 and Nef. U87-CD4-CXCR4 cells were
co-transfected with pCZ-Cav-1 and HA tagged Nef(pCI NL4-3
Nef-HA-VVT) or NefG2A(pCI NL4-3 NefG2A-HA). Twenty-four hours
post transfection the cells were cultured in the presence of
cholesterol (30 pg/ml) for 48 hours followed by apoA-I (20 pg/ml)
for 10 min. (A) The cells were harvested and subjected to
immunoprecipitation using anti-HA antibody and immunoblots
using anti-Cav-1 or anti-Nef antibody. (B) Alternatively,
immunoprecipitaion was performed using anti-Cav-1 and
immunoblotting using anti-Nef antibody.


Cav-1 reduces HIV-1 infectivity by reducing the
cholesterol content of virus particles
HIV is well known to rely on the host cellular cholesterol
machinery for efficient replication and particle formation
[25,26,28,30,44]. Since our results show that Cav-1 res-
tores Nef impaired cholesterol efflux, we sought to deter-
mine if the promotion of this efflux by Cav-1 would have
an impact on the infectivity of released virus particles. In
order to demonstrate whether Cav-1 influences HIV in-
fectivity, primary macrophages (MDMs) were transduced
with Ad-Cav-1 or the control Ad-GFP. The level of GFP
and Cav-1 expression in macrophages from which culture
supernatant harvested is shown in Figure 6A. Twenty-four
hours post transduction the macrophages were infected
with HIV AD8. Virions produced from these infected
macrophages were titered using the TZM-bl indicator cell
line and normalized for the infectivity studies. As shown
in Figure 6B infectivity of virus harvested from Cav-1 trea-
ted macrophages was reduced by 46% compared to Cav-1
untreated HIV infected cells. There was no significant
difference in infectivity of virus particles obtained from


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B.
120%
Ad-GFP Ad-Cav-1 100%
J2 s 80%
8 60%
-actin 40%
20%
S 0%


2500
S2000
S1500

I
2 1000
3 500




Figure 6 Cav-1 over-expression inhibits HIV-1 infectivity. MDMs were infected with AD8 o
combination with Ad-Cav-1 or Ad-GFP. (A) The levels of GFP and Cav-1 expression are shown.
culture supernatants were titered and normalized. Level of infectivity was measured by infectir
experiments were performed in triplicate and results shown are mean + SD with P values.


Ad-GFP transduced cells when compared to that of virus
harvested from Cav-1 untreated HIV infected cells. Simi-
lar experiments were performed using ADnefmut infec-
tions. Contrary to what was observed with AD8 HIV the
level of infectivity of ADnefmut virus harvested from
Ad-Cav-1 or Ad-GFP treated cells remained the same
(Figure 6C). This, therefore, establishes that Cav-1 impairs
HIV infectivity implicating that this may be linked to Cav-
1 mediated promotion of cholesterol efflux by apoA-I that
is impaired by Nef during HIV infection. Since Cholesterol
within the HIV particle is strictly required for infection,
our next set of experiments were aimed at investigating
whether the reduction of HIV infectivity is related to the
modulation of lipid content of the virions. HIV provirus
DNA was co-transfected into U87 cells with pCZ-Cav-1
or pCZ-vector. Virus harvested from transfected cells was
concentrated and normalized by a p24 ELISA assay. Equal
amounts of virus particles were used to measure the cho-
lesterol composition in the virion by the Amplex Red
cholesterol Assay Kit. As shown in Figure 7A the cho-
lesterol content of virus particles harvested from cells re-
ceiving Cav-1 was reduced by 48% compared to cells
transfected with pCZ-vector. In addition, cholesterol was
replenished within concentrated virus that was normalized
and equal amounts were treated with (2-Hydroxypropyl)-
B-Cyclodextrin (CD) and saturated exogenous cholesterol
to see if infectivity could be restored. Infectivity of choles-
terol replenished virus was measured by luciferase activity
in infected TZM-bl cells. The infectivity of virus particles


0 i0i
_


r ADnefmut at an moi of 0.1 alone or in
(B) and (C) Infectious particles harvested from
g TZM-bl cells and subsequent luciferase assay. AI


collected on Cav-1 treated cells was reduced by 58% as
compared to those collected on cells treated by pCZ-
vector (Figure 7B). There was no difference in infectivity
of cholesterol replenished and control viral particles col-
lected from pCZ-vector treated cells (Figure 7B). As might
be expected the infectivity of cholesterol replenished virus
particles collected from Cav-1 treated cells was increased
by 37% compared to the virus particles harvested from
Cav-1 treated control (Figure 7B). In addition, we exa-
mined whether Cav-1 is incorporated into the virion to
make sure that such incorporation has not affected infec-
tivity directly rather than influencing the cholesterol con-
tent of the HIV virus particles. As shown in Figure 7C, we
observed that Cav-1 protein is not incorporated in the
virus particles as determined by Western blot analysis.
Therefore, Cav-1 reduces virus infectivity by promoting
cholesterol efflux which consequently decreased the avai-
lability of cholesterol during viral particle formation.

Discussion
HIV has been indicated to manipulate host cholesterol
metabolism, leading to excessive cholesterol accumula-
tion in infected T cells or macrophages [38,45], thereby
supporting efficient viral replication. In the absence of
proper esterification to fatty acid and efflux, cholesterol
accumulates in the endoplasmic reticulum eventually lea-
ding to ER dysfunction and the activation of an ER stress
associated apoptosis pathway [46-48]. Cav-1 is an impor-
tant cellular cholesterol regulator, and its expression is


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S HI
HIV/pCZ-vector HIV/pCZ-Cav-1


gag -24KD

Nef V! -Z6KD
64 -17KD


Cav-1


120%
100%
80%
60%
40%
20%
0%



12000


-22KD


P<0.05


* Control
O Replenished chol


S /


P<0.05


10000
-
-
C 8000

w
E 6000

3 4000

2000


0 1 I 1 I
HIV/pCZ-vector HIV/pCZ-Cav-1
Figure 7 Cav-1 over-expression reduces the cholesterol content in HIV-1 virus particles. (A) Proviral DNA genome NL4-3 was transfected
into U87 cells either with pCZ-vector or with pCZ-Cav-1. Virus particles generated were concentrated and normalized by p24 assay. Cholesterol
contents were measured using the Amplex Red cholesterol Assay Kit. The results are presented as percentage of cholesterol content relative to
control (HIV/pCZ-vector, set as 100%) and are the mean SD of triplicate experiments with P values. (B) Normalized samples were replenished
with exogenous cholesterol, and the level of infectivity was measured by infection of TZM-b cells and luciferase assay. All experiments were
performed in triplicate, and results shown are mean SD with P values. (C) Normalized samples from samples of (7A) were subjected to Wester
blot analysis using antibody to Gag, Nef, and Cav-1 to determine whether Cav-1 protein is incorporated in virus particles.


dramatically enhanced in HIV infected macrophages
[17], implicating a role for Cav-1 in HIV associated
cholesterol alterations. Cav-1 is a structural component
of Caveolae membrane microdomains, which have been
suggested to play an important role in cholesterol traf-
ficking and efflux. In this study, we investigate the effect
of Cav-1 on the cholesterol efflux in HIV infected macro-
phages and human astrocytes-derived glioblastoma U87
cells. Our results show that Cav-1 restores the Nef
induced impairment of cholesterol efflux by apoA-I. Fur-
thermore, this restoration causes a reduction in the chol-
esterol composition of virus particles leading to decreased
HIV infectivity. This suggests a role for Cav-1 in macro-
phage HIV persistent infection by enhancing cholesterol
efflux.
Our results show neither Nef nor Cav-1 had significant
effect on HDL mediated cholesterol efflux. HDL plays an
important role in reverse cholesterol transport (RCT), in
which HDL transports cholesterol from peripheral tissues
to liver for excretion. RCT is a multifaceted, dynamic
pathway which is involved with multiple molecules and
effectors. The first step in RCT is ABCA-1 dependent ef-
flux of cholesterol and phospholipids to apoA-I, the major


component of HDL. ABCA-1 interacts with apoA-I and
stimulates free cholesterol and phospholipids efflux re-
sponsible for nascent HDL formation [49]. Wang et al.
[50] reported that ABCA-1 expression markedly increases
apoA-I but not HDL mediated lipid efflux; the reason
could be that compared with HDL, apoA-I is the preferred
acceptor for ABCAl-promoted cholesterol and phospho-
lipid efflux. We also found upon HIV infection Nef down
regulates ABCA-1 expression, which dramatically inhibits
apoA-I mediated cholesterol efflux, whereas HDL me-
diated cholesterol efflux was not affected by HIV infection.
Over-expression of Cav-1 restores the impaired choles-
terol efflux to apoA-I significantly, but not so much on in-
tact HDL cholesterol efflux.
Promotion of cholesterol efflux by over-expression of
Cav-1 is observed in hepatic cells [9]. Cav-1 can enhance
the transfer of cholesterol to cholesterol-rich domains
in the plasma membrane, where it is accessible to efflux.
Multiple mechanisms are proposed for Cav-l's regula-
tion of cholesterol homeostasis. These include the mo-
dulation of the expression of lipoprotein receptors and
the activity of proteins involved in lipid metabolism as
well as interactions with lipid transport or transport of


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cholesterol to the plasma membrane facilitating choles-
terol efflux [6-8,22,43]. ABCA-1 expression is important
in regulating cholesterol efflux to apoA-I and it has been
implicated that ABCA-1 stimulates the reorganization of
plasma membrane microdomains to facilitate cholesterol
efflux to apoA-I [51,52]. Cav-1 can regulate cholesterol
homeostasis by modulating the expression of lipid regu-
lators. Reduced levels of ABCA-1 have been observed in
macrophages of Cav-1 knockout mice [43]. Our results
show that we observe no change in the level of ABCA-1
expression when Cav-1 is over-expressed suggesting that
the endogenous Cav-1 expression is sufficient enough to
maintain physiologically relevant levels of ABCA-1 and
that ..i-, 1..... i amounts of Cav-1 does not have an im-
pact on ABCA-1 expression. The reduced level of
ABCA-1 observed in the knockout mice is in complete
absence of Cav-1 expression. ABCA-1 dependent choles-
terol efflux can be impaired by HIV Nef mediated down
modulation and di. i,:i-. of the intracellular :i .-i !:Ii,,..
of ABCA-1 ""1: "1 Similarly we observed a 69% decrease
in ABCA-1 expression in the presence of Nef. Interes-
tingly the decrease in ABCA-1 remains the same when
additional amounts of Cav-1 are provided indicating that
the reversal of Nef's effect on cholesterol efflux by Cav-1
is not related to the level of ABCA-1 expression. Inhib-
ition of ABCA-1 protein expression, as it pertains to Nef,
in part depends upon the ER associated proteasomal deg-
radation mechanism [42]. An unknown ..:l.:i] ..:.! path-
way unrelated to proteasomal activity is also suggested to
contribute to ABCA-1 degradation. .i.... i, ABCA-1 is
shown to interact with Nef the physical association is not
essential for Nef mediated down-regulation of ABCA-1 ef-
flux activity .. i:-. However, the influence of cellular dis-
tribution of ABCA-1 by Nef has been determined using
confocal microscopy with Nef causing a prominent trap-
ping of ABCA-1 in the ER [42]. ABCA-1 expression has
been implicated in .11,, ..... ... the J..'. t i.. ... of cho-
lesterol and Cav-1 [52]. Redistribution of Cav-1 from
punctate caveolae-like structures to the general area of the
plasma membrane is observed upon ABCA-1 expression.
Our co-immunoprecipitation study reveals an interaction
between Cav-1 and Nef. Furthermore, our observation
that Cav-1 does not interact with the myristoylation de-
fective Nef 1'i .-_ F,.-_. mutant) implicates an association of
these proteins at the plasma membrane. These observa-
tions .* i that the interplay of Cav-1 with Nef and
cholesterol subsequently counters Nef induced impair-
ment of cholesterol efflux by apoA-1. In addition, since
caveolae is a major source and platform for cholesterol
efflux [4] over-expression of Cav-1 may induce the forma-
tion of more caveolae, which should subsequently enhance
cholesterol efflux. The presence of Cav-1 in macrophages
and its '!I- -' li: .." upon HIV infection, therefore, can
contribute to increased cholesterol efflux in these cells.


Cholesterol is an important structural component of
HIV particles and their cholesterol content is tightly lin-
ked to HIV infectivity [25,27,44]. Cholesterol depletion
significantly reduces HIV-1 particle production [29-34,44].
There is also a marked decrease in infectivity of virions
produced from such cells [26]. The significant reduction
correlates with the amount of virion-associated choles-
terol [35]. In the current study, we clearly established that
Cav-1 significantly reduces infection with virions pro-
duced from Cav-1 treated cells when compared to that of
the same number of virions obtained from untreated cells.
We have previously shown that Cav-1 represses HIV gene
expression by blocking the NF-iB pathway thus subse-
quently affecting virus production [18]. The decrease in
virus production is therefore in part due to transcriptional
suppression of HIV gene expression. Here, we examined
the cholesterol content of HIV particles produced from
Cav-1 treated cells and clearly established a significant
cholesterol decrease in virus particles. Furthermore, nor-
malized amounts of virus in the infectivity assay of HIV
released from Cav-1 treated cells shows that infectivity is
.. n, dII; reduced. Normalized amounts of virus to assay
for infectivity, rules out any concern regarding the level of
virus release i..fi..l.,:ii: to the reduction of infectivity.
The major step that causes a decrease in virion infectivity
related to cholesterol depletion is the fusion steps of infec-
tion [30]. In i..... i of this notion, we previously demon-
strated that Cav-1 significantly suppressed Env-induced
membrane hemifusion [16], indicating that the decrease
in fusion partly involves a reduction in the cholesterol
S...M.!... i;... of the plasma membrane. Cav-1 can counter
the influence of HIV on cholesterol metabolism by pro-
moting cholesterol i' :r; 1;..L to the membrane subse-
quently enhancing cholesterol efflux, therefore, depriving
the HIV virion of cholesterol. Since Cav-1 is involved in
cholesterol metabolism the up-regulation of Cav-1 can
have an impact on the level of cellular cholesterol thereby
i .i.. .I.,. ..... to a reduction in virus production and infec-
tivity, .*!!. !!i1' contributing to a persistent infection
of macrophages.

Conclusion
Infected macrophages are relatively resistant to cyto-
pathic effect and consequently play an essential role in
viral dissemination to host tissues and organs [53-55].
Furthermore, in this viral reservoir HIV infection ap-
pears not to be associated with apoptosis but with a
chronic productively infected phenotype [56,57]. Ati....
several mechanisms have been proposed we still don't
have a clear picture as to the mechanisms of persistent in-
fection in macrophages. The relationship between HIV-1
and host factors determines the modulation of both cellu-
lar functions and virus **i,, i r.... within an infected indi-
vidual, and with the interaction of these viral and cellular


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factors being evident in all steps of virus replication
[58-62]. Their associations may be an important fac-
tor in the modification of host cell processes during a
chronic viral infection. This suggests that a persistent
infection is regulated by cellular factors at different steps
in virus replication. Gene expression profiles that are uni-
que to macrophages [63-65] when compared to that of
activated CD4+ T cells should help determine the mech-
anism of persistent infection in macrophages. Cav-1 is
highly expressed in terminally differentiated or quiescent
cells including dendritic cells and monocytes/macro-
phages [2,66]. While macrophages express Cav-1,
human T cells are generally believed to lack the Cav-
1 protein [67-69]. The lack of Cav-1 in T cells, our dis-
covery that HIV infection enhances Cav-1 expression
mediated by Tat in macrophages and that Cav-1 reduces
HIV replication [17] suggests a role for Cav-1 in an HIV
persistent infection of macrophages. Here we have shown
that Cav-1, by restoring cholesterol efflux impaired by Nef
and subsequently influencing the cholesterol content of
HIV particles which negatively affects virus infectivity, ef-
fectively inhibits HIV replication contributing to macro-
phage HIV persistent infection (Figure 8).


Methods
Plasmids
The HIV-1 proviral constructs pNL4-3 (T-tropic), pNL-
AD8 (M-tropic), pWT/Bal (M-tropic), pNL4-3.Luc.R-E-,
and pSG3Aenv were kindly provided by NIH AIDS Re-
search and Reference Reagent Program [70-76]. The con-
struct pNL4-3.Luc.R-E- is defective for env and nef where
as pSG3Aenv has intact nef, but a deletion in env. An ex-
pression plasmid for vesicular stomatitis virus envelope
G protein (pCI-VSV) was kindly provided by Jiing-Kuan
Yee of City of Hope National Medical Center, Duarte,
California. A Cav-1 expressing plasmid, pCZ-cav-1, was
generated as described previously [16]. pCZ-vector is the
same as pCZ-cav-1 except it lacks the coding sequence of
cav-1. The Nef expression plasmid pcDNA3.1SF2Nef was
provided by NIH AIDS Research and Reference Reagent
Program [77,78]. A construct expressing Nef tagged with
HA (pCI NL4-3 Nef-HA-WT) was purchased from
Addgene Inc (Cambridge, MA). The NefG2A mutation
plasmid was generated using a site-directed mutagenesis
kit according to the manufacturer's protocol (Strategene).
Briefly, the mutation was generated by PCR amplifica-
tion using pCI NL4-3 Nef-HA-WT as template and the


HfV infection ll
~jj Tat
Nef^ (ID 7^^^^^^^^^^"1^^^^^^^^^^^^


ABCA1



ABCA-1 down regulation
or land redistribution


Cav-1 upregulation





4I


Impair Stimulate
cholesterol efflux _---- i cholesterol efflux


Lipids accumulation in cells


Efficient viral production
Efficient viral production


Reduced viral cholesterol content


Reduced viral infectivity
Reduced viral infectivity


Virus Balance Host

Cholesterol machinery
Figure 8 A model for the interplay of Cav-1 with Nef that counters Nef induced impairment of cholesterol efflux by apoA-l and
contribution to persistent infection. Upon HIV infection, Nef protein interacts with ABCA-1 and down-regulates and/or redistributes ABCA-1
expression which results in cholesterol efflux impairment. This creates a micro-environment in which HIV can replicate efficiently. On the other
hand, in cells which express Cav-1, such as macrophages, Tat induces an up-regulation of Cav-1. The overabundance of Cav-1 then leads to botl
its binding to Nef and ability to activate cholesterol efflux. This, therefore, leads to less cholesterol accumulation which in turn reduces the
amount that can be incorporated into viral particles and thereby reducing HIV infectivity.


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following pair of primers: 5'-ggattttgctataagatggctggcaag
tggtcaaaaagt-3' and 5'-actttttgaccacttgccagccatcttatagcaa
aatcc-3'. The PCR products were digested with the restric-
tion enzyme DpnI to destroy template plasmids and were
then transformed into DH5a competent cells. Introduc-
tion of the mutation (pCI NL4-3 NefG2A-HA) was con-
firmed by sequence analysis. Wild type AD8 and a
replication competent nef defective AD8 derived HIV
provirus DNA construct ADnefmut [79] were provided by
Dr. Maureen Goodenow of the University of Florida.
Adenovirus particles (Ad) for expressing Cav-1 (Ad-Cav-1)
and GFP (Ad-GFP) were obtained from Vector Biolabs
(Philadelphia, PA).

Cell cultures
Human U87MG-CD4 cells stably transfected with CXCR4
(U87-CD4-CXCR4) or CCR5 (U87-CD4-CCR5), human
acute monocytic leukemia (THP-1),and an indicator cell
line for tittering HIV (TZM-bl) was .1I iI: provided by
the NIH AIDS Research and Reference Reagent Program.
U87-CD4-CXCR4 were maintained in DMEM containing
15% FBS, penicillin-streptomycin (100 ;. '.l i glutamine,
puromycin (1pg/ml; Sigma Cl. l, 1i', and neomycin
(G418; 300pg/ml; Sigma). THP-1 cells were grown in
RPMI-1640 containing 10% FBS, 1.0mM sodium pyruvate,
and 0.05 mM 2-mercaptoethanol. For ,11. .. '. .*i.. into
macrophages, THP-1 11 were treated with 50 ng/ml of
phorbol 12-myristate 13-acetate (PMA, Sigma Chemical)
for 5 days until the cells adhered and exhibited ma-
crophage-like morphology. TZM-bl and 293T cells were
grown in DMEM medium supplemented with 10% FBS
and penicillin-streptomycin (ilp ,,i,'!: i All cultures were
maintained at 37C in a humidified atmosphere with 5%
C02.
Peripheral blood mononuclear cells (PBMCs) were iso-
lated from buffy coats prepared from healthy donors by
centrifugation through a Ficoll gradient '*",...i .-Aldrich,
St. Louis, MO). Monocytes were isolated by negative se-
lection with a human monocyte enrichment kit accor-
ding to the manufacturer's instructions (EasySep' Human
Monocyte Enrichment Kit, Stemcell T 1,.!1..L;. The
monocyte preparations contained 97% CD14' cells, as de-
termined by flow cytometry. For iii... ii. il.... of mono-
cytes into macrophages (MDMs), monocytes were seeded
into Biocoat poly-D-lysine plates (B.D. Bioscience), and
cultured in 1 :,.i I supplemented with 10% heat-inacti-
vated human serum, gentamicin -.iiy, (, -mi ciprofloxacin
(i i., i,,:!i. and M-CSF (1000U/ml) for 7 days. MDM cul-
ture medium was half-exchanged every 2 to 3 days.

Transfection of siRNA
Small interfering RNA (siRNA) targeting Cav-1 and con-
trol siRNA were purchased from Santa Cruz Biotechno-
logy, Inc. Transfection of siRNA was performed using


OligofectaminrT Reagent (Invitrogen Corp., Carlsbad,
( J:') according to the manufacturer's protocol. Briefly,
the day before transfection, U87cells were seeded into a
24 well plate and cultured with antibiotics free medium
to 30% confluence. Cells were washed and resuspended
in 200ul serum free medium. Transfection mixture was
prepared by incubating 50pmol of siRNA duplexes with
3ul of (.li...I. i..,... in a final volume of 50ul Opti-
MEM I Medium. After a 5 hour incubation, 125ul of
growth medium with 3 times the normal concentration
of serum was added to cells. Transfection was repeated
once the next day. For THP1 macrophages, cells were
first transfected with siRNA followed by HIV infection,
and cells were then transfected again with siRNA the
day after infection. The efficiency of Cav-1 knock-down
by the siRNA transfection was monitored using Western
blot analysis.

Virus production and concentration
Infectious virus HIV-1 AD8, ADnefmut, Bal, and NL4-3
were generated by calcium phosphate transfection of
monolayers of 293T cells in 75-cm2 flasks with 25Lg
provirus DNA. Supernatants containing virus were har-
vested 4 days after transfection and :,n,'(r-..:l using the
TZM-bl indicator cells as well as by measuring reverse
transcriptase and a p24 ELISA method as described pre-
viously [17]. When required, virus was produced from
U87-CD4-CXCR4 cells transfected with 18[g proviral
HIV NL4-3 along with 9pg pCZ-Cav-1 or pCZ-vector.
To generate pseudotyped HIV particles 20ug pSG3A e.
or pNL4-3.Luc.R-E- was co-transfected with 3pg pCI-
VSV into monolayers of 293T cells in 75-cm2 culture
flasks by the calcium phosphate method. Pseudotyped
viral supernatants were harvested 4 days post-transfec-
tion and were I I,:..-1 by .. .. tFi,. 'i,.. at 3,000 rpm for
20 min and then by : .!r.i through a 0.45 mu-pore size
filter. Virus particles were concentrated using virus preci-
pitation reagent Retro-ConcentinTM (System Biosciences)
according to the manufacturer's protocol.

Oil red 0 staining
To determine the influence of Cav-1 on the level of lipid
accumulation in HIV infected and uninfected li. oil
red O staining was performed. THP-1 cells were differ-
entiated into macrophages by treatment with 50 ',.1
PMA for 5 days then infected with HIV AD8 (moi, 0.01)
or Bal (moi, 0.001). On day 10 post infection, cells were
loaded with cholesterol by incubating with =ipt ':'l Ac-
LDL i:'. ..I.. .i.!. Technologies Inc., ;~,.,-i ....' M A) for
48 h followed by .'iq. ..i apoA-I stimulation for 18
hours. D1il. .. li, it. TH P-1 II i 11- 1I ii t, .i m acro-
phage cells were also infected with Ad-Cav-1 or Ad-GFP
at an moi (multiplicity of infection) of 100. Twenty-four
hours later they were infected with VSV pseudotyped


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HIV pSG3sen" or pNL4-3.Luc.R-E- at an moi of 3 and
incubated for 5 days. Oil red O staining was performed
as previously described [80]. Briefly, cells were rinsed
with PBS, followed by fixation with 3.7% paraformalde-
hyde for 60 min. The cells were stained using freshly
prepared Oil red O (Sigma) working solution at room
temperature for 10min. Intensity of cell staining was
observed using a light microscope.

Virus infectivity assay
To test Cav-l's influence on HIV-1 infectivity, TZM-bl
cells were infected with virus harvested from Cav-1 trea-
ted cells and the infectivity levels were measured by
luciferase activity. MDMs were first infected with adeno-
virus expressing Cav-1 or GFP at an moi of 100 in serum
free medium for 6 hours. The cells were then washed
and incubated in serum-containing medium over-night,
after which cells were infected with HIV AD8 at an moi
of 0.1 for 6 hours, at which point they were washed and
refreshed with new medium. On day 6 post infection,
supernatants were subjected to RT assay or titered using
the ..1i. h'.- TZM-bl cell line. Virus amounts were nor-
malized with level of infectivity being assayed by mea-
suring luciferase within TZM-bl cells [74]. Normalized
amounts of virus were used for subsequent infections.

Determination of cholesterol content and cholesterol
replenishment assay
Equivalent amounts of virions were quantified by ..2i
assay and tested for cholesterol content using the Amplex
Red cholesterol Assay Kit ,i-.. ;.,. i Carlsbad, CA) ac-
cording to the manufacturer's protocol. To replenish cho-
lesterol virus amounts were also normalized by p24 assay
and incubated in 0.5mM (2-Hydroxypropyl)-fi-Cyclodex-
trin solution (Sigma Aldrich) with 1.5mM cholesterol
(Sigma Aldrich) at 37C for 1 hour. These .,i r ir-.:_ and
normalized amounts of virus were used to infect TZM-bl
cells and monitored for luciferase activity.

Cholesterol efflux
U87 cells were transfected with pcDNA3.1 and pCZ-
vector (mock), pcDNA3.1SF2Nef and pCZ-vector (Nef),
pCZ-cav-1 and pcDNA3.1 (Cav-1), pcDNA3.1SF2Nef
and pCZ-cav-1 (Nef plus Cav-1), pCI NL4-3 NefG2A-
HA (NefG2A) and pCZ-vector, or pCI NL4-3 NefG2A-
HA and pCZ-cav-1 (NefG2A plus Cav-1). Twenty-four
hours after transfection cell culture medium was re-
placed with serum free medium containing 2 pCi/mL
[3H] cholesterol and 1.5% BSA and incubated for 36
hours. Radioisotope-containing medium was then re-
moved and cells were washed twice with PBS and cul-
tured for an additional 18 hours in serum free medium
in the presence or absence of 50 ,-',1l ApoA-1 l '
dical Technologies Inc., Stoughton, MA). Cholesterol


content was measured in the cell free media as well as
within cells after losing using 0.1N NaOH. ApoA-1 .i'. : 1-
cholesterol efflux was determined using the formula: apoA-
1 ... ii,. efflux = % cholesterol efflux with apoA-1 % cho-
lesterol efflux without apoA-1 (blank); cholesterol efflux=
[cpm(supernatants)/, 1""( "i-... In I I .1, 1 )] x100%. HDL
mediated cholesterol efflux is also examined by incubating
cells for 18 hours in the presence or absence of 50 pg/ml
HDL (Biomedical Technologies Inc., Stoughton, MA).
To determine cholesterol efflux from macrophages,
MDMs were first infected with Ad-Cav-1 or Ad-GFP at
an moi of 50 for 24 hours, which was followed by infec-
tion of pseudotyped HIV pSG3Aenv (psHIVwtNef) or
pNL4-3.Luc.R-E- (psHIVANef). Five days post infection
cells were then labeled with 1 VCi/mL [3H] cholesterol
for 48 hours and apoA-l mediated cholesterol efflux was
determined as described above. Similarly cholesterol ef-
flux from THP-1 cell- i,. i.. nii, i macrophages was
determined 21 days after infecting with HIV AD8 at an
moi of 0.001. Cholesterol efflux was also determined 14
days after THP-1 I. I'-. ,. 'entiated macrophages in-
fected with an moi of 0.001, 0.01, or 0.1. In .,I.la..I, pri-
mary macrophages (MDMs) were infected with AD8 or
ADnefmut HIV with an moi 0.01 and then cultured cells
were subjected to cholesterol efflux assay 15 days after
infection. ABCA-1 expression was determined by West-
ern blots in MDMs 14 days after co-infection with AD8
or ADnefmut HIV and with Ad-Cav-1 or Ad-GFP. In-
hibition of HIV replication was i:.. .i.- by treating
infected cells with 5 uM azidothymidine (AZT)
Aldrich, St. Louis, MO).

Immunoprecipitation and Immunoblotting analyses
U87 cells were transfected with pCZ-Cav-1 and HA-
tagged Nef (pCI NL4-3 Nef-HA-WT) or HA-tagged
NefG2A (pCI NL4-3 NefG2A-HA), :..!! ...:1 by incuba-
tion of medium containing cholesterol i, :. ,* "11 for 48
hours. Cells were then treated with apoA-I (20pg/ml) for
30 min. ( 1:. were put on ice, washed twice with cold
PBS and total cellular protein was extracted in lysis buf-
fer (50 mM Tris pH 7.5,100 mM NaC1, 1 mM EDTA,
0.1% (v/v) Triton X-100, 10 mM NaF, 1 mM phenyl-
methyl sulfonyl fluoride, and 1 mmol/L vanadate) with a
complete protease Inhibitor mixture (Roche Diagnostics,
Indianapolis, IN). The concentration of extracted protein
was determined and adjusted to 1 ug/ul. A total of 500
ul was used for each immunoprecipitation, to which 2tig
of antibodies (anti-Cav-1 or anti-HA) or normal IgG
were added. The mixtures were incubated at 4C over-
night. F. 1il ..... the overnight incubation, 25 pl of pro-
tein A,' -- !.. beads (Santa Cruz F...t li... % .
Santa Cruz, CA) were added and the mixtures were then
rotated for 2 hours at 4C. The beads were harvested by
centrifugation and washed five times with lysis buffer.


Page 13 of 16







Lin et al. Retrovirology 2012, 9:85
http://www.retrovirology.com/content/9/1/85


Loading buffer was added and boiled for 5 min. The sam-
ples were subjected to SDS-PAGE and analyzed by im-
munoblotting as described previously [81]. The primary
,.:iii.,.... used for immunoblotting were rabbit poly-
clonal anti-Cav-1(Santa Cruz F.,..!,,.,. Santa Cruz,
CA), mouse monoclonal anti-Nef, rabbit Nef antiserum,
and human monoclonal anti-Gag (NIH AIDS Research
and Reference T .. :t Program), goat polyclonal anti-HA
(Genescript), mouse monoclonal anti-ABCA1 (abcam),
and f-actin protein antibody (Sigma, St. Louis, MO). The
secondary .,,!i..i. were HRP-linked anti-rabbit, anti-
mouse (Cell Signaling Technology, Inc., Danvers, MA),
anti-human IgG (Sigma, St. Louis, MO) or anti-goat IgG
(Santa Cruz Biotechnology, Santa Cruz, CA).


Statistical analysis
Student's t test was ,i... .1 to analyze the differences bet-
ween sets of data. All analyses were performed with SPSS
12.0.1 for Windows, and were considered significant at
p < 0.05.

Competing interests
The authors declare that they have no competing interests

Authors' contributions
SL Participated in the design of experiments, carried out most of the
experiment, prepared samples and conducted cholesterol analysis and
Western blots, analyzed data and contributed lo manuscript preparahon PN
participated and assisted in sample collections and experiments XW was
involved in the design of constructs used for tee study as well as establish
efficient methodology to deliver of protein of interest in primary
macrophages AM conceived the study, designed and coordinated
experiments, participated in data analysis and prepared contributed the
manuscript Al authors read and approved the final manuscript

Acknowledgements
This research was supported by a grant from the National Institutes of
Health (Ai39126) to AM

Received: 18 June 2012 Accepted: 26 September 2012
Published: 15 October 2012

References
1 Rothberg KG, Heuser JE, Donzell WC, Yng YS, Glenney JR, Anderson RG'
Caveolin, a protein component of caveolae membrane coats. Celi 1992,
68:673-682
2 Harris Werling D, Hope JC, Iaylor G, Howard .J Caveolae and caveolin
in immune cells: distribution and functions. Trends imr unol 2002
23:158-164
3 Galbiati F, Volonte D, Liu j, Capozza F, Frank PG, Zhu L, Pestell RG, Lisanti
MP Caveolin-1 expression negatively regulates cell cycle progression by
inducing G(0)/G(1) arrest via a p53/p21(WAF1/Cipl)-dependent
mechanism. Mo! Bio Ce; 2001, 12:2229-2244
4 Fielding PE, Russel JS, Spencer TA, I akamaa ii, Nagao K, Fielding CO Sterol
efflux to apolipoprotein A-1 originates from caveolin-rich microdomains
and potentiates PDGF-dependent protein kinase activity. Biocheirstiy
2002, 41:4929-4937
5 Gargalovic P, Dory L Cellular apoptosis is associated with increased
caveolin-1 expression in macrophages. Lipid Res 2003, 44:1622-1632
6 Gargalovic P, Dory I Caveolins and macrophage lipid metabolism. J Lipid
Rei 2003, 44:11-21
7 Le PU, Guay G, Altschuler Y, Nabi IR Caveolin-1 is a negative regulator of
caveolae-mediated endocytosis to the endoplasmic reticulum. j Biol
Chem 2002, 277:3371-3379


8 Chao VvT, Fan 55, Chen JK, Yang VC Visualizing caveolin-1 and HDL in
cholesterol-loaded aortic endothelial cells. J Lipid Res 2003, 44:1094-1099
9 Fu Y, long A, Escher G, Parton RG, Krozowski Z, Sviridov D Expression of
caveolin-1 enhances cholesterol efflux in hepatic cells. J Biol Chem 2004,
279:14140-14146
10 Couet j, Li S, Okamoto T, Ikezu T, Lisanti MP Identification of peptide and
protein ligands for the .*. ... i domain Implications for the
interaction of caveolin with caveolae-associated proteins. Biol hern
1997, 272:6525-6533
11 Quest AF, I yton Parraga M' Caveolins, caveolae, and lipid rafts in
cellular transport, signaling, and disease. Biochem Cel Bio! 2004,
82:129-144
12 Williams TM, Lisanti MP Caveolin-1 in oncogenic transformation, cancer,
and metastasis. Am Physio Cell Physioi 2005, 288:C494-C506
13 Huang H, I u i u H, Chen X, iang S, Chen YH Identification of the HIV-1
gp41 core-binding motif in the .. "_ ., i domain of caveolin-1. J Biol
Chemo 2007, 282:6143-6152
14 lovanessian AG, Briand JP, Said EA, Svab J, Ferris S, Dali Il, Muller 5,
Desgranges C, Krust B The caveolin-1 binding domain of HIV-1
glycoprotein gp41 is an efficient B cell epitope vaccine candidate
against virus infection. immunity 2004, 21:6 7-627
15 Benferhat R Krust B Rey Cuille MA, Hovanessian AG The caveolin-1
binding domain of HIV-1 glycoprotein gp41 (CBD1) contains several
overlapping neutralizing epitopes. Vaccine 2009, 27:3620-3630
16 Wang XM, Nadeau PE I o YI Mergia A Caveolin-1 modulates HIV-1
envelope-induced bystander apoptosis through gp41. Vi ro 2010,
84:6515-6526
17 Lin S, Wang XM, Nadeau PE, Mergia A HIV infection upregulates caveolin
1 expression to restrict virus production. J Virol 010, 84:9487-9496
18 Wang XM, Nadeau PE, Lin S, Abbott JR, Mergia A Caveolin 1 inhibits HIV
replication by transcriptional repression mediated through NF-kappaB.
1 Virol 2011, 85:5483-5493
19 Lin VC, Ma C, su WC, Lo I F, Yang VC Molecular interaction between
caveolin-1 and ABCA1 on high-density lipoprotein-mediated cholesterol
efflux in aortic endothelial cells. Cardiovasc Res 2007, 75:575-583
20 Ikonen E, Parton RG' Caveolins and cellular cholesterol balance. Traffic
2000, 1:2 12- 17
21 Truong TQ, Brodeur MR, Falstrault L, Rhainds D, Brissette L Expression of
caveolin-1 in hepatic cells increases oxidized LDL uptake and preserves
the expression of lipoprotein receptors. i Cell Biochem 2009, 108:906-915
22 Smart F Ying Y, Donzell WC, Anderson RG' A role for caveolin in transport
of cholesterol from endoplasmic reticulum to plasma membrane. i Biol
Chemo 1996, 271:29427-29435
23 Uittenbogaard A, Ving Y, mrart E' Characterization of a cytosolic heat-
shock protein-caveolin chaperone complex Involvement in cholesterol
..I B ., i Biol Chem 1998, 273:6525-6532
24 Truong TQ, Aubin D, Falstrault L, Brodeur MR, Brissette L SR-BI, CD36, and
caveolin-1 contribute positively to cholesterol efflux in hepatic cells. Cehl
Biochern Funr 2010, 28:480-489
25 Liao Z, Graham DR, I ildreth JE Lipid rafts and HIV pathogenesis: virion-
associated cholesterol is required for fusion and infection of susceptible
cells. A!DS Res Hum Reiroviruses 2003, 19:675-687
26 Zheng YH, Plemenitas A, Linnemann T, Fackler OT, Peterlin BM Nef
increases infectivity of HIV via lipid rafts. Curr Biol 2001, 11:875-879
27 Maziere JC, Landureau JC, Giral P, Auclair M, Fall L, Lachgar A, Achour A,
Zagury D Lovastatin inhibits HIV-1 expression in H9 human T
lymphocytes cultured in cholesterol-poor medium. Biomed Phaimacoiher
1994, 48:63-67
28 Carter GC, Berntone L, Sangani D, Bee JW, Harder 1, James W' HIV entry
in macrophages is dependent on intact lipid rafts. Virology 2009,
386:192-202
29 Aloia RC, Tian Ii, Jensen FC Lipid composition and fluidity of the human
immunodeficiency virus envelope and host cell plasma membranes. Proc
Nail Acad Sci USA 1993, 90:5181-5185
30 Bukrinsky M, Sviridov D Human immunodeficiency virus infection and
macrophage cholesterol metabolism. j Leukoc Biol2006, 80:1044-1051
31 Ding L, Derdowski A, Wang JJ, Spearman P Independent segregation of
human immunodeficiency virus type 1 Gag protein complexes and lipid
rafts. J Viroi 2003, 77:1916-1926
32 Holm K, Weclewicz K, Hevvson R, Suomalainen M Human
immunodeficiency virus type 1 assembly and lipid rafts: Pr55(gag)


Page 14 of 16








Lin et al. Retrovirology 2012, 9:85
http://www.retrovirology.com/content/9/1/85


associates with membrane domains that are largely resistant to Brij98
but sensitive to Triton X-100. J iro1 2003, 77:4805-4817
33 Nguyen DI Hildreth JE Evidence for budding of human
immunodeficiency virus type 1 selectively from glycolipid-enriched
membrane lipid rafts. J '/toi 2000, 74:3264-3272
34 Ono A, Freed EO Plasma membrane rafts play a critical role in HIV-1
assembly and release. Proc Nat Acad Sc USA 2001, 98:13925- 3930
35 Guyader M, Kiyokawa E, Abrami L, Turelli P, Trono D Role for human
immunodeficiency virus type 1 membrane cholesterol in viral
internalization. i Vio 2002, 76: 0356-10364
36 Zheng VI Plemenitas A, Fielding C, Peterlin BM Nef increases the
synthesis of and transports cholesterol to lipid rafts and HIV-1 progeny
virions. Proc Noi Acad Si USA 2003, 100:8460-8465
37 Wout AB v 't, Swain JV, Schindler M, Rao U, Pathmajeyan MS, Mullins JI,
Kirchhoif F Nef induces multiple genes involved in cholesterol synthesis
and uptake in human immunodeficiency virus type 1-infected T cells.
J Vi'ol2005, 79:10053-10058
38 Mujawar Z, Rose H, Morrow iMP, Pushkarsky 1, Dubrovsky L, MukhJamedova N,
Fu Y, Dart A, Orenstein JM, Bobryshev Y, et a' Human immunodeficiency
virus impairs reverse cholesterol transport from macrophages. PLoS Bio
2006, 4:e365
39 lian X, Kitamoto S, Lian Q, Boisvert WA Interleukin-10 facilitates both
cholesterol uptake and efflux in macrophages. J Bio Chem 2009,
284:32950-32958
4 Welker R, I arris M, Cardel B, Krausslich I G Virion incorporation of human
immunodeficiency virus type 1 Nef is mediated by a bipartite
membrane-targeting signal: analysis of its role in enhancement of viral
infectivity. J Viro/ 1998, 72:8833-8840
4I DjordJevic IJ, Schibeci SD, Stewart GJ, Wiliamsion P HIV type 1 Nef
increases the association of T cell receptor (TCR)-signaling molecules
with T cell rafts and promotes activation-induced raft fusion. ADS Res
Slum Retroviruses 2004, 20:547-555
42 Mujawar Z, Tamehiro N, Grant A, Sviridov D, Bukrinsky M, Fitzgeraldl ML'
Mutation of the ABCA1 C-terminus disrupts HIV-1 Nef binding but does
not block the Nef enhancement of ABCA1 protein degradation.
Biochemisty 20! 0, 49:8338-8349
43 Frank PG, Cheung MW, Pavlides S, Llaverias G, Park DS, Lisanti MP Caveolin-
1 and regulation of cellular cholesterol homeostasis. Am Physiol Heart
Crc Physio/ 2006, 291 :H677-H686
44 Hamard Peron E, Muriaux D Retroviral matrix and lipids, the intimate
interaction. Retrovirology 2011, 8:15
45 Morrow MP, Grant A, Mujawar Z, Dubrovsky L, Pushkarsky T, Kiselyeva Y,
Jennelle L, Mukhamredova N, iRemaley AT, Kashanchi F, et a!
Stimulation of the liver X receptor pathway inhibits HIV-1 replication
via induction of ATP-binding cassette transporter Al. Mo/ Pharmoco!
2010, 78:215-225
46 Crowe SM, Westhorpe CL, Mukhamedova N, Jaworowski A, 5viridov D,
Bukrinsky M The macrophage: the intersection between HIV infection
and atherosclerosis. 1 !eukoc Bi 201 0, 87:589-598
47 Choudhury RP, Lee JM, Greaves DR' Mechanisms of disease: macrophage-
derived foam cells emerging as therapeutic targets in atherosclerosis.
No! Clin Pract Cardiovac Med 2005, 2:309-315
48 Feng B, Yao PM, Li Y, Devlin CM, Zhang D, Harding HP, Sweeney M, Rong
JX, Kurakose G, Fisher EA, e! a! The endoplasmic reticulum is the site of
cholesterol-induced cytotoxicity in macrophages. Naor Ce Bioi 2003,
5:781-792
19 Ohashi R, Mu H, Wang X, Yao QI Chen C Reverse cholesterol transport
and cholesterol efflux in atherosclerosis. QJM 2005, 98:845-856
50 Wang N Silver DL, Costet P, Iall AR Specific binding of ApoA-I, enhanced
cholesterol efflux, and altered plasma membrane morphology in cells
expressing ABC1. J Biol Chem 2000, 275:33053-33058
51 Argrann CA, Edwards Y, Sawyez CG, O'Neil CH, Hegele RA, Pickering JG,
Huff MW' Regulation of macrophage cholesterol efflux through
hydroxymethylglutaryl-CoA reductase inhibition: a role for RhoA in
ABCAl-mediated cholesterol efflux. Biol Chem 2005, 280:222 12-22221
52 Landry YD, Denis M, Nandi S, Bell S, Vaughan AM, Zha X ATP-binding
cassette transporter Al expression disrupts raft membrane
microdomains through its ATPase-related functions. J Bii Chemr 2006,
281:36091-36 10
53 Bergamaschi A, Pancino G Host hindrance to HIV-1 replication in
monocytes and macrophages. Retiovirology 2010, 7:31


54 E erbein G, Varin A The macrophage in HIV-1 infection: from activation to
deactivation? Retroviology 2010, 7:33
55 Le Douce \V I erbein G, Rohr 0, Schwartz C Molecular mechanisms of
HIV-1 persistence in the monocyte-macrophage lineage. Retroviology
20 0, 7:32
56 Guillemard E, Jacquemot C, Aillet F, Schmitt N, Barre-Sinoussi F,
Israel N' Human immunodeficiency virus 1 favors the persistence of
infection by activating macrophages through TNF. ViiClogy 2004,
329:371-380
57 Salahuddin 5Z, Pose RM, Groopman ;J, Markham PD, Gallo RC Human T
lymphotropic virus type III infection of human alveolar macrophages.
Blood 1986, 68:281-284
58 Gartner S, Markovits P, Markovilz DM, Kaplan MH, Gallo RC, Popovic M The
role of mononuclear phagocytes in HTLV-III/LAV infection. Sdence 1986,
233:215-219
59 Sharova N, Swingler C, Sharkey M, Stevenson M Macrophages
archive HIV-1 virions for dissemination in trans. EMBO J 2005,
24:2481-2489
60 Freed EO HIV-1 and the host cell: an intimate association. Tends
Microbio 2004, 12:170-177
61 Malim MI, Emerman M HIV-1 Accessory Proteins. Ensuring Viral Survival
in a Hostile Environment. Cel Host Microbe 2008, 3:388-398
62 Liu I Oliveira NM, Cheney KM, Pade C, Dreja H, Bergin AM, ,
Beach DI, Bishop CL, Dittmar MT, McKnight A A whole genome screen
for HIV restriction factors. Retroviroogy 2011, 8:94
63 Giri MS, Nebozhyn M, Showe L, Montaner U Microarray data on gene
modulation by HIV-1 in immune cells: 2000-2006. J Leukoc Bio 2006,
80:1031-1043
64 Giri MS, Nebozyhn M, Raymond A, Gekonge B, I ancock A, Creer S, Nicols C,
Yousef M, Foulkes AS, Mounzer K, et ao Circulating Monocytes in HIV-1-
Infected Viremic Subjects Exhibit an Antiapoptosis Gene Signature and
Virus- and Host-Mediated Apoptosis Resistancel. J Immuno! 2009,
182:4459-4470
65 Vazquez N, Greenwell Wild T Marinos Ni, Swaim WD, Nares S, Ot DE,
Schubert U, Elenklein P, Orenstein JM, Sporn MB, Wahl SM' HIV-1 induced
macrophage gene expression includes p21, a target for viral regulation.
Vir/ 2005, 79:4479-4491
66 Parton RG, Simons K The multiple faces of caveolae. Nt Rev 2007, 8:185-194
67 Fra AM, Williamson E, Simons K, Parton RG RG De novo formation of
caveolae in lymphocytes by expression of VIP21-caveolin. Proc Not Acod
Sri US4 1995, 92:8655-8659
68 Hatanaka M, Maeda T Ikemoto T, Mori H, Seya T, Shimizu A Expression of
Caveolin-1 in Human T Cell Leukemia Cell Lines. Bioch Bioph Res Corim
998, 253:382-387
69 Vallejo lardin CD Expression of caveolin-1 in lymphocytes induces
caveolae formation and recruitment of phosphofructokinase to the
plasma membrane. FAEBi 2005, 16:586-587
70 Adachi A, Gendelman HE, Koenig S, Folks I, Willey I, Pabson A, Martin MA
Production of acquired immunodeficiency syndrome-associated
retrovirus in human and nonhuman cells transfected with an infectious
molecular clone. J Vro/ 1986, 59:284-291
71 Freed EO, Englund G, Martin MA Role of the basic domain of human
immunodeficiency virus type 1 matrix in macrophage infection. J Viro
1995, 69:3949-3954
72 Hwang SS, Boyle TJ, Lyerly K, Cullen BR' Identification of the envelope V3
loop as the primary determinant of cell tropism in HIV-1. Science 1991
253:71-74
73 lie J, Choe S, Walker R, Di Marzio P, Morgan DO, Landau NR Human
immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the
G2 phase of the cell cycle by inhibiting p34cdc2 activity. J Viral 1995,
69:6705-6711
74 Connor FI, Chen BK Choe S, Landau NR Vpr is required for efficient
replication of human immunodeficiency virus type-1 in mononuclear
phagocytes. Virology 1995, 206:935-944
75 Wei X, Decker JM, Liu H, Zhang Z, Aiani RB, Kilby JM, Saag MS, Wu X, Shaw
GM, Kappes JC Emergence of resistant human immunodeficiency virus
type 1 in patients receiving fusion inhibitor (T-20) monotherapy.
Antimicrob Agents rhemoher 2002, 46:1896-1905
76 Wei X, Decker JM, Wang S, Hui ., Kappes JC, Wu X, Salazar-Gonzalez JF,
Salazar MG, Kilby M, Saag MS, et ol Antibody neutralization and escape
by HIV-1. Nature 2003, 422:307-312


Page 15 of 16








Lin et al. Retrovirology 2012, 9:85 Page 16 of 16
http://www.retrovirology.com/content/9/1/85





77 Raney A, Kuo LS, Baugh LL, Foster JL, Garcia JV Reconstitution and
molecular analysis of an active human immunodeficiency virus type 1
Nef/p21-activated kinase 2 complex. Virol 2005, 79:12732-12741
78 O'Neill E, Kuo LS, Krisko JF, Tomchick DR, Garcia JV, Foste~rJL Dynamic
evolution of the human immunodeficiency virus type 1 pathogenic
factor, Nef. J Viro 2006, 80:1311-1320
79 Theodore TS, Englund G, Buckler-White A, Buckler CE, Martin MA, Peden KW
Construction and characterization of a stable full-length macrophage-
tropic HIV type 1 molecular clone that directs the production of high
titers of progeny virions. AIDS Res Hum Retroviruses 1996, 12:191-194
80 Howell KW, Meng X, Fullerton DA, Jin C, Reece TB, Cleveland JC Jr Toll-like
Receptor 4 Mediates Oxidized LDL-Induced Macrophage Differentiation
to Foam Cells. J Surg Res 2011, 171:e27-e31
81 Lin S, Wu M, Xu Y, Xiong W, Yi Z, Zhang X, Zhenghong Y Inhibition of
hepatitis B virus replication by MyD88 is mediated by nuclear factor-
kappaB activation. Biochim Biophys Acta 2007, 1772:1150-1157

doi:10.1186/1742-4690-9-85
Cite this article as: Lin et al Caveolin-1 reduces HIV-1 infectivity by
restoration of HIV Nef mediated impairment of cholesterol efflux by
apoA-l. Retrovirology 2012 9'85


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Full Text
!DOCTYPE art SYSTEM 'http:www.biomedcentral.comxmlarticle.dtd'
ui 1742-4690-9-85
ji 1742-4690
fm
dochead Research
bibl
title
p Caveolin-1 reduces HIV-1 infectivity by restoration of HIV Nef mediated impairment of cholesterol efflux by apoA-I
aug
au id A1 snm Linfnm Shanshaninsr iid I1 email shanlin@ufl.edu
A2 Nadeaumi EPeterpnadeau@ufl.edu
A3 WangXiaomeiwangxiaomei@ufl.edu
A4 ca yes MergiaAyalewmergiaa@ufl.edu
insg
ins Department of Infectious Disease and Pathology, University of Florida, Gainesville, Florida, 32611, USA
source Retrovirology
issn 1742-4690
pubdate 2012
volume 9
issue 1
fpage 85
url http://www.retrovirology.com/content/9/1/85
xrefbib pubidlist pubid idtype doi 10.1186/1742-4690-9-85pmpid 23067370
history rec date day 18month 6year 2012acc 2692012pub 15102012
cpyrt 2012collab Lin et al.; licensee BioMed Central Ltd.note This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
kwdg
kwd HIV
Caveolin-1
Cholesterol efflux
Nef
Apolipoprotein A-I
abs
sec
st
Abstract
Background
HIV infection results in inhibited cholesterol efflux by apolipoprotein A-I (apoA-I) in macrophages, and this impairment involves Nef mediated down-regulation and redistribution of ATP-binding cassette transporter A1 (ABCA-1). We investigated the effect of caveolin-1 (Cav-1) on the cholesterol efflux by apoA-I in HIV infected primary and THP-1 cell-differentiated macrophages as well as astrocyte derived glioblastoma U87 cells.
Results
Our results reveal that Cav-1 restores the Nef -mediated impairment of cholesterol efflux by apoA-I in both cell types. Co-immunoprecipitation studies indicate a physical association of Cav-1 and Nef. The level of ABCA-1 expression remains the same whether Cav-1 is over-expressed or not. In addition, we examined the cholesterol composition of HIV particles released from Cav-1 treated cells and identified that the cholesterol content is dramatically reduced. The infectivity level of these virus particles is also significantly decreased.
Conclusions
These observations suggest that the interplay of Cav-1 with Nef and cholesterol subsequently counters Nef induced impairment of cholesterol efflux by apoA-l. The findings provide a cellular mechanism by which Cav-1 has an ability to restore HIV mediated impairment of cholesterol efflux in macrophages. This subsequently influences the cholesterol content incorporated into virus particles thereby inhibiting HIV infectivity and contributing to HIV’s persistent infection of macrophages.
bdy
Background
Caveolin 1 (Cav-1), a 21~24-kDa scaffolding protein, is an important structural component of caveolae abbrgrp
abbr bid B1 1
, small invaginations of the plasma membrane, which are enriched in cholesterol, phospholipids, and sphingolipids. This protein is highly expressed in terminally differentiated cells including endothelial cells, macrophages, dendritic cells and adipocytes
B2 2
B3 3
. Functional studies have shown that Cav-1 is involved in a wide range of cellular processes, including cell cycle regulation, signal transduction, endocytosis, cholesterol trafficking and efflux
3
B4 4
B5 5
B6 6
B7 7
B8 8
B9 9
. Multiple lines of evidence indicate that Cav-1 acts as a scaffolding protein capable of directly interacting with and modulating the activity of caveolin-bound signaling molecules. The Cav-1 scaffolding domain (CSD), residues 82 to 101, is essential for both Cav-1 oligomerization and the interaction of caveolin with other proteins
B10 10
. Associations with other proteins through the CSD help provide coordinated and efficient signal transduction
B11 11
B12 12
. The CSD serves as a receptor for binding proteins containing the sequence φXφXXXXφ, φXXXXφXXφ, or φXφXXXXφXXφ (φ representing any aromatic amino acid and X any other amino acid)
10
. HIV Env has been shown to interact with Cav-1 via a motif (WNNMTWMQW) localized within the ectodomain (the C-terminal heptad repeats) of HIV-1 gp41
B13 13
B14 14
B15 15
. Our group has shown the binding of Cav-1 with HIV Env in the lipid rafts which subsequently blocks cell fusion and innocent bystander killing mediated by HIV envelope
B16 16
. We have also demonstrated that HIV infection in primary human monocyte derived macrophages (MDMs) results in a dramatic up-regulation of Cav-1 expression mediated by HIV Tat
B17 17
. Furthermore, over-expression of Cav-1 causes significant reduction in HIV replication in macrophages. Cav-1 inhibits HIV replication through transcriptional repression of viral gene expression by modulating the NF-κB pathway
B18 18
. The up-regulation of Cav-1 by HIV infection and subsequent inhibition of HIV replication suggest a role for Cav-1 in macrophage persistent infection.Cav-1 plays an important role in cellular cholesterol homeostasis, a process that controls intracellular lipid composition and prevents cholesterol accumulation. Cav-1 has been implicated in modulating the expression of lipoprotein receptors and interacts with many lipid transporter molecules
11
B19 19
B20 20
B21 21
. Furthermore, it is involved in the transport of newly synthesized cholesterol from the endoplasmic reticulum (ER) to the plasma membrane
11
B22 22
B23 23
and promotes cholesterol efflux in hepatic cells
9
B24 24
. HIV appears to manipulate cellular cholesterol metabolism to ensure that there is a sufficient supply of cholesterol and that it is located in the appropriate compartments such as lipid rafts for efficient virus release and subsequent infectivity
B25 25
B26 26
B27 27
B28 28
. Cholesterol is an important component that influences HIV production and efficient virus infectivity. Cholesterol depletion significantly reduces HIV-1 particle production
B29 29
B30 30
B31 31
B32 32
B33 33
B34 34
. Virus infectivity is also negatively affected when HIV is produced from cholesterol depleted cells
26
B35 35
.The HIV accessory protein Nef has an ability to exploit cholesterol metabolism. Proposed mechanisms for this strategy include binding to cholesterol and aiding the transport of newly synthesized cholesterol into lipid rafts and viral particles as well as enhancing cholesterol synthesis
B36 36
B37 37
. Nef has also been shown to impair ATP binding cassette transporter protein 1 (ABCA-1)-dependent cholesterol efflux from human macrophages by down-regulation and redistribution of ABCA-1
B38 38
. This suggests that Nef is involved in HIV mediated cholesterol accumulation. Since Cav-1 has a high affinity for cholesterol and aids in the transport of newly synthesized cholesterol from the ER to the plasma membrane and indirectly promoting the transfer to extracellular acceptors such as lipid free apolipoprotein A-I (apoA-I) we hypothesize it would influence the level of cholesterol accumulation as well as virus production and infectivity. Macrophages are major targets for HIV infection and also play an important role in its pathogenesis. The up-regulation of Cav-1 by HIV infection and the role of Cav-1 in cholesterol trafficking suggest a mechanism for a Cav-1/cholesterol mediated impact on HIV replication in macrophages. In this report, we establish evidence for a Cav-1/cholesterol mediated mechanism of inhibition of HIV replication for the first time providing a new angle in understanding HIV’s persistent infection of macrophages.
Results
Cav-1 restores HIV Nef mediated impairment of cholesterol efflux by apoA-I in U87 cells and macrophages
HIV infection impairs ATP-binding cassette transporter A1 (ABCA-1) dependent cholesterol efflux by apoA-l. The Nef protein is identified as the key molecule responsible for this effect
38
. Since Cav-1 is an important regulator of cholesterol trafficking and is involved in the transport of newly synthesized cholesterol from the ER to the plasma membrane, it is likely to influence Nef mediated ABCA-1 dependent down modulation of cholesterol efflux. To determine whether Cav-1 counters the influence of Nef on cholesterol trafficking, first, we tested the impairment of cholesterol efflux in HIV infected THP-1 cell-differentiated macrophages. HIV AD8 infected THP-1 cells were exposed to lipid-free apoA-I or HDL treatment to induce cholesterol efflux. Cholesterol efflux was measured as the fraction of total radiolabeled cholesterol appearing in the medium in the presence of apoA-I after subtraction of values for apoA-I-free medium
B39 39
. ApoA-I stimulated cholesterol efflux from HIV infected THP-1 cell-differentiated macrophages was markedly decreased in a dose dependent manner with the reduction reaching 71.6% as compared to uninfected cells (Figure figr fid F1 1A). No significant difference was observed between HIV infected and uninfected cells in HDL mediated cholesterol efflux (Figure 1B). The decrease in cholesterol efflux to apoA-I by HIV infection was not present in the presence of AZT, an inhibitor of the HIV replication (Figure 1C). These results suggest that HIV infection decreased the apoA-l mediated cholesterol efflux substantially and are in accordance with previous findings
38
. To further examine impairment of cholesterol efflux due to HIV infection the level of cholesterol accumulation was tested by oil red O staining of infected macrophages. As shown in Figure 1D, accumulated cholesterol was markedly increased in AD8 or Bal infected macrophages as compared to uninfected cells which is similar to previous findings
38
.
fig Figure 1 caption HIV-1 impairs apoA-I mediated cholesterol efflux from THP-1 cell-differentiated macrophagestext
b HIV-1 impairs apoA-I mediated cholesterol efflux from THP-1 cell-differentiated macrophages. (A) HIV AD8 infected and uninfected THP-1 cell-differentiated macrophages were cultured for 14 days. MOI represents multiplicity of infection. Cells were labeled with [sup 3H] cholesterol, and subsequently incubated with media in the presence and absence of apoA-I (50 μg/ml). ApoA-I-induced cholesterol efflux was measured and analyzed as described in the Materials and Methods. (B) Cells were treated the same as above but incubated with medium in the presence or in absence of HDL (50 μg/ml) and HDL mediated cholesterol efflux was measured. (C) ApoA-I mediated cholesterol efflux was determined in the presence and absence of the HIV replication inhibitor AZT (5uM). All experiments were performed in triplicate, and results shown are mean ± SD with P values. (D) Uninfected or infected with HIV AD8 (moi 0.01) and HIV Bal (moi 0.001) THP-1 cell-differentiated macrophages cultured for 10 days then incubated with AcLDL (50μg/ml) for 48 h followed by 30μg/ml apoA-I stimulation for 18 hours. Lipid accumulation was determined by Oil red O staining by light microscopy. HIV reverse transcriptase activity in culture medium is shown in the bottom panel.
graphic file 1742-4690-9-85-1 To address the influence of Cav-1 on cholesterol efflux of HIV infected cells, we examined whether Cav-1 can restore Nef mediated impairment of cholesterol efflux. First, U87-CD4-CXCR4 cells were transfected with a Cav-1 expression construct (pCZ-Cav-1) in the presence or absence of a Nef expression plasmid (pcDNA3.1SF2Nef). ApoA-l or HDL mediated cholesterol efflux was measured by harvesting the culture media as well as cell lysate samples. As a control U87-CD4-CXCR4 cells were also transfected with expression vector lacking Cav-1 (pCZ-vector) or Nef (pcDNA3.1). The expression of Cav-1 and Nef in transfected cells was determined by Western blot analysis (Figure F2 2A). As expected apoA-l mediated cholesterol efflux from cells transfected with the Nef expression construct alone was reduced by 77% compared to cells transfected with the plasmid construct devoid of nef or cells that received pCZ-Cav-1 (Figure 2B). Interestingly, apoA-l mediated cholesterol efflux from cells co-transfected with pCZ-Cav-1 and pcDNA3.1SF2Nef was comparable, and even slightly higher, to that of mock which did not receive Nef treatment, suggesting that Cav-1 can restore the impairment of cholesterol efflux caused by Nef. Neither Nef nor Cav-1 had significant effect on HDL mediated cholesterol efflux (data not shown). We confirmed our findings by conducting these studies in physiologically relevant primary monocyte derived macrophages (MDMs). We infected MDMs with vesicular stomatitis virus glycoprotein (VSV-G) Env pseudotyped pSG3Δenv HIV provirus carrying wild type nef (psHIVwtNef) or defective nef (psHIVΔNef) for one round of replication. Infection of MDMs with psHIVwtNef showed a significant reduction (70%) in apoA-l mediated cholesterol efflux as compared to uninfected cells (Figure 2C) similar to what is observed in the U87-CD4-CXCR4 cells (Figure 2B). The reduction in cholesterol efflux was 75% when compared to Nef defective HIV. There was no significant difference in cholesterol efflux between Nef defective HIV infected and uninfected MDMs. Introduction of exogenous Cav-1 into psHIVwtNef MDMs using adenovirus expressing Cav-1 (Ad-Cav-1) increased cholesterol efflux by 43% compared to cells only infected with psHIVwtNef. The control adenovirus carrying GFP (Ad-GFP) was not able to counter cholesterol efflux impairment induced by psHIVwtNef infection of the MDMs (Figure 2C). As expected, Western blot analysis of MDMs transduced with Ad-GFP revealed endogenous Cav-1 expression, with increased amounts of Cav-1 in MDMs treated with Ad-Cav-1 (Figure 2D). Furthermore, introduction of exogenous Cav-1 or GFP using adenovirus did not alter cholesterol efflux from MDMs infected with the Nef defective HIV. In addition, we compared apoA-I mediated cholesterol efflux in MDMs infected with wild type AD8 and replication competent nef deleted AD8 (ADnefmut) viruses. Cholesterol efflux was decreased by 56.3% in wild type infected macrophages compared to uninfected cells while ADnefmut infection had no significant effect (Figure 2E). These results taken together suggest that Cav-1 is capable of restoring HIV induced impairment of apoA-1 mediated cholesterol efflux. To confirm our findings, we further examined the affect of Cav-1 on cholesterol accumulation by oil red O staining in HIV infected THP-1 cell-differentiated macrophage cells. As shown in Figure 2F, psHIVwtNef infected cells had significantly increased cholesterol accumulation compared to uninfected cells (mock) which is similar to HIV AD8 infected THP-1 cell-differentiated macrophages (Figure 1D). The co-infection of macrophages with Ad-Cav-1 and psHIVwtNef, on the other hand, showed a dramatic reduction of intracellular cholesterol inclusions when compared to psHIVwtNef only infected cells. As expected, no significant influence on intracellular cholesterol accumulation was observed when THP-1 cell-differentiated macrophages were infected with Nef defective HIV (psHIVΔNef). In addition, over-expression of Cav-1 by Ad-Cav-1 infection did not alter intracellular cholesterol inclusions in cells infected with psHIVΔNef.
Figure 2 Cav-1 restores HIV −1 Nef mediated impairment of cholesterol efflux to apoA-I
Cav-1 restores HIV −1 Nef mediated impairment of cholesterol efflux to apoA-I. U87-CD4-CXCR4 cells were transfected with Nef expression plasmid (pcDNA3.1SF2Nef) along with pCZ-cav-1 or pCZ vector. Cells were labeled with [3H] cholesterol for 36 h. (A) The level of Nef and Cav-1 expression was determined by Western blot analysis. (B) ApoA-I induced cholesterol efflux is shown. All experiments were performed in triplicate, and results shown are mean ± SD with P values. (C) Primary monocyte derived macrophages (MDMs) were infected with Ad-Cav-1 or Ad-GFP, followed by infection with VSV-G pseudotyped HIV, either carrying Nef (psHIVwtNef) or defective Nef (psHIVΔNef) at an moi of 5. ApoA-I mediated cholesterol efflux was performed and analyzed by incubating cells in medium in the presence or absence of 50 μg/ml apoA-I. The results are presented as a percentage of cholesterol efflux to apoA-I from control (set as 100%), and are the mean± SD of triplicate determinations. The expression levels of Cav-1 and GFP are shown in (D). (E) Primary macrophages were infected with AD8 or nef defective AD8 (ADnefmut) at an moi of 0.01. ApoA-I mediated cholesterol efflux was measured 15 days post infection. (F) THP-1 cell-differentiated macrophages were infected with psHIVwtNef or psHIVΔNef at an moi 3 with or without Ad-Cav-1. On day 5 after infection, Oil red O staining was performed and a representative area in each well is shown.
1742-4690-9-85-2 We further determined whether endogenous Cav-1 has an effect on Nef mediated suppression of cholesterol efflux to apoA-1 in U87 cells and THP-1 cell-differentiated macrophages. The expression of endogenous Cav-1 was knocked down using specific siRNA, and the expression of Nef was accomplished by transfecting U87 cells with pCDNA3.1SF2Nef or pseudotyped HIV (psHIVwtNef) infection of THP-1 cell-differentiated macrophages. The siRNA treatment reduced the expression of Cav-1 by 76% in U87 cells and 38% in THP-1 cell-differentiated macrophages (Figure F3 3A and 3B, respectively). Cholesterol efflux to apoA1 was reduced by 61% in Nef expressing U87 cells in the absence of siRNA targeting Cav-1 (Figure 3A). The level of cholesterol was further reduced (96%) when Cav-1 expression was knocked-down with Cav-1 specific siRNA. Similar results were observed in THP-1 cell-differentiated macrophages showing a decrease in cholesterol efflux by 50% in Nef expressing cells and by 79% in Cav-1 siRNA treated Nef expressing cells (Figure 3B). Furthermore, the levels of cholesterol efflux correlate with the efficiency of siRNA knock-down in U87 and THP-1 cells (Figure 3A and 3B).
Figure 3 siRNA knockdown of Cav-1 and its effect on cholesterol of cells expressing wild type Nef or NefG2A
siRNA knockdown of Cav-1 and its effect on cholesterol of cells expressing wild type Nef or NefG2A. (A). U87-CD4-CXCR4 cells were transfected with Cav-1 siRNA or control siRNA (CTL-siRNA) followed by transfection with Nef expressing plasmid (pSHIVwtNef) or pCDNA3.1 vector. The level of Cav-1 expression in siRNA treated cells was detected by Western blotting analysis. Cells were labelled with [3H] cholesterol for 36 hours and apoA-I induced cholesterol efflux was measured. Results are shown as the percentage of cholesterol efflux relative to control. (B) THP-1 cell-differentiated macrophages were first transfected with Cav-1 siRNA or CTL-siRNA which was followed by infection with VSV-G pseudotyped HIV (psHIVwtNef) at an moi of 3. Cells were again treated with siRNA the day after infection. Cholestrol efflux was measuered as described above four days post-infection. (C) U87-CD4-CXCR4 cells were transfected with pCI (Mock), pCI NL4-3 Nef-HA-WT (Nef), pCI NL4-3 NefG2A-HA (NefG2A), or pCI NL4-3 NefG2A-HA along with pCZ-cav-1 (Nef plus Cav-1). Cholestrol efflux to apoA-I was determined. All experiments were performed in triplicate and results shown are mean ± SD with P values.
1742-4690-9-85-3 In addition, to determine whether Cav-1 specifically restores Nef mediated impairment of cholesterol efflux to apoA-l, U87 cells were co-transfected with a Nef mutant (NefG2A) and Cav-1 expressing plasmids. The NefG2A is a Nef mutant that cannot undergo myristoylation
B40 40
. Its association with the plasma membrane is impaired
26
35
, and it lacks the ability to decrease apoA-l stimulated cholesterol efflux
36
B41 41
. As shown in Figure 3C, cells expressing Nef experienced 62% less cholesterol efflux to apoA-I compared to Mock. In contrast, NefG2A had no effect on apoA-l mediated cholesterol efflux in the presence of either endogenous or over-expressing Cav-1 cells. These studies, therefore, clearly establish that Cav-1 counters Nef mediated impairment of cholesterol efflux by apoA-l.
Cav-1 over-expression has no effect on ABCA-1 expression
Nef has been shown to impair ABCA1-dependent cholesterol efflux from human macrophages, and the expression of ABCA-1 is shown to be down regulated by HIV infection or Nef expression
38
B42 42
. Cav-1 is implicated in positive regulation of ABCA-1 expression and ABCA-1 expression is down regulated in Cav-1 knockout mice
B43 43
. In order to understand the mechanism responsible for Cav-1 mediated restoration of cholesterol efflux upon HIV infection we examined the expression of ABCA-1 in Cav-1 over-expressing cells. U87-CD4-CXCR4 cells were transfected with pCZ-Cav-1 in a dose-dependent manner, and the level of ABCA-1 expression was monitored by Western blot analysis. As shown in Figure F4 4A, Cav-1 over-expression did not alter the level of ABCA-1 expression. To further confirm our findings, we co-transfected U87 cells with pCZ-Cav-1 and pcDNA3.1SF2Nef, and then analyzed ABCA-1 expression by Western blot in the presence and absence of Cav-1 or Nef. Although Nef down-regulated ABCA-1 (by 69%) the level of ABCA-1 expression remained the same in Nef treated cells whether Cav-1 is over-expressed or not (Figure 4B), suggesting that Cav-1 mediated restoration of cholesterol efflux is not related to the regulation of ABCA-1 expression. Likewise, VSV-G pseudotyped HIV (psHIVwtNef) infection down-regulated ABCA-1 expression in MDMs, and co-infection of MDMs with psHIVwtNef and Ad-Cav-1 did not restore the reduced ABCA-1 levels (Figure 4C). MDMs were also infected with AD8 or ADnefmut virus along with infection of Ad-Cav-1 or Ad-GFP to determine the level of ABCA-1 expression. ADnefmut HIV infection did not affect the expression of ABCA-1 with either Ad-Cav-1 or Ad-GFP co-infection (Figure 4D). ABCA-1 expression, however, was decreased when MDMs were co-infected with AD8 and Ad-Cav-1 or Ad-GFP confirming the role of Nef in the reduction of ABCA-1 expression while over expression of Cav-1 has no impact on ABCA-1 expression. Furthermore, we examined the level of ABCA-1 expression in U87 or THP-1 cells where the expression of endogenous Cav-1 was knocked down with siRNA using samples described for results in Figure 3A and 3B. As shown in Figure 4E reduced endogenous Cav-1 expression by siRNA treatment did not alter the level of ABCA-1 expression.
Figure 4 Cav-1 over-expression has no influence on ABCA-1 expression
Cav-1 over-expression has no influence on ABCA-1 expression. (A) U87-CD4-CXCR4 cells were transiently transfected either with pCZ-vector (mock) or with different doses of Cav-1 (pCZ-Cav-1). Expression of ABCA-1 protein was examined by Western blot analysis. (B) Cells were transfected with pCZ-vector (mock), Nef expressing construct (pcDNA3.1SF2Nef) and pCZ-Cav-1 or pCZ-Cav-1 only. The expression levels of ABCA-1 in the presence or absence of Nef were determined in the cells with endogenous or over-expressing Cav-1. (C) MDMs were infected with VSV-G pseudotyped HIV (psHIVwtNef), Ad-Cav-1, or both. The level of Cav-1 and ABCA-1 expression was examined by Western blot analysis in the presence and absence of Cav-1 and/or Nef. Representative results from three experiments are shown in A, B and C. (D) MDMs were co-infected with wild type AD8 or nef defective AD8 (ADnefmut) and Ad-Cav-1 or Ad-GFP. The level of expression of ABCA-1, Nef, and Cav-1 was examined by Western blot analysis. (E) To determine whether siRNA knock-down of Cav-1 affects the expression of ABCA-1 samples used in experiment 3A and B were used to measure the level of ABCA-1 expression. Note that the siRNA Cav-1 knock out and β-actin are the same bands shown in Figure 3 because the same samples were used to demonstrate the level of ABCA-1 expression. The densities of bands corresponding to each protein were quantified using image densitometer analysis. The numbers at the bottom of each blot are the relative values of ABCA1 expression in transfected or transduced cells compared to those in control cells (mock).
1742-4690-9-85-4
Interaction of Nef and Cav-1
Since over-expression of Cav-1 does not alter ABCA-1 expression, the mechanism of restoration of cholesterol efflux by Cav-1 that is impaired by Nef may not involve the level of ABCA-1 expression. Nef has been shown to bind to ABCA-1
38
42
, and it is not known whether Cav-1 interacts with Nef. Cav-1 may associate, either directly or indirectly, with Nef thereby countering the impairment of cholesterol efflux. To determine whether there is a physical association between Cav-1 and Nef, we performed co-immunoprecipitation and immunoblotting experiments. U87-CD4-CXCR4 cells were co-transfected with pCZ-Cav-1 and HA-tagged wild type Nef (pCI NL4-3 Nef-HA-WT) or the NefG2A mutant (pCI NL4-3 NefG2A-HA). Transfected cells were then cultured in medium containing cholesterol (30μg/ml) for 48 hours followed by treatment with apoA-I (20μg/ml) for 30min. Cell lysates were then subjected to co-immunoprecipitation and analyzed for Cav-1 and Nef interactions by immunoblotting. As shown in Figures F5 5A and 5B the association of Cav-1 and Nef is evident in U87 cells. Interestingly, there was no NefG2A mutant interaction with Cav-1 implicating the association of Nef and Cav-1 is at the cell membrane. We examined the endogenous interaction of Cav-1 and ABCA-1 in U87 cells and were unable to show ABCA-1 association with Cav-1 by co-immunopreciptation and immunoblotting analysis (data not shown). The interaction of Cav-1 with Nef suggests that Cav-1 by associating with Nef blocks the activity of Nef and subsequently helps restore cholesterol efflux impaired by Nef.
Figure 5 Interaction of Cav-1 and Nef
Interaction of Cav-1 and Nef. U87-CD4-CXCR4 cells were co-transfected with pCZ-Cav-1 and HA tagged Nef (pCI NL4-3 Nef-HA-WT) or NefG2A(pCI NL4-3 NefG2A-HA). Twenty-four hours post transfection the cells were cultured in the presence of cholesterol (30 μg/ml) for 48 hours followed by apoA-I (20 μg/ml) for 10 min. (A) The cells were harvested and subjected to immunoprecipitation using anti-HA antibody and immunoblots using anti-Cav-1 or anti-Nef antibody. (B) Alternatively, immunoprecipitaion was performed using anti-Cav-1 and immunoblotting using anti-Nef antibody.
1742-4690-9-85-5
Cav-1 reduces HIV-1 infectivity by reducing the cholesterol content of virus particles
HIV is well known to rely on the host cellular cholesterol machinery for efficient replication and particle formation
25
26
28
30
B44 44
. Since our results show that Cav-1 restores Nef impaired cholesterol efflux, we sought to determine if the promotion of this efflux by Cav-1 would have an impact on the infectivity of released virus particles. In order to demonstrate whether Cav-1 influences HIV infectivity, primary macrophages (MDMs) were transduced with Ad-Cav-1 or the control Ad-GFP. The level of GFP and Cav-1 expression in macrophages from which culture supernatant harvested is shown in Figure F6 6A. Twenty-four hours post transduction the macrophages were infected with HIV AD8. Virions produced from these infected macrophages were titered using the TZM-bl indicator cell line and normalized for the infectivity studies. As shown in Figure 6B infectivity of virus harvested from Cav-1 treated macrophages was reduced by 46% compared to Cav-1 untreated HIV infected cells. There was no significant difference in infectivity of virus particles obtained from Ad-GFP transduced cells when compared to that of virus harvested from Cav-1 untreated HIV infected cells. Similar experiments were performed using ADnefmut infections. Contrary to what was observed with AD8 HIV the level of infectivity of ADnefmut virus harvested from Ad-Cav-1 or Ad-GFP treated cells remained the same (Figure 6C). This, therefore, establishes that Cav-1 impairs HIV infectivity implicating that this may be linked to Cav-1 mediated promotion of cholesterol efflux by apoA-I that is impaired by Nef during HIV infection. Since Cholesterol within the HIV particle is strictly required for infection, our next set of experiments were aimed at investigating whether the reduction of HIV infectivity is related to the modulation of lipid content of the virions. HIV provirus DNA was co-transfected into U87 cells with pCZ-Cav-1 or pCZ-vector. Virus harvested from transfected cells was concentrated and normalized by a p24 ELISA assay. Equal amounts of virus particles were used to measure the cholesterol composition in the virion by the Amplex Red cholesterol Assay Kit. As shown in Figure F7 7A the cholesterol content of virus particles harvested from cells receiving Cav-1 was reduced by 48% compared to cells transfected with pCZ-vector. In addition, cholesterol was replenished within concentrated virus that was normalized and equal amounts were treated with (2-Hydroxypropyl)-ß-Cyclodextrin (CD) and saturated exogenous cholesterol to see if infectivity could be restored. Infectivity of cholesterol replenished virus was measured by luciferase activity in infected TZM-bl cells. The infectivity of virus particles collected on Cav-1 treated cells was reduced by 58% as compared to those collected on cells treated by pCZ-vector (Figure 7B). There was no difference in infectivity of cholesterol replenished and control viral particles collected from pCZ-vector treated cells (Figure 7B). As might be expected the infectivity of cholesterol replenished virus particles collected from Cav-1 treated cells was increased by 37% compared to the virus particles harvested from Cav-1 treated control (Figure 7B). In addition, we examined whether Cav-1 is incorporated into the virion to make sure that such incorporation has not affected infectivity directly rather than influencing the cholesterol content of the HIV virus particles. As shown in Figure 7C, we observed that Cav-1 protein is not incorporated in the virus particles as determined by Western blot analysis. Therefore, Cav-1 reduces virus infectivity by promoting cholesterol efflux which consequently decreased the availability of cholesterol during viral particle formation.
Figure 6 Cav-1 over-expression inhibits HIV-1 infectivity
Cav-1 over-expression inhibits HIV-1 infectivity. MDMs were infected with AD8 or ADnefmut at an moi of 0.1 alone or in combination with Ad-Cav-1 or Ad-GFP. (A) The levels of GFP and Cav-1 expression are shown. (B) and (C) Infectious particles harvested from culture supernatants were titered and normalized. Level of infectivity was measured by infecting TZM-bl cells and subsequent luciferase assay. All experiments were performed in triplicate and results shown are mean ± SD with P values.
1742-4690-9-85-6
Figure 7 Cav-1 over-expression reduces the cholesterol content in HIV-1 virus particles
Cav-1 over-expression reduces the cholesterol content in HIV-1 virus particles. (A) Proviral DNA genome NL4-3 was transfected into U87 cells either with pCZ-vector or with pCZ-Cav-1. Virus particles generated were concentrated and normalized by p24 assay. Cholesterol contents were measured using the Amplex Red cholesterol Assay Kit. The results are presented as percentage of cholesterol content relative to control (HIV/pCZ-vector, set as 100%) and are the mean± SD of triplicate experiments with P values. (B) Normalized samples were replenished with exogenous cholesterol, and the level of infectivity was measured by infection of TZM-bl cells and luciferase assay. All experiments were performed in triplicate, and results shown are mean ± SD with P values. (C) Normalized samples from samples of (7A) were subjected to Western blot analysis using antibody to Gag, Nef, and Cav-1 to determine whether Cav-1 protein is incorporated in virus particles.
1742-4690-9-85-7
Discussion
HIV has been indicated to manipulate host cholesterol metabolism, leading to excessive cholesterol accumulation in infected T cells or macrophages
38
B45 45
, thereby supporting efficient viral replication. In the absence of proper esterification to fatty acid and efflux, cholesterol accumulates in the endoplasmic reticulum eventually leading to ER dysfunction and the activation of an ER stress associated apoptosis pathway
B46 46
B47 47
B48 48
. Cav-1 is an important cellular cholesterol regulator, and its expression is dramatically enhanced in HIV infected macrophages
17
, implicating a role for Cav-1 in HIV associated cholesterol alterations. Cav-1 is a structural component of Caveolae membrane microdomains, which have been suggested to play an important role in cholesterol trafficking and efflux. In this study, we investigate the effect of Cav-1 on the cholesterol efflux in HIV infected macrophages and human astrocytes-derived glioblastoma U87 cells. Our results show that Cav-1 restores the Nef induced impairment of cholesterol efflux by apoA-I. Furthermore, this restoration causes a reduction in the cholesterol composition of virus particles leading to decreased HIV infectivity. This suggests a role for Cav-1 in macrophage HIV persistent infection by enhancing cholesterol efflux.Our results show neither Nef nor Cav-1 had significant effect on HDL mediated cholesterol efflux. HDL plays an important role in reverse cholesterol transport (RCT), in which HDL transports cholesterol from peripheral tissues to liver for excretion. RCT is a multifaceted, dynamic pathway which is involved with multiple molecules and effectors. The first step in RCT is ABCA-1 dependent efflux of cholesterol and phospholipids to apoA-I, the major component of HDL. ABCA-1 interacts with apoA-I and stimulates free cholesterol and phospholipids efflux responsible for nascent HDL formation
B49 49
. Wang it et al.
B50 50
reported that ABCA-1 expression markedly increases apoA-I but not HDL mediated lipid efflux; the reason could be that compared with HDL, apoA-I is the preferred acceptor for ABCA1-promoted cholesterol and phospholipid efflux. We also found upon HIV infection Nef down regulates ABCA-1 expression, which dramatically inhibits apoA-I mediated cholesterol efflux, whereas HDL mediated cholesterol efflux was not affected by HIV infection. Over-expression of Cav-1 restores the impaired cholesterol efflux to apoA-I significantly, but not so much on intact HDL cholesterol efflux.Promotion of cholesterol efflux by over-expression of Cav-1 is observed in hepatic cells
9
. Cav-1 can enhance the transfer of cholesterol to cholesterol-rich domains in the plasma membrane, where it is accessible to efflux. Multiple mechanisms are proposed for Cav-1’s regulation of cholesterol homeostasis. These include the modulation of the expression of lipoprotein receptors and the activity of proteins involved in lipid metabolism as well as interactions with lipid transport or transport of cholesterol to the plasma membrane facilitating cholesterol efflux
6
7
8
22
43
. ABCA-1 expression is important in regulating cholesterol efflux to apoA-I and it has been implicated that ABCA-1 stimulates the reorganization of plasma membrane microdomains to facilitate cholesterol efflux to apoA-I
B51 51
B52 52
. Cav-1 can regulate cholesterol homeostasis by modulating the expression of lipid regulators. Reduced levels of ABCA-1 have been observed in macrophages of Cav-1 knockout mice
43
. Our results show that we observe no change in the level of ABCA-1 expression when Cav-1 is over-expressed suggesting that the endogenous Cav-1 expression is sufficient enough to maintain physiologically relevant levels of ABCA-1 and that additional amounts of Cav-1 does not have an impact on ABCA-1 expression. The reduced level of ABCA-1 observed in the knockout mice is in complete absence of Cav-1 expression. ABCA-1 dependent cholesterol efflux can be impaired by HIV Nef mediated down modulation and altering of the intracellular distribution of ABCA-1
38
42
. Similarly we observed a 69% decrease in ABCA-1 expression in the presence of Nef. Interestingly the decrease in ABCA-1 remains the same when additional amounts of Cav-1 are provided indicating that the reversal of Nef’s effect on cholesterol efflux by Cav-1 is not related to the level of ABCA-1 expression. Inhibition of ABCA-1 protein expression, as it pertains to Nef, in part depends upon the ER associated proteasomal degradation mechanism
42
. An unknown additional pathway unrelated to proteasomal activity is also suggested to contribute to ABCA-1 degradation. Although ABCA-1 is shown to interact with Nef the physical association is not essential for Nef mediated down-regulation of ABCA-1 efflux activity
38
42
. However, the influence of cellular distribution of ABCA-1 by Nef has been determined using confocal microscopy with Nef causing a prominent trapping of ABCA-1 in the ER
42
. ABCA-1 expression has been implicated in influencing the redistribution of cholesterol and Cav-1
52
. Redistribution of Cav-1 from punctate caveolae-like structures to the general area of the plasma membrane is observed upon ABCA-1 expression. Our co-immunoprecipitation study reveals an interaction between Cav-1 and Nef. Furthermore, our observation that Cav-1 does not interact with the myristoylation defective Nef (NefG2A mutant) implicates an association of these proteins at the plasma membrane. These observations suggest that the interplay of Cav-1 with Nef and cholesterol subsequently counters Nef induced impairment of cholesterol efflux by apoA-l. In addition, since caveolae is a major source and platform for cholesterol efflux
4
over-expression of Cav-1 may induce the formation of more caveolae, which should subsequently enhance cholesterol efflux. The presence of Cav-1 in macrophages and its up-regulation upon HIV infection, therefore, can contribute to increased cholesterol efflux in these cells.Cholesterol is an important structural component of HIV particles and their cholesterol content is tightly linked to HIV infectivity
25
27
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. Cholesterol depletion significantly reduces HIV-1 particle production
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. There is also a marked decrease in infectivity of virions produced from such cells
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. The significant reduction correlates with the amount of virion-associated cholesterol
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. In the current study, we clearly established that Cav-1 significantly reduces infection with virions produced from Cav-1 treated cells when compared to that of the same number of virions obtained from untreated cells. We have previously shown that Cav-1 represses HIV gene expression by blocking the NF-κB pathway thus subsequently affecting virus production
18
. The decrease in virus production is therefore in part due to transcriptional suppression of HIV gene expression. Here, we examined the cholesterol content of HIV particles produced from Cav-1 treated cells and clearly established a significant cholesterol decrease in virus particles. Furthermore, normalized amounts of virus in the infectivity assay of HIV released from Cav-1 treated cells shows that infectivity is markedly reduced. Normalized amounts of virus to assay for infectivity, rules out any concern regarding the level of virus release contributing to the reduction of infectivity. The major step that causes a decrease in virion infectivity related to cholesterol depletion is the fusion steps of infection
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. In support of this notion, we previously demonstrated that Cav-1 significantly suppressed Env-induced membrane hemifusion
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, indicating that the decrease in fusion partly involves a reduction in the cholesterol composition of the plasma membrane. Cav-1 can counter the influence of HIV on cholesterol metabolism by promoting cholesterol trafficking to the membrane subsequently enhancing cholesterol efflux, therefore, depriving the HIV virion of cholesterol. Since Cav-1 is involved in cholesterol metabolism the up-regulation of Cav-1 can have an impact on the level of cellular cholesterol thereby contributing to a reduction in virus production and infectivity, consequently contributing to a persistent infection of macrophages.
Conclusion
Infected macrophages are relatively resistant to cytopathic effect and consequently play an essential role in viral dissemination to host tissues and organs
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. Furthermore, in this viral reservoir HIV infection appears not to be associated with apoptosis but with a chronic productively infected phenotype
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. Although several mechanisms have been proposed we still don’t have a clear picture as to the mechanisms of persistent infection in macrophages. The relationship between HIV-1 and host factors determines the modulation of both cellular functions and virus replication within an infected individual, and with the interaction of these viral and cellular factors being evident in all steps of virus replication
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. Their associations may be an important factor in the modification of host cell processes during a chronic viral infection. This suggests that a persistent infection is regulated by cellular factors at different steps in virus replication. Gene expression profiles that are unique to macrophages
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when compared to that of activated CD4+ T cells should help determine the mechanism of persistent infection in macrophages. Cav-1 is highly expressed in terminally differentiated or quiescent cells including dendritic cells and monocytes/macrophages
2
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. While macrophages express Cav-1, human T cells are generally believed to lack the Cav-1 protein
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. The lack of Cav-1 in T cells, our discovery that HIV infection enhances Cav-1 expression mediated by Tat in macrophages and that Cav-1 reduces HIV replication
17
suggests a role for Cav-1 in an HIV persistent infection of macrophages. Here we have shown that Cav-1, by restoring cholesterol efflux impaired by Nef and subsequently influencing the cholesterol content of HIV particles which negatively affects virus infectivity, effectively inhibits HIV replication contributing to macrophage HIV persistent infection (Figure F8 8).
Figure 8 A model for the interplay of Cav-1 with Nef that counters Nef induced impairment of cholesterol efflux by apoA-l and contribution to persistent infection
A model for the interplay of Cav-1 with Nef that counters Nef induced impairment of cholesterol efflux by apoA-l and contribution to persistent infection. Upon HIV infection, Nef protein interacts with ABCA-1 and down-regulates and/or redistributes ABCA-1 expression which results in cholesterol efflux impairment. This creates a micro-environment in which HIV can replicate efficiently. On the other hand, in cells which express Cav-1, such as macrophages, Tat induces an up-regulation of Cav-1. The overabundance of Cav-1 then leads to both its binding to Nef and ability to activate cholesterol efflux. This, therefore, leads to less cholesterol accumulation which in turn reduces the amount that can be incorporated into viral particles and thereby reducing HIV infectivity.
1742-4690-9-85-8
Methods
Plasmids
The HIV-1 proviral constructs pNL4-3 (T-tropic), pNL-AD8 (M-tropic), pWT/Bal (M-tropic), pNL4-3.Luc.R-E-, and pSG3Δenv were kindly provided by NIH AIDS Research and Reference Reagent Program
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. The construct pNL4-3.Luc.R-E- is defective for env and nef where as pSG3Δenv has intact nef, but a deletion in env. An expression plasmid for vesicular stomatitis virus envelope G protein (pCI-VSV) was kindly provided by Jiing-Kuan Yee of City of Hope National Medical Center, Duarte, California. A Cav-1 expressing plasmid, pCZ-cav-1, was generated as described previously
16
. pCZ-vector is the same as pCZ-cav-1 except it lacks the coding sequence of cav-1. The Nef expression plasmid pcDNA3.1SF2Nef was provided by NIH AIDS Research and Reference Reagent Program
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. A construct expressing Nef tagged with HA (pCI NL4-3 Nef-HA-WT) was purchased from Addgene Inc (Cambridge, MA). The NefG2A mutation plasmid was generated using a site-directed mutagenesis kit according to the manufacturer’s protocol (Strategene). Briefly, the mutation was generated by PCR amplification using pCI NL4-3 Nef-HA-WT as template and the following pair of primers: 5′-ggattttgctataagatggctggcaagtggtcaaaaagt-3′ and 5′-actttttgaccacttgccagccatcttatagcaaaatcc-3′. The PCR products were digested with the restriction enzyme DpnI to destroy template plasmids and were then transformed into DH5α competent cells. Introduction of the mutation (pCI NL4-3 NefG2A-HA) was confirmed by sequence analysis. Wild type AD8 and a replication competent nef defective AD8 derived HIV provirus DNA construct ADnefmut
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were provided by Dr. Maureen Goodenow of the University of Florida. Adenovirus particles (Ad) for expressing Cav-1 (Ad-Cav-1) and GFP (Ad-GFP) were obtained from Vector Biolabs (Philadelphia, PA).
Cell cultures
Human U87MG-CD4 cells stably transfected with CXCR4 (U87-CD4-CXCR4) or CCR5 (U87-CD4-CCR5), human acute monocytic leukemia (THP-1),and an indicator cell line for tittering HIV (TZM-bl) was kindly provided by the NIH AIDS Research and Reference Reagent Program. U87-CD4-CXCR4 were maintained in DMEM containing 15% FBS, penicillin-streptomycin (100 μg/mL), glutamine, puromycin (1μg/ml; Sigma Chemical), and neomycin (G418; 300μg/ml; Sigma). THP-1 cells were grown in RPMI-1640 containing 10% FBS, 1.0mM sodium pyruvate, and 0.05 mM 2-mercaptoethanol. For differentiation into macrophages, THP-1 cells were treated with 50 ng/ml of phorbol 12-myristate 13-acetate (PMA, Sigma Chemical) for 5 days until the cells adhered and exhibited macrophage-like morphology. TZM-bl and 293T cells were grown in DMEM medium supplemented with 10% FBS and penicillin-streptomycin (100μg/ml). All cultures were maintained at 37°C in a humidified atmosphere with 5% CO2.Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats prepared from healthy donors by centrifugation through a Ficoll gradient (Sigma-Aldrich, St. Louis, MO). Monocytes were isolated by negative selection with a human monocyte enrichment kit according to the manufacturer’s instructions (EasySep® Human Monocyte Enrichment Kit, Stemcell Technologies). The monocyte preparations contained 97% CD14+ cells, as determined by flow cytometry. For differentiation of monocytes into macrophages (MDMs), monocytes were seeded into Biocoat poly-D-lysine plates (B.D. Bioscience), and cultured in DMEM, supplemented with 10% heat-inactivated human serum, gentamicin (50μg/ml), ciprofloxacin (10μg/ml), and M-CSF (1000U/ml) for 7 days. MDM culture medium was half-exchanged every 2 to 3 days.
Transfection of siRNA
Small interfering RNA (siRNA) targeting Cav-1 and control siRNA were purchased from Santa Cruz Biotechnology, Inc. Transfection of siRNA was performed using OligofectaminTM Reagent (Invitrogen Corp., Carlsbad, Calif.) according to the manufacturer’s protocol. Briefly, the day before transfection, U87cells were seeded into a 24 well plate and cultured with antibiotics free medium to 30% confluency. Cells were washed and resuspended in 200ul serum free medium. Transfection mixture was prepared by incubating 50pmol of siRNA duplexes with 3ul of Oligofectamin in a final volume of 50ul Opti-MEM I Medium. After a 5 hour incubation, 125ul of growth medium with 3 times the normal concentration of serum was added to cells. Transfection was repeated once the next day. For THP1 macrophages, cells were first transfected with siRNA followed by HIV infection, and cells were then transfected again with siRNA the day after infection. The efficiency of Cav-1 knock-down by the siRNA transfection was monitored using Western blot analysis.
Virus production and concentration
Infectious virus HIV-1 AD8, ADnefmut, Bal, and NL4-3 were generated by calcium phosphate transfection of monolayers of 293T cells in 75-cm2 flasks with 25μg provirus DNA. Supernatants containing virus were harvested 4 days after transfection and quantified using the TZM-bl indicator cells as well as by measuring reverse transcriptase and a p24 ELISA method as described previously
17
. When required, virus was produced from U87-CD4-CXCR4 cells transfected with 18μg proviral HIV NL4-3 along with 9μg pCZ-Cav-1 or pCZ-vector. To generate pseudotyped HIV particles 20μg pSG3Δenv or pNL4-3.Luc.R-E- was co-transfected with 3μg pCI-VSV into monolayers of 293T cells in 75-cm2 culture flasks by the calcium phosphate method. Pseudotyped viral supernatants were harvested 4 days post-transfection and were clarified by centrifugation at 3,000 rpm for 20 min and then by filtering through a 0.45 μm-pore size filter. Virus particles were concentrated using virus precipitation reagent Retro-ConcentinTM (System Biosciences) according to the manufacturer’s protocol.
Oil red O staining
To determine the influence of Cav-1 on the level of lipid accumulation in HIV infected and uninfected cells oil red O staining was performed. THP-1 cells were differentiated into macrophages by treatment with 50 ng/ml PMA for 5 days then infected with HIV AD8 (moi, 0.01) or Bal (moi, 0.001). On day 10 post infection, cells were loaded with cholesterol by incubating with 50μg/ml Ac-LDL (Biomedical Technologies Inc., Stoughton, MA) for 48 h followed by 30μg/ml apoA-I stimulation for 18 hours. Differentiated THP-1 cell-differentiated macrophage cells were also infected with Ad-Cav-1 or Ad-GFP at an moi (multiplicity of infection) of 100. Twenty-four hours later they were infected with VSV pseudotyped HIV pSG3Δenv or pNL4-3.Luc.R-E- at an moi of 3 and incubated for 5 days. Oil red O staining was performed as previously described
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. Briefly, cells were rinsed with PBS, followed by fixation with 3.7% paraformaldehyde for 60 min. The cells were stained using freshly prepared Oil red O (Sigma) working solution at room temperature for 10min. Intensity of cell staining was observed using a light microscope.
Virus infectivity assay
To test Cav-1’s influence on HIV-1 infectivity, TZM-bl cells were infected with virus harvested from Cav-1 treated cells and the infectivity levels were measured by luciferase activity. MDMs were first infected with adenovirus expressing Cav-1 or GFP at an moi of 100 in serum free medium for 6 hours. The cells were then washed and incubated in serum-containing medium over-night, after which cells were infected with HIV AD8 at an moi of 0.1 for 6 hours, at which point they were washed and refreshed with new medium. On day 6 post infection, supernatants were subjected to RT assay or titered using the indicator TZM-bl cell line. Virus amounts were normalized with level of infectivity being assayed by measuring luciferase within TZM-bl cells
74
. Normalized amounts of virus were used for subsequent infections.
Determination of cholesterol content and cholesterol replenishment assay
Equivalent amounts of virions were quantified by p24 assay and tested for cholesterol content using the Amplex Red cholesterol Assay Kit (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. To replenish cholesterol virus amounts were also normalized by p24 assay and incubated in 0.5mM (2-Hydroxypropyl)-ß-Cyclodextrin solution (Sigma Aldrich) with 1.5mM cholesterol (Sigma Aldrich) at 37°C for 1 hour. These quantified and normalized amounts of virus were used to infect TZM-bl cells and monitored for luciferase activity.
Cholesterol efflux
U87 cells were transfected with pcDNA3.1 and pCZ-vector (mock), pcDNA3.1SF2Nef and pCZ-vector (Nef), pCZ-cav-1 and pcDNA3.1 (Cav-1), pcDNA3.1SF2Nef and pCZ-cav-1 (Nef plus Cav-1), pCI NL4-3 NefG2A-HA (NefG2A) and pCZ-vector, or pCI NL4-3 NefG2A-HA and pCZ-cav-1 (NefG2A plus Cav-1). Twenty-four hours after transfection cell culture medium was replaced with serum free medium containing 2 μCi/mL [3H] cholesterol and 1.5% BSA and incubated for 36 hours. Radioisotope-containing medium was then removed and cells were washed twice with PBS and cultured for an additional 18 hours in serum free medium in the presence or absence of 50 μg/ml ApoA-l (Biomedical Technologies Inc., Stoughton, MA). Cholesterol content was measured in the cell free media as well as within cells after lysing using 0.1N NaOH. ApoA-l specific cholesterol efflux was determined using the formula: apoA-l specific efflux = % cholesterol efflux with apoA-l % cholesterol efflux without apoA-l (blank); cholesterol efflux= [cpm(supernatants)/cpm(supernatants+cells)] ×100%. HDL mediated cholesterol efflux is also examined by incubating cells for 18 hours in the presence or absence of 50 μg/ml HDL (Biomedical Technologies Inc., Stoughton, MA).To determine cholesterol efflux from macrophages, MDMs were first infected with Ad-Cav-1 or Ad-GFP at an moi of 50 for 24 hours, which was followed by infection of pseudotyped HIV pSG3Δenv (psHIVwtNef) or pNL4-3.Luc.R-E- (psHIVΔNef). Five days post infection cells were then labeled with 1 μCi/mL [3H] cholesterol for 48 hours and apoA-l mediated cholesterol efflux was determined as described above. Similarly cholesterol efflux from THP-1 cell-differentiated macrophages was determined 21 days after infecting with HIV AD8 at an moi of 0.001. Cholesterol efflux was also determined 14 days after THP-1 cell-differentiated macrophages infected with an moi of 0.001, 0.01, or 0.1. In addition, primary macrophages (MDMs) were infected with AD8 or ADnefmut HIV with an moi 0.01 and then cultured cells were subjected to cholesterol efflux assay 15 days after infection. ABCA-1 expression was determined by Western blots in MDMs 14 days after co-infection with AD8 or ADnefmut HIV and with Ad-Cav-1 or Ad-GFP. Inhibition of HIV replication was performed by treating infected cells with 5 uM azidothymidine (AZT) (Sigma-Aldrich, St. Louis, MO).
Immunoprecipitation and Immunoblotting analyses
U87 cells were transfected with pCZ-Cav-1 and HA-tagged Nef (pCI NL4-3 Nef-HA-WT) or HA-tagged NefG2A (pCI NL4-3 NefG2A-HA), followed by incubation of medium containing cholesterol (30μg/ml) for 48 hours. Cells were then treated with apoA-I (20μg/ml) for 30 min. Cells were put on ice, washed twice with cold PBS and total cellular protein was extracted in lysis buffer (50 mM Tris pH 7.5,100 mM NaCl, 1 mM EDTA, 0.1% (v/v) Triton X-100, 10 mM NaF, 1 mM phenylmethyl sulfonyl fluoride, and 1 mmol/L vanadate) with a complete protease Inhibitor mixture (Roche Diagnostics, Indianapolis, IN). The concentration of extracted protein was determined and adjusted to 1 ug/ul. A total of 500 ul was used for each immunoprecipitation, to which 2μg of antibodies (anti-Cav-1 or anti-HA) or normal IgG were added. The mixtures were incubated at 4°C overnight. Following the overnight incubation, 25 μl of protein A/G-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) were added and the mixtures were then rotated for 2 hours at 4°C. The beads were harvested by centrifugation and washed five times with lysis buffer. Loading buffer was added and boiled for 5 min. The samples were subjected to SDS-PAGE and analyzed by immunoblotting as described previously
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. The primary antibodies used for immunoblotting were rabbit polyclonal anti-Cav-1(Santa Cruz Biotechnology, Santa Cruz, CA), mouse monoclonal anti-Nef, rabbit Nef antiserum, and human monoclonal anti-Gag (NIH AIDS Research and Reference Reagent Program), goat polyclonal anti-HA (Genescript), mouse monoclonal anti-ABCA1 (abcam), and ß-actin protein antibody (Sigma, St. Louis, MO). The secondary antibodies were HRP-linked anti-rabbit, anti-mouse (Cell Signaling Technology, Inc., Danvers, MA), anti-human IgG (Sigma, St. Louis, MO) or anti-goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA).
Statistical analysis
Student’s t test was applied to analyze the differences between sets of data. All analyses were performed with SPSS 12.0.1 for Windows, and were considered significant at p ≤ 0.05.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SL Participated in the design of experiments, carried out most of the experiment, prepared samples and conducted cholesterol analysis and Western blots, analyzed data and contributed to manuscript preparation. PN participated and assisted in sample collections and experiments. XW was involved in the design of constructs used for the study as well as establish efficient methodology to deliver of protein of interest in primary macrophages. AM conceived the study, designed and coordinated experiments, participated in data analysis and prepared contributed the manuscript. All authors read and approved the final manuscript.
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Acknowledgements
This research was supported by a grant from the National Institutes of Health (AI39126) to A.M.
refgrp Caveolin, a protein component of caveolae membrane coatsRothbergKGHeuserJEDonzellWCYingYSGlenneyJRAndersonRGCell199268673lpage 68210.1016/0092-8674(92)90143-Zlink fulltext 1739974Caveolae and caveolin in immune cells: distribution and functionsHarrisJWerlingDHopeJCTaylorGHowardCJTrends Immunol20022315816410.1016/S1471-4906(01)02161-511864845Caveolin-1 expression negatively regulates cell cycle progression by inducing G(0)/G(1) arrest via a p53/p21(WAF1/Cip1)-dependent mechanismGalbiatiFVolonteDLiuJCapozzaFFrankPGZhuLPestellRGLisantiMPMol Biol Cell20011222292244pmcid 5859111514613Sterol efflux to apolipoprotein A-I originates from caveolin-rich microdomains and potentiates PDGF-dependent protein kinase activityFieldingPERusselJSSpencerTAHakamataHNagaoKFieldingCJBiochemistry2002414929493710.1021/bi012091y11939788Cellular apoptosis is associated with increased caveolin-1 expression in macrophagesGargalovicPDoryLJ Lipid Res2003441622163210.1194/jlr.M300140-JLR20012777465Caveolins and macrophage lipid metabolismGargalovicPDoryLJ Lipid Res200344112110.1194/jlr.R200005-JLR20012518018Caveolin-1 is a negative regulator of caveolae-mediated endocytosis to the endoplasmic reticulumLePUGuayGAltschulerYNabiIRJ Biol Chem20022773371337910.1074/jbc.M11124020011724808Visualizing caveolin-1 and HDL in cholesterol-loaded aortic endothelial cellsChaoWTFanSSChenJKYangVCJ Lipid Res2003441094109910.1194/jlr.M300033-JLR20012639973Expression of caveolin-1 enhances cholesterol efflux in hepatic cellsFuYHoangAEscherGPartonRGKrozowskiZSviridovDJ Biol Chem2004279141401414610.1074/jbc.M31106120014729661Identification of peptide and protein ligands for the caveolin-scaffolding domain Implications for the interaction of caveolin with caveolae-associated proteinsCouetJLiSOkamotoTIkezuTLisantiMPJ Biol Chem19972726525653310.1074/jbc.272.10.65259045678Caveolins, caveolae, and lipid rafts in cellular transport, signaling, and diseaseQuestAFLeytonLParragaMBiochem Cell Biol20048212914410.1139/o03-07115052333Caveolin-1 in oncogenic transformation, cancer, and metastasisWilliamsTMLisantiMPAm J Physiol Cell Physiol2005288C494C50615692148Identification of the HIV-1 gp41 core-binding motif in the scaffolding domain of caveolin-1HuangJHLuLLuHChenXJiangSChenYHJ Biol Chem20072826143615217197700The caveolin-1 binding domain of HIV-1 glycoprotein gp41 is an efficient B cell epitope vaccine candidate against virus infectionHovanessianAGBriandJPSaidEASvabJFerrisSDaliHMullerSDesgrangesCKrustBImmunity20042161762710.1016/j.immuni.2004.08.01515539149The caveolin-1 binding domain of HIV-1 glycoprotein gp41 (CBD1) contains several overlapping neutralizing epitopesBenferhatRKrustBRey-CuilleMAHovanessianAGVaccine2009273620363010.1016/j.vaccine.2009.03.05719464543Caveolin-1 modulates HIV-1 envelope-induced bystander apoptosis through gp41WangXMNadeauPELoYTMergiaAJ Virol2010846515652610.1128/JVI.02722-09290324220392844HIV infection upregulates caveolin 1 expression to restrict virus productionLinSWangXMNadeauPEMergiaAJ Virol2010849487949610.1128/JVI.00763-10293762320610713Caveolin 1 inhibits HIV replication by transcriptional repression mediated through NF-kappaBWangXMNadeauPELinSAbbottJRMergiaAJ Virol2011855483549310.1128/JVI.00254-11309498121430048Molecular interaction between caveolin-1 and ABCA1 on high-density lipoprotein-mediated cholesterol efflux in aortic endothelial cellsLinYCMaCHsuWCLoHFYangVCCardiovasc Res20077557558310.1016/j.cardiores.2007.04.01217499231Caveolins and cellular cholesterol balanceIkonenEPartonRGTraffic2000121221710.1034/j.1600-0854.2000.010303.x11208104Expression of caveolin-1 in hepatic cells increases oxidized LDL uptake and preserves the expression of lipoprotein receptorsTruongTQBrodeurMRFalstraultLRhaindsDBrissetteLJ Cell Biochem200910890691510.1002/jcb.2232119718657A role for caveolin in transport of cholesterol from endoplasmic reticulum to plasma membraneSmartEJYingYDonzellWCAndersonRGJ Biol Chem1996271294272943510.1074/jbc.271.46.294278910609Characterization of a cytosolic heat-shock protein-caveolin chaperone complex Involvement in cholesterol traffickingUittenbogaardAYingYSmartEJJ Biol Chem19982736525653210.1074/jbc.273.11.65259497388SR-BI, CD36, and caveolin-1 contribute positively to cholesterol efflux in hepatic cellsTruongTQAubinDFalstraultLBrodeurMRBrissetteLCell Biochem Funct20102848048910.1002/cbf.168020629037Lipid rafts and HIV pathogenesis: virion-associated cholesterol is required for fusion and infection of susceptible cellsLiaoZGrahamDRHildrethJEAIDS Res Hum Retroviruses20031967568710.1089/08892220332228090013678470Nef increases infectivity of HIV via lipid raftsZhengYHPlemenitasALinnemannTFacklerOTPeterlinBMCurr Biol20011187587910.1016/S0960-9822(01)00237-811516650Lovastatin inhibits HIV-1 expression in H9 human T lymphocytes cultured in cholesterol-poor mediumMaziereJCLandureauJCGiralPAuclairMFallLLachgarAAchourAZaguryDBiomed Pharmacother199448636710.1016/0753-3322(94)90077-97522603HIV entry in macrophages is dependent on intact lipid raftsCarterGCBernstoneLSanganiDBeeJWHarderTJamesWVirology200938619220210.1016/j.virol.2008.12.03119185899Lipid composition and fluidity of the human immunodeficiency virus envelope and host cell plasma membranesAloiaRCTianHJensenFCProc Natl Acad Sci USA1993905181518510.1073/pnas.90.11.5181466798389472Human immunodeficiency virus infection and macrophage cholesterol metabolismBukrinskyMSviridovDJ Leukoc Biol2006801044105110.1189/jlb.020611317056763Independent segregation of human immunodeficiency virus type 1 Gag protein complexes and lipid raftsDingLDerdowskiAWangJJSpearmanPJ Virol2003771916192610.1128/JVI.77.3.1916-1926.200314087512525626Human immunodeficiency virus type 1 assembly and lipid rafts: Pr55(gag) associates with membrane domains that are largely resistant to Brij98 but sensitive to Triton X-100HolmKWeclewiczKHewsonRSuomalainenMJ Virol2003774805481710.1128/JVI.77.8.4805-4817.200315212212663787Evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid raftsNguyenDHHildrethJEJ Virol2000743264327210.1128/JVI.74.7.3264-3272.200011182710708443Plasma membrane rafts play a critical role in HIV-1 assembly and releaseOnoAFreedEOProc Natl Acad Sci USA200198139251393010.1073/pnas.2413202986114311717449Role for human immunodeficiency virus type 1 membrane cholesterol in viral internalizationGuyaderMKiyokawaEAbramiLTurelliPTronoDJ Virol200276103561036410.1128/JVI.76.20.10356-10364.200213659012239312Nef increases the synthesis of and transports cholesterol to lipid rafts and HIV-1 progeny virionsZhengYHPlemenitasAFieldingCJPeterlinBMProc Natl Acad Sci USA20031008460846510.1073/pnas.143745310016625112824470Nef induces multiple genes involved in cholesterol synthesis and uptake in human immunodeficiency virus type 1-infected T cellsWout ABv ’tSwainJVSchindlerMRaoUPathmajeyanMSMullinsJIKirchhoffFJ Virol200579100531005810.1128/JVI.79.15.10053-10058.2005118159716014965Human immunodeficiency virus impairs reverse cholesterol transport from macrophagesMujawarZRoseHMorrowMPPushkarskyTDubrovskyLMukhamedovaNFuYDartAOrensteinJMBobryshevYVetal PLoS Biol20064e36510.1371/journal.pbio.0040365162903417076584Interleukin-10 facilitates both cholesterol uptake and efflux in macrophagesHanXKitamotoSLianQBoisvertWAJ Biol Chem2009284329503295810.1074/jbc.M109.040899278171019776020Virion incorporation of human immunodeficiency virus type 1 Nef is mediated by a bipartite membrane-targeting signal: analysis of its role in enhancement of viral infectivityWelkerRHarrisMCardelBKrausslichHGJ Virol199872883388401103009765428HIV type 1 Nef increases the association of T cell receptor (TCR)-signaling molecules with T cell rafts and promotes activation-induced raft fusionDjordjevicJTSchibeciSDStewartGJWilliamsonPAIDS Res Hum Retroviruses20042054755510.1089/08892220432308780415186530Mutation of the ABCA1 C-terminus disrupts HIV-1 Nef binding but does not block the Nef enhancement of ABCA1 protein degradationMujawarZTamehiroNGrantASviridovDBukrinskyMFitzgerald1MLBiochemistry2010498338834910.1021/bi100466q294356820731376Caveolin-1 and regulation of cellular cholesterol homeostasisFrankPGCheungMWPavlidesSLlaveriasGParkDSLisantiMPAm J Physiol Heart Circ Physiol2006291H677H68610.1152/ajpheart.01092.200516603689Retroviral matrix and lipids, the intimate interactionHamard-PeronEMuriauxDRetrovirology201181510.1186/1742-4690-8-15305929821385335Stimulation of the liver X receptor pathway inhibits HIV-1 replication via induction of ATP-binding cassette transporter A1MorrowMPGrantAMujawarZDubrovskyLPushkarskyTKiselyevaYJennelleLMukhamedovaNRemaleyATKashanchiFMol Pharmacol20107821522510.1124/mol.110.065029291785920479131The macrophage: the intersection between HIV infection and atherosclerosisCroweSMWesthorpeCLMukhamedovaNJaworowskiASviridovDBukrinskyMJ Leukoc Biol20108758959810.1189/jlb.0809580308548319952353Mechanisms of disease: macrophage-derived foam cells emerging as therapeutic targets in atherosclerosisChoudhuryRPLeeJMGreavesDRNat Clin Pract Cardiovasc Med2005230931510.1038/ncpcardio019516265535The endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophagesFengBYaoPMLiYDevlinCMZhangDHardingHPSweeneyMRongJXKuriakoseGFisherEANat Cell Biol2003578179210.1038/ncb103512907943Reverse cholesterol transport and cholesterol efflux in atherosclerosisOhashiRMuHWangXYaoQChenCQJM20059884585610.1093/qjmed/hci13616258026Specific binding of ApoA-I, enhanced cholesterol efflux, and altered plasma membrane morphology in cells expressing ABC1WangNSilverDLCostetPTallARJ Biol Chem2000275330533305810918065Regulation of macrophage cholesterol efflux through hydroxymethylglutaryl-CoA reductase inhibition: a role for RhoA in ABCA1-mediated cholesterol effluxArgmannCAEdwardsJYSawyezCGO'NeilCHHegeleRAPickeringJGHuffMWJ Biol Chem2005280222122222110.1074/jbc.M50276120015817453ATP-binding cassette transporter A1 expression disrupts raft membrane microdomains through its ATPase-related functionsLandryYDDenisMNandiSBellSVaughanAMZhaXJ Biol Chem2006281360913610110.1074/jbc.M60224720016984907Host hindrance to HIV-1 replication in monocytes and macrophagesBergamaschiAPancinoGRetrovirology201073110.1186/1742-4690-7-31286879720374633The macrophage in HIV-1 infection: from activation to deactivation?HerbeinGVarinARetrovirology201073310.1186/1742-4690-7-33285975220380696Molecular mechanisms of HIV-1 persistence in the monocyte-macrophage lineageLe DouceVHerbeinGRohrOSchwartzCRetrovirology201073210.1186/1742-4690-7-32287350620380694Human immunodeficiency virus 1 favors the persistence of infection by activating macrophages through TNFGuillemardEJacquemotCAilletFSchmittNBarre-SinoussiFIsraelNVirology200432937138010.1016/j.virol.2004.08.03015518816Human T lymphotropic virus type III infection of human alveolar macrophagesSalahuddinSZRoseRMGroopmanJEMarkhamPDGalloRCBlood1986682812843013342The role of mononuclear phagocytes in HTLV-III/LAV infectionGartnerSMarkovitsPMarkovitzDMKaplanMHGalloRCPopovicMScience198623321521910.1126/science.30146483014648Macrophages archive HIV-1 virions for dissemination in transSharovaNSwinglerCSharkeyMStevensonMEMBO J2005242481248910.1038/sj.emboj.7600707117314815920469HIV-1 and the host cell: an intimate associationFreedEOTrends Microbiol20041217017710.1016/j.tim.2004.02.00115051067HIV-1 Accessory Proteins. Ensuring Viral Survival in a Hostile EnvironmentMalimMHEmermanMCell Host Microbe2008338839810.1016/j.chom.2008.04.00818541215A whole genome screen for HIV restriction factorsLiuLOliveiraNMCheneyKMPadeCDrejaHBerginAMBorgdorffVBeachDHBishopCLDittmarMTMcKnightARetrovirology201189410.1186/1742-4690-8-94322884522082156Microarray data on gene modulation by HIV-1 in immune cells: 2000–2006GiriMSNebozhynMShoweLMontanerLJJ Leukoc Biol2006801031104310.1189/jlb.030615716940334Circulating Monocytes in HIV-1-Infected Viremic Subjects Exhibit an Antiapoptosis Gene Signature and Virus- and Host-Mediated Apoptosis Resistance1GiriMSNebozyhnMRaymondAGekongeBHancockACreerSNicolsCYousefMFoulkesASMounzerKJ Immunol20091824459447010.4049/jimmunol.0801450277606419299747HIV-1 induced macrophage gene expression includes p21, a target for viral regulationVazquezNGreenwell-WildTMarinosNJSwaimWDNaresSOttDESchubertUHenkleinPOrensteinJMSpornMBWahlSMJ Virol2005794479449110.1128/JVI.79.7.4479-4491.2005106152215767448The multiple faces of caveolaePartonRGSimonsKNat Rev20078185194De novo formation of caveolae in lymphocytes by expression of VIP21-caveolinFraAMWilliamsonESimonsKParton RGRGProc Natl Acad Sci USA1995928655865910.1073/pnas.92.19.8655410257567992Expression of Caveolin-1 in Human T Cell Leukemia Cell LinesHatanakaMMaedaTIkemotoTMoriHSeyaTShimizuABioch Bioph Res Comm199825338238710.1006/bbrc.1998.9744Expression of caveolin-1 in lymphocytes induces caveolae formation and recruitment of phosphofructokinase to the plasma membraneVallejoJHardinCDFASEB J200516586587Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular cloneAdachiAGendelmanHEKoenigSFolksTWilleyRRabsonAMartinMAJ Virol1986592842912530773016298Role of the basic domain of human immunodeficiency virus type 1 matrix in macrophage infectionFreedEOEnglundGMartinMAJ Virol199569394939541891247745752Identification of the envelope V3 loop as the primary determinant of cell tropism in HIV-1HwangSSBoyleTJLyerlyHKCullenBRScience1991253717410.1126/science.19058421905842Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phase of the cell cycle by inhibiting p34cdc2 activityHeJChoeSWalkerRDi MarzioPMorganDOLandauNRJ Virol199569670567111895807474080Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytesConnorRIChenBKChoeSLandauNRVirology199520693594410.1006/viro.1995.10167531918Emergence of resistant human immunodeficiency virus type 1 in patients receiving fusion inhibitor (T-20) monotherapyWeiXDeckerJMLiuHZhangZAraniRBKilbyJMSaagMSWuXShawGMKappesJCAntimicrob Agents Chemother2002461896190510.1128/AAC.46.6.1896-1905.200212724212019106Antibody neutralization and escape by HIV-1WeiXDeckerJMWangSHuiHKappesJCWuXSalazar-GonzalezJFSalazarMGKilbyJMSaagMSNature200342230731210.1038/nature0147012646921Reconstitution and molecular analysis of an active human immunodeficiency virus type 1 Nef/p21-activated kinase 2 complexRaneyAKuoLSBaughLLFosterJLGarciaJVJ Virol200579127321274110.1128/JVI.79.20.12732-12741.2005123586416188976Dynamic evolution of the human immunodeficiency virus type 1 pathogenic factor, NefO'NeillEKuoLSKriskoJFTomchickDRGarciaJVFosterJLJ Virol2006801311132010.1128/JVI.80.3.1311-1320.2006134696216415008Construction and characterization of a stable full-length macrophage-tropic HIV type 1 molecular clone that directs the production of high titers of progeny virionsTheodoreTSEnglundGBuckler-WhiteABucklerCEMartinMAPedenKWAIDS Res Hum Retroviruses19961219119410.1089/aid.1996.12.1918835195Toll-like Receptor 4 Mediates Oxidized LDL-Induced Macrophage Differentiation to Foam CellsHowellKWMengXFullertonDAJinCReeceTBClevelandJCsuf JrJ Surg Res2011171e27e3110.1016/j.jss.2011.06.03321920554Inhibition of hepatitis B virus replication by MyD88 is mediated by nuclear factor-kappaB activationLinSWuMXuYXiongWYiZZhangXZhenghongYBiochim Biophys Acta200717721150115710.1016/j.bbadis.2007.08.00117935950


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Abstract
Background
HIV infection results in inhibited cholesterol efflux by apolipoprotein A-I (apoA-I) in macrophages, and this impairment involves Nef mediated down-regulation and redistribution of ATP-binding cassette transporter A1 (ABCA-1). We investigated the effect of caveolin-1 (Cav-1) on the cholesterol efflux by apoA-I in HIV infected primary and THP-1 cell-differentiated macrophages as well as astrocyte derived glioblastoma U87 cells.
Results
Our results reveal that Cav-1 restores the Nef -mediated impairment of cholesterol efflux by apoA-I in both cell types. Co-immunoprecipitation studies indicate a physical association of Cav-1 and Nef. The level of ABCA-1 expression remains the same whether Cav-1 is over-expressed or not. In addition, we examined the cholesterol composition of HIV particles released from Cav-1 treated cells and identified that the cholesterol content is dramatically reduced. The infectivity level of these virus particles is also significantly decreased.
Conclusions
These observations suggest that the interplay of Cav-1 with Nef and cholesterol subsequently counters Nef induced impairment of cholesterol efflux by apoA-l. The findings provide a cellular mechanism by which Cav-1 has an ability to restore HIV mediated impairment of cholesterol efflux in macrophages. This subsequently influences the cholesterol content incorporated into virus particles thereby inhibiting HIV infectivity and contributing to HIV’s persistent infection of macrophages.
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Lin, Shanshan
Nadeau, Peter E
Wang, Xiaomei
Mergia, Ayalew
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Shanshan Lin et al.; licensee BioMed Central Ltd.
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Retrovirology. 2012 Oct 15;9(1):85
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RESEARCHOpenAccessCaveolin-1reducesHIV-1infectivitybyrestoration ofHIVNefmediatedimpairmentofcholesterol effluxbyapoA-IShanshanLin,PeterENadeau,XiaomeiWangandAyalewMergia*AbstractBackground: HIVinfectionresultsininhibitedcholesteroleffluxbyapolipoproteinA-I(apoA-I)inmacrophages,and thisimpairmentinvolvesNefmediateddown-regulationandredistributionofATP-bindingcassettetransporterA1 (ABCA-1).Weinvestigatedtheeffectofcaveolin-1(Cav-1)onthecholesteroleffluxbyapoA-IinHIVinfected primaryandTHP-1cell-differentiatedmacrophagesaswellasastrocytederivedglioblastomaU87cells. Results: OurresultsrevealthatCav-1restorestheNef-mediatedimpairmentofcholesteroleffluxbyapoA-Iinboth celltypes.Co-immunoprecipitationstudiesindicateaphysicalassociationofCav-1andNef.ThelevelofABCA-1 expressionremainsthesamewhetherCav-1isover-expressedornot.Inaddition,weexaminedthecholesterol compositionofHIVparticlesreleasedfromCav-1treatedcellsandidentifiedthatthecholesterolcontentis dramaticallyreduced.Theinfectivitylevelofthesevirusparticlesisalsosignificantlydecreased. Conclusions: TheseobservationssuggestthattheinterplayofCav-1withNefandcholesterolsubsequently countersNefinducedimpairmentofcholesteroleffluxbyapoA-l.Thefindingsprovideacellularmechanismby whichCav-1hasanabilitytorestoreHIVmediatedimpairmentofcholesteroleffluxinmacrophages.This subsequentlyinfluencesthecholesterolcontentincorporatedintovirusparticlestherebyinhibitingHIVinfectivity andcontributingtoHIV ’ spersistentinfectionofmacrophages. Keywords: HIV,Caveolin-1,Cholesterolefflux,Nef,ApolipoproteinA-IBackgroundCaveolin1(Cav-1),a21~24-kDascaffoldingprotein,is animportantstructuralcomponentofcaveolae[1],small invaginationsoftheplasmamembrane,whichareenrichedincholesterol,phospholipids,andsphingolipids. Thisproteinishighlyexpressedinterminallydifferentiatedcellsincludingendothelialcells,macrophages,dendriticcellsandadipocytes[2,3].Functionalstudieshave shownthatCav-1isinvolvedinawiderangeofcellular processes,includingcellcycleregulation,signaltransduction,endocytosis,cholesteroltraffickingandefflux[3-9]. MultiplelinesofevidenceindicatethatCav-1actsasa scaffoldingproteincapableofdirectlyinteractingwithand modulatingtheactivityofcaveolin-boundsignalingmolecules.TheCav-1scaffoldingdomain(CSD),residues82to 101,isessentialforbothCav-1oligomerizationandthe interactionofcaveolinwithotherproteins[10].AssociationswithotherproteinsthroughtheCSDhelpprovide coordinatedandefficientsignaltransduction[11,12].The CSDservesasareceptorforbindingproteinscontaining thesequence X XXXX XXXX XX ,or X XXX X XX ( representinganyaromaticaminoacidandX anyotheraminoacid)[10].HIVEnvhasbeenshown tointeractwithCav-1viaamotif(WNNMTWMQW) localizedwithintheectodomain(theC-terminalheptad repeats)ofHIV-1gp41[13-15].Ourgrouphasshownthe bindingofCav-1withHIVEnvinthelipidraftswhich subsequentlyblockscellfusionandinnocentbystander killingmediatedbyHIVenvelope[16].Wehavealso demonstratedthatHIVinfectioninprimaryhuman monocytederivedmacrophages(MDMs)resultsina dramaticup-regulationofCav-1expressionmediated byHIVTat[17].Furthermore,over-expressionofCav-1 *Correspondence: mergiaa@ufl.edu DepartmentofInfectiousDiseaseandPathology,UniversityofFlorida, Gainesville,Florida32611,USA 2012Linetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and reproductioninanymedium,providedtheoriginalworkisproperlycited.Lin etal.Retrovirology 2012, 9 :85 http://www.retrovirology.com/content/9/1/85

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causessignificantreductioninHIVreplicationinmacrophages.Cav-1inhibitsHIVreplicationthroughtranscriptionalrepressionofviralgeneexpressionbymodulating theNFBpathway[18].Theup-regulationofCav-1by HIVinfectionandsubsequentinhibitionofHIVreplicationsuggestaroleforCav-1inmacrophagepersistent infection. Cav-1playsanimportantroleincellularcholesterol homeostasis,aprocessthatcontrolsintracellularlipid compositionandpreventscholesterolaccumulation. Cav-1hasbeenimplicated inmodulatingtheexpressionoflipoproteinreceptor sandinteractswithmany lipidtransportermolecules[11,19-21].Furthermore,it isinvolvedinthetransportofnewlysynthesizedcholesterolfromtheendoplasmicreticulum(ER)tothe plasmamembrane[11,22,23]andpromotescholesterol effluxinhepaticcells[9,24].HIVappearstomanipulate cellularcholesterolmetabolismtoensurethatthereisa sufficientsupplyofcholesterolandthatitislocatedin theappropriatecompartmentssuchaslipidraftsforefficientvirusreleaseandsubsequentinfectivity[25-28]. Cholesterolisanimportantcomponentthatinfluences HIVproductionandefficientvirusinfectivity.CholesteroldepletionsignificantlyreducesHIV-1particle production[29-34].Virusinfectivityisalsonegatively affectedwhenHIVisproducedfromcholesteroldepleted cells[26,35]. TheHIVaccessoryproteinNefhasanabilitytoexploit cholesterolmetabolism.Proposedmechanismsforthis strategyincludebindingtocholesterolandaidingthe transportofnewlysynthesizedcholesterolintolipid raftsandviralparticlesaswellasenhancingcholesterol synthesis[36,37].Nefhasalsobeenshowntoimpair ATPbindingcassettetransporterprotein1(ABCA-1)dependentcholesteroleffluxfromhumanmacrophages bydown-regulationandredistributionofABCA-1[38]. ThissuggeststhatNefisinvolvedinHIVmediated cholesterolaccumulation.SinceCav-1hasahighaffinityforcholesterolandaidsinthetransportof newlysynthesizedcholesterolfromtheERtotheplasma membraneandindirectlypromotingthetransfertoextracellularacceptorssuchaslipidfreeapolipoproteinA-I (apoA-I)wehypothesizeitwouldinfluencethelevelof cholesterolaccumulationaswellasvirusproduction andinfectivity.Macrophagesaremajortargetsfor HIVinfectionandalsoplayanimportantroleinits pathogenesis.Theup-regulationofCav-1byHIVinfectionandtheroleofCav-1incholesteroltrafficking suggestamechanismforaCav-1/cholesterolmediated impactonHIVreplicationinmacrophages.Inthisreport, weestablishevidenceforaCav-1/cholesterolmediated mechanismofinhibitionofHIVreplicationforthefirst timeprovidinganewangleinunderstandingHIV ’ spersistentinfectionofmacrophages.ResultsCav-1restoresHIVNefmediatedimpairmentofcholesterol effluxbyapoA-IinU87cellsandmacrophagesHIVinfectionimpairsATP-bindingcassettetransporter A1(ABCA-1)dependentcholesteroleffluxbyapoA-l. TheNefproteinisidentifiedasthekeymoleculeresponsibleforthiseffect[38].SinceCav-1isanimportant regulatorofcholesteroltraffickingandisinvolvedinthe transportofnewlysynthesizedcholesterolfromtheER totheplasmamembrane,itislikelytoinfluenceNef mediatedABCA-1dependentdownmodulationofcholesterolefflux.TodeterminewhetherCav-1countersthe influenceofNefoncholesteroltrafficking,first,wetestedtheimpairmentofcholesteroleffluxinHIVinfected THP-1cell-differentiatedmacrophages.HIVAD8infectedTHP-1cellswereexposedtolipid-freeapoA-Ior HDLtreatmenttoinducecholesterolefflux.Cholesterol effluxwasmeasuredasthefractionoftotalradiolabeled cholesterolappearinginthemediuminthepresence ofapoA-IaftersubtractionofvaluesforapoA-I-free medium[39].ApoA-Istimulatedcholesteroleffluxfrom HIVinfectedTHP-1cell-differentiatedmacrophageswas markedlydecreasedinadosedependentmannerwith thereductionreaching71.6%ascomparedtouninfected cells(Figure1A).Nosignificantdifferencewasobserved betweenHIVinfectedanduninfectedcellsinHDL mediatedcholesterolefflux(Figure1B).Thedecreasein cholesteroleffluxtoapoA-IbyHIVinfectionwasnot presentinthepresenceofAZT,aninhibitoroftheHIV replication(Figure1C).TheseresultssuggestthatHIV infectiondecreasedtheapoA-lmediatedcholesteroleffluxsubstantiallyandareinaccordancewithprevious findings[38].TofurtherexamineimpairmentofcholesteroleffluxduetoHIVinfectionthelevelofcholesterol accumulationwastestedbyoilredOstainingofinfected macrophages.AsshowninFigure1D,accumulatedcholesterolwasmarkedlyincreasedinAD8orBalinfected macrophagesascomparedtouninfectedcellswhichis similartopreviousfindings[38]. ToaddresstheinfluenceofCav-1oncholesterolefflux ofHIVinfectedcells,weexaminedwhetherCav-1canrestoreNefmediatedimpairmentofcholesterolefflux.First, U87-CD4-CXCR4cellsweretransfectedwithaCav-1expressionconstruct(pCZ-Cav-1)inthepresenceorabsenceofaNefexpressionplasmid(pcDNA3.1SF2Nef). ApoA-lorHDLmediatedcholesteroleffluxwasmeasured byharvestingtheculturemediaaswellascelllysatesamples.AsacontrolU87-CD4-CXCR4cellswerealsotransfectedwithexpressionvectorlackingCav-1(pCZ-vector) orNef(pcDNA3.1).TheexpressionofCav-1andNefin transfectedcellswasdeterminedbyWesternblotanalysis(Figure2A).AsexpectedapoA-lmediatedcholesteroleffluxfromcellstransfectedwiththeNefexpression constructalonewasreducedby77%comparedtocellsLin etal.Retrovirology 2012, 9 :85 Page2of16 http://www.retrovirology.com/content/9/1/85

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transfectedwiththeplasmidconstructdevoidofnef orcellsthatreceivedpCZ-Cav-1(Figure2B).Interestingly,apoA-lmediatedcholesteroleffluxfromcellscotransfectedwithpCZ-Cav-1andpcDNA3.1SF2Nefwas comparable,andevenslightlyhigher,tothatofmock whichdidnotreceiveNeftreatment,suggestingthat Cav-1canrestoretheimpairmentofcholesterolefflux causedbyNef.NeitherNefnorCav-1hadsignificanteffectonHDLmediatedcholesterolefflux(datanotshown). Weconfirmedourfindingsbyconductingthesestudies inphysiologicallyrelevantprimarymonocytederived macrophages(MDMs).WeinfectedMDMswithvesicular stomatitisvirusglycoprotein(VSV-G)Envpseudotyped pSG3 envHIVproviruscarryingwildtypenef(psHIVwtNef)ordefectivenef(psHIV Nef)foroneroundofreplication.InfectionofMDMswithpsHIVwtNefshowed asignificantreduction(70%)inapoA-lmediatedcholesteroleffluxascomparedtouninfectedcells(Figure2C) similartowhatisobservedintheU87-CD4-CXCR4cells (Figure2B).Thereductionincholesteroleffluxwas75% whencomparedtoNefdefectiveHIV.TherewasnosignificantdifferenceincholesteroleffluxbetweenNefdefectiveHIVinfectedanduninfectedMDMs.Introduction ofexogenousCav-1intopsHIVwtNefMDMsusing adenovirusexpressingCav-1(Ad-Cav-1)increasedcholesteroleffluxby43%comparedtocellsonlyinfectedwith psHIVwtNef.ThecontroladenoviruscarryingGFP(AdGFP)wasnotabletocountercholesteroleffluximpairmentinducedbypsHIVwtNefinfectionoftheMDMs (Figure2C).Asexpected,WesternblotanalysisofMDMs transducedwithAd-GFPrevealedendogenousCav-1 expression,withincreasedamountsofCav-1inMDMs treatedwithAd-Cav-1(Figure2D).Furthermore,introductionofexogenousCav-1orGFPusingadenovirusdid notaltercholesteroleffluxfromMDMsinfectedwiththe NefdefectiveHIV.Inaddition,wecomparedapoA-I mediatedcholesteroleffluxinMDMsinfectedwithwild typeAD8andreplicationcompetentnefdeletedAD8 Figure1 HIV-1impairsapoA-ImediatedcholesteroleffluxfromTHP-1cell-differentiatedmacrophages. ( A )HIVAD8infectedand uninfectedTHP-1cell-differentiatedmacrophageswereculturedfor14days.MOIrepresentsmultiplicityofinfection.Cellswerelabeledwith[3H] cholesterol,andsubsequentlyincubatedwithmediainthepresenceandabsenceofapoA-I(50 g/ml).ApoA-I-inducedcholesteroleffluxwas measuredandanalyzedasdescribedintheMaterialsandMethods.( B )Cellsweretreatedthesameasabovebutincubatedwithmediuminthe presenceorinabsenceofHDL(50 g/ml)andHDLmediatedcholesteroleffluxwasmeasured.( C )ApoA-Imediatedcholesteroleffluxwas determinedinthepresenceandabsenceoftheHIVreplicationinhibitorAZT(5uM).Allexperimentswereperformedintriplicate,andresults shownaremeanSDwithPvalues.( D )UninfectedorinfectedwithHIVAD8(moi0.01)andHIVBal(moi0.001)THP-1cell-differentiated macrophagesculturedfor10daysthenincubatedwithAcLDL(50 g/ml)for48hfollowedby30 g/mlapoA-Istimulationfor18hours.Lipid accumulationwasdeterminedbyOilredOstainingbylightmicroscopy.HIVreversetranscriptaseactivityinculturemediumisshowninthe bottompanel. Lin etal.Retrovirology 2012, 9 :85 Page3of16 http://www.retrovirology.com/content/9/1/85

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(ADnefmut)viruses.Cholesteroleffluxwasdecreasedby 56.3%inwildtypeinfectedmacrophagescomparedtouninfectedcellswhileADnefmutinfectionhadnosignificant effect(Figure2E).Theseresultstakentogethersuggest thatCav-1iscapableofrestoringHIVinducedimpairmentofapoA-1mediatedcholesterolefflux.Toconfirm ourfindings,wefurtherexaminedtheaffectofCav-1 oncholesterolaccumulationbyoilredOstainingin HIVinfectedTHP-1cell-differentiatedmacrophage cells.AsshowninFigure2F,psHIVwtNefinfectedcells hadsignificantlyincreasedcholesterolaccumulationcomparedtouninfectedcells(mock)whichissimilartoHIV AD8infectedTHP-1cell-differentiatedmacrophages (Figure1D).Theco-infectionofmacrophageswithAdCav-1andpsHIVwtNef,ontheotherhand,showedadramaticreductionofintracellularcholesterolinclusions whencomparedtopsHIVwtNefonlyinfectedcells.As expected,nosignificantinfluenceonintracellularcholesterolaccumulationwasobservedwhenTHP-1cell-differentiatedmacrophageswereinfectedwithNefdefective HIV(psHIV Nef).Inaddition,over-expressionofCav-1 byAd-Cav-1infectiondidnotalterintracellularcholesterolinclusionsincellsinfectedwithpsHIV Nef. WefurtherdeterminedwhetherendogenousCav-1 hasaneffectonNefmediatedsuppressionofcholesterol effluxtoapoA-1inU87cellsandTHP-1cell-differentiated macrophages.TheexpressionofendogenousCav-1was knockeddownusingspecificsiRNA,andtheexpressionof Figure2 Cav-1restoresHIV 1NefmediatedimpairmentofcholesteroleffluxtoapoA-I. U87-CD4-CXCR4cellsweretransfectedwithNef expressionplasmid(pcDNA3.1SF2Nef)alongwithpCZ-cav-1orpCZvector.Cellswerelabeledwith[3H]cholesterolfor36h.( A )ThelevelofNef andCav-1expressionwasdeterminedbyWesternblotanalysis.( B )ApoA-Iinducedcholesteroleffluxisshown.Allexperimentswereperformed intriplicate,andresultsshownaremeanSDwithPvalues.( C )Primarymonocytederivedmacrophages(MDMs)wereinfectedwithAd-Cav-1or Ad-GFP,followedbyinfectionwithVSV-GpseudotypedHIV,eithercarryingNef(psHIVwtNef)ordefectiveNef(psHIV Nef)atanmoiof5.ApoA-I mediatedcholesteroleffluxwasperformedandanalyzedbyincubatingcellsinmediuminthepresenceorabsenceof50 g/mlapoA-I.The resultsarepresentedasapercentageofcholesteroleffluxtoapoA-Ifromcontrol(setas100%),andarethemeanSDoftriplicate determinations.TheexpressionlevelsofCav-1andGFPareshownin( D ).( E )PrimarymacrophageswereinfectedwithAD8ornefdefectiveAD8 (ADnefmut)atanmoiof0.01.ApoA-Imediatedcholesteroleffluxwasmeasured15dayspostinfection.( F )THP-1cell-differentiatedmacrophages wereinfectedwithpsHIVwtNeforpsHIV Nefatanmoi3withorwithoutAd-Cav-1.Onday5afterinfection,OilredOstainingwasperformed andarepresentativeareaineachwellisshown. Lin etal.Retrovirology 2012, 9 :85 Page4of16 http://www.retrovirology.com/content/9/1/85

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NefwasaccomplishedbytransfectingU87cellswith pCDNA3.1SF2NeforpseudotypedHIV(psHIVwtNef)infectionofTHP-1cell-differentiatedmacrophages.The siRNAtreatmentreducedtheexpressionofCav-1by76% inU87cellsand38%inTHP-1cell-differentiatedmacrophages(Figure3Aand3B,respectively).Cholesterolefflux toapoA1wasreducedby61%inNefexpressingU87cells intheabsenceofsiRNAtargetingCav-1(Figure3A).The levelofcholesterolwasfurtherreduced(96%)whenCav-1 expressionwasknocked-downwithCav-1specificsiRNA. SimilarresultswereobservedinTHP-1cell-differentiated macrophagesshowingadecreaseincholesteroleffluxby 50%inNefexpressingcellsandby79%inCav-1siRNA treatedNefexpressingcells(Figure3B).Furthermore,the levelsofcholesteroleffluxcorrelatewiththeefficiencyof siRNAknock-downinU87andTHP-1cells(Figure3A and3B). Inaddition,todeterminewhetherCav-1specifically restoresNefmediatedimpairmentofcholesterolefflux toapoA-l,U87cellswereco-transfectedwithaNef mutant(NefG2A)andCav-1expressingplasmids.The NefG2AisaNefmutantthatcannotundergomyristoylation[40].Itsassociationwiththeplasmamembraneis impaired[26,35],anditlackstheabilitytodecrease Figure3 siRNAknockdownofCav-1anditseffectoncholesterolofcellsexpressingwildtypeNeforNefG2A. ( A ).U87-CD4-CXCR4cells weretransfectedwithCav-1siRNAorcontrolsiRNA(CTL-siRNA)followedbytransfectionwithNefexpressingplasmid(pSHIVwtNef)orpCDNA3.1 vector.ThelevelofCav-1expressioninsiRNAtreatedcellswasdetectedbyWesternblottinganalysis.Cellswerelabelledwith[3H]cholesterolfor 36hoursandapoA-Iinducedcholesteroleffluxwasmeasured.Resultsareshownasthepercentageofcholesteroleffluxrelativetocontrol. ( B )THP-1cell-differentiatedmacrophageswerefirsttransfectedwithCav-1siRNAorCTL-siRNAwhichwasfollowedbyinfectionwithVSV-G pseudotypedHIV(psHIVwtNef)atanmoiof3.CellswereagaintreatedwithsiRNAthedayafterinfection.Cholestroleffluxwasmeasueredas describedabovefourdayspost-infection.( C )U87-CD4-CXCR4cellsweretransfectedwithpCI(Mock),pCINL4-3Nef-HA-WT(Nef),pCINL4-3 NefG2A-HA(NefG2A),orpCINL4-3NefG2A-HAalongwithpCZ-cav-1(NefplusCav-1).CholestroleffluxtoapoA-Iwasdetermined.Allexperiments wereperformedintriplicateandresultsshownaremeanSDwithPvalues. Lin etal.Retrovirology 2012, 9 :85 Page5of16 http://www.retrovirology.com/content/9/1/85

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apoA-lstimulatedcholesterolefflux[36,41].Asshown inFigure3C,cellsexpressingNefexperienced62%less cholesteroleffluxtoapoA-IcomparedtoMock.Incontrast,NefG2AhadnoeffectonapoA-lmediatedcholesteroleffluxinthepresenceofeitherendogenousoroverexpressingCav-1cells.Thesestudies,therefore,clearlyestablishthatCav-1countersNefmediatedimpairmentof cholesteroleffluxbyapoA-l.Cav-1over-expressionhasnoeffectonABCA-1 expressionNefhasbeenshowntoimpairABCA1-dependentcholesteroleffluxfromhumanmacrophages,andtheexpressionofABCA-1isshowntobedownregulatedby HIVinfectionorNefexpression[38,42].Cav-1isimplicatedinpositiveregulationofABCA-1expression andABCA-1expressionisdownregulatedinCav-1 knockoutmice[43].InordertounderstandthemechanismresponsibleforCav-1mediatedrestorationofcholesteroleffluxuponHIVinfectionweexaminedthe expressionofABCA-1inCav-1over-expressingcells. U87-CD4-CXCR4cellsweretransfectedwithpCZ-Cav-1 inadose-dependentmanner,andthelevelofABCA-1 expressionwasmonitoredbyWesternblotanalysis.As showninFigure4A,Cav-1over-expressiondidnotalter thelevelofABCA-1expression.Tofurtherconfirmour findings,weco-transfectedU87cellswithpCZ-Cav-1 andpcDNA3.1SF2Nef,andthenanalyzedABCA-1expressionbyWesternblotinthepresenceandabsenceof Cav-1orNef.AlthoughNefdown-regulatedABCA-1 (by69%)thelevelofABCA-1expressionremainedthe sameinNeftreatedcellswhetherCav-1isover-expressed ornot(Figure4B),suggestingthatCav-1mediatedrestorationofcholesteroleffluxisnotrelatedtotheregulation Figure4 Cav-1over-expressionhasnoinfluenceonABCA-1expression. ( A )U87-CD4-CXCR4cellsweretransientlytransfectedeitherwith pCZ-vector(mock)orwithdifferentdosesofCav-1(pCZ-Cav-1).ExpressionofABCA-1proteinwasexaminedbyWesternblotanalysis.( B )Cells weretransfectedwithpCZ-vector(mock),Nefexpressingconstruct(pcDNA3.1SF2Nef)andpCZ-Cav-1orpCZ-Cav-1only.Theexpressionlevelsof ABCA-1inthepresenceorabsenceofNefweredeterminedinthecellswithendogenousorover-expressingCav-1.( C )MDMswereinfectedwith VSV-GpseudotypedHIV(psHIVwtNef),Ad-Cav-1,orboth.ThelevelofCav-1andABCA-1expressionwasexaminedbyWesternblotanalysisinthe presenceandabsenceofCav-1and/orNef.Representativeresultsfromthreeexperimentsareshownin A B and C .( D )MDMswereco-infected withwildtypeAD8ornefdefectiveAD8(ADnefmut)andAd-Cav-1orAd-GFP.ThelevelofexpressionofABCA-1,Nef,andCav-1wasexamined byWesternblotanalysis.( E )TodeterminewhethersiRNAknock-downofCav-1affectstheexpressionofABCA-1samplesusedinexperiment3A andBwereusedtomeasurethelevelofABCA-1expression.NotethatthesiRNACav-1knockoutand -actinarethesamebandsshownin Figure3becausethesamesampleswereusedtodemonstratethelevelofABCA-1expression.Thedensitiesofbandscorrespondingtoeach proteinwerequantifiedusingimagedensitometeranalysis.ThenumbersatthebottomofeachblotaretherelativevaluesofABCA1expression intransfectedortransducedcellscomparedtothoseincontrolcells(mock). Lin etal.Retrovirology 2012, 9 :85 Page6of16 http://www.retrovirology.com/content/9/1/85

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ofABCA-1expression.Likewise,VSV-Gpseudotyped HIV(psHIVwtNef)infectiondown-regulatedABCA-1expressioninMDMs,andco-infectionofMDMswith psHIVwtNefandAd-Cav-1didnotrestorethereduced ABCA-1levels(Figure4C).MDMswerealsoinfected withAD8orADnefmutvirusalongwithinfectionofAdCav-1orAd-GFPtodeterminethelevelofABCA-1expression.ADnefmutHIVinfectiondidnotaffecttheexpressionofABCA-1witheitherAd-Cav-1orAd-GFPcoinfection(Figure4D).ABCA-1expression,however,was decreasedwhenMDMswereco-infectedwithAD8and Ad-Cav-1orAd-GFPconfirmingtheroleofNefinthereductionofABCA-1expressionwhileoverexpressionof Cav-1hasnoimpactonABCA-1expression.Furthermore,weexaminedthelevelofABCA-1expressionin U87orTHP-1cellswheretheexpressionofendogenous Cav-1wasknockeddownwithsiRNAusingsamples describedforresultsinFigure3Aand3B.Asshown inFigure4EreducedendogenousCav-1expressionby siRNAtreatmentdidnotalterthelevelofABCA-1 expression.InteractionofNefandCav-1Sinceover-expressionofCav-1doesnotalterABCA-1 expression,themechanismofrestorationofcholesterol effluxbyCav-1thatisimpairedbyNefmaynotinvolve thelevelofABCA-1expression.Nefhasbeenshownto bindtoABCA-1[38,42],anditisnotknownwhether Cav-1interactswithNef.Cav-1mayassociate,either directlyorindirectly,withNeftherebycounteringthe impairmentofcholesterolefflux.Todeterminewhether thereisaphysicalassociationbetweenCav-1andNef, weperformedco-immunoprecipitationandimmunoblottingexperiments.U87-CD4-CXCR4cellswerecotransfectedwithpCZ-Cav-1andHA-taggedwildtype Nef(pCINL4-3Nef-HA-WT)ortheNefG2Amutant (pCINL4-3NefG2A-HA).Transfectedcellswerethen culturedinmediumcontainingcholesterol(30 g/ml)for 48hoursfollowedbytreatmentwithapoA-I(20 g/ml) for30min.Celllysateswerethensubjectedtoco-immunoprecipitationandanalyzedforCav-1andNefinteractionsbyimmunoblotting.AsshowninFigures5Aand 5BtheassociationofCav-1andNefisevidentinU87 cells.Interestingly,therewasnoNefG2AmutantinteractionwithCav-1implicatingtheassociationofNefand Cav-1isatthecellmembrane.WeexaminedtheendogenousinteractionofCav-1andABCA-1inU87cells andwereunabletoshowABCA-1associationwithCav1byco-immunopreciptationandimmunoblottinganalysis(datanotshown).TheinteractionofCav-1with NefsuggeststhatCav-1byassociatingwithNefblocks theactivityofNefandsubsequentlyhelpsrestorecholesteroleffluximpairedbyNef.Cav-1reducesHIV-1infectivitybyreducingthe cholesterolcontentofvirusparticlesHIViswellknowntorelyonthehostcellularcholesterol machineryforefficientreplicationandparticleformation [25,26,28,30,44].SinceourresultsshowthatCav-1restoresNefimpairedcholesterolefflux,wesoughttodetermineifthepromotionofthiseffluxbyCav-1wouldhave animpactontheinfectivityofreleasedvirusparticles.In ordertodemonstratewhetherCav-1influencesHIVinfectivity,primarymacrophages(MDMs)weretransduced withAd-Cav-1orthecontrolAd-GFP.ThelevelofGFP andCav-1expressioninmacrophagesfromwhichculture supernatantharvestedisshowninFigure6A.Twenty-four hoursposttransductionthemacrophageswereinfected withHIVAD8.Virionsproducedfromtheseinfected macrophagesweretiteredusingtheTZM-blindicatorcell lineandnormalizedfortheinfectivitystudies.Asshown inFigure6BinfectivityofvirusharvestedfromCav-1treatedmacrophageswasreducedby46%comparedtoCav-1 untreatedHIVinfectedcells.Therewasnosignificant differenceininfectivityofvirusparticlesobtainedfrom Figure5 InteractionofCav-1andNef. U87-CD4-CXCR4cellswere co-transfectedwithpCZ-Cav-1andHAtaggedNef(pCINL4-3 Nef-HA-WT)orNefG2A(pCINL4-3NefG2A-HA).Twenty-fourhours posttransfectionthecellswereculturedinthepresenceof cholesterol(30 g/ml)for48hoursfollowedbyapoA-I(20 g/ml) for10min.( A )Thecellswereharvestedandsubjectedto immunoprecipitationusinganti-HAantibodyandimmunoblots usinganti-Cav-1oranti-Nefantibody.( B )Alternatively, immunoprecipitaionwasperformedusinganti-Cav-1and immunoblottingusinganti-Nefantibody. Lin etal.Retrovirology 2012, 9 :85 Page7of16 http://www.retrovirology.com/content/9/1/85

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Ad-GFPtransducedcellswhencomparedtothatofvirus harvestedfromCav-1untreatedHIVinfectedcells.SimilarexperimentswereperformedusingADnefmutinfections.ContrarytowhatwasobservedwithAD8HIVthe levelofinfectivityofADnefmutvirusharvestedfrom Ad-Cav-1orAd-GFPtreatedcellsremainedthesame (Figure6C).This,therefore,establishesthatCav-1impairs HIVinfectivityimplicatingthatthismaybelinkedtoCav1mediatedpromotionofcholesteroleffluxbyapoA-Ithat isimpairedbyNefduringHIVinfection.SinceCholesterol withintheHIVparticleisstrictlyrequiredforinfection, ournextsetofexperimentswereaimedatinvestigating whetherthereductionofHIVinfectivityisrelatedtothe modulationoflipidcontentofthevirions.HIVprovirus DNAwasco-transfectedintoU87cellswithpCZ-Cav-1 orpCZ-vector.Virusharvestedfromtransfectedcellswas concentratedandnormalizedbyap24ELISAassay.Equal amountsofvirusparticleswereusedtomeasurethecholesterolcompositioninthevirionbytheAmplexRed cholesterolAssayKit.AsshowninFigure7AthecholesterolcontentofvirusparticlesharvestedfromcellsreceivingCav-1wasreducedby48%comparedtocells transfectedwithpCZ-vector.Inaddition,cholesterolwas replenishedwithinconcentratedvirusthatwasnormalized andequalamountsweretreatedwith(2-Hydroxypropyl)-Cyclodextrin(CD)andsaturatedexogenouscholesterol toseeifinfectivitycouldberestored.Infectivityofcholesterolreplenishedviruswasmeasuredbyluciferaseactivity ininfectedTZM-blcells.Theinfectivityofvirusparticles collectedonCav-1treatedcellswasreducedby58%as comparedtothosecollectedoncellstreatedbypCZvector(Figure7B).Therewasnodifferenceininfectivity ofcholesterolreplenishedandcontrolviralparticlescollectedfrompCZ-vectortreatedcells(Figure7B).Asmight beexpectedtheinfectivityofcholesterolreplenishedvirus particlescollectedfromCav-1treatedcellswasincreased by37%comparedtothevirusparticlesharvestedfrom Cav-1treatedcontrol(Figure7B).Inaddition,weexaminedwhetherCav-1isincorporatedintothevirionto makesurethatsuchincorporationhasnotaffectedinfectivitydirectlyratherthaninfluencingthecholesterolcontentoftheHIVvirusparticles.AsshowninFigure7C,we observedthatCav-1proteinisnotincorporatedinthe virusparticlesasdeterminedbyWesternblotanalysis. Therefore,Cav-1reducesvirusinfectivitybypromoting cholesteroleffluxwhichconsequentlydecreasedtheavailabilityofcholesterolduringviralparticleformation.DiscussionHIVhasbeenindicatedtomanipulatehostcholesterol metabolism,leadingtoexcessivecholesterolaccumulationininfectedTcellsormacrophages[38,45],thereby supportingefficientviralreplication.Intheabsenceof properesterificationtofattyacidandefflux,cholesterol accumulatesintheendoplasmicreticulumeventuallyleadingtoERdysfunctionandtheactivationofanERstress associatedapoptosispathway[46-48].Cav-1isanimportantcellularcholesterolregulator,anditsexpressionis Figure6 Cav-1over-expressioninhibitsHIV-1infectivity. MDMswereinfectedwithAD8orADnefmutatanmoiof0.1aloneorin combinationwithAd-Cav-1orAd-GFP.( A )ThelevelsofGFPandCav-1expressionareshown.( B )and( C )Infectiousparticlesharvestedfrom culturesupernatantsweretiteredandnormalized.LevelofinfectivitywasmeasuredbyinfectingTZM-blcellsandsubsequentluciferaseassay.All experimentswereperformedintriplicateandresultsshownaremeanSDwithPvalues. Lin etal.Retrovirology 2012, 9 :85 Page8of16 http://www.retrovirology.com/content/9/1/85

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dramaticallyenhancedinHIVinfectedmacrophages [17],implicatingaroleforCav-1inHIVassociated cholesterolalterations.Cav-1isastructuralcomponent ofCaveolaemembranemicrodomains,whichhavebeen suggestedtoplayanimportantroleincholesteroltraffickingandefflux.Inthisstudy,weinvestigatetheeffect ofCav-1onthecholesteroleffluxinHIVinfectedmacrophagesandhumanastrocytes-derivedglioblastomaU87 cells.OurresultsshowthatCav-1restorestheNef inducedimpairmentofcholesteroleffluxbyapoA-I.Furthermore,thisrestorationcausesareductioninthecholesterolcompositionofvirusparticlesleadingtodecreased HIVinfectivity.ThissuggestsaroleforCav-1inmacrophageHIVpersistentinfectionbyenhancingcholesterol efflux. OurresultsshowneitherNefnorCav-1hadsignificant effectonHDLmediatedcholesterolefflux.HDLplaysan importantroleinreversecholesteroltransport(RCT),in whichHDLtransportscholesterolfromperipheraltissues toliverforexcretion.RCTisamultifaceted,dynamic pathwaywhichisinvolvedwithmultiplemoleculesand effectors.ThefirststepinRCTisABCA-1dependenteffluxofcholesterolandphospholipidstoapoA-I,themajor componentofHDL.ABCA-1interactswithapoA-Iand stimulatesfreecholesterolandphospholipidseffluxresponsiblefornascentHDLformation[49].Wang etal. [50]reportedthatABCA-1expressionmarkedlyincreases apoA-IbutnotHDLmediatedlipidefflux;thereason couldbethatcomparedwithHDL,apoA-Iisthepreferred acceptorforABCA1-promotedcholesterolandphospholipidefflux.WealsofounduponHIVinfectionNefdown regulatesABCA-1expression,whichdramaticallyinhibits apoA-Imediatedcholesterolefflux,whereasHDLmediatedcholesteroleffluxwasnotaffectedbyHIVinfection. Over-expressionofCav-1restorestheimpairedcholesteroleffluxtoapoA-Isignificantly,butnotsomuchonintactHDLcholesterolefflux. Promotionofcholesteroleffluxbyover-expressionof Cav-1isobservedinhepaticcells[9].Cav-1canenhance thetransferofcholesteroltocholesterol-richdomains intheplasmamembrane,whereitisaccessibletoefflux. MultiplemechanismsareproposedforCav-1 ’ sregulationofcholesterolhomeostasis.Theseincludethemodulationoftheexpressionoflipoproteinreceptorsand theactivityofproteinsinvolvedinlipidmetabolismas wellasinteractionswithlipidtransportortransportof Figure7 Cav-1over-expressionreducesthecholesterolcontentinHIV-1virusparticles. ( A )ProviralDNAgenomeNL4-3wastransfected intoU87cellseitherwithpCZ-vectororwithpCZ-Cav-1.Virusparticlesgeneratedwereconcentratedandnormalizedbyp24assay.Cholesterol contentsweremeasuredusingtheAmplexRedcholesterolAssayKit.Theresultsarepresentedaspercentageofcholesterolcontentrelativeto control(HIV/pCZ-vector,setas100%)andarethemeanSDoftriplicateexperimentswithPvalues.( B )Normalizedsampleswerereplenished withexogenouscholesterol,andthelevelofinfectivitywasmeasuredbyinfectionofTZM-blcellsandluciferaseassay.Allexperimentswere performedintriplicate,andresultsshownaremeanSDwithPvalues.( C )Normalizedsamplesfromsamplesof(7 A )weresubjectedtoWestern blotanalysisusingantibodytoGag,Nef,andCav-1todeterminewhetherCav-1proteinisincorporatedinvirusparticles. Lin etal.Retrovirology 2012, 9 :85 Page9of16 http://www.retrovirology.com/content/9/1/85

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cholesteroltotheplasmamembranefacilitatingcholesterolefflux[6-8,22,43].ABCA-1expressionisimportant inregulatingcholesteroleffluxtoapoA-Iandithasbeen implicatedthatABCA-1stimulatesthereorganizationof plasmamembranemicrodomainstofacilitatecholesterol effluxtoapoA-I[51,52].Cav-1canregulatecholesterol homeostasisbymodulatingtheexpressionoflipidregulators.ReducedlevelsofABCA-1havebeenobservedin macrophagesofCav-1knockoutmice[43].Ourresults showthatweobservenochangeinthelevelofABCA-1 expressionwhenCav-1isover-expressedsuggestingthat theendogenousCav-1expressionissufficientenoughto maintainphysiologicallyrelevantlevelsofABCA-1and thatadditionalamountsofCav-1doesnothaveanimpactonABCA-1expression.Thereducedlevelof ABCA-1observedintheknockoutmiceisincomplete absenceofCav-1expression.ABCA-1dependentcholesteroleffluxcanbeimpairedbyHIVNefmediateddown modulationandalteringoftheintracellulardistribution ofABCA-1[38,42].Similarlyweobserveda69%decrease inABCA-1expressioninthepresenceofNef.InterestinglythedecreaseinABCA-1remainsthesamewhen additionalamountsofCav-1areprovidedindicatingthat thereversalofNef ’ seffectoncholesteroleffluxbyCav-1 isnotrelatedtothelevelofABCA-1expression.InhibitionofABCA-1proteinexpression,asitpertainstoNef, inpartdependsupontheERassociatedproteasomaldegradationmechanism[42].Anunknownadditionalpathwayunrelatedtoproteasomalactivityisalsosuggestedto contributetoABCA-1degradation.AlthoughABCA-1is showntointeractwithNefthephysicalassociationisnot essentialforNefmediateddown-regulationofABCA-1effluxactivity[38,42].However,theinfluenceofcellulardistributionofABCA-1byNefhasbeendeterminedusing confocalmicroscopywithNefcausingaprominenttrappingofABCA-1intheER[42].ABCA-1expressionhas beenimplicatedininfluencingtheredistributionofcholesterolandCav-1[52].RedistributionofCav-1from punctatecaveolae-likestructurestothegeneralareaofthe plasmamembraneisobserveduponABCA-1expression. Ourco-immunoprecipitationstudyrevealsaninteraction betweenCav-1andNef.Furthermore,ourobservation thatCav-1doesnotinteractwiththemyristoylationdefectiveNef(NefG2Amutant)implicatesanassociationof theseproteinsattheplasmamembrane.TheseobservationssuggestthattheinterplayofCav-1withNefand cholesterolsubsequentlycountersNefinducedimpairmentofcholesteroleffluxbyapoA-l.Inaddition,since caveolaeisamajorsourceandplatformforcholesterol efflux[4]over-expressionofCav-1mayinducetheformationofmorecaveolae,whichshouldsubsequentlyenhance cholesterolefflux.ThepresenceofCav-1inmacrophages anditsup-regulationuponHIVinfection,therefore,can contributetoincreasedcholesteroleffluxinthesecells. Cholesterolisanimportantstructuralcomponentof HIVparticlesandtheircholesterolcontentistightlylinkedtoHIVinfectivity[25,27,44].Cholesteroldepletion significantlyreducesHIV-1particleproduction[29-34,44]. Thereisalsoamarkeddecreaseininfectivityofvirions producedfromsuchcells[26].Thesignificantreduction correlateswiththeamountofvirion-associatedcholesterol[35].Inthecurrentstudy,weclearlyestablishedthat Cav-1significantlyreducesinfectionwithvirionsproducedfromCav-1treatedcellswhencomparedtothatof thesamenumberofvirionsobtainedfromuntreatedcells. WehavepreviouslyshownthatCav-1repressesHIVgene expressionbyblockingtheNFBpathwaythussubsequentlyaffectingvirusproduction[18].Thedecreasein virusproductionisthereforeinpartduetotranscriptional suppressionofHIVgeneexpression.Here,weexamined thecholesterolcontentofHIVparticlesproducedfrom Cav-1treatedcellsandclearlyestablishedasignificant cholesteroldecreaseinvirusparticles.Furthermore,normalizedamountsofvirusintheinfectivityassayofHIV releasedfromCav-1treatedcellsshowsthatinfectivityis markedlyreduced.Normalizedamountsofvirustoassay forinfectivity,rulesoutanyconcernregardingthelevelof virusreleasecontributingtothereductionofinfectivity. Themajorstepthatcausesadecreaseinvirioninfectivity relatedtocholesteroldepletionisthefusionstepsofinfection[30].Insupportofthisnotion,wepreviouslydemonstratedthatCav-1significantlysuppressedEnv-induced membranehemifusion[16],indicatingthatthedecrease infusionpartlyinvolvesareductioninthecholesterol compositionoftheplasmamembrane.Cav-1cancounter theinfluenceofHIVoncholesterolmetabolismbypromotingcholesteroltraffickingtothemembranesubsequentlyenhancingcholesterolefflux,therefore,depriving theHIVvirionofcholesterol.SinceCav-1isinvolvedin cholesterolmetabolismtheup-regulationofCav-1can haveanimpactonthelevelofcellularcholesterolthereby contributingtoareductioninvirusproductionandinfectivity,consequentlycontributingtoapersistentinfection ofmacrophages.ConclusionInfectedmacrophagesarerelativelyresistanttocytopathiceffectandconsequentlyplayanessentialrolein viraldisseminationtohosttissuesandorgans[53-55]. Furthermore,inthisviralreservoirHIVinfectionappearsnottobeassociatedwithapoptosisbutwitha chronicproductivelyinfectedphenotype[56,57].Although severalmechanismshavebeenproposedwestilldon ’ t haveaclearpictureastothemechanismsofpersistentinfectioninmacrophages.TherelationshipbetweenHIV-1 andhostfactorsdeterminesthemodulationofbothcellularfunctionsandvirusreplicationwithinaninfectedindividual,andwiththeinteractionoftheseviralandcellularLin etal.Retrovirology 2012, 9 :85 Page10of16 http://www.retrovirology.com/content/9/1/85

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factorsbeingevidentinallstepsofvirusreplication [58-62].Theirassociationsmaybeanimportantfactorinthemodificationofhostcellprocessesduringa chronicviralinfection.This suggeststhatapersistent infectionisregulatedbycellularfactorsatdifferentsteps invirusreplication.Geneexpressionprofilesthatareuniquetomacrophages[63-65]whencomparedtothatof activatedCD4+Tcellsshouldhelpdeterminethemechanismofpersistentinfectioninmacrophages.Cav-1is highlyexpressedinterminallydifferentiatedorquiescent cellsincludingdendriticcellsandmonocytes/macrophages[2,66].WhilemacrophagesexpressCav-1, humanTcellsaregenerallybelievedtolacktheCav1protein[67-69].ThelackofCav-1inTcells,ourdiscoverythatHIVinfectionenhancesCav-1expression mediatedbyTatinmacrophagesandthatCav-1reduces HIVreplication[17]suggestsaroleforCav-1inanHIV persistentinfectionofmacrophages.Herewehaveshown thatCav-1,byrestoringcholesteroleffluximpairedbyNef andsubsequentlyinfluencingthecholesterolcontentof HIVparticleswhichnegativelyaffectsvirusinfectivity,effectivelyinhibitsHIVreplicationcontributingtomacrophageHIVpersistentinfection(Figure8).MethodsPlasmidsTheHIV-1proviralconstructspNL4-3(T-tropic),pNLAD8(M-tropic),pWT/Bal(M-tropic),pNL4-3.Luc.R-E-, andpSG3 envwerekindlyprovidedbyNIHAIDSResearchandReferenceReagentProgram[70-76].TheconstructpNL4-3.Luc.R-E-isdefectiveforenvandnefwhere aspSG3 envhasintactnef,butadeletioninenv.Anexpressionplasmidforvesicularstomatitisvirusenvelope Gprotein(pCI-VSV)waskindlyprovidedbyJiing-Kuan YeeofCityofHopeNationalMedicalCenter,Duarte, California.ACav-1expressingplasmid,pCZ-cav-1,was generatedasdescribedpreviously[16].pCZ-vectoristhe sameaspCZ-cav-1exceptitlacksthecodingsequenceof cav-1.TheNefexpressionplasmidpcDNA3.1SF2Nefwas providedbyNIHAIDSResearchandReferenceReagent Program[77,78].AconstructexpressingNeftaggedwith HA(pCINL4-3Nef-HA-WT)waspurchasedfrom AddgeneInc(Cambridge,MA).TheNefG2Amutation plasmidwasgeneratedusingasite-directedmutagenesis kitaccordingtothemanufacturer ’ sprotocol(Strategene). Briefly,themutationwasgeneratedbyPCRamplificationusingpCINL4-3Nef-HA-WTastemplateandthe Figure8 AmodelfortheinterplayofCav-1withNefthatcountersNefinducedimpairmentofcholesteroleffluxbyapoA-land contributiontopersistentinfection. UponHIVinfection,NefproteininteractswithABCA-1anddown-regulatesand/orredistributesABCA-1 expressionwhichresultsincholesteroleffluximpairment.Thiscreatesamicro-environmentinwhichHIVcanreplicateefficiently.Ontheother hand,incellswhichexpressCav-1,suchasmacrophages,Tatinducesanup-regulationofCav-1.TheoverabundanceofCav-1thenleadstoboth itsbindingtoNefandabilitytoactivatecholesterolefflux.This,therefore,leadstolesscholesterolaccumulationwhichinturnreducesthe amountthatcanbeincorporatedintoviralparticlesandtherebyreducingHIVinfectivity. Lin etal.Retrovirology 2012, 9 :85 Page11of16 http://www.retrovirology.com/content/9/1/85

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followingpairofprimers:50-ggattttgctataagatggctggcaag tggtcaaaaagt-30and50-actttttgaccacttgccagccatcttatagcaa aatcc-30.ThePCRproductsweredigestedwiththerestrictionenzyme DpnI todestroytemplateplasmidsandwere thentransformedintoDH5 competentcells.Introductionofthemutation(pCINL4-3NefG2A-HA)wasconfirmedbysequenceanalysis.WildtypeAD8anda replicationcompetentnefdefectiveAD8derivedHIV provirusDNAconstructADnefmut[79]wereprovidedby Dr.MaureenGoodenowoftheUniversityofFlorida. Adenovirusparticles(Ad)forexpressingCav-1(Ad-Cav-1) andGFP(Ad-GFP)wereobtainedfromVectorBiolabs (Philadelphia,PA).CellculturesHumanU87MG-CD4cellsstablytransfectedwithCXCR4 (U87-CD4-CXCR4)orCCR5(U87-CD4-CCR5),human acutemonocyticleukemia(THP-1),andanindicatorcell linefortitteringHIV(TZM-bl)waskindlyprovidedby theNIHAIDSResearchandReferenceReagentProgram. U87-CD4-CXCR4weremaintainedinDMEMcontaining 15%FBS,penicillin-streptomycin(100 g/mL),glutamine, puromycin(1 g/ml;SigmaChemical),andneomycin (G418;300 g/ml;Sigma).THP-1cellsweregrownin RPMI-1640containing10%FBS,1.0mMsodiumpyruvate, and0.05mM2-mercaptoethanol.Fordifferentiationinto macrophages,THP-1cellsweretreatedwith50ng/mlof phorbol12-myristate13-acetate(PMA,SigmaChemical) for5daysuntilthecellsadheredandexhibitedmacrophage-likemorphology.TZM-bland293Tcellswere growninDMEMmediumsupplementedwith10%FBS andpenicillin-streptomycin(100 g/ml).Allcultureswere maintainedat37Cinahumidifiedatmospherewith5% CO2. Peripheralbloodmononuclearcells(PBMCs)wereisolatedfrombuffycoatspreparedfromhealthydonorsby centrifugationthroughaFicollgradient(Sigma-Aldrich, St.Louis,MO).Monocyteswereisolatedbynegativeselectionwithahumanmonocyteenrichmentkitaccordingtothemanufacturer ’ sinstructions(EasySepHuman MonocyteEnrichmentKit,StemcellTechnologies).The monocytepreparationscontained97%CD14+cells,asdeterminedbyflowcytometry.Fordifferentiationofmonocytesintomacrophages(MDMs),monocyteswereseeded intoBiocoatpoly-D-lysineplates(B.D.Bioscience),and culturedinDMEM,supplementedwith10%heat-inactivatedhumanserum,gentamicin(50 g/ml),ciprofloxacin (10 g/ml),andM-CSF(1000U/ml)for7days.MDMculturemediumwashalf-exchangedevery2to3days.TransfectionofsiRNASmallinterferingRNA(siRNA)targetingCav-1andcontrolsiRNAwerepurchasedfromSantaCruzBiotechnology,Inc.TransfectionofsiRNAwasperformedusing OligofectaminTMReagent(InvitrogenCorp.,Carlsbad, Calif.)accordingtothemanufacturer ’ sprotocol.Briefly, thedaybeforetransfection,U87cellswereseededintoa 24wellplateandculturedwithantibioticsfreemedium to30%confluency.Cellswerewashedandresuspended in200ulserumfreemedium.Transfectionmixturewas preparedbyincubating50pmolofsiRNAduplexeswith 3ulofOligofectamininafinalvolumeof50ulOptiMEMIMedium.Aftera5hourincubation,125ulof growthmediumwith3timesthenormalconcentration ofserumwasaddedtocells.Transfectionwasrepeated oncethenextday.ForTHP1macrophages,cellswere firsttransfectedwithsiRNAfollowedbyHIVinfection, andcellswerethentransfectedagainwithsiRNAthe dayafterinfection.TheefficiencyofCav-1knock-down bythesiRNAtransfectionwasmonitoredusingWestern blotanalysis.VirusproductionandconcentrationInfectiousvirusHIV-1AD8,ADnefmut,Bal,andNL4-3 weregeneratedbycalciumphosphatetransfectionof monolayersof293Tcellsin75-cm2flaskswith25 g provirusDNA.Supernatantscontainingviruswereharvested4daysaftertransfectionandquantifiedusingthe TZM-blindicatorcellsaswellasbymeasuringreverse transcriptaseandap24ELISAmethodasdescribedpreviously[17].Whenrequired,viruswasproducedfrom U87-CD4-CXCR4cellstransfectedwith18 gproviral HIVNL4-3alongwith9 gpCZ-Cav-1orpCZ-vector. TogeneratepseudotypedHIVparticles20 gpSG3 envorpNL4-3.Luc.R-E-wasco-transfectedwith3 gpCIVSVintomonolayersof293Tcellsin75-cm2culture flasksbythecalciumphosphatemethod.Pseudotyped viralsupernatantswereharvested4dayspost-transfectionandwereclarifiedbycentrifugationat3,000rpmfor 20minandthenbyfilteringthrougha0.45 m-poresize filter.VirusparticleswereconcentratedusingvirusprecipitationreagentRetro-ConcentinTM(SystemBiosciences) accordingtothemanufacturer ’ sprotocol.OilredOstainingTodeterminetheinfluenceofCav-1ontheleveloflipid accumulationinHIVinfectedanduninfectedcellsoil redOstainingwasperformed.THP-1cellsweredifferentiatedintomacrophagesbytreatmentwith50ng/ml PMAfor5daystheninfectedwithHIVAD8(moi,0.01) orBal(moi,0.001).Onday10postinfection,cellswere loadedwithcholesterolbyincubatingwith50 g/mlAcLDL(BiomedicalTechnologiesInc.,Stoughton,MA)for 48hfollowedby30 g/mlapoA-Istimulationfor18 hours.DifferentiatedTHP-1cell-differentiatedmacrophagecellswerealsoinfectedwithAd-Cav-1orAd-GFP atanmoi(multiplicityofinfection)of100.Twenty-four hourslatertheywereinfectedwithVSVpseudotypedLin etal.Retrovirology 2012, 9 :85 Page12of16 http://www.retrovirology.com/content/9/1/85

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HIVpSG3 envorpNL4-3.Luc.R-E-atanmoiof3and incubatedfor5days.OilredOstainingwasperformed aspreviouslydescribed[80].Briefly,cellswererinsed withPBS,followedbyfixationwith3.7%paraformaldehydefor60min.Thecellswerestainedusingfreshly preparedOilredO(Sigma)workingsolutionatroom temperaturefor10min.Intensityofcellstainingwas observedusingalightmicroscope.VirusinfectivityassayTotestCav-1 ’ sinfluenceonHIV-1infectivity,TZM-bl cellswereinfectedwithvirusharvestedfromCav-1treatedcellsandtheinfectivitylevelsweremeasuredby luciferaseactivity.MDMswerefirstinfectedwithadenovirusexpressingCav-1orGFPatanmoiof100inserum freemediumfor6hours.Thecellswerethenwashed andincubatedinserum-containingmediumover-night, afterwhichcellswereinfectedwithHIVAD8atanmoi of0.1for6hours,atwhichpointtheywerewashedand refreshedwithnewmedium.Onday6postinfection, supernatantsweresubjectedtoRTassayortiteredusing theindicatorTZM-blcellline.VirusamountswerenormalizedwithlevelofinfectivitybeingassayedbymeasuringluciferasewithinTZM-blcells[74].Normalized amountsofviruswereusedforsubsequentinfections.Determinationofcholesterolcontentandcholesterol replenishmentassayEquivalentamountsofvirionswerequantifiedbyp24 assayandtestedforcholesterolcontentusingtheAmplex RedcholesterolAssayKit(Invitrogen,Carlsbad,CA)accordingtothemanufacturer ’ sprotocol.Toreplenishcholesterolvirusamountswerealsonormalizedbyp24assay andincubatedin0.5mM(2-Hydroxypropyl)--Cyclodextrinsolution(SigmaAldrich)with1.5mMcholesterol (SigmaAldrich)at37Cfor1hour.Thesequantifiedand normalizedamountsofviruswereusedtoinfectTZM-bl cellsandmonitoredforluciferaseactivity.CholesteroleffluxU87cellsweretransfectedwithpcDNA3.1andpCZvector(mock),pcDNA3.1SF2NefandpCZ-vector(Nef), pCZ-cav-1andpcDNA3.1(Cav-1),pcDNA3.1SF2Nef andpCZ-cav-1(NefplusCav-1),pCINL4-3NefG2AHA(NefG2A)andpCZ-vector,orpCINL4-3NefG2AHAandpCZ-cav-1(NefG2AplusCav-1).Twenty-four hoursaftertransfectioncellculturemediumwasreplacedwithserumfreemediumcontaining2 Ci/mL [3H]cholesteroland1.5%BSAandincubatedfor36 hours.Radioisotope-containingmediumwasthenremovedandcellswerewashedtwicewithPBSandculturedforanadditional18hoursinserumfreemedium inthepresenceorabsenceof50 g/mlApoA-l(BiomedicalTechnologiesInc.,Stoughton,MA).Cholesterol contentwasmeasuredinthecellfreemediaaswellas withincellsafterlysingusing0.1NNaOH.ApoA-lspecific cholesteroleffluxwasdeterminedusingtheformula:apoAlspecificefflux=%cholesteroleffluxwithapoA-l-%cholesteroleffluxwithoutapoA-l(blank);cholesterolefflux= [cpm(supernatants)/cpm(sup ernatants+cells)]100%.HDL mediatedcholesteroleffluxisalsoexaminedbyincubating cellsfor18hoursinthepresenceorabsenceof50 g/ml HDL(BiomedicalTechnologiesInc.,Stoughton,MA). Todeterminecholesteroleffluxfrommacrophages, MDMswerefirstinfectedwithAd-Cav-1orAd-GFPat anmoiof50for24hours,whichwasfollowedbyinfectionofpseudotypedHIVpSG3 env(psHIVwtNef)or pNL4-3.Luc.R-E-(psHIV Nef).Fivedayspostinfection cellswerethenlabeledwith1 Ci/mL[3H]cholesterol for48hoursandapoA-lmediatedcholesteroleffluxwas determinedasdescribedabove.SimilarlycholesteroleffluxfromTHP-1cell-differentiatedmacrophageswas determined21daysafterinfectingwithHIVAD8atan moiof0.001.Cholesteroleffluxwasalsodetermined14 daysafterTHP-1cell-differentiatedmacrophagesinfectedwithanmoiof0.001,0.01,or0.1.Inaddition,primarymacrophages(MDMs)wereinfectedwithAD8or ADnefmutHIVwithanmoi0.01andthenculturedcells weresubjectedtocholesteroleffluxassay15daysafter infection.ABCA-1expressionwasdeterminedbyWesternblotsinMDMs14daysafterco-infectionwithAD8 orADnefmutHIVandwithAd-Cav-1orAd-GFP.InhibitionofHIVreplicationwasperformedbytreating infectedcellswith5uMazidothymidine(AZT)(SigmaAldrich,St.Louis,MO).ImmunoprecipitationandImmunoblottinganalysesU87cellsweretransfectedwithpCZ-Cav-1andHAtaggedNef(pCINL4-3Nef-HA-WT)orHA-tagged NefG2A(pCINL4-3NefG2A-HA),followedbyincubationofmediumcontainingcholesterol(30 g/ml)for48 hours.CellswerethentreatedwithapoA-I(20 g/ml)for 30min.Cellswereputonice,washedtwicewithcold PBSandtotalcellularproteinwasextractedinlysisbuffer(50mMTrispH7.5,100mMNaCl,1mMEDTA, 0.1%(v/v)TritonX-100,10mMNaF,1mMphenylmethylsulfonylfluoride,and1mmol/Lvanadate)witha completeproteaseInhibitormixture(RocheDiagnostics, Indianapolis,IN).Theconcentrationofextractedprotein wasdeterminedandadjustedto1ug/ul.Atotalof500 ulwasusedforeachimmunoprecipitation,towhich2 g ofantibodies(anti-Cav-1oranti-HA)ornormalIgG wereadded.Themixtureswereincubatedat4Covernight.Followingtheovernightincubation,25 lofproteinA/G-agarosebeads(SantaCruzBiotechnology, SantaCruz,CA)wereaddedandthemixtureswerethen rotatedfor2hoursat4C.Thebeadswereharvestedby centrifugationandwashedfivetimeswithlysisbuffer.Lin etal.Retrovirology 2012, 9 :85 Page13of16 http://www.retrovirology.com/content/9/1/85

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Loadingbufferwasaddedandboiledfor5min.ThesamplesweresubjectedtoSDS-PAGEandanalyzedbyimmunoblottingasdescribedpreviously[81].Theprimary antibodiesusedforimmunoblottingwererabbitpolyclonalanti-Cav-1(SantaCruzBiotechnology,SantaCruz, CA),mousemonoclonalanti-Nef,rabbitNefantiserum, andhumanmonoclonalanti-Gag(NIHAIDSResearch andReferenceReagentProgram),goatpolyclonalanti-HA (Genescript),mousemonoclonalanti-ABCA1(abcam), and-actinproteinantibody(Sigma,St.Louis,MO).The secondaryantibodieswereHRP-linkedanti-rabbit,antimouse(CellSignalingTechnology,Inc.,Danvers,MA), anti-humanIgG(Sigma,St.Louis,MO)oranti-goatIgG (SantaCruzBiotechnology,SantaCruz,CA).StatisticalanalysisStudent ’ sttestwasappliedtoanalyzethedifferencesbetweensetsofdata.AllanalyseswereperformedwithSPSS 12.0.1forWindows,andwereconsideredsignificantat p 0.05.Competinginterests Theauthorsdeclarethattheyhavenocompetinginterests. Authors ’ contributions SLParticipatedinthedesignofexperiments,carriedoutmostofthe experiment,preparedsamplesandconductedcholesterolanalysisand Westernblots,analyzeddataandcontributedtomanuscriptpreparation.PN participatedandassistedinsamplecollectionsandexperiments.XWwas involvedinthedesignofconstructsusedforthestudyaswellasestablish efficientmethodologytodeliverofproteinofinterestinprimary macrophages.AMconceivedthestudy,designedandcoordinated experiments,participatedindataanalysisandpreparedcontributedthe manuscript.Allauthorsreadandapprovedthefinalmanuscript. Acknowledgements ThisresearchwassupportedbyagrantfromtheNationalInstitutesof Health(AI39126)toA.M. Received:18June2012Accepted:26September2012 Published:15October2012 References1.RothbergKG,HeuserJE,DonzellWC,YingYS,GlenneyJR,AndersonRG: Caveolin,aproteincomponentofcaveolaemembranecoats. Cell 1992, 68: 673 – 682. 2.HarrisJ,WerlingD,HopeJC,TaylorG,HowardCJ: Caveolaeandcaveolin inimmunecells:distributionandfunctions. TrendsImmunol 2002, 23: 158 – 164. 3.GalbiatiF,VolonteD,LiuJ,CapozzaF,FrankPG,ZhuL,PestellRG,Lisanti MP: Caveolin-1expressionnegativelyregulatescellcycleprogressionby inducingG(0)/G(1)arrestviaap53/p21(WAF1/Cip1)-dependent mechanism. MolBiolCell 2001, 12: 2229 – 2244. 4.FieldingPE,RusselJS,SpencerTA,HakamataH,NagaoK,FieldingCJ: Sterol effluxtoapolipoproteinA-Ioriginatesfromcaveolin-richmicrodomains andpotentiatesPDGF-dependentproteinkinaseactivity. Biochemistry 2002, 41: 4929 – 4937. 5.GargalovicP,DoryL: Cellularapoptosisisassociatedwithincreased caveolin-1expressioninmacrophages. JLipidRes 2003, 44: 1622 – 1632. 6.GargalovicP,DoryL: Caveolinsandmacrophagelipidmetabolism. JLipid Res 2003, 44: 11 – 21. 7.LePU,GuayG,AltschulerY,NabiIR: Caveolin-1isanegativeregulatorof caveolae-mediatedendocytosistotheendoplasmicreticulum. JBiol Chem 2002, 277: 3371 – 3379. 8.ChaoWT,FanSS,ChenJK,YangVC: Visualizingcaveolin-1andHDLin cholesterol-loadedaorticendothelialcells. JLipidRes 2003, 44: 1094 – 1099. 9.FuY,HoangA,EscherG,PartonRG,KrozowskiZ,SviridovD: Expressionof caveolin-1enhancescholesteroleffluxinhepaticcells. JBiolChem 2004, 279: 14140 – 14146. 10.CouetJ,LiS,OkamotoT,IkezuT,LisantiMP: Identificationofpeptideand proteinligandsforthecaveolin-scaffoldingdomainImplicationsforthe interactionofcaveolinwithcaveolae-associatedproteins. JBiolChem 1997, 272: 6525 – 6533. 11.QuestAF,LeytonL,ParragaM: Caveolins,caveolae,andlipidraftsin cellulartransport,signaling,anddisease. BiochemCellBiol 2004, 82: 129 – 144. 12.WilliamsTM,LisantiMP:Caveolin-1inoncogenictransformation,cancer, andmetastasis. AmJPhysiolCellPhysiol 2005, 288: C494 – C506. 13.HuangJH,LuL,LuH,ChenX,JiangS,ChenYH: IdentificationoftheHIV-1 gp41core-bindingmotifinthescaffoldingdomainofcaveolin-1. JBiol Chem 2007, 282: 6143 – 6152. 14.HovanessianAG,BriandJP,SaidEA,SvabJ,FerrisS,DaliH,MullerS, DesgrangesC,KrustB: Thecaveolin-1bindingdomainofHIV-1 glycoproteingp41isanefficientBcellepitopevaccinecandidate againstvirusinfection. Immunity 2004, 21: 617 – 627. 15.BenferhatR,KrustB,Rey-CuilleMA,HovanessianAG: Thecaveolin-1 bindingdomainofHIV-1glycoproteingp41(CBD1)containsseveral overlappingneutralizingepitopes. Vaccine 2009, 27: 3620 – 3630. 16.WangXM,NadeauPE,LoYT,MergiaA: Caveolin-1modulatesHIV-1 envelope-inducedbystanderapoptosisthroughgp41. JVirol 2010, 84: 6515 – 6526. 17.LinS,WangXM,NadeauPE,MergiaA: HIVinfectionupregulatescaveolin 1expressiontorestrictvirusproduction. JVirol 2010, 84: 9487 – 9496. 18.WangXM,NadeauPE,LinS,AbbottJR,MergiaA: Caveolin1inhibitsHIV replicationbytranscriptionalrepressionmediatedthroughNF-kappaB. JVirol 2011, 85: 5483 – 5493. 19.LinYC,MaC,HsuWC,LoHF,YangVC: Molecularinteractionbetween caveolin-1andABCA1onhigh-densitylipoprotein-mediatedcholesterol effluxinaorticendothelialcells. CardiovascRes 2007, 75: 575 – 583. 20.IkonenE,PartonRG: Caveolinsandcellularcholesterolbalance. Traffic 2000, 1: 212 – 217. 21.TruongTQ,BrodeurMR,FalstraultL,RhaindsD,BrissetteL: Expressionof caveolin-1inhepaticcellsincreasesoxidizedLDLuptakeandpreserves theexpressionoflipoproteinreceptors. JCellBiochem 2009, 108: 906 – 915. 22.SmartEJ,YingY,DonzellWC,AndersonRG: Aroleforcaveolinintransport ofcholesterolfromendoplasmicreticulumtoplasmamembrane. JBiol Chem 1996, 271: 29427 – 29435. 23.UittenbogaardA,YingY,SmartEJ:Characterizationofacytosolicheatshockprotein-caveolinchaperonecomplexInvolvementincholesterol trafficking. JBiolChem 1998, 273: 6525 – 6532. 24.TruongTQ,AubinD,FalstraultL,BrodeurMR,BrissetteL: SR-BI,CD36,and caveolin-1contributepositivelytocholesteroleffluxinhepaticcells. Cell BiochemFunct 2010, 28: 480 – 489. 25.LiaoZ,GrahamDR,HildrethJE: LipidraftsandHIVpathogenesis:virionassociatedcholesterolisrequiredforfusionandinfectionofsusceptible cells. AIDSResHumRetroviruses 2003, 19: 675 – 687. 26.ZhengYH,PlemenitasA,LinnemannT,FacklerOT,PeterlinBM: Nef increasesinfectivityofHIVvialipidrafts. CurrBiol 2001, 11: 875 – 879. 27.MaziereJC,LandureauJC,GiralP,AuclairM,FallL,LachgarA,AchourA, ZaguryD: LovastatininhibitsHIV-1expressioninH9humanT lymphocytesculturedincholesterol-poormedium. BiomedPharmacother 1994, 48: 63 – 67. 28.CarterGC,BernstoneL,SanganiD,BeeJW,HarderT,JamesW: HIVentry inmacrophagesisdependentonintactlipidrafts. Virology 2009, 386: 192 – 202. 29.AloiaRC,TianH,JensenFC: Lipidcompositionandfluidityofthehuman immunodeficiencyvirusenvelopeandhostcellplasmamembranes. Proc NatlAcadSciUSA 1993, 90: 5181 – 5185. 30.BukrinskyM,SviridovD: Humanimmunodeficiencyvirusinfectionand macrophagecholesterolmetabolism. JLeukocBiol 2006, 80: 1044 – 1051. 31.DingL,DerdowskiA,WangJJ,SpearmanP: Independentsegregationof humanimmunodeficiencyvirustype1Gagproteincomplexesandlipid rafts. JVirol 2003, 77: 1916 – 1926. 32.HolmK,WeclewiczK,HewsonR,SuomalainenM: Human immunodeficiencyvirustype1assemblyandlipidrafts:Pr55(gag)Lin etal.Retrovirology 2012, 9 :85 Page14of16 http://www.retrovirology.com/content/9/1/85

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associateswithmembranedomainsthatarelargelyresistanttoBrij98 butsensitivetoTritonX-100. JVirol 2003, 77: 4805 – 4817. 33.NguyenDH,HildrethJE: Evidenceforbuddingofhuman immunodeficiencyvirustype1selectivelyfromglycolipid-enriched membranelipidrafts. JVirol 2000, 74: 3264 – 3272. 34.OnoA,FreedEO: PlasmamembraneraftsplayacriticalroleinHIV-1 assemblyandrelease. ProcNatlAcadSciUSA 2001, 98: 13925 – 13930. 35.GuyaderM,KiyokawaE,AbramiL,TurelliP,TronoD: Roleforhuman immunodeficiencyvirustype1membranecholesterolinviral internalization. JVirol 2002, 76: 10356 – 10364. 36.ZhengYH,PlemenitasA,FieldingCJ,PeterlinBM: Nefincreasesthe synthesisofandtransportscholesteroltolipidraftsandHIV-1progeny virions. ProcNatlAcadSciUSA 2003, 100: 8460 – 8465. 37.WoutABv ’ t,SwainJV,SchindlerM,RaoU,PathmajeyanMS,MullinsJI, KirchhoffF: Nefinducesmultiplegenesinvolvedincholesterolsynthesis anduptakeinhumanimmunodeficiencyvirustype1-infectedTcells. JVirol 2005, 79: 10053 – 10058. 38.MujawarZ,RoseH,MorrowMP,PushkarskyT,DubrovskyL,MukhamedovaN, FuY,DartA,OrensteinJM,BobryshevYV, etal : Humanimmunodeficiency virusimpairsreversecholesteroltransportfrommacrophages. PLoSBiol 2006, 4: e365. 39.HanX,KitamotoS,LianQ,BoisvertWA: Interleukin-10facilitatesboth cholesteroluptakeandeffluxinmacrophages. JBiolChem 2009, 284: 32950 – 32958. 40.WelkerR,HarrisM,CardelB,KrausslichHG: Virionincorporationofhuman immunodeficiencyvirustype1Nefismediatedbyabipartite membrane-targetingsignal:analysisofitsroleinenhancementofviral infectivity. JVirol 1998, 72: 8833 – 8840. 41.DjordjevicJT,SchibeciSD,StewartGJ,WilliamsonP: HIVtype1Nef increasestheassociationofTcellreceptor(TCR)-signalingmolecules withTcellraftsandpromotesactivation-inducedraftfusion. AIDSRes HumRetroviruses 2004, 20: 547 – 555. 42.MujawarZ,TamehiroN,GrantA,SviridovD,BukrinskyM,Fitzgerald1ML: MutationoftheABCA1C-terminusdisruptsHIV-1Nefbindingbutdoes notblocktheNefenhancementofABCA1proteindegradation. Biochemistry2010, 49: 8338 – 8349. 43.FrankPG,CheungMW,PavlidesS,LlaveriasG,ParkDS,LisantiMP: Caveolin1andregulationofcellularcholesterolhomeostasis. AmJPhysiolHeart CircPhysiol 2006, 291: H677 – H686. 44.Hamard-PeronE,MuriauxD: Retroviralmatrixandlipids,theintimate interaction. Retrovirology 2011, 8: 15. 45.MorrowMP,GrantA,MujawarZ,DubrovskyL,PushkarskyT,KiselyevaY, JennelleL,MukhamedovaN,RemaleyAT,KashanchiF, etal : StimulationoftheliverXreceptorpathwayinhibitsHIV-1replication viainductionofATP-bindingcassettetransporterA1. MolPharmacol 2010, 78: 215 – 225. 46.CroweSM,WesthorpeCL,MukhamedovaN,JaworowskiA,SviridovD, BukrinskyM: Themacrophage:theintersectionbetweenHIVinfection andatherosclerosis. JLeukocBiol 2010, 87: 589 – 598. 47.ChoudhuryRP,LeeJM,GreavesDR: Mechanismsofdisease:macrophagederivedfoamcellsemergingastherapeutictargetsinatherosclerosis. NatClinPractCardiovascMed 2005, 2: 309 – 315. 48.FengB,YaoPM,LiY,DevlinCM,ZhangD,HardingHP,SweeneyM,Rong JX,KuriakoseG,FisherEA, etal : Theendoplasmicreticulumisthesiteof cholesterol-inducedcytotoxicityinmacrophages. NatCellBiol 2003, 5: 781 – 792. 49.OhashiR,MuH,WangX,YaoQ,ChenC: Reversecholesteroltransport andcholesteroleffluxinatherosclerosis. QJM 2005, 98: 845 – 856. 50.WangN,SilverDL,CostetP,TallAR: SpecificbindingofApoA-I,enhanced cholesterolefflux,andalteredplasmamembranemorphologyincells expressingABC1. JBiolChem 2000, 275: 33053 – 33058. 51.ArgmannCA,EdwardsJY,SawyezCG,O'NeilCH,HegeleRA,PickeringJG, HuffMW: Regulationofmacrophagecholesteroleffluxthrough hydroxymethylglutaryl-CoAreductaseinhibition:aroleforRhoAin ABCA1-mediatedcholesterolefflux. JBiolChem 2005, 280: 22212 – 22221. 52.LandryYD,DenisM,NandiS,BellS,VaughanAM,ZhaX: ATP-binding cassettetransporterA1expressiondisruptsraftmembrane microdomainsthroughitsATPase-relatedfunctions. JBiolChem 2006, 281:36091 – 36101. 53.BergamaschiA,PancinoG: HosthindrancetoHIV-1replicationin monocytesandmacrophages. Retrovirology 2010, 7: 31. 54.HerbeinG,VarinA: ThemacrophageinHIV-1infection:fromactivationto deactivation? Retrovirology 2010, 7: 33. 55.LeDouceV,HerbeinG,RohrO,SchwartzC: Molecularmechanismsof HIV-1persistenceinthemonocyte-macrophagelineage. Retrovirology 2010, 7: 32. 56.GuillemardE,JacquemotC,AilletF,SchmittN,Barre-SinoussiF, IsraelN: Humanimmunodeficiencyvirus1favorsthepersistenceof infectionbyactivatingmacrophagesthroughTNF. Virology 2004, 329: 371 – 380. 57.SalahuddinSZ,RoseRM,GroopmanJE,MarkhamPD,GalloRC: HumanT lymphotropicvirustypeIIIinfectionofhumanalveolarmacrophages. Blood 1986, 68: 281 – 284. 58.GartnerS,MarkovitsP,MarkovitzDM,KaplanMH,GalloRC,PopovicM: The roleofmononuclearphagocytesinHTLV-III/LAVinfection. Science 1986, 233: 215 – 219. 59.SharovaN,SwinglerC,SharkeyM,StevensonM: Macrophages archiveHIV-1virionsfordisseminationintrans. EMBOJ 2005, 24: 2481 – 2489. 60.FreedEO: HIV-1andthehostcell:anintimateassociation. Trends Microbiol 2004, 12: 170 – 177. 61.MalimMH,EmermanM: HIV-1AccessoryProteins.EnsuringViralSurvival inaHostileEnvironment. CellHostMicrobe 2008, 3: 388 – 398. 62.LiuL,OliveiraNM,CheneyKM,PadeC,DrejaH,BerginAM,BorgdorffV, BeachDH,BishopCL,DittmarMT,McKnightA: Awholegenomescreen forHIVrestrictionfactors. Retrovirology 2011, 8: 94. 63.GiriMS,NebozhynM,ShoweL,MontanerLJ: Microarraydataongene modulationbyHIV-1inimmunecells:2000 – 2006. JLeukocBiol 2006, 80: 1031 – 1043. 64.GiriMS,NebozyhnM,RaymondA,GekongeB,HancockA,CreerS,NicolsC, YousefM,FoulkesAS,MounzerK, etal: CirculatingMonocytesinHIV-1InfectedViremicSubjectsExhibitanAntiapoptosisGeneSignatureand Virus-andHost-MediatedApoptosisResistance1. JImmunol 2009, 182: 4459 – 4470. 65.VazquezN,Greenwell-WildT,MarinosNJ,SwaimWD,NaresS,OttDE, SchubertU,HenkleinP,OrensteinJM,SpornMB,WahlSM: HIV-1induced macrophagegeneexpressionincludesp21,atargetforviralregulation. JVirol 2005, 79: 4479 – 4491. 66.PartonRG,SimonsK: Themultiplefacesofcaveolae. NatRev 2007, 8: 185 – 194. 67.FraAM,WilliamsonE,SimonsK,PartonRGRG: Denovo formationof caveolaeinlymphocytesbyexpressionofVIP21-caveolin. ProcNatlAcad SciUSA 1995, 92: 8655 – 8659. 68.HatanakaM,MaedaT,IkemotoT,MoriH,SeyaT,ShimizuA: Expressionof Caveolin-1inHumanTCellLeukemiaCellLines. BiochBiophResComm 1998, 253: 382 – 387. 69.VallejoJ,HardinCD: Expressionofcaveolin-1inlymphocytesinduces caveolaeformationandrecruitmentofphosphofructokinasetothe plasmamembrane. FASEBJ 2005, 16: 586 – 587. 70.AdachiA,GendelmanHE,KoenigS,FolksT,WilleyR,RabsonA,MartinMA: Productionofacquiredimmunodeficiencysyndrome-associated retrovirusinhumanandnonhumancellstransfectedwithaninfectious molecularclone. JVirol 1986, 59: 284 – 291. 71.FreedEO,EnglundG,MartinMA: Roleofthebasicdomainofhuman immunodeficiencyvirustype1matrixinmacrophageinfection. JVirol 1995, 69: 3949 – 3954. 72.HwangSS,BoyleTJ,LyerlyHK,CullenBR: IdentificationoftheenvelopeV3 loopastheprimarydeterminantofcelltropisminHIV-1. Science 1991, 253: 71 – 74. 73.HeJ,ChoeS,WalkerR,DiMarzioP,MorganDO,LandauNR: Human immunodeficiencyvirustype1viralproteinR(Vpr)arrestscellsinthe G2phaseofthecellcyclebyinhibitingp34cdc2activity. JVirol 1995, 69: 6705 – 6711. 74.ConnorRI,ChenBK,ChoeS,LandauNR: Vprisrequiredforefficient replicationofhumanimmunodeficiencyvirustype-1inmononuclear phagocytes. Virology 1995,206: 935 – 944. 75.WeiX,DeckerJM,LiuH,ZhangZ,AraniRB,KilbyJM,SaagMS,WuX,Shaw GM,KappesJC: Emergenceofresistanthumanimmunodeficiencyvirus type1inpatientsreceivingfusioninhibitor(T-20)monotherapy. AntimicrobAgentsChemother 2002, 46: 1896 – 1905. 76.WeiX,DeckerJM,WangS,HuiH,KappesJC,WuX,Salazar-GonzalezJF, SalazarMG,KilbyJM,SaagMS, etal : Antibodyneutralizationandescape byHIV-1. Nature 2003, 422: 307 – 312.Lin etal.Retrovirology 2012, 9 :85 Page15of16 http://www.retrovirology.com/content/9/1/85

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77.RaneyA,KuoLS,BaughLL,FosterJL,GarciaJV: Reconstitutionand molecularanalysisofanactivehumanimmunodeficiencyvirustype1 Nef/p21-activatedkinase2complex. JVirol 2005, 79: 12732 – 12741. 78.O'NeillE,KuoLS,KriskoJF,TomchickDR,GarciaJV,FosterJL: Dynamic evolutionofthehumanimmunodeficiencyvirustype1pathogenic factor,Nef. JVirol 2006, 80: 1311 – 1320. 79.TheodoreTS,EnglundG,Buckler-WhiteA,BucklerCE,MartinMA,PedenKW: Constructionandcharacterizationofastablefull-lengthmacrophagetropicHIVtype1molecularclonethatdirectstheproductionofhigh titersofprogenyvirions. AIDSResHumRetroviruses 1996, 12: 191 – 194. 80.HowellKW,MengX,FullertonDA,JinC,ReeceTB,ClevelandJCJr: Toll-like Receptor4MediatesOxidizedLDL-InducedMacrophageDifferentiation toFoamCells. JSurgRes 2011, 171: e27 – e31. 81.LinS,WuM,XuY,XiongW,YiZ,ZhangX,ZhenghongY: Inhibitionof hepatitisBvirusreplicationbyMyD88ismediatedbynuclearfactorkappaBactivation. BiochimBiophysActa 2007, 1772: 1150 – 1157.doi:10.1186/1742-4690-9-85 Citethisarticleas: Lin etal. : Caveolin-1reducesHIV-1infectivityby restorationofHIVNefmediatedimpairmentofcholesteroleffluxby apoA-I. Retrovirology 2012 9 :85. Submit your next manuscript to BioMed Central and take full advantage of: € Convenient online submission € Thorough peer review € No space constraints or color “gure charges € Immediate publication on acceptance € Inclusion in PubMed, CAS, Scopus and Google Scholar € Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Lin etal.Retrovirology 2012, 9 :85 Page16of16 http://www.retrovirology.com/content/9/1/85


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