Group Title: Retrovirology 2009, 6:32
Title: Accuracy estimation of foamy virus genome copying
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Title: Accuracy estimation of foamy virus genome copying
Series Title: Retrovirology 2009, 6:32
Physical Description: Archival
Creator: Gärtner K
Wiktorowicz T
Park J
Mergia A
Rethwilm A
Scheller C
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Source Institution: University of Florida
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Accuracy estimation of foamy virus genome copying
Kathleen Gartnerl, Tatiana Wiktorowicz1, Jeonghae Park2, Ayalew Mergia2,
Axel Rethwilm*I and Carsten Scheller1

Address: 'Universitit Wiirzburg, Institut fir Virologie und Immunbiologie, Versbacher Str 7, 97078, Wiirzburg, Germany and 2Department of
Infectious Disease and Pathology, College of Veterinary Medicine, University of Florida, Gainesville, FL, USA
Email: Kathleen Girtner; Tatiana Wiktorowicz;
Jeonghae Park; Ayalew Mergia; Axel Rethwilm*;
Carsten Scheller
* Corresponding author

Published: 6 April 2009 Received: 4 November 2008
Retrovirology 2009, 6:32 doi:10.1 186/1742-4690-6-32 Accepted: 6 April 2009
This article is available from:
2009 Gartner et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background: Foamy viruses (FVs) are the most genetically stable viruses of the retrovirus family.
This is in contrast to the in vitro error rate found for recombinant FV reverse transcriptase (RT).
To investigate the accuracy of FV genome copying in vivo we analyzed the occurrence of mutations
in HEK 293T cell culture after a single round of reverse transcription using a replication-deficient
vector system. Furthermore, the frequency of FV recombination by template switching (TS) and
the cross-packaging ability of different FV strains were analyzed.
Results: We initially sequenced 90,000 nucleotides and detected 39 mutations, corresponding to
an in vivo error rate of approximately 4 x 10-4 per site per replication cycle. Surprisingly, all
mutations were transitions from G to A, suggesting that APOBEC3 activity is the driving force for
the majority of mutations detected in our experimental system. In line with this, we detected a late
but significant APOBEC3G and 3F mRNA by quantitative PCR in the cells. We then analyzed
170,000 additional nucleotides from experiments in which we co-transfected the APOBEC3-
interfering foamy viral bet gene and observed a significant 50% drop in G to A mutations, indicating
that APOBEC activity indeed contributes substantially to the foamy viral replication error rate in
vivo. However, even in the presence of Bet, 35 out of 37 substitutions were G to A, suggesting that
residual APOBEC activity accounted for most of the observed mutations. If we subtract these
APOBEC-like mutations from the total number of mutations, we calculate a maximal intrinsic in vivo
error rate of I. x 10-5 per site per replication. In addition to the point mutations, we detected one
49 bp deletion within the analyzed 260000 nucleotides.
Analysis of the recombination frequency of FV vector genomes revealed a 27% probability for a
template switching (TS) event within a I kilobase (kb) region. This corresponds to a 98% probability
that FVs undergo at least one additional TS event per replication cycle. We also show that a given
FV particle is able to cross-transfer a heterologous FV genome, although at reduced efficiency than
the homologous vector.
Conclusion: Our results indicate that the copying of the FV genome is more accurate than
previously thought. On the other hand recombination among FV genomes appears to be a frequent

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Open Access


Retroviral genomes are highly susceptible to the introduc-
tion of mutations, most of which are assumed to result
from the action of the viral RT. While the contribution of
the host RNA polymerase II to retroviral mutations has
long been speculated [1], RNA polymerase II is now
assumed to be a high fidelity polymerase because of its 3'
to 5' repair activity [2,3]. Moreover, the significant varia-
tion in the in vivo mutation rates of different retroviruses
suggests a host-independent source of mutations [4,5].

Retroviruses are pseudo-diploid and usually generate one
DNA copy from the two RNA copies that are packaged
into the viral particle. During reverse transcription, the RT
enzyme can jump from one template strand to the other,
thereby generating a hybrid transcript. If the two RNA
templates are not identical, these template switching (TS)
events can contribute to the overall retroviral mutation
rate [6-10]. These TS events can occur even between dis-
tantly related retroviruses, provided that the different
viruses can cross-package the heterologous viral genomes

FVs, the only genus in the spumaretrovirus subfamily of
Retroviridae, are known to be genetically extremely stable
and have co-evolved with their host species [12-19] over
more than 60 million years and represent the genetically
most stable viruses which have an RNA phase in replica-
tion [12-19]. The biochemical and biological reasons for
this stability have yet to be determined. It may be that the
error rate of the FV RT is exceptionally low among retrovi-
ruses. However, the in vitro error rates of the human
immunodeficiency virus (HIV) type 1 and of the proto-
type FV (PFV) RTs have recently been compared [20]. The
overall probability of generating mutations was deter-
mined to be 7.5 x 10-5 mutations per nucleotide per repli-
cation cycle for HIV-1 RT and 1.7 x 10-4 for PFV RT [20].
Single nucleotide substitutions contributed to this with
6.3 x 10-5 (HIV-1) and 5.8 x 10-5 (PFV) mutations/nt per
replication cycle. The remaining mutations were found to
be due to insertions and deletions [20]. Thus, it appears
that the rate of point-mutation during HIV-1 and PFV rep-
lication are remarkably similar, raising the possibility that
a very low in vivo FV replication rate is the main reason for
their genetic stability. Alternatively, the relatively high FV
RT error rate that has been reported may be due to the spe-
cific in vitro assay conditions. This prompted us to analyze
the in vivo mutation rate of PFV RT.

TS events happen frequently among plus-strand RNA-con-
taining viruses and require the simultaneous infection of
one cell by two parental viruses. Frequencies of TS have
been studied for several retroviruses, particularly for HIV
[7,21-26]. For instance, the development of resistance to
antiviral therapy can be a consequence of recombination

events during reverse transcription [27,28]. Through the
use of retroviral vectors in single replication assays, the TS
rates of HIV-1 and murine leukemia virus (MLV) were
determined to be in the range of 3-4 crossovers per
genome per replication cycle [26,29]. This is not necessar-
ily reflected by the in vivo recombination rate that differs
between HIV-1 and MLV [26,29]. Recombination rates
were found experimentally to be 42.4% and 4.7% per 1 kb
for HIV-1 and MLV, respectively [21,24].

Previous in vitro analysis has shown that the PFV RT cre-
ates a large number of small and large deletions, which
suggest that the PFV RT jumps to an upstream site of the
same strand during polymerization [20]. A high processiv-
ity of PFV RT [30] was proposed to be responsible for this
slippage [20]. However, jumping to an upstream site in
vivo may also result in a template switch. Since the in vivo
TS rate of FVs has not thus far been determined, we have
examined this mechanism using PFV in single replication
assays. In addition, we investigated the ability of PFV par-
ticles to cross-transfer the genome of the related simian FV
from macaque (SFVmac) and vice versa to estimate the
probability and biological significance ofTS in FVs. This
may be particularly relevant in the light of recent findings
on trans-species SFV infections of humans in non-occupa-
tional settings and in the case of HIV/FV double infections
in humans [31-33].

Retroviral vectors are frequently used in gene transfer pro-
tocols and have been applied successfully in the clinical
setting [34]. Considering an average clinical preparation
of approximately 109 vector particles of a 10 kb vector,
such a pharmaceutical product would harbor approxi-
mately 108 variants, assuming an RT point-mutation rate
of 10-5 per nucleotide per replication round. Thus, the
accuracy of a retroviral RT enzyme and, furthermore, the
chances to mobilize an integrated vector genome by
superinfection with a homologous or heterologous virus
appear to be critical factors to examine, especially since FV
vectors are close to being used for clinical applications in
humans [35-37].

Analysis of FV mutation frequencies in the absence of Bet
A previous analysis of the fidelity of the PFV RT used a
recombinant enzyme and an assay that depended on the
functionality of an indicator gene [20]. This raises the pos-
sibility that silent mutations could have been missed. To
estimate the in vivo PFV RT mutation rate, we produced
the replication deficient FVvector KG83 in HEK 293T cells
(Fig. 1A). Following the transduction of HeLa recipient
cells, we sorted single EGFP-positive cells into 96 well
plates to obtain monoclonal cell cultures that each carries
one FV provirus. The individual proviral sequences from a
total of 346 clones were amplified by PCR using Pwo

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Retrovirology 2009, 6:32


lacZa ALTR

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-IN eukaryotic
#4250 Promoter &


0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48

relative APOBEC3G expression


0% -


Figure I
Analysis of the FV in vivo mutation rate and APOBEC3G expression. (A) Construct pKG83 used to evaluate the FV
mutation rate in vivo. Marker gene EGFP was used for identification of infected cells. The locations of the primers (#4250 and
#4254) used to amplify proviral sequences for sequencing are indicated. (B) Quantitative determination of APOBEC3G mRNA
in HEK 293T (three runs) and HeLa cells as well as in PMBCs (two runs of two different PBMC preparations). H20 served as
negative control. (C) Relative amounts of APOBEC3G mRNA in HEK 293T cells and in PBMCs (set to 100%) with respect to
the amounts detected for the three housekeeping genes P-actin, GAPDH, and SDHA.

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Retrovirology 2009, 6:32



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polymerase, and the resulting PCR products were
sequenced. As PCR-introduced errors are randomly dis-
tributed over the transcript and the amplicon pool, it is
unlikely that an individual PCR-introduced mutation will
be detected in the sequencing reaction. However, in order
to exclude such false positive events, we amplified and
sequenced each mutation-carrying provirus twice. For a
direct comparison of results we included the same genetic
element in our analysis that has been used in the previous
study by Boyer et al. [20].

Initially, we sequenced a total of 93,003 bases from 110
single cell-derived colonies and detected 39 point muta-
tions, resulting in an error rate of approx. 4.2 x 10-4 per
base per replication cycle (Table 1).

APOBEC3 expression in HEK 293T cells
All detected mutations were G to A transitions suggesting
that APOBEC3 activity may be the driving force for muta-
tions in our experimental system. This was surprising as

previous studies documented the absence of APOBEC3G
in HEK 293T cells by Western blotting [38]. Similarly, we
could not detect APOBEC3G protein in these cells by
Western blotting (data not shown). However, using the
more sensitive quantitative RT-PCR for APOBEC3G, we
measured a late (compared to PBMC) but significant PCR
signal in these cells, whereas HeLa cells were negative for
APOBEC message (Fig. 1B). APOBEC3G levels in 293T
cells were about 25% of the amount detected in PBMC
(Fig. 1C). APOBEC3F mRNA was even more abundant
and was found to be almost four-fifths of the PBMC level
(Additional file 1, Fig S3). To exclude the possibility that
this feature was unique to the HEK 293T cells used in our
laboratory, we also analyzed HEK 293T cells directly pur-
chased from the American Type Culture Collection and
detected the same signal. Thus, HEK 293T cells appear to
express restriction factors that may influence the genera-
tion of foamy viral and other retroviral vectors produced
in these cells.

Table I: PFV point mutations identified after a single round of replication in the absence or presence of Bet.

vector packaging in 293T cells w/o Bet

vector packaging in 293T cells with Bet

number of mutations in a total of 93,003 nucleotides





number of mutations in a total of 172,368 nucleotides








error rate

4.2 x 10-4

2.1 x 10-4

Vector particles were packaged in HEK 293T cells in the absence or presence of a bet expression plasmid. Vector preparations were used to
transduce HeLa cells. Singly transduced cells were sorted into 96 well plates to form monoclonal cell cultures that carry a single provirus. Proviral
DNA was amplified by PCR and sequenced in order to assess the in vivo error rate of FV replication.

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type of mutation

Retrovirology 2009, 6:32


Analysis of FV mutation frequencies in the presence of Bet
In order to challenge the hypothesis that APOBEC activity
is the driving force for the mutations in our experimental
system, we analyzed 172,368 additional nucleotides from
236 individual cell clones from experiments in which we
co-transfected 293T cells with the APOBEC3-inhibiting
foamy viral bet gene. Bet has been demonstrated to inhibit
APOBEC3G-triggered G-to-A mutations [38-40]. One
group, however, has shown that Bet does not counteract
APOBEC3G-mediated block of FV infectivity, as wildtype
FV strains were similarly susceptible to APOBEC when
compared with strains with a delta-bet mutation [41].
Introducing Bet into our model system, we observed a sig-
nificant (X2 = 10.13) drop in mutations by 50%, indicat-
ing that APOBEC activity indeed contributed substantially
to the foamy viral replication error rate in HEK 293 T cells
(Table 1). Bet co-transfection reduced G to A exchanges
not only in the GG context that was reported to be a pref-
erential target site for APOBEC3G in the HIV genome
[42], but also in other sequence contexts (Table 2). In line
with this, a study published by Delebecque and colleagues
did not identify such GG hotspots for APOBEC3G-muta-
genesis in the foamy viral genome [41].

As the total amount of residual APOBEC activity in our
bet-cotransfection experiments is unclear, we cannot
exactly pin down the APOBEC-independent error rate of
foamy viral replication. However, it seems very unlikely
that the majority of the G to A mutations in the presence
of Bet is caused by an intrinsic activity of FV RT as the
study of Boyer et al. found that only 1 out of 3 nucleotide
substitutions caused by FV RT is a G to A exchange [20].
As we detected only 2 non-G-to-A mutations within the
total of 265,371 sequenced nucleotides in our study, one
would expect to find no more than one additional G to A
transition intrinsically caused by foamy viral RT. In this
light, it seems very likely to us that the remaining G to A
mutations that we detected in the presence of Bet were
caused by residual APOBEC enzyme activity. This scenario
results in a corrected error rate of 3 mutations in 265,371

nucleotides (i.e. an error rate of 1.1 x 10-5 per site per rep-

Within the 265,371 nucleotides that we sequenced, we
identified only a single deletion (which occurred in the
experiments with Bet expression) and no insertion (see
also Additional file 1, Fig. S4). This is in sharp contrast to
the situation reported with recombinant FV RT where
such mutations accounted for the majority of all muta-
tions identified [20]. This probably indicates differences
between the in vitro and the in vivo accuracy of FV reverse
transcription. The one 49 bp deletion we observed took
place at a DNA stretch with no obviously repeated
sequence (Additional file 1, Fig. S4). Boyer et al. also
reported that deletions occurring during FV reverse tran-
scription do not necessarily involve repeated sequences

Experimental design to determine FV TS
We determined the TS rate of FV by a phenotypic resist-
ance assay described by Anderson et al. for MLV [21]. As
an internal quality control for the assay, we similarly
determined the MLVTS rate and found it to be in the same
range (7.1% + 2.0% SE for a 1 kb fragment, data not
shown) as what has been reported previously (4.7%,

For the determination of the foamy viral TS rate, we con-
structed the vectors KG81 and KG82 that carry the resist-
ance genes for hygromycin and neomycin (Fig. 2A). KG81
carries a mutation in the Hygro resistance gene that
destroys both the resistance activity as well as a pre-exist-
ing SacII restriction site. KG82 has a mutation in the Neo
resistance gene that destroys both the resistance activity as
well as a preexisting EheI site (the function of the restric-
tion sites will be discussed later). Both mutations are 1 kb

Vector production was performed by transfection of 293T
cells with a mixture of KG81 and KG82 together with gag-

Table 2: Sequence context of G to A mutations in the absence or presence of Bet.

vector packaging in 293T cells w/o Bet

G to A mutation with sequence context




number of mutations in a total of 93,003



vector packaging in 293T cells with Bet

number of mutations in a total of 172,368




G to A mutations from Table I categorized according to their sequence context.

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Retrovirology 2009, 6:32


ALTR CAS 1/11 U3 Hygro IRES Neo ALTR

-I I r m*1- m

Restriction pattern
of PCR product

c N

PCR product

TS genotype Scl Ehe
iSacll Ehel

with 2 non-TS

2200 bp
1700 bp
1550 bp

650 bp
500 bp

2200 bp
1700 bp
1550 bp

650 bp
500 bp



1 2 3 4 5 6 7 8 9 10
2300 -
2000 --
Sm - -1550

- 650

1 2 3 4 5 6 7 8 9 10
2300 -
2000 - wm, 1700


Figure 2
Template Switching (TS) rate of foamy viral replication. (A) MD9-derived PFV vector viruses (KG81 and KG82)
expressing hygromycin and neomycin resistance genes under control of a SFFV U3 promoter. CASI/II are cis-acting sequences
required for FV vector transfer [71]. KG81 carries a point mutation in the hygromycin resistance gene that abolishes its func-
tion and destroys a Sacll restriction site. KG812 carries a point mutation in the neomycin resistance gene that abolishes its
function and destroys an Ehell restriction site. The two mutations are I kb apart. (B) Distinction of TS events from superinfec-
tion with KG81 and KG82 by restriction pattern: amplification of the proviral sequences by PCR generates a 2.2 kb fragment
with a Sad site at position 500 and an Ehell site at position 1550. Amplicons of TS events carry the two intact restriction sites
and show a restriction pattern depicted in the upper box, whereas amplicons of superinfected cells show the restriction pat-
tern depicted in the lower box. (C) Representative digests of 10 clones from the TS experiment. All 10 clones show the
expected pattern for TS events. Upper lane 3 and lower lane 2 show incomplete digests.

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Retrovirology 2009, 6:32

m m


pol-env-helper plasmids. The resulting vector preparation
consisted of virions that contained either the original
KG81 or KG82 sequences or KG81/KG82 hybrid
sequences as a result of template switching. This vector
preparation was used to transduce HEK 293 cells that were
grown in the presence of neomycin and/or hygromycin.
The resulting colonies were quantified.

Colonies carrying the KG81 provirus are resistant to neo-
mycin (Neor) but sensitive to hygromycin (Hygros). Col-
onies carrying the KG82 provirus are sensitive to
neomycin (Neos) but resistant to hygromycin (Hygror).
These phenotypes will be referred to as non-TS pheno-
types. A double-resistant colony (Neor Hygror) carries a
provirus that is the result of a TS event within the 1 kb
region between the mutation sites, a phenotype that will
be referred to as TS-phenotype.

The TS rate in this 1 kb region can be calculated from the
ratio of TS-phenotype colonies versus the total number
(TS and non-TS phenotypes) of colonies. If no TS
occurred, vectors displayed either the phenotype Neos and
Hygror or Neor and Hygro". In the case of a TS event, vec-
tors were either Neor plus Hygror or Neos plus Hygro". The
number of TS events can be quantified on culture plates
supplemented with the two antibiotics, allowing the out-
growth of one of the two TS phenotypes (Neor plus
Hygror). Since the other TS phenotype (Neos plus Hygro")
will be suppressed, the number of TS events is twice as
high as the number of colonies on the double antibiotics
plate. On plates supplemented with only one of the two
antibiotics, the respective non-TS phenotypes will grow
out as well as the double resistant TS-phenotype. The
number of non-TS colonies on these plates can therefore

be calculated by subtracting the number of double resist-
ant colonies (counted from the double antibiotics plate)
from the total number of colonies visible on each single
antibiotic plate.

Double resistant colonies can not only result from the
transduction with TS-genotypes but can also result from a
superinfection with both KG81 and KG82. (To minimize
superinfection we transduced the cells with an m.o.i. <
0.01). Superinfections can easily be distinguished from
transductions with TS-genotypes by amplification of the
proviral DNA and subsequent digestion with SacII and
Ehel; whereas the PCR product of a TS genotype carries
both the intact SacHI and the intact Ehel site on a single
molecule (and will therefore generate a positive restric-
tion pattern with the two enzymes); the PCR products
from superinfected cells carry the restriction sites on dif-
ferent molecules (and will therefore produce a different
restriction pattern). The digestion of the 2.2 kb amplicons
with EheI and SacII should result in bands of 1.55 and
0.65 kb or 1.7 and 0.5 kb respectively if a recombination
event had occurred between the two sites 1 kb apart (Fig.
2B). If double resistance was caused by superinfection
with two viruses, the digestions would show a third band.
This latter band would have been caused by the uncut 2.2
kb amplicon of one provirus (Fig. 2B).

Analysis of the FV TS rate
Table 3 summarizes the values obtained for the FV TS rate
in the transient assay. Within the investigated 1 kb region
we calculated an average recombination rate of 22.3% +
0.27% SE from the results of three independent experi-
ments. When the proviruses of 26 cell colonies were ana-
lyzed by PCR and restriction enzyme digestion for the

Table 3: Calculation of the PFV TS rate.

Column I Column 2 Column 3 Column 4

Column 5

Column 6

Column 7

Selection Medium Neo + Hygro Neo

Hygro TS Phenotype

Phenotype (Neo) Phenotype (Hygro)

Experiment I

Experiment 2

Experiment 3


7,000 21,000

4,300 16,500 22,500

600 4,100 1,400















KG81 and KG82 (Fig. 2A) were combined and packaged in 293T cells. Recipient HEK 293 cells were transduced with vector preparations and cells
were grown on culture plates supplemented with either neomycin, hygromycin, or both. Colonies were quantified. Column I: number of colonies
counted on plates with Neomycin and Hygromycin; column 2: number of colonies counted on plates with Neomycin; column 3: number of colonies
counted on plates with Hygromycin; column 4: TS phenotypes (including the double sensitive phenotype that does not grow on the double
antibiotics plate) calculated as 2x column I (e.g. 2 x 3,200 = 6,400); column 5: Non-TS phenotypes (NeoR, Hygros) calculated as column 2 column
I (e.g. 7,000 3,200 = 3,800); column 6: Non-TS phenotypes (Neos, HygroR) calculated as column 3 column I (e.g. 21,000 3,200 = 17,800);
column 7: TS rate within I kb fragment calculated as 100x(column 4/[column 4 + column 5 + column 6]) (e.g. 100 x (6400/[6400+3800+1780]) =

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TS rate per I kb

Retrovirology 2009, 6:32


presence of superinfections with two vectors, we exclu-
sively found evidence for recombinant proviruses, dem-
onstrating that the calculated TS rate is not biased by false-
positive colonies. A representative digestion pattern is
shown in Fig. 2C.

As the TS rate is in the range of 20%, one would assume
that one out of 5 TS-viruses would undergo a second TS
event, resulting again in a mixed resistant phenotype
(Neos and Hygror or Neor and Hygros) that would not
show up on the double antibiotics plate. The real, partly
hidden TS rate is therefore about 20% higher than the
observable TS rate, so that the true PFVTS rate is probably
in the range of 27% within the 1 kb region. For a typical
FV RNA (pre-) genome of 12 kb, this would correspond to
a 98% probability (calculated as P = 1-[1-0.27]12) to
undergo at least one internal TS event per replication

Recombination may not be relevant to the application of
FV-derived vectors, as both templates are identical in
sequence, but it is clearly relevant for the generation of
new viruses or new viral variants [14,27,28]. The recent
analyses of SFV sequences from wild chimpanzees dem-
onstrated the presence of frequent FV recombinants and
the infection of chimpanzees by FVs from lower monkeys
[14,43]. We therefore tried to estimate the probability of
generating new FV recombinants by packaging heterolo-
gous viral sequences.

Investigation of the transfer of FV vector genomes
To analyze the transfer of FV vector genomes, we investi-
gated whether a given FV capsid is able to package and
transfer a related, but clearly different FV genome. We
used the vectors KG84 and EGFPD that were derived from
PFV (from chimpanzee isolate) and SFVmac (from Asian
monkeys) [44], respectively (Fig. 3A). To analyze the cross
packaging activity of the two viruses, we packed the PFV
vector with Gag Pol proteins derived from SFVmac and
the SFVmac vector with Gag Pol proteins derived from the
PFV vector. As an internal control, each vector was also
packaged with its homologous Gag Pol proteins. There is
evidence that (pre-) genomic RNA may have a structure-
forming capability in FV particle assembly [45,46]. To
exclude that this feature inhibits proper particle assembly
in a cross packaging situation, we determined the protein
composition of the vector particles being released into the
supernatant by the packaging cells. As shown in Fig. 3B,
we did not detect significant differences in the Gag and
Pol protein composition between homologous and heter-
ologous vector particles.

In order to determine the infectivity of the produced vec-
tors, we transduced HT1080 fibroblastoid recipient cells
with the supematants from the different packaging cul-
tures and determined the amount of EGFP-positive cells

by flow cytometry. Table 4 summarizes the data obtained
from the cross-packaging experiments. The results show
that both PFV and SFVmac can be effectively cross-packed
by heterologous Gag Pol proteins, although at a slightly
lower efficiency.

In contrast to the experimental setup described above, the
in vivo situation in which cross-packaging could occur a
co-infection of a cell with two different viruses would
represent an environment in which two viral genomes
would compete to be packaged by one viral capsid. In
order to determine the cross-packaging activity in the
presence of the competing homologous system, we con-
structed another PFV vector (TWO5) that expresses mRFP
instead of EGFP (Fig. 3C) so that the PFV vector can be
easily distinguished from the SFVmac vector EGFPD that
encodes for EGFP. We then co-transfected HEK 293T cells
with both vectors and packaged them with either PFV or
SFVmac helper plasmids. Vector production was quanti-
fied by transduction of HT1080 cells and flow cytometric
analysis of EGFP and mRFP expression. As depicted in
Table 5, each of the two vectors was packaged with high
efficiency by its homologous Gag Pol proteins in the pres-
ence of the competing heterologous vector. More impor-
tantly, however, both packaging systems allowed for the
simultaneous packaging of the heterologous vector with a
relative efficiency of 1.3% (SFVmac vector plus PFV parti-
cles) and 15% (PFV vector plus SFV particles).

These results show that FV particles are in principle able to
transfer heterologous but related sequences, albeit at a
considerably lower efficiency in relation to the homolo-
gous vector genome. Furthermore, the transfer of heterol-
ogous genomes may not be reciprocal between different

FVs appear to be an exception to the majority of retrovi-
ruses in respect to their genome conservation. This virus is
genetically very stable and, with the exception of trans-
species transmissions, has co-evolved with its hosts [17].
Their high genome conservation often allows the designa-
tion to a particular monkey or ape subspecies through the
analysis of the appropriate FV sequence [14,19]. Further-
more, in trans-species transmissions to humans or apes
the transmitted virus can be easily traced back to the trans-
mitting monkey species and appears to be genetically sta-
ble in the new host for decades [16,47,48].

We have demonstrated that G to A transitions dominate
the error rate in foamy viral vector production in HEK
293T cells and that the bet gene has a substantial influence
on the overall FV mutation rate in vivo as its presence
reduced the number of mutations in our assay system by
50%. Our experimental data suggest that members of the
APOBEC family rather than an intrinsic activity of FV RT

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KG84 / M



F7 ] M_

homo hetero homo hetero

PFV gag pol
PFV vector (KG84)
SFVma, gag pol
SFVmao vector (EGFPD)

Gag antibodies

Pol antibodies

+ +
+ +
+ +
+ +


q_ P. 4- Pol-precursor

nI am d -+ PR/RT/RNaseH


+- IN

a-PFV a-SFVmac



r\--1 1 _

Figure 3
Analysis of cross packaging. (A) KG84 (PFV) and EGFPD (SFVmac) vector viruses used in this analysis. "Gag/
Pol" of the EGFPD vector corresponds to the CASI/II region found in KG84. Due to point mutations of start and internal
ATGs of the gag and pol ORFs no viral proteins are translated from the EGFPD vector virus. (B) Gag and Pol protein composi-
tion of PFV vector (KG84) and SFVmac vector (EGFPD) particles produced in the presence of homologous (homo) and heter-
ologous (hetero) gag pol proteins. (C) Structure of the TW05 (PFV) vector virus expressing mRFP used to analyze the
simultaneous transfer of PFV and SFVmac FV genomes.

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" IU

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Table 4: Transfer rates of PFV vector (KG84) and SFVma vector (EGFPD) viruses on HT 1080 fibroblastoid cells after cross-packaging
with homologous and heterologous FV gag pol proteins.

vectors and packaging systems

EGFP-positive target cells

Cross-packaging efficiency

PFV gag pol +
PFV vector

PFV gag pol +
SFVmac vector

SFVmac gag pol +
SFVmac vector

SFVmac gag pol +
PFV vector






Vectors were packaged together with the indicated packaging proteins in 293T cells. Supernatants from packaging cells were used for transduction
of HT1080 cells. Transduction rates were measured by detection of EGFP by flow cytometry. The cross-packaging efficiency is calculated from the
ratio of EGFP-positive cells of the heterologous system versus the homologous system (e.g. 26.4% = 13.5%/51.3%).

are responsible for this mutation hotspot, so that the error
rate of foamy viral replication would be in the range of 1.1
x 10-5.

This contrasts to what has been published previously for a
recombinant assay system in which an overall error rate of
1.7 x 10-4 has been determined. In this case, the error rate
was dominated by deletions and insertions whereas
nucleotide substitutions contributed to the error rate only
with a factor of 5.8 x 10-5 [20]. Within the 265,371 nude-
otides that we sequenced we identified only a single dele-
tion and no insertion (see also Additional file 1, Fig. S4).
This probably indicates differences between the in vitro
and the in vivo accuracy of FV reverse transcription. The
one 49 bp deletion we observed took place at a DNA

stretch with no obviously repeated sequence (Additional
file 1, Fig. S4). Boyer et al. also reported that deletions
occurring during FV reverse transcription do not necessar-
ily involve repeated sequences [20]. Our results suggest a
much higher in vivo accuracy of FV genome replication
than has previously been thought. These findings are rem-
iniscent of previous studies comparing the in vivo and in
vitro mutation rates of HIV-1 [49,50].

Although this low mutation rate would be more consist-
ent with FV genome conservation, it does not completely
explain the genomic stability of FVs. For instance, even the
extrapolated FV point-mutation rate is slightly higher than
the point-mutation rate reported for primate T-lympho-
tropic virus type I (PTLV-I) [4]. However, FV evolved

Table 5: Competitive transfer rates of PFV (TW05, ref fluorescence) and SFVmac (EGFPD, green fluorescence) vectors following
simultaneous packaging into PFV or SFVmac particles.

vectors and packaging systems

fluorescence-positive cells

competitive cross-packaging efficiency

PFV vector (red) +
PFV gag pol

SFVmac vector (green) +
SFVmac gag pol

PFV vector (red) +
SFVmac vector (green) +
PFV gag pol

PFV vector (red) +
SFVmac gag pol +
SFVmac vector (green)

68% red cells

67% green cells

63% red cells
0,85% green cells 1.3%

6,8% green cells
1,2% red cells 15%

Vectors were packaged together with the indicated packaging proteins in 293T cells. Supernatants from packaging cells were used for transduction
of HT1080 cells. Transduction rates were measured by detection of EGFP and mRFP expression by flow cytometry. The competitive cross-
packaging efficiency is calculated from the ratio of fluorescence-positive cells of the heterologous system versus the homologous + heterologous
system (e.g. 1.3% = 0.85%/(63% + 0.85%)).

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approximately ten times slower than PTLV-I in the living
host, implying less in vivo genetic stability in the latter
[17,51]. Thus, additional factors probably contribute to
FV genome conservation.

The findings of this study suggest that the HEK 293T cell
line, which is widely used in laboratories, exerts APOBEC
activity that easily overrides the intrinsic error rate of
foamy viruses. Our data suggest thatAPOBEC3G could be
involved in the observed G-to-A hypermutating activity,
although other members of the APOBEC family may con-
tribute to this phenomenon as well. Russell et al. have
published that foamy viral replication is vulnerable to a
broad variety of APOBEC isoforms, including hA3A,
hA3C, hA3F, and hA3G [52]. It is beyond the scope of this
study to identify which of these isoforms are expressed in
HEK 293T cells, but a further characterization of this cell
line in this regard remains a desirable task.

To reverse transcribe the virus RNA, the retroviral RT must
naturally perform the switches of templates either intra-
or inter-molecularly. Therefore, the aspect ofTS is not easy
to accurately analyze. Furthermore, different conditions
exist that have major influences on the recombination
rate by TS, such as the role of nucleocapsid (NC) protein
[53,54], a preferential homodimer packaging of the
genomic RNA [55-57], the accessibility to the recombina-
tion machinery [29], the possible presence of recombina-
tion hotspots [58], properties of the RT enzyme complex
[29], and the overall homology of the two packaged
genomes. On the other hand, HIV-1 and -2, which make
use of different mechanisms of genome packaging [59],
recombine at similar rates [22]. Recombination between
these two viruses which have a relatively low overall
homology has also been reported [11]. FVs do not have a
NC protein [60,61] and all the other points, in particular
whether homo- or hetero-dimers are preferentially pack-
aged, are still unknown. In addition, their replication
pathway diverges from the orthoretroviral strategy
[60,61]. However, they package two copies of (pre-)
genomic RNA [62,63], and therefore are theoretically
suited to recombine by TS. Recombination of FV genomes
has been shown recently for wild chimpanzee FVs [14]
and superinfection by SFVs from lower monkeys to chim-
panzees has been documented [14,43]. To get a first
insight into the frequency of FV TS we analyzed this by
methods which have been established to measure MLV
recombination [21]. Our results indicate that recombina-
tion of FVs by TS occurs at a higher frequency than MLV
[21], but at a lower frequency than HIV-1 [24]. Clearly,
additional studies will be required to more precisely deter-
mine the FV TS rate.

An issue which becomes relevant for recombination by TS
is the packaging of two different genomes in one capsid.

Thus, we investigated the probability of cross-vector trans-
fer of two related but clearly different FVs in the presence
or absence of the homologous genome. The ability to
cross-package a heterologous retroviral genome has been
extensively investigated among lentiviruses, with the like-
lihood of cross-packaging increasing with the relatedness
of the viruses in question. However, a non-reciprocal
packaging was found when for instance HIV-1 and -2 were
investigated [64-68]. Our results show that in the FV sys-
tem, heterologous vector transfer is possible although less
efficient than the homologous transfer. This implies the
packaging of heterologous (pre-) genomic RNAs and may
have consequences for the possibility of generating new
FVs by recombination. Given the differences in spuma-
and ortho-retroviral replication pathways in general
[60,61], and especially in the packaging, assembly, and
conditions of reverse transcription [61], a recombination
by TS requiring co-packaging of spuma- and ortho-retro-
viral genomes appears unlikely. However, we did not
investigate this issue, and it may be worth doing so in
future experiments.

Although heterologous vector transfer was reduced, parti-
cle composition was found to be unaltered. In particular,
the cleavage of the Gag precursor proteins indicative of
Pol protein incorporation was found to be unaffected by
the type of vector genome packaged. It is known that Pol
encapsidation appears to somehow depend on the pres-
ence of viral RNA which is packaged by a still unknown
mechanism [45,46]. Our results show that we may have
established an experimental system that will allow this to
be more accurately defined.

In terms of genome conservation, our results show that
FVs undergo recombination by TS during reverse tran-
scription relatively easily, but they have a much lower
mutation rate than previously thought. This is clearly the
case for deletions that occurred only at a very low fre-
quency in the in vivo assay but probably also pertain to the
frequency of point-mutations. In order to avoid altera-
tions to their genome, FVs appear to have developed a par-
ticular strategy through the generation of the Bet protein.
This may help us to better understand the conundrum of
error prone reverse transcription and FV genome conser-

Recombinant DNA
Standard techniques in molecular biology [69,70] were
used to generate the plasmids described below. Vector
viruses were abbreviated with the plasmid name lacking
the 'p'. Sequence data of oligonucleotide primers are
available as supporting online material (Additional file 1,
Fig. S1 at

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(i) The pKG83 vector, used to determine the FV point-
mutation rate, is shown in Fig. 1A. In a pMD9-derived
backbone it contains [71] within the PFV internally-
deleted LTRs (lack of U3 region), the cassette with the bac-
terial origin of replication, the ampicillin resistance gene,
a eukaryotic SFFV-derived promoter which directs gene
expression of EGFP, and the a-fragment of the lacZ gene.
It was constructed by generating the intermediate vector
pMH119 from pMH118 [72] by lighting the pLIB-(Clon-
tech) derived BsmBI/blunt 1.65 kb PCR product (ampli-
fled with primers #4300/#4301 and representing the
bacterial origin of replication and the ampicillin resist-
ance gene) with the 6.78 kb pMH118 BsmBI/SalI and the
4.59 kb pMH 118 Sall/blunt fragments in a three-fragment
ligation. Exchange of the gag/pol part of pMH 119 for the
cis-acting sequences (CAS I/II) ofpMD9 as a 2.6 kb EcoRI/
MluI fragment and insertion of a pUC19-derived PCR cas-
sette as a 0.45 kb fragment (amplified with primers
#4229/#4230 and representing the a-fragment of the bac-
terial lacZ gene) into the single NotI site of this vector gen-
erated pKG83. Transfer of pKG83 allowed the sorting of
transduced eukaryotic cells via their green fluorescence
and the sequencing of the bacterial parts, which were not
under selective pressure in eukaryotes.

(ii) To create the PFV vectors pKG80, pKG81, and pKG82,
the Hygro-IRES-Neo gene cassettes from the parental plas-
mids pJS30, pJA31-1 kb, and pJA32-1 kb [21] were ampli-
fied with the primers #1854/#1856 in the presence of 5%
dimethylsulfoxide (DMSO), due to very high G-C content
of the target DNA. The amplicons were inserted into the
pCR2.1-Topo vector (Invitrogen). Following DNA
sequencing the resistance gene cassettes were excised by
digestion with BstXI and EcoRV. Subsequently they were
blunt end-inserted downstream of the internal SFFV U3
promoter into the pMD9 vector [71] that was digested
with BamHI and NotI to release the gene coding for EGFP.
This resulted in plasmids pKG80, pKG81, and pKG82,
which were completely sequenced in the amplified parts
to exclude any unwanted nucleotide changes.

(iii) The SFVmac vector (pEGFPD) and packaging plas-
mids (pCIgag-1, pCIpol, and pCIenv3.5) will be described
in detail elsewhere. In brief, the complete open reading
frames (ORFs) of SFVmac gag, pol and env were inserted
separately downstream of the chimeric 3-globin-IgG
intron of the pCI expression vector (Promega). In the SFV-
mac vector pEGFPD the ATG initiation codons of the gag
and pol ORFs were inactivated by in vitro mutagenesis to
TAG and additional stop codons were introduced into the
gag ORF to abrogate translation from internal ATGs. Sub-
stitution of the SFFV U3 promoter in pMD9 [71] by the
human cytomegalovirus (CMV) immediate early gene
enhancer/promoter of pEGFPD generated pKG84. To cre-
ate this, the CMV/EGFP expression cassette was released
from pEGFPD as a 1.56 kb KpnI/NotI fragment and blunt

end inserted into pMD9 from which the SFFV U3/EGFP
expression cassette had been excised as a 1.2 kb NotI/
Eco47III fragment. The PFV packaging plasmids pCZIgag-
2, pCZIpol, and pCZenvEM002 were described previously

(iv) The pTW05 vector was made by amplifying the mRFP
gene with primers #4404 and #4405 from plasmid 11935
mRFP-Ub (Addgene). Following sequence determination
the resulting 0.67 kb fragment, digested with Agel/NotI,
was inserted behind the CMV promoter of pKG84 in place
of the EGFP gene which had been excised as a 0.73 kb frag-
ment using the same restriction enzymes.

Determination of FV in vivo mutation rate
2 x 106 HEK 293T cells [74], seeded in a 6 cm dish, were
transfected with a total amount of 6 jig DNA (1.5 jig of
pKG83 vector and the three packaging plasmids pCZIgag-2,
pCZIpol, and pCZenvEM002 [71,73] or with 1.5 jig vector
and the packaging plasmids plus the 1.5 jig pLENbet con-
struct [75]) using a polyethylenimine (PEI) protocol [73].
Following enhancement of viral transcription by the addi-
tion of 10 mM sodium butyrate for 8 hrs at 1 day post-
transfection (d.p.t), the vector-containing supernatant was
harvested after 48 hrs. After filtration through a 450 nm fil-
ter (Schleicher & Schuell) it was aliquoted and stored at -
80C. HeLa recipient cells were transduced with an effi-
ciency below 5% [as determined by EGFP-detection in flu-
orescence-activated cell sorting (FACS)] to avoid
superinfection of cells. Positive-scoring cells were sorted on
a FACSDiVa (Beckton Dickinson) into 96 well plates at a
rate of 1 EGFP-positive cell per well to allow the generation
of monoclonal cell cultures. Cells were grown to conflu-
ence, and transferred to 12 well plates. Total DNA was har-
vested with the Qiagen Blood and Tissue kit and subjected
to a PCR reaction with primers #4250 and #4254 and Pwo
polymerase (PeqLab). The resulting 2.2 kb amplicon was
purified with the GenElute PCR Clean-Up kit (Sigma) and
the prokaryotic parts were directly sequenced with BigDye
Terminator vl.1 on an ABI PRISM 3100 Genetic Analyzer
(Applied Biosystems) using primers #4257 or #4265. A
total of 110 cell colonies from KG83 vector-transduced
cells in the absence of Bet protein and 236 colonies from
cells transduced with KG83 vector and transient Bet pro-
tein-expression were analyzed this way.

Quantitative RT-PCR
Total cellular RNA was harvested from 5 x 106 HEK 293T,
HeLa, and PBMCs, obtained from the Department of
Transfusion Medicine (Universitat Wilrzburg). RNA was
extracted using the RNeasy kit (Qiagen) following the
manufacturers instructions. After elution in 50 il RNase-
free water, 1 jig of RNA was used for cDNA synthesis
using the iScript cDNA Synthesis kit (BioRad) that
primes the cDNA synthesis with oligo(dT). Real time
PCR was performed on an iCycleriQ Multicolor Real-

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Time PCR detection system (BioRad) utilizing 1 il of the
cDNA reaction, SYBRGreen as fluorophore, and primer
pairs able to amplify transcripts ofAPOBEC3G (#4302)
and 3F (#4286/#4287), B-actin (#4304), GAPDH
(#4305), and the A subunit of SDHA (#4303). The levels
of APOBEC mRNA in the individual cells were normal-
ized to the levels of the housekeeping genes (B-actin,
GAPDH, A subunit of SDHA) using the geNorm VBA
Applet for Excel [76]. All assays were independently car-
ried out at least three times.

TS assay
Vector virus-containing supernatants were obtained by
transfection of 2 x 106 HEK 293T cells, seeded in 6 cm
dishes, with a total amount of 6 jig DNA (0.75 jig KG81,
0.75 _ig KG82, and 1.5 jig of each of the three gag, pol,
env helper plasmids) using PEI [73]. Transcription was
enhanced by the addition of 10 mM sodium butyrate for
8 hrs at 1 d.p.t. The supernatants were harvested 2 d.p.t,
passed through a 450 nm filter (Schleicher & Schuell),
and stored at -80 C. The vector titers were determined
on HEK 293 target cells by single selection with 600 jig/
ml G418 (Roth) or 250 lig/ml Hygro (Roth) and double
selection with 400 lig/ml G418 plus 150 lig/ml Hygro.
The TS rate was calculated by the equation: TS rate =
number of TS phenotypes/(number of TS phenotypes +
number of non-TS Phenotypes). For production of single
cell colonies, HEK 293 target cells were transduced at a
multiplicity of infection (MOI) below 0.01 to avoid dou-
ble infections and selected for two weeks with medium
containing the antibiotic concentrations as described. To
characterize recombinant proviruses, we amplified the
proviral vector genomes from double resistant colonies
by a nested PCR using the first round primers #1854/
#4294 and the second round primers #1855/#1857. One
sixth of the reaction from the first PCR cycle was
employed in the nested reaction. Both amplification
cycles were performed in the presence of 5% DMSO due
to very high G-C content of the target DNA. The ampli-
cons were analyzed by agarose gel electrophoresis fol-
lowing digestion with EheI and SacII.

Vector transfer assays
6 x 106 HEK 293T cells seeded in 10 cm dishes, were trans-
fected with a total amount of 16 jig DNA (4 jig of the PFV
vector pKG84 or the SFVmac vector pEGFPD and 4 jig of
each of the three packaging constructs for PFV or SFVmac)
using a PEI protocol [73]. After 48 hrs, HT1080 fibroblas-
toid target cells were exposed to a 1:100 dilution of 0.45
_im filtered supernatant (Schleicher & Schuell) and ana-
lyzed two days later for vector transfer by flow cytometry
with FACSCalibur (Beckton&Dickinson) using the Cell
Quest Pro software. The normalized cross-transfer rate
was calculated as the percentage of SFVmac vector transfer
packaged with PFV constructs in relation to the authentic
PFV vector transfer and vice versa.

For the investigation of the simultaneous packaging of
two FV genomes 2 x 106 HEK 293T cells seeded in a 6 cm
dish, were transfected with 2 jig of each vector (pTW05
and/or pEGFPD) as well as 2 jig of each packaging plas-
mid using a PEI protocol. The total amount of DNA in the
transfection mix was adjusted to 20 jig using
pcDNA3.lzeo(+) plasmid (Invitrogen). The empty vector
was also used to adjust for DNA differences resulting from
omitting one vector. Following enhancement of transcrip-
tion by the addition of 10 mM sodium butyrate for 8 hrs
at 1 d.p.t the supernatant was harvested at 2 d.p.t, passed
through a 0.45 jim filter (Millipore), diluted 1:10, and
applied to 1.5 x 104 HT1080 fibroblastoid target cells.
These were analyzed for EGFP and/or mRFP expression by
FACS two days after transduction. Vector transfer assays
were performed at least three times using different plas-
mid preparations.

Lysis of HEK 293T cells was performed with lysis buffer in
6 cm dishes. Extracellular virions were prepared from the
filtered supernatant of transfected cell cultures by ultra-
centrifugation through a 20% sucrose cushion in a Sorvall
Surespin 630 rotor at 25,000 rpm, 4 C for 3 hrs. Viral par-
ticles and cellular lysates were resolved in protein sample
buffer, separated in sodium dodecylsulfate-containing
protein gels, blotted to nitrocellulose, and reacted with
PFV Gag and Pol monoclonal antibodies [71,77], SFVmac
Gag and Pol polyclonal rabbit antisera [78] or
APOBEC3G monoclonal antibodies (Santa Cruz) as
described previously [46,79,80].

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
KG, TW, JP, and AM performed the experiments. AR and
CS designed the study. KG, AR, and CS wrote the manu-
script. All authors read and approved the final manu-

Additional material

Additional file 1
Supplementary figures. Figure S1 -Oligonucleotide primer sequences.
Figure S2 Point mutations found after FV vector transfer. The vectors
were produced in the absence or presence (mutations in boldface) of Bet
protein. The two A to G transitions are in italics. Figure S3 -Level of
human APOBEC3F (A3F) mRNA expression in 293T cells in relation to
PBMCs. Figure 4 One deletion detected among over 265,000 nucle-
otides analysed. Figure S5 -Number of colonies resistant to Hygro and/
or Neo upon transfer of MLV vectors. Figure S6 Number of colonies
resistant to Hygro and/or Neo upon transfer of PFV vectors.
Click here for file

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We are indebted to Wei-Shau Hu (Frederick, Washington D.C., USA) for
the gift of vector plasmids, Benedikt WeiBbrich (Wurzburg, Germany) and
the Institute's DNA sequencing team, Michael Bock (Hannover, Germany)
for help with plasmid constructions, Helmut Hanenberg (Dusseldorf, Ger-
many) for HEK 293T cells, Andreas Opitz (Department of Transfusion
Medicine) for PBMCs, and Birgitta Wohrl (Bayreuth, Germany), Jochen
Bodem (Wurzburg, Germany) and Martin Ludlow (Great Britain) for criti-
cal reading of the manuscript and Eleni Koutsilieri (WOrzburg, Germany)
for assistance in statistics. This work was supported by grants from the
DFG (SFB479, RE627/7 and RE627/8) to AR and by grants from the NIH
(A1039126) to AM.

I. Preston BD, Dougherty JP: Mechanisms of retroviral mutation.
Trends Microbiol 1996, 4:16-2 1.
2. Gnatt AL, Cramer P, Fu J, Bushnell DA, Kornberg RD: Structural
basis of transcription: an RNA polymerase II elongation
complex at 3.3 A resolution. Science 2001, 292:1876-1882.
3. Thomas MJ, Platas AA, Hawley DK: Transcriptional fidelity and
proofreading by RNA polymerase II. Cell 1998, 93:627-637.
4. Mansky LM: In vivo analysis of human T-cell leukemia virus
type I reverse transcription accuracy. j Virol 2000,
5. Preston BD, Poiesz BJ, Loeb LA: Fidelity of HIV- I reverse tran-
scriptase. Science 1988, 242: 168- 1171.
6. Goodrich DW, Duesberg PH: Retroviral recombination during
reverse transcription. Proc Nat Acad Sci USA 1990, 87:2052-2056.
7. Hu WS, Temin HM: Genetic consequences of packaging two
RNA genomes in one retroviral particle: pseudodiploidy and
high rate of genetic recombination. Proc NatlAcad Sci USA 1990,
8. Hu WS, Temin HM: Retroviral recombination and reverse
transcription. Science 1990, 250:1227-1233.
9. Luo GX, Taylor J: Template switching by reverse transcriptase
during DNA synthesis. j Virol 1990, 64:4321-4328.
10. Stuhlmann H, Berg P: Homologous recombination of copack-
aged retrovirus RNAs during reverse transcription. J Virol
1992, 66:2378-2388.
II. Motomura K, Chen J, Hu WS: Genetic recombination between
human immunodeficiency virus type I (HIV-1) and HIV-2,
two distinct human lentiviruses. J Virol 2008, 82:1923-1933.
12. Blewett EL, Black DH, Lerche NW, White G, Eberle R: Simian
foamy virus infections in a baboon breeding colony. Virology
2000, 278:183-193.
13. Broussard SR, Comuzzie AG, Leighton KL, Leland MM, Whitehead
EM, Allan JS: Characterization of new simian foamy viruses
from African nonhuman primates. Virology 1997, 237:349-359.
14. Liu W, Worobey M, Li Y, Keele B, Bibollet-Ruche F, Guo Y, Goepfert
P, Santiago M, Ndjango J-B, Neel C, et a.: Molecular ecology and
natural history of simian foamy virus infection in wild-living
chimpanzees. PLoS Pathogens 2008, 4:e 1000097.
15. Schweizer M, Neumann-Haefelin D: Phylogenetic analysis of pri-
mate foamy viruses by comparison of pol sequences. Virology
1995, 207:577-582.
16. Schweizer M, Schleer H, Pietrek M, Liegibel J, Falcone V, Neumann-
Haefelin D: Genetic stability of foamy viruses: long-term study
in an African green monkey population. j Virol 1999,
17. Switzer WM, Salemi M, Shanmugam V, Gao F, Cong ME, Kuiken C,
Bhullar V, Beer BE, Vallet D, Gautier-Hion A, et al.: Ancient co-spe-
ciation of simian foamy viruses and primates. Nature 2005,
18. Thumer L, Rethwilm A, Holmes EC, Bodem J: The complete nucle-
otide sequence of a New World simian foamy virus. Virology
2007, 369:191-197.
19. Verschoor EJ, Langenhuijzen S, Bontjer I, Fagrouch Z, Niphuis H,
Warren KS, Eulenberger K, Heeney J: The phylogeography of
orangutan foamy viruses supports the theory of ancient
repopulation of Sumatra, J Virol 2004, 78:12712-12716.
20. Boyer PL, Stenbak CR, Hoberman D, Linial ML, Hughes SH: In vitro
fidelity of the prototype primate foamy virus (PFV) RT com-
pared to HIV-I RT. Virology 2007, 367:253-264.

21. Anderson JA, Bowman EH, Hu WS: Retroviral recombination
rates do not increase linearly with marker distance and are
limited by the size of the recombining subpopulation. j Virol
1998, 72:1195-1202.
22. Chen J, Powell D, Hu WS: High frequency of genetic recombi-
nation is a common feature of primate lentivirus replication.
J Virol 2006, 80:9651-9658.
23. JetztAE, Yu H, Klarmann GJ, Ron Y, Preston BD, DoughertyJP: High
rate of recombination throughout the human immunodefi-
ciency virus type I genome. j Virol 2000, 74:1234-1240.
24. Rhodes T, Wargo H, Hu WS: High rates of human immunodefi-
ciency virus type I recombination: near-random segregation
of markers one kilobase apart in one round of viral replica-
tion. J Virol 2003, 77:11193-1 1200.
25. Zhuang J, Jetzt AE, Sun G, Yu H, Klarmann G, Ron Y, Preston BD,
Dougherty JP: Human immunodeficiency virus type I recom-
bination: rate, fidelity, and putative hot spots. j Virol 2002,
26. Zhuang J, Mukherjee S, Ron Y, Dougherty JP: High rate of genetic
recombination in marine leukemia virus: implications for
influencing proviral ploidy. J Virol 2006, 80:6706-671 I.
27. Charpentier C, Nora T, Tenaillon O, Clavel F, Hance AJ: Extensive
recombination among human immunodeficiency virus type
I quasispecies makes an important contribution to viral
diversity in individual patients. J Virol 2006, 80:2472-2482.
28. Nora T, Charpentier C, Tenaillon O, Hoede C, Clavel F, Hance AJ:
Contribution of recombination to the evolution of human
immunodeficiency viruses expressing resistance to antiret-
roviral treatment. J Virol 2007, 81:7620-7628.
29. Onafuwa A, An W, Robson ND, Telesnitsky A: Human immuno-
deficiency virus type I genetic recombination is more fre-
quent than that of Moloney marine leukemia virus despite
similar template switching rates. J Virol 2003, 77:4577-4587.
30. Rinke CS, Boyer PL, Sullivan MD, Hughes SH, Linial ML: Mutation of
the catalytic domain of the foamy virus reverse transcriptase
leads to loss of processivity and infectivity. j Virol 2002,
3 I. Calattini S, Betsem EB, Froment A, Mauclere P, Tortevoye P, Schmitt
C, Njouom R, Saib A, Gessain A: Simian foamy virus transmis-
sion from apes to humans, rural Cameroon. Emerg Infect Dis
2007, 13:1314-1320.
32. Jones-Engel L, May CC, Engel GA, Steinkraus KA, Schillaci MA,
Fuentes A, Rompis A, Chalise MK, Aggimarangsee N, Feeroz MM, et
al.: Diverse contexts of zoonotic transmission of simian
foamy viruses in Asia. Emerg Infect Dis 2008, 14:1200-1208.
33. Switzer WM, Garcia AD, Yang C, Wright A, Kalish ML, Folks TM,
Heneine W: Coinfection with HIV-I and Simian Foamy Virus
in West Central Africans. j Infect Dis 2008, 197:1389-1393.
34. Edelstein ML, Abedi MR, Wixon J: Gene therapy clinical trials
worldwide to 2007 an update. j Gene Med 2007, 9:833-842.
35. Bauer TRJr, Allen JM, Hai M, Tuschong LM, Khan IF, Olson EM, Adler
RL, Burkholder TH, Gu YC, Russell DW, Hickstein DD: Successful
treatment of canine leukocyte adhesion deficiency by foamy
virus vectors. Not Med 2008, 14:93-97.
36. Vassilopoulos G, Rethwilm A: The usefulness of a perfect para-
site. Gene Ther 2008, 15:1299-130 I.
37. Williams DA: Foamy virus vectors come of age. Mol Ther 2008,
38. Russell RA, Wiegand HL, Moore MD, Schafer A, McClure MO, Cullen
BR: Foamy virus Bet proteins function as novel inhibitors of
the APOBEC3 family of innate antiretroviral defense fac-
tors. J Virol 2005, 79:8724-873 I.
39. Lochelt M, Romen F, Bastone P, Muckenfuss H, Kirchner N, Kim YB,
Truyen U, Rosier U, Battenberg M, Saib A, et al.: The antiretroviral
activity of APOBEC3 is inhibited by the foamy virus acces-
sory Bet protein. Proc Natl Acad Sci USA 2005, 102:7982-7987.
40. Perkovic M, Schmidt S, Marino D, Russell RA, Stauch B, Hofmann H,
Kopietz F, Kloke BP, Zielonka J, Strover H, et al.: Species-specific
Inhibition ofAPOBEC3C by the Prototype Foamy Virus Pro-
tein Bet. j Biol Chem 2009, 284:5819-5826.
41. Delebecque F, Suspene R, Calattini S, Casartelli N, Saib A, Froment A,
Wain-Hobson S, Gessain A, Vartanian JP, Schwartz O: Restriction
of foamy viruses by APOBEC cytidine deaminases. j Virol
2006, 80:605-614.
42. Yu Q, Konig R, Pillai S, Chiles K, Kearney M, Palmer S, Richman D,
Coffin JM, Landau NR: Single-strand specificity of APOBEC3G

Page 14 of 15
(page number not for citation purposes)

Retrovirology 2009, 6:32


accounts for minus-strand deamination of the HIV genome.
Nat Struct Mol Biol 2004, I 1:435-442.
43. Leendertz FH, Zirkel F, Couacy-Hymann E, Ellerbrok H, Morozov VA,
Pauli G, Hedemann C, Formenty P, Jensen SA, Boesch C, Junglen S:
Interspecies transmission of simian foamy virus in a natural
predator-prey system. J Virol 2008, 82:7741-7744.
44. Mergia A, Shaw KE, Lackner JE, Luciw PA: Relationship of the env
genes and the endonuclease domain of the pol genes of sim-
ian foamy virus type I and human foamy virus. j Virol 1990,
45. Heinkelein M, Leurs C, Rammling M, Peters K, Hanenberg H, Reth-
wilm A: Pregenomic RNA is required for efficient incorpora-
tion of pol polyprotein into foamy virus capsids. j Virol 2002,
46. Peters K, Wiktorowicz T, Heinkelein M, Rethwilm A: RNA and pro-
tein requirements for incorporation of the pol protein into
foamy virus particles. j Virol 2005, 79:7005-7013.
47. Boneva RS, Switzer WM, Spira TJ, Bhullar VB, Shanmugam V, Cong
ME, Lam L, Heneine W, Folks TM, Chapman LE: Clinical and viro-
logical characterization of persistent human infection with
simian foamy viruses. AIDS Res Hum Retroviruses 2007,
48. Heneine W, Schweizer M, Sandstrom P, Folks T: Human infection
with foamy viruses. Curr Top Microbiol Immunol 2003, 277:181-196.
49. Mansky LM, Temin HM: Lower in vivo mutation rate of human
immunodeficiency virus type I than that predicted from the
fidelity of purified reverse transcriptase. j Virol 1995,
50. Roberts JD, Bebenek K, Kunkel TA: The accuracy of reverse tran-
scriptase from HIV-1. Science 1988, 242:1171-1173.
51. Lemey P, Pybus OG, Van Dooren S, Vandamme AM: A Bayesian
statistical analysis of human T-cell lymphotropic virus evolu-
tionary rates. Infect Genet Evol 2005, 5:291-298.
52. Russell RA, Wiegand HL, Moore MD, Schafer A, McClure MO, Cullen
BR: Foamy virus Bet proteins function as novel inhibitors of
the APOBEC3 family of innate antretroviral defense factors.
J Virol 2005, 79:8724-8731.
53. Darlix JL, Vincent A, Gabus C, de Rocquigny H, Roques B: Trans-
activation of the 5' to 3' viral DNA strand transfer by nucle-
ocapsid protein during reverse transcription of HIVI RNA. C
R Acad Sci III 1993, 3 16:763-771.
54. Weiss S, Konig B, Morikawa Y,Jones I: Recombinant HIV-I nucle-
ocapsid protein pl5 produced as a fusion protein with glu-
tathione S-transferase in Escherichia coli mediates
dimerization and enhances reverse transcription of retrovi-
ral RNA. Gene 1992, 121:203-212.
55. Dorman N, Lever A: Comparison of viral genomic RNA sorting
mechanisms in human immunodeficiency virus type I (HIV-
I), HIV-2, and Moloney murine leukemia virus. j Virol 2000,
56. Flynn JA, An W, King SR, Telesnitsky A: Nonrandom dimerization
of murine leukemia virus genomic RNAs. j Virol 2004,
57. Levin JG, Grimley PM, RamseurJM, Berezesky IK: Deficiency of 60
to 70S RNA in murine leukemia virus particles assembled in
cells treated with actinomycin D. j Virol 1974, 14:152-161.
58. Pathak VK, Temin HM: Broad spectrum of in vivo forward
mutations, hypermutations, and mutational hotspots in a
retroviral shuttle vector after a single replication cycle: sub-
stitutions, frameshifts, and hypermutations. Proc NatlAcad Sci
USA 1990, 87:6019-6023.
59. KayeJF, Lever AM: Human immunodeficiency virus types I and
2 differ in the predominant mechanism used for selection of
genomic RNA for encapsidation. j Virol 1999, 73:3023-3031.
60. Linial M: Foamy Viruses. In Fields Virology Volume 2. 5th edition.
Edited by: Kniepe DM, PM Howley E. Philadelphia: Lippincot Williams
& Wilkins; 2007:2245-2262.
61. Rethwilm A: Foamy Viruses. In Virology. Topley & Wilson's Microbiol-
ogy and Microbial Infections Volume 2. 10th edition. Edited by: Mahy
BWJ, ter Meulen V. London: Hodder Arnold; 2005:1304-1321.
62. Cain D, Erlwein O, Grigg A, Russell RA, McClure MO: Palindromic
sequence plays a critical role in human foamy virus dimeri-
zation. j Virol 2001, 75:3731-3739.
63. Erlwein O, Cain D, Fischer N, Rethwilm A, McClure MO: Identifica-
tion of sites that act together to direct dimerization of
human foamy virus RNA in vitro. Virology 1997, 229:251-258.

64. Browning MT, Schmidt RD, Lew KA, Rizvi TA: Primate and feline
lentivirus vector RNA packaging and propagation by heter-
ologous lentivirus virions. j Virol 2001, 75:5129-5140.
65. Kaye JF, Lever AM: Nonreciprocal packaging of human immu-
nodeficiency virus type I and type 2 RNA: a possible role for
the p2 domain of Gag in RNA encapsidation. j Virol 1998,
66. Rizvi TA, Panganiban AT: Simian immunodeficiency virus RNA
is efficiently encapsidated by human immunodeficiency virus
type I particles. j Virol 1993, 67:2681-2688.
67. Strappe PM, Hampton DW, Brown D, Cachon-Gonzalez B, Caldwell
M, Fawcett JW, Lever AM: Identification of unique reciprocal
and non reciprocal cross packaging relationships between
HIV- I, HIV-2 and SIV reveals an efficient SIV/HIV-2 lentiviral
vector system with highly favourable features for in vivo test-
ing and clinical usage. Retrovirology 2005, 2:55.
68. White SM, Renda M, Nam NY, Klimatcheva E, Zhu Y, Fisk Halter-
man M, Rimel BJ, Federoff H, Pandya S, et al.: Lentivirus vectors
using human and simian immunodeficiency virus elements. j
Virol 1999, 73:2832-2840.
69. Ausubel FM, Brent R, Kingston RE, Moore D, Seidman JG, Smith JA,
K S: Current protocols in molecular biology New York, N.Y.: John Wiley;
70. Sambrook J, Russell DW: Molecular cloning: a laboratory manual 3rd
edition. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory
Press; 2001.
71. Heinkelein M, Dressier M, Jarmy G, Rammling M, Imrich H, Thurow
J, Lindemann D, Rethwilm A: Improved primate foamy virus vec-
tors and packaging constructs. j Virol 2002, 76:3774-3783.
72. Leurs C, Jansen M, Pollok KE, Heinkelein M, Schmidt M, Wissler M,
Lindemann D, Von Kalle C, Rethwilm A, Williams DA, Hanenberg H:
Comparison of three retroviral vector systems for transduc-
tion of nonobese diabetic/severe combined immunodefi-
ciency mice repopulating human CD34+ cord blood cells.
Hum Gene Ther 2003, 14:509-5 19.
73. Stange A, Mannigel I, Peters K, Heinkelein M, Stanke N, Cartellieri M,
Gottlinger H, Rethwilm A, Zentgraf H, Lindemann D: Characteriza-
tion of prototype foamy virus gag late assembly domain
motifs and their role in particle egress and infectivity. j Virol
2005, 79:5466-5476.
74. DuBridge RB, Tang P, Hsia HC, Leong PM, MillerJH, Calos MP: Anal-
ysis of mutation in human cells by using an Epstein-Barr virus
shuttle system. Mol Cell Biol 1987, 7:379-387.
75. Bock M, Heinkelein M, Lindemann D, Rethwilm A: Cells expressing
the human foamy virus (HFV) accessory Bet protein are
resistant to productive HFV superinfection. Virology 1998,
76. Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, De
Paepe A, Speleman F: Accurate normalization of real-time
quantitative RT-PCR data by geometric averaging of multi-
ple internal control genes. Genome Biol 2002, 3:RESEARCH0034.
77. Imrich H, Heinkelein M, Herchenroder O, Rethwilm A: Primate
foamy virus Pol proteins are imported into the nucleus. J Gen
Virol 2000, 81:2941-2947.
78. Kretzschmar B, Nowrouzi A, Hartl MJ, Gartner K, Wiktorowicz T,
Herchenroder O, Kanzler S, Rudolph W, Mergia A, Wohrl B, Reth-
wilm A: AZT-resistant foamy virus. Virology 2008, 370:15 1-157.
79. Cartellieri M, Herchenroder O, Rudolph W, Heinkelein M, Linde-
mann D, Zentgraf H, Rethwilm A: N-terminal Gag domain
required for foamy virus particle assembly and export. j Virol
2005, 79:12464-12476.
80. Heinkelein M, Schmidt M, Fischer N, Moebes A, Lindemann D, Enssle
J, Rethwilm A: Characterization of a cis-acting sequence in the
Pol region required to transfer human foamy virus vectors.
j Virol 1998, 72:6307-6314.

Page 15 of 15
(page number not for citation purposes)

Retrovirology 2009, 6:32

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