Group Title: Retrovirology 2009, 6:9
Title: Ancient, independent evolution and distinct molecular features of the novel human T-lymphotropic virus type 4
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Title: Ancient, independent evolution and distinct molecular features of the novel human T-lymphotropic virus type 4
Series Title: Retrovirology 2009, 6:9
Physical Description: Archival
Creator: Switzer WM
Salemi M
Qari SH
Jia H
Gray RR
Katzourakis A
Marriott SJ
Pryor KN
Wolfe ND
Burke DS
Folks TM
Heneine W
Publication Date: 39846
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Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
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Ancient, independent evolution and distinct molecular features of
the novel human T-lymphotropic virus type 4
William M Switzer*', Marco Salemi2, Shoukat H Qarit1, Hongwei Jiati,
Rebecca R Gray2, Aris Katzourakis3, Susan J Marriott4, Kendle N Pryor4,
Nathan D Wolfe5'6, Donald S Burke7, Thomas M Folkss and Walid Heneinel


Address: 'Laboratory Branch, Division of HIV/AIDS Prevention, National Center for HIV/AIDS, Viral Hepatitis, STD, and TB Prevention, Centers
for Disease Control and Prevention, Atlanta, GA 30333, USA, 2Department of Pathology, Immunology and Laboratory Medicine, College of
Medicine, University of Florida, Gainesville, FL 32610, USA, 3Department of Zoology, University of Oxford, Oxford, OX1 3PS, UK, 4Department
of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, Texas 77030, USA, 5Stanford University, Program in Human Biology,
Stanford, CA 94305, USA, 6Global Viral Forecasting Initiative, San Francisco, CA 94105, USA, 7Graduate School of Public Health, University of
Pittsburgh, Pittsburgh, PA 15261, USA and 8Southwest National Primate Research Center, San Antonio, TX 78227, USA
Email: William M Switzer* bis3@cdc.gov; Marco Salemi salemi@pathology.ufl.edu; Shoukat H Qari sqari@cdc.gov;
Hongwei Jia hjia@cdc.gov; Rebecca R Gray rgray@ufl.edu; Aris Katzourakis aris.katzourakis@zoology.oxford.ac.uk;
Susan J Marriott susanm@bcm.tmc.edu; Kendle N Pryor pryor@bcm.tmc.edu; Nathan D Wolfe nwolfe@stanford.edu;
Donald S Burke donburke@pitt.edu; Thomas M Folks tfolks@sfbrgenetics.org; Walid Heneine wheneine@cdc.gov
* Corresponding author tEqual contributors



Published: 2 February 2009 Received: 23 October 2008
Retrovirology 2009, 6:9 doi: 10.1 186/1742-4690-6-9 Accepted: 2 February 2009
This article is available from: http://www.retrovirology.com/content/6/l/9
2009 Switzer et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.ore/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.



Abstract
Background: Human T-lymphotropic virus type 4 (HTLV-4) is a new deltaretrovirus recently
identified in a primate hunter in Cameroon. Limited sequence analysis previously showed that
HTLV-4 may be distinct from HTLV- I, HTLV-2, and HTLV-3, and their simian counterparts, STLV-
I, STLV-2, and STLV-3, respectively. Analysis of full-length genomes can provide basic information
on the evolutionary history and replication and pathogenic potential of new viruses.
Results: We report here the first complete HTLV-4 sequence obtained by PCR-based genome
walking using uncultured peripheral blood lymphocyte DNA from an HTLV-4-infected person. The
HTLV-4(1863LE) genome is 8791-bp long and is equidistant from HTLV-1, HTLV-2, and HTLV-3
sharing only 62-71% nucleotide identity. HTLV-4 has a prototypic genomic structure with all
enzymatic, regulatory, and structural proteins preserved. Like STLV-2, STLV-3, and HTLV-3,
HTLV-4 is missing a third 21 -bp transcription element found in the long terminal repeats of HTLV-
I and HTLV-2 but instead contains unique c-Myb and pre B-cell leukemic transcription factor
binding sites. Like HTLV-2, the PDZ motif important for cellular signal transduction and
transformation in HTLV-I and HTLV-3 is missing in the C-terminus of the HTLV-4 Tax protein. A
basic leucine zipper (b-ZIP) region located in the antisense strand of HTLV-I and believed to play
a role in viral replication and oncogenesis, was also found in the complementary strand of HTLV-
4. Detailed phylogenetic analysis shows that HTLV-4 is clearly a monophyletic viral group. Dating
using a relaxed molecular clock inferred that the most recent common ancestor of HTLV-4 and
HTLV-2/STLV-2 occurred 49,800 to 378,000 years ago making this the oldest known PTLV lineage.
Interestingly, this period coincides with the emergence of Homo sapiens sapiens during the Middle
Pleistocene suggesting that early humans may have been susceptible hosts for the ancestral HTLV-
4.


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Conclusion: The inferred ancient origin of HTLV-4 coinciding with the appearance of Homo
sapiens, the propensity of STLVs to cross-species into humans, the fact that HTLV- I and -2 spread
globally following migrations of ancient populations, all suggest that HTLV-4 may be prevalent.
Expanded surveillance and clinical studies are needed to better define the epidemiology and public
health importance of HTLV-4 infection.


Background
Deltaretroviruses are a diverse group of human and sim-
ian T-lymphotropic viruses (HTLV and STLV, respectively)
that until lately were composed of only two distinct
human groups called HTLV types 1 and 2 [1-7]. Two new
HTLVs, HTLV-3 and HTLV-4, were recently identified in
primate hunters in Cameroon effectively doubling the
genetic diversity of deltaretroviruses in humans [6,8]. Col-
lectively, members of the HTLV groups and their STLV
analogues are called primate T-lymphotropic viruses
(PTLV) with PTLV-1, PTLV-2, and PTLV-3 being composed
of HTLV-1/STLV-1, HTLV-2/STLV-2, and HTLV-3/STLV-3,
respectively. The PTLV-4 group currently has only one
member, HTLV-4, since a simian counterpart has yet to be
identified [6].

STLV-1 has a broad geographic distribution in nonhuman
primates (NHPs) in both Asia and Africa thus providing
humans with historical and contemporaneous opportuni-
ties for exposure to this virus [2,4,5,9,10]. Indeed, phylo-
genetic analysis of simian T-lymphotropic viruses type 1
(STLV-1) and global HTLV-1 sequences suggests that dif-
ferent STLV-ls were introduced into humans multiple
times in the past resulting in at least six phylogenetically
distinct HTLV-1 subtypes [1-5,11]. Recently, a new HTLV-
1 subtype was found in Cameroon that was closest phylo-
genetically to STLV-1 from monkeys hunted in this region
and which shared greater that 99 % nucleotide identity [6].
Since similar high sequence identities are typically seen in
both vertical and horizontal linked transmission cases of
HTLV-1 [12-14], the finding of this new HTLV-1 subtype
in Cameroon suggests a relatively recent cross-species
transmission of STLV-1 to this primate hunter and that
these zoonotic infections continue to occur in persons
naturally exposed to NHPs.

Although a simian T-lymphotropic virus type 2 (STLV-2)
has been identified in two troops of captive bonobos (Pan
paniscus), the zoonotic relationship of this divergent virus
to HTLV-2 is less clear [15-17]. Like STLV-1, STLV-3 also
has a broad and ancient geographic distribution across
Africa [9,10,18-23]. Thus, while only three distinct HTLV-
3 strains have been identified to date in Cameroon
[6,8,24], it is conceivable that HTLV-3 may be prevalent
throughout Africa and, like HTLV-1 and HTLV-2, poten-
tially could be spread globally through migrations of


infected human populations. Expanded screening is
needed to define the prevalence of HTLV-3 in human pop-
ulations. Likewise, the epidemiology of HTLV-4 is not
well understood since only a single human infection has
been reported and a simian counterpart has yet to be iden-
tified [6]. Although limited sequencing of very small gene
regions showed that HTLV-4 is most genetically related to
STLV-2 and HTLV-2, but is a distinct lineage separate from
all known PTLVs [6], understanding the evolutionary rela-
tionship of HTLV-4 to known PTLVs requires additional
phylogenetic analyses using longer sequences or the com-
plete viral genome.

Like HIV, both HTLV-1 and -2 have spread globally and
are pathogenic human viruses [1,2,5,7,25]. HTLV-1 causes
adult T-cell leukemia/lymphoma (ATL), HTLV-1 associ-
ated myelopathy/tropical spastic paraperesis (HAM/TSP),
and other inflammatory diseases in less than 5% of those
infected [2,5,7]. HTLV-2 is less pathogenic than HTLV-1
and has been associated with a neurologic disease similar
to HAM/TSP [1]. The recent identification of HTLV-3 and
HTLV-4 in only four persons limits an evaluation of the
disease potential and secondary transmissibility of these
novel viruses [6,8,24]. However, complete genomic
sequences of these viruses can provide insights on the
genetic structure and whether functional motifs that are
important for viral expression and HTLV-induced leuke-
mogenesis are preserved [6,8,24,26-30]. In addition,
determination of the viral sequence will be important to
develop improved diagnostic assays to better understand
the epidemiology of this novel human virus.

In this paper, we report the first full-length sequence of
HTLV-4 and demonstrate by detailed phylogenetic analy-
sis that this virus clearly falls outside the diversity of all
other PTLVs. The observed low nucleotide substitution
rate, absence of evident genetic recombination, and con-
served genomic structure of HTLV-4 demonstrate the
genetic stability of this virus. In addition, molecular dat-
ing suggests that the HTLV-4 lineage split from the pro-
genitor of PTLV-2 about 200 millennia ago and is older
than the ancestors of HTLV-1, HTLV-2, and HTLV-3. We
also highlight biologically important molecular features
in HTLV-4 that are unique or common to HTLV-1, HTLV-
2, and HTLV-3.




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Results
Comparison of the HTLV-4(1863LE) proviral genome with
prototypical PTLVs
The complete genome of HTLV-4(1863LE) was obtained
using a PCR strategy as depicted in Fig. 1 and was deter-
mined to be 8791-bp in length. Comparison of the HTLV-
4(1863LE) sequence with prototypical PTLV genomes
demonstrates that this newly identified human virus is
nearly equidistant from HTLV-1 (62% identityy, PTLV-2
(70.7% identity), and PTLV-3 (63.4% identity) groups
across the genome (Table 1). The most genetic divergence
between HTLV-4 and the other PTLV groups was seen in
the LTR (43-65%) and protease (pro) gene (59-70%),
while the greatest nucleotide identity and amino acid sim-
ilarity was observed within the highly conserved regula-
tory genes, tax and rex (73-81% and 58-91%,
respectively). This relationship was highlighted further by
comparing HTLV-4(1863LE) with prototypical full-length
STLV and HTLV genomes in a similarity plot analysis,
where the highest similarity was seen in the highly con-
served tax gene, which is located at the 5' end of the pX
region of the genome (Fig. 2). As seen within other PTLV
groups [31], no clear evidence of genetic recombination
of HTLV-4(1863LE) with prototypical HTLV and STLV
proviral sequences was observed using bootscanning
analysis in the SimPlot program (data not shown).

Phylogenetic analysis
The unique genetic relationship of HTLV-4(1863LE) to
other PTLVs was confirmed by Bayesian phylogenetic


analysis that inferred trees using alignments of each major
viral gene in the PTLV genome after excluding 3rd codon
positions (cdp) which were significantly saturated as
determined by pair-wise transition and transversion ver-
sus genetic divergence plots using the DAMBE program
(Additional file 1, Fig. Si). At the 3rd cdp transitions and
transversions plateaued indicating sequence saturation
(Additional file 1, Fig. Si). In contrast, transitions and
transversions increased linearly for the 1st and 2nd cdp
without reaching a plateau indicating they still retained
enough phylogenetic signal (Additional file 1, Fig. Sl).
Maximum clade credibility trees inferred by using a
Markov Chain Monte Carlo (MCMC) sampler showed
three major, well supported, monophyletic PTLV groups
(posterior probability p = 1.0) with HTLV-1, HTLV-2, and
HTLV-3, each clustering in separate clades (Figs. 3, 4, 5
and 6). For each gene region analyzed, HTLV-4 appears as
an independent and highly divergent monophyletic line-
age sharing a common ancestor with the PTLV-2 clade (p
= 1.0). The phylogenetic relationships among PTLV line-
ages inferred from different gene regions were also similar
(Figs. 3, 4, 5 and 6). The only exception was the mono-
phyletic PTLV-3 lineage which was either a sister lineage
to PTLV-4/PTLV-2 or PTLV-5/PTLV-1 [10] in the gag (Fig.
3) and env (Fig. 5) or pol (Fig. 4) and tax (Fig. 6) tree topol-
ogies, respectively, but in each case with weak posterior
probabilities (p < 0.75) (Figs 3, 4, 5 and 6). Similarly, the
position of the PTLV-3 phylogroup was unresolved using
both the maximum likelihood (ML) and Neighbor Join-
ing (NJ) methods (Additional file 1, Fig. S2). The long


Table I: Percent Nucleotide Identity and Amino Acid Similarity of HTLV4(1863LE) with other PTLV Prototypes'.


HTLV-I (ATK) HTLV-2 (MoT)


HTLV-2 (Efe) STLV-2 (PanP) STLV-2 (PPI664)


HTLV-3 (2026ND)


STLV-3 (TGE2117)


Genome 62.0

LTR 45.2


gag
pl9M2
p24C2
pl5NC2


70.4 (82.0)
(68.5)
(90.2)
(81.5)


73.2 (85.9)
(77.5)
(91.6)
(84.0)


70.8

62.5

74.6 (85.9)
(79.1)
(91.6)
(81.5)


70.4

62.5

74.7 (85.0)
(79.1)
(91.5)
(81.5)


70.8

64.7

74.8 (86.3)
(79.1)
(92.6)
(84.0)


pro 59.0 (61.9) 66.7 (70.8) 66.5 (59.3) 70.0 (59.6) 67.0 (60.0)

pol 63.9 (68.0) 71.4 (78.7) 71.5 (78.6) 71.1 (73.3) 71.2 (78.7)


65.8 (75.9)
(70.4)
(84.7)


73.1 (85.3)
(80.1)
(94.4)


72.8 (86.0)
(81.4)
(93.8)


72.0 (84.9)
(80.4)
(92.7)


72.0 (85.5)
(81.4)
(92.7)


rex 76.0 (63.9) 79.5 (74.1) 81.1 (75.3) 78.7 (65.2) 80.7 (68.8)

tax 75.9 (82.6) 80.0 (90.9) 80.1 (89.5) 76.7 (85.5) 77.2 (90.0)


I amino acid similarity in parentheses; strain names given in parentheses
2 M, matrix protein; C, capsid; NC, nucleocapsid
3 SU, surface protein; TM, transmembrane


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68.3 (83.6)
(78.2)
(89.3)
(76.5)

64.1 (65.5.)

64.9 (71.4)

68.5 (79.4)
(72.9)
(90.5)

72.7 (59.4)

74.2 (82.6)


68.6 (82.7)
(71.9)
(90.2)
(74.1)

64.7 (60.0)

65.5 (71.2)

67.2 (78.9)
(71.7)
(91.0)

72.5 (57.7)

74.1 (82.6)


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rex



HTLV-4 (1863LE) M I


Ci\: HBZ
I ORFI
IORFII


OI RFIV

IIO ORFV
St t


sd-LTR
(414)


sd-Env
(5105)


sa-pX2
(7274)
sa-pX3
(7645)
sa-T/R
(7119)


LF2-*
LF3 -*


PF3-*
PF5 -*
PR4
PR5


662-bp

I I I I
0 1 2 3


pXF1-* *- LR1
PGTAXTF7a+b PGTATA1+2R1
I-ER TF8 -- -PGTATA1+2R1


ER_04 ER3
EF2 -*
EF2 -*


319-bp

4 5
4 5


4- TR1
4- TR2


730-bp


I I 8 I
6 7 8 9 kB


Figure I
Organization of the HTLV-4 genome (a) and schematic representation of the PCR-based genome walking
strategy (b). (a) shown are non-coding long terminal repeats (LTR), coding regions for all major proteins (gag, group specific
antigen; pro, protease; pol, polymerase; env, envelope; rex, regulator of expression; tax, transactivator), HTLV basic leucine zip-
per (HBZ), and 3' genomic open reading frames (ORF) of unknown function. Putative splice donor (sd) and splice acceptor (sa)
sites are indicated. (b) Small proviral sequences (purple bars) were first amplified from each major gene region and the long
terminal repeat using generic primers as described in methods. The complete proviral sequence was then obtained by using
PCR primers located within each major gene region by genome walking as indicated with arrows and orange bars.


branch length leading to the HTLV-4 strain suggests an
ancient separation of this lineage from PTLV-2. Similarly,
STLV-1(MarB43) and STLV-2 each formed distinct line-
ages from PTLV-1 and HTLV-2, respectively, with long
branch lengths (Figs. 3, 4, 5 and 6). These findings sup-
port further the recent re-classification of STLV-
1(MarB43) as a new PTLV lineage called STLV-5 and the
need to re-classify STLV-2 as a distinct PTLV group [10].
The unequivocal monophyletic relationship of HTLV-4 to
other PTLVs was supported further by phylogenetic infer-
ence of similar tree topologies with robust statistical sup-
port obtained with NJ and ML analysis, using both
separate alignments for each genes and the full-length
genome without LTRs (Additional file 1, Fig. S2).

Dating the origin of HTLV-4(1863LE) and other PTLVs
The long branch leading to the HTLV-4 strain suggests an
ancient, independent evolution of this human retrovirus.


Hence, additional molecular analyses were performed to
estimate the divergence times of the HTLV and PTLV line-
ages. Although we and others have reported finding a
clock-like behavior of PTLV sequences using partial LTR or
env sequences [3,18-20], we were unable to confirm these
results. Instead, the clock hypothesis was strongly rejected
(p < 0.00001) for the 1st + 2nd codon position alignment
of full-length PTLV genomes without LTRs, as well as for
separate alignments of full-length gag, pol, env and tax
genes (p < 0.00001 in each case) suggesting significant
evolutionary rate heterogeneity among the different viral
lineages. Indeed, sequence analysis showed unequal base
composition for some lineages and substitution satura-
tion at the 3rd codon position (cdp) for all PTLVs (Addi-
tional file 1, Fig. Sl). Substitution saturation was not
observed in the 1st and 2nd cdps (Additional file 1, Fig. S1)
and these sites were thus suitable for estimating posterior



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Primer Positions


(8791-bp)


I


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o 80
78
S76
74
= 72
E 70
Ct 68
66
64
62
60
58
56
54
52
50
0 1,000 2,000 3,000 4,000 5,000 6,000 7,000 8,000 9,000
Position (bp)

Window: 200 bp, Step: 20 bp, GapStrip: On, F84 ("Maximum Likelihood"), T/t: 2.28

Figure 2
Similarity plot analysis of the full-length HTLV-4(1863LE) and PTLV genomes using a 200-bp window size in 20
step increments on gap-stripped sequences. The F84 (maximum likelihood) model was used with a transition-to-trans-
version ratio of 2.28.


evolutionary rates and divergence dates of PTLV by using
Bayesian analysis with a MCMC algorithm.

The relaxed molecular clock was calibrated with two inde-
pendent molecular calibration points; 12,000 30,000 ya
as confidence intervals for the origin of HTLV-2 as it
migrated out of Africa and Asia and into the Americas via
the Bering land bridge and 40,000 60,000 ya as confi-
dence intervals for the origin of HTLV-1 in Melanesia as it
became populated with people from Asia [23,32,33]. The
use of two calibration points has previously been shown
to provide more reliable estimates of PTLV substitution
rates than a single calibration date [3,32]. Using these
methods we found that the PTLV posterior mean evolu-
tionary rates differed for each of the four major coding
regions and ranged from 2.89 x 10-7 to 7.92 x 10-7 substi-
tutions/site/year (Table 2). The highest mean evolution-
ary rate was seen in pol while the lowest rate was observed
in gag (Table 2). These rates are consistent with those cal-
culated previously using the same calibration points with
and without enforcing a molecular clock [3,4,18-
20,23,31,32], including those of Lemey et al. who also
found disparate PTLV evolutionary rates across the PTLV
genome [33].


Median estimates and 95% high posterior density (95%
HPD) intervals for the time of the most recent common
ancestor (tMRCA) of the major PTLV clades according to
different gene regions are given in Table 3. The tMRCA of
the PTLV tree ranged between 214,650 (tax gene) and
385,100 ya (env gene) confirming an ancient evolution of
the primate deltaretroviruses [3]. These dates are lower
than those reported previously for the PTLV cenancestor
which were inferred using methods less accurate than the
Bayesian analyses employed here [3,4]. Remarkably, the
inferred PTLV divergence dates were very similar for each
gene region with those estimated for the highly conserved
tax gene being slightly lower (Table 3). Nevertheless, the
95% HPD intervals overlapped for all four genes (Table 3)
supporting the strength of the inferred PTLV divergence
dates. Estimates for the PTLV-4 progenitor split from
PTLV-2 ranged between 124,250 ya (c.i., 49,800 -
218,250 ya) in the tax gene to 221,650 ya (c.i., 89,650 -
378,000 ya) in the env gene and were comparatively ear-
lier than the median tMRCA of PTLV-1 (54,250-75,100
ya), PTLV-2 (75,200-128,600 ya), and PTLV-3 (40,850-
71,700 ya) clades (Table 3). These results suggest that the
HTLV-4/PTLV-2 ancestor may represent the oldest PTLV
identified to date.




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STLV-5(MarB43)
1 STLV-1(TE4)
1 HTLV-1 (Mel5)
1 STLV-1 (Tan90)
0.77 HTLV-1 (ATK)
0 HTLV-1 (Boi)
HTLV-1 (ATL-YS)
1 STLV-3(Ph969)
STLV-3(TGE2117)
1 HTLV-3(Pyl43)
STLV-3(CT0604)
046 HTLV-3(2026ND)
1 STLV-3(NG409)
STLV-3(Ppaf3)
HTLV-4(1863LE)
STLV-2(pp1664)
STLV-2(Pan-p)
1 HTLV-2d(Efe)
HTLV-2a(MoT)
SHTLV-2a(SP-WV)
S HTLV-2a(Kay96)
HTLV-2b(G2)
.56 HTLV-2b(G12)
g 56 HTLV-2b(Gab)
100000.0

Figure 3
Phylogenetic relationship of HTLV-4(1863LE) to other PTLVs in gag using Bayesian inference. First and second
codon positions of gag were used to generate PTLV phylogenies by sampling 10,000 trees with a Markov Chain Monte Carlo
method under a relaxed clock model, and the maximum clade credibility tree, i.e. the tree with the maximum product of the
posterior clade probabilities, was chosen. Branch lengths are proportional to median divergence times in years estimated from
the post-burn in trees with the scale at the bottom indicating 100,000 years. Posterior probabilities for each node are indi-
cated. Branches leading to PTLV- I, HTLV-2 and PTLV-3 sequences are drawn in red, blue and green respectively. The branch
leading to HTLV-4(1863LE), STLV-2, and to the divergent MarB43 strain are drawn in magenta, purple, and yellow respectively.


Genomic organization and characterization of the HTLV-
4(1863LE) structural and enzymatic proteins, and the LTR
The genomic structure of HTLV-4(1863LE) was similar to
that of other PTLVs and included the structural, enzy-
matic, and regulatory proteins all flanked by long termi-
nal repeats (LTRs) (Fig. 1). Like HTLV-3 (697-bp), the
HTLV-4(1863LE) LTR (696-bp) was smaller than that of
HTLV-1 (756-bp) and HTLV-2 (764-bp), by having two
rather than the typical three 21-bp transcription regula-
tory repeat sequences in the U3 region of HTLV-1 and
HTLV-2 (Fig. 7) [18-20,23,31,34,35]. The distal 21-bp
repeat element found in HTLV-1 and HTLV-2 is absent
from the HTLV-4(1863LE) genome (Fig. 7). Others have
shown that deletion of the middle, rather than the distal
21-bp element, is more critical for the loss of basal HTLV-
1 transcription levels [36]. In addition, the lack of the dis-
tal 21-bp repeat does not seem to affect viral expression of
PTLV-3 [35,37]. Nonetheless, additional studies are
needed to determine what effect the absence of a 21-bp
element has on HTLV-4(1863LE) gene expression and
replication.


Other regulatory motifs such as the polyadenylation sig-
nal, TATA box, and cap site were all conserved in the
HTLV-4(1863LE) LTR (Fig. 7). Highly conserved pre-B cell
leukemia (Pbx-1, TGACAG) and c-Myb (YAACKG) tran-
scription factor binding sites were also identified at posi-
tions 1-6 and 86-91 of the LTR, respectively, upstream of
the first 21-bp repeat element (Fig. 7). The Pbx-1 and c-
Myb sites are also conserved in the LTRs of STLV-2 and
two nearly identical PTLV-3 strains (STLV-3(CTO604) and
HTLV-3(Pyl43)) [15,16,19,34], respectively, but are
absent in other PTLV LTRs. Binding to the predicted c-Myb
target sequence within the HTLV-4 LTR oligonucleotide
was observed and was specific based upon banding pat-
terns observed in the presence of specific and non-specific
oligonucleotide competitors in an electrophoretic mobil-
ity shift assay (EMSA). The shifted band was identified as
c-Myb since an anti-c-Myb antibody supershifted the com-
plex while an unrelated antibody did not (Fig 8). While
this analysis confirms the specificity of the putative c-Myb
binding site in the HTLV-4 LTR oligonucleotide and likely
reflects binding of c-Myb to the HTLV-4 LTR, this remains


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0.42
0.39


0.91


.0.45


STLV-5(MarB43)
- STLV-1 (TE4)
- HTLV-1(Mel5)
- STLV-1 (Tan90)
SHTLV-1 (ATL-YS)
o9 HTLV-1 (ATK)
HTLV-1(Boi)
STLV-3(Ppaf3)
HTLV-3(2026ND)
STLV-3(NG409)
STLV-3(TGE2117)
STLV-3(Ph969)
HTLV-3(Pyl43)
STLV-3(CT0604)
HTLV-4(1863LE)
STLV-2(pp1664)
STLV-2(Pan-p)
HTLV-2d(Efe)
"- HTLV-2a(MoT)


pol
100000.0


Figure 4
Phylogenetic relationship of HTLV-4(1863LE) to other PTLVs in pol using Bayesian inference. First and second
codon positions of pol sequences were used to generate PTLV phylogenies by sampling 10,000 trees with a Markov Chain
Monte Carlo method under a relaxed clock model, and the maximum clade credibility tree, i.e. the tree with the maximum
product of the posterior clade probabilities, was chosen. Branch lengths are proportional to median divergence times in years
estimated from the post-burn in trees with the scale at the bottom indicating 100,000 years. Posterior probabilities for each
node are indicated. Branches leading to PTLV-I, HTLV-2 and PTLV-3 sequences are drawn in red, blue and green respectively.
The branch leading to HTLV-4(1863LE), STLV-2, and to the divergent MarB43 strain are drawn in magenta, purple, and yellow
respectively.


to be tested in vivo. Secondary structure analysis of the LTR
RNA sequence predicted a stable stem loop structure from
nucleotides 425 466 (Fig 9) similar to that shown to be
essential for Rex-responsive viral gene expression in both
HTLV-1 and HTLV-2.

Translation of predicted protein open reading frames
(ORFs) across the viral genome identified all major Gag,
Pro (protease), Pol, and Env proteins, as well as the regu-
latory proteins, Tax and Rex (Fig. 1). Translation of the
overlapping gag and pro and pro and pol ORFs occurs by
one or more successive -1 ribosomal frameshifts that align
the different ORFs. The conserved slippage nucleotide
sequence 6(A)-8nt-6(G)-llnt-6(C) is present in the Gag-
Pro overlap starting at nucleotide 1997. Similarly, the Pro-
Pol overlap slippage sequence (TITAAAC) was identical
to that seen in HTLV-1 and HTLV-2 but which is different
from that found in HTLV-3 by a single nucleotide substi-
tution at the beginning of this motif (GTTAAAC) [31].


Importantly, the asparagine codon (AAC) crucial for the
slippage mechanism is conserved in all HTLVs.

The structural and group-specific precursor Gag protein
consisted of 424 amino acids (aa), and is predicted to be
cleaved into the three core proteins p19 (matrix), p24
capsidd), and p15 (nucleocapsid) similar to HTLV-1,
HTLV-2, and HTLV-3. Across PTLVs, Gag is one of the
most conserved proteins, with the HTLV-4 Gag having
82% to 86% similarity to HTLV-1, PTLV-2, and PTLV-3
(Table 1). The Gag capsid protein (214 aa) showed about
90% to 93% similarity to other PTLV capsids, while the
matrix (129 aa) and nucleocapsid (81 aa) proteins were
somewhat less conserved, showing less than 85% similar-
ity to HTLV-1, PTLV-2, and PTLV-3 (Table 1). The conser-
vation of the capsid protein supports the observed cross-
reactivity to Gag seen with plasma from the HTLV-4-
infected person in Western blot (WB) assays employing
HTLV-1 antigens [6,38].




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STLV-5(MarB43)
STLV-1(TE4)
HTLV-1(Mel5)
STLV-1(Tan90)


0.94 STLV-3(TGE2117)
STLV-3(Ph969)
1 1 HTLV-3(Pyl43)
STLV-3(CT0604)
0.9 HTLV-3(2026ND)
0.64 1 62 STLV-3(Ppaf3)
STLV-3(NG409)
HTLV-4(1863LE)
STLV-2(pp1664)
STLV-2(Pan-p)
1 HTLV-2d(Efe)
HTLV-2b(Gab)
HTLV-2b(G2)
1 HTLV-2b(G12)
HTLV-2a(SP-WV)
en 0.2 HTLV-2a(Kay96)
env 0.92 HTLV-2a(MoT)
100000.0

Figure 5
Phylogenetic relationship of HTLV-4(1863LE) to other PTLVs in env using Bayesian inference. First and second
codon positions of env sequences were used to generate PTLV phylogenies by sampling 10,000 trees with a Markov Chain
Monte Carlo method under a relaxed clock model, and the maximum clade credibility tree, i.e. the tree with the maximum
product of the posterior clade probabilities, was chosen. Branch lengths are proportional to median divergence times in years
estimated from the post-burn in trees with the scale at the bottom indicating 100,000 years. Posterior probabilities for each
node are indicated. Branches leading to PTLV-I, HTLV-2 and PTLV-3 sequences are drawn in red, blue and green respectively.
The branch leading to HTLV-4(1863LE), STLV-2, and to the divergent MarB43 strain are drawn in magenta, purple, and yellow
respectively.


The predicted size of the HTLV-4 (1863LE) Env polypro-
tein is 485 aa, which is slightly shorter than the Env of
PTLV-2 (486 aa), PTLV-1 (488 aa), and PTLV-3 (491-492
aa). The Env surface (SU) protein (307 aa) showed the
most genetic divergence from other PTLVs with only 70%
- 81% similarity, while the transmembrane (TM) protein
(178 aa) was highly conserved across all PTLVs, sharing
85% 94% similarity, supporting the use of recombinant
HTLV-1 TM protein (GD21) on WB strips to identify
divergent PTLVs, including HTLV-4. The HTLV-4(1863LE)
SU showed about 86% similarity to the HTLV-2 type spe-
cific SU peptide (K55) despite the observed weak reactiv-
ity of anti-HTLV-4(1863LE) antibodies to [6,38] K55
spiked onto WB strips. This amino acid similarity is some-
what greater than the 67.4% and 72.1% similarity of the
HTLV-1 and HTLV-3 SUs to K55, respectively, allowing
serologic discrimination of HTLV-2 from HTLV-1 in this
region. In contrast, the HTLV-4(1863LE), HTLV-2, and
HTLV-3 SUs share from 68.8% to 70.8% similarity to the
HTLV-1 type specific SU peptide (MTA-1). Although these


results are limited to testing the sera of a single HTLV-4-
infected individual, they suggest that higher antibody
reactivity to the HTLV-2-type specific peptide may be
observed in HTLV-4-infected persons [38].

The glucose transporter GLUT1 has been shown to be the
HTLV-1 and -2 envelope receptor and a retrovirus binding
domain (RBD) for GLUT1 has been identified in the SU of
these viruses [39,40]. Analysis of the HTLV-4 Env protein
revealed a putative RBD located at positions 85 138 of
the SU that shared about 80%, 78%, and 87% amino acid
similarity with the RBDs of HTLV-1(ATK), HTLV-2(MoT),
and that identified by analysis of the HTLV-3(2026ND)
Env, respectively. In addition, both aspartic acid and the
tyrosine residues located as positions 106 and 114 of
HTLV-1 (ATK) are highly conserved in the putative HTLV-
4 RBD and all other PTLV RBDs (data not shown), sup-
porting a critical role for these residues as the receptor
binding core as previously suggested [41].


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HTLV-4(1863LE)
STLV-2(Pan-p)
STLV-2(pp1664)
HTLV-2d(Efe)
HTLV-2a(MoT)
HTLV-2a(SP-WV)
HTLV-2a(Kay96)


0.64


0.76


STLV-1 (TE4)
HTLV-1(Mel5)
STLV-1 (Tan90)
SHTLV-1(Boi)
-0..7 HTLV-1(ATL-YS)
HTLV-1 (ATK)
HTLV-3(2026ND)
0.98 STLV-3(NG409)
STLV-3(Ppaf3)
STLV-3(Ph969)
STLV-3(TGE2117)
STLV-3(CTO604)
HTLV-3(Pyl43)


100000.0

Figure 6
Phylogenetic relationship of HTLV-4(1863LE) to other PTLVs tax using Bayesian inference. First and second
codon positions of tax sequences were used to generate PTLV phylogenies by sampling 10,000 trees with a Markov Chain
Monte Carlo method under a relaxed clock model, and the maximum clade credibility tree, i.e. the tree with the maximum
product of the posterior clade probabilities, was chosen. Branch lengths are proportional to median divergence times in years
estimated from the post-burn in trees with the scale at the bottom indicating 100,000 years. Posterior probabilities for each
node are indicated. Branches leading to PTLV-I, HTLV-2 and PTLV-3 sequences are drawn in red, blue and green respectively.
The branch leading to HTLV-4(1863LE), STLV-2, and to the divergent MarB43 strain are drawn in magenta, purple, and yellow
respectively.


Characterization of Regulatory and Accessory Proteins of
HTLV-4(1863LE)
The HTLV-1, HTLV-2, and HTLV-3 Tax proteins (Taxl,
Tax2, and Tax3, respectively) transactivate initiation of
viral gene expression from the promoter located in the 5'
LTR and are thus essential for viral replication [27,30,42].
Taxi and Tax2 have also been shown to be important for
T-cell immortalization [27,30]. To characterize the HTLV-
4 Tax (Tax4) we compared the sequence of Tax4 with


those ofprototypic HTLV-1, PTLV-2, and PTLV-3s to deter-
mine if motifs associated with specific Tax functions were
preserved between each group. Alignment of the predicted
Tax4 sequence shows excellent conservation of the critical
functional regions, including the nuclear localization sig-
nal (NLS), cAMP response element (CREB) binding pro-
tein (CBP)/P300 binding motifs, and nuclear export
signal (NES) (Fig. 10). Three sets of amino acids (Ml,
M22, M47) shown to be important for Taxi transactiva-


Table 2: PTLV evolutionary rates' at I s + 2nd codon positions of different gene regions assuming a Bayesian relaxed molecular clock.


a-parameter
(F-distribution)2


0.23 (0.168 0.303)
0.417 (0.356 0. 475)
0.29 (0.228 0.359)
0.311 (0.215 -0.421)


Mean rate


3.02 x 10-7
7.92 x 10-7
4.08 x 10-7
4.32 x 10-7


Median rate


2.89 x 10-7
7.57 x 10-7
3.9 x 10-7
4.17x 10-7


95% HPD


1.65 -4.78 x 10-7
3.93 12.7 x 10-7
2.25- 6.44 x 10-7
2.34 6.47 x 10-7


I Evolutionary rate is in nucleotide substitutions/site/year.
295% HPD (high posterior density) intervals are given in parentheses.


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Table 3: PTLV evolutionary time-scale calculated with a Bayesian relaxed molecular clock using Ist + 2nd codon positions of different
gene regions'.


Clade


env


PTLV root


STLV-5 (MarB43)/PTLV- I

PTLV-I

HTLV-l(Mel)/PTLVIa, b 2


HTLV-4(1863)/PTLV-2

PTLV-2

STLV-2

HTLV-2

HTLV-2a, b 3


PTLV-3


358,500
(169,200- 600,200)

121,850
(68,650- 201,300)
75,100
(50,200- 1 15,200)
46,350
(40,000 57,900)

187,500
(85,050- 321,800)
128,600
(57,000 226,550)
42,350
(1 1,650- 87,100)
33,600
(15,750-58,200)
23,000
(14,350- 30,000)

71,700
(28,800 120,700)


308,500
(136,400 -559,900)

121,450
(60,450 220,600)
54,250
(40,410 79,340)
47,450
(40,000 58,400)

175,100
(63,850- 334,750)
103,700
(41,300 205,100)
37,200
(9,800 82,800)
30,100
(13,900 -54,900)
20,400
(12,000 -28,700)

64,550
(25,010- 129,800)


385,100
(172,300- 638,900)

147,850
(72,450 244,800)
58,250
(41,600- 84,000)
47,550
(40,000 58,400)

221,650
(89,650 378,000)
126,850
(51,850- 223,350)
27,700
(8,150 58,100)
30,600
(13,750-54,100)
20,000
(12,000 -28,350)

60,050
(32,950 122,200)


214,650
(104,050- 353,100)

87,500
(50,400- 143,250)
54,800
(40,900- 76,100)
48,200
(40,000 58,500)

124,250
(49,800-218,250)
75,200
(29,850 135,200)
35,550
(12,100 70,050)
23,500
(12,800- 41,050)
18,350
(12,000 27,950)

40,850
(16,400- 81,150)


I The most recent common ancestor (tMRCA) is the median Bayesian estimate in years ago (ya); 95% HPD intervals are given in parentheses.
2 The tMRCA for this node was constrained by using a uniform distribution prior of 40,000-60,000 ya.
3 The tMRCA for this node was constrained by using a uniform distribution prior of 12,000-30,000 ya.


tion and activation of the nuclear factor (NF)-kp pathway
are also highly conserved in Tax4 (Fig. 10) [43]. The C-ter-
minal transcriptional activating domain (CR2), essential
for CBP/p300 binding, was also conserved within Tax4,
except for two mutations, N to T and I/V to F, at positions
two and five of the motif, respectively (Fig. 10). However,
the CR2 binding domain of the STLV-3 Tax, which con-
tains these identical mutations, has been shown recently
to retain its ability to bind CBP and to a lesser extent p300
with no deleterious effect on transactivation of the viral
promoter [42].

Although important functional motifs are highly con-
served in PTLVs, phenotypic differences between HTLV-1
and HTLV-2 Tax proteins have lead to speculation that
these differences account for the different pathologies
associated with both HTLVs [27]. Recently, the C-termi-
nus of Taxi, but not Tax2, has been shown to contain a
conserved PDZ binding domain present in cellular pro-
teins involved in signal transduction and induction of IL-
2-independent growth required for T-cell transformation
[29,44,45] and may contribute to the phenotypic differ-
ences between these two viral groups. The consensus PDZ
domain has been defined as S/TXV-COOH, where the first
amino acid is serine or threonine, X is any amino acid, fol-
lowed by valine and the carboxyl terminus. Tax4 does not
contain a PDZ domain (Fig. 10), suggesting that like


HTLV-2, HTLV-4 may possibly be less pathogenic than
HTLV-1.

Besides Tax and Rex, two additional ORFs encoding four
proteins, p27', p12', p3011, and p13" (where I and II
denote ORFI and ORFII, respectively), have been identi-
fied in the pX region of HTLV-1 and are important in viral
infectivity and replication, T-cell activation, and cellular
gene expression [26]. Analysis of the pX region of HTLV-
4(1863LE) revealed a total of five additional putative
ORFs (named I-V, respectively) encoding predicted pro-
teins of 101, 161, 99, 133, and 115 aa in length (Fig la).
Since none of the potential ORFs begin with methionine
start codons, we determined potential splice junctions in
the HTLV-4 genome to ascertain the potential for novel
ORFs via complex splicing mechanisms. Prediction of
splice junction positions in HTLV-4 identified only two
donor sites with high confidence, one at nucleotide 414 in
the LTR (sd-LTR) and one at nucleotide 5105 in Env (sd-
Env) (Fig. la). Three additional putative splice acceptor
sites were identified at nucleotides 7274 (sa-pX2) and
7645 (sa-pX3), and in Tax/Rex at nucleotide 7245 (sa-T/
R). The sa-T/R is used with the sd-Env to generate the Tax
and Rex proteins via complex splicing mechanisms (Fig.
1). Rex mRNA is predicted to be spliced using sd/sa sites
in a different reading frame than Tax and with a different
methionine start codon (nucleotide positions 5043 -



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Pbx-1
GGACAACGACCCTCTCCCAGGGGCGACAGCAAGCCCCCAAGGACAAAA
c-Myb
CTAGCAGGGACTAGTCATCAGCCAAAAGG TTCACA CAP TAAGGAT
bZIP910 ----- - - -
bZIP910 Distal 21R in PTLV-1/2
CC AGGTT CCAGCC AGCCTCAAAACCAGGAAATCCATAGAAATGC
Middle 21R
ACCTCGCCCTTACCCACTTCCCCTATCATGAAAAAC GGCTGTGACGATACCC
Proximal 21R
TTCCCCAAAAAATTTGCTTAAACCATCAATAAAGACAGCCTAGCCTATATAAGC
U3IR poly (A) signal TATA box
ATGAGGATGGTTCAGGAGG GCTCGCTCTCTTGCCGATCGCCCTGCTCACCTCGA
cap
GTGTCCATCTCCTGGTCAATCAGTTGAGACGCCGCCGGCTGCCGGTCTCCTGGTTGT
CGCACCTCCTGAACCACCCCTT GTAAGCCCCCCTTGGTCCGAGCTTGGCTACGG
LRex core
sd-LTR Rexcore
TTTCTGTAGTCGCTCCCAGGGAAGTCTCCGAGACTGCCCAAGCCTCTGCTTGCAAGG
Rex coreR I U5
CTACGGCCCTCCACCTC TTCCGCGTCCGTGTTAATCTCTTCGCGCCAACCGAAAA
CGAAAGCGCCTCCAGCTCTCTTGGCCCGGGGCCAGGCCTGAGCCGCGCGGGCGCAC
CACCTTAAGCCCGCTGTACTCAAACCCCTCCGGGAGGGGCCCTTTACAGTAGGCGCC
CGTCCCCCCGGGGGAAACATACAAGTGGGGGCTCGTCCGGGATCTGTCCTCCGCTTCG
PBS
CCGTTCCCCCCCTCCCACT

Figure 7
Nucleotide sequence of the HTLV-4(1863LE) LTR
and pre-gag region. The U3-R-U5 locations (vertical lines),
the pre-B cell leukemia (Pbx-1, TGACAG) and c-Myb
(YAACKG) transcription factor binding sites, approximate
cap site (cap), polyadenylation (poly(A)) signal, TATA box,
predicted splice donor site (sd-LTR), and two 21 -bp repeat
elements (middle and proximal based on positions in HTLV- I
and -2), as well as the location of the distal 21 -bp repeat in
HTLV-I and -2 (dashed lines), are indicated. In the R and US
regions, the predicted Rex core elements and nuclear ribo-
protein Al binding sites are underlined. The pre-gag region
and primer binding site (PBS, underlined) are in italics.


5105 and 7120 7566) to generate a 170 aa protein. Tax
mRNA is spliced from nucleotide positions 5102 5105
and 7120 8150 to generate a protein predicted to be 345
aa in length. Two potential accessory proteins 68 and 93
aa in length are then predicted using the sd-Env and either
the sa-pX2 or sa-pX3 in ORFIV or ORFV, respectively (Figs.
1 and 11). The HTLV-4 ORFIV protein shared 75% simi-
larity with the HTLV-lpl311 and HTLV-2 p28" accessory
proteins but was missing the mitochondrial targeting
sequence and the active region typically located at the
amino-terminus of the protein (Fig. 11). Interestingly, 19
of 26 (73%) amino acids in the HTLV-4 ORFIV (positions
4-29) were identical to similar ORFs from all other major
PTLVs, suggesting a conserved functionality of this motif
(Fig. 11). The predicted HTLV-4 ORFV protein shared only
weak similarity (41%) to the carboxyl-terminus of the
HTLV-2 p28X" protein (Fig. 11). In contrast to the HTLV-
4 ORFIV and ORFV proteins, the predicted HTLV-4 ORFI,
ORFII, and ORFIII proteins did not share significant
sequence identity with any PTLV accessory proteins, but
shared weak sequence similarity with only miscellaneous
microbial proteins available in GenBank such as Pseu-
domonas histidine kinase (37% similarity) (data not
shown). Analysis of alternatively spliced messenger RNA


expression in viable cells or tissue culture, and/or in vitro
characterization, will be required to investigate the expres-
sion and functionality of these putative accessory pro-
teins.

A novel protein termed the HTLV-1 basic leucine zipper
ZIP (bZIP) factor (HBZ) was recently found to be encoded
on the complementary strand of the viral RNA genome
between the env and tax/rex genes which was shown to
negatively regulate viral replication and to enhance viral
infectivity and persistence [28,46]. The recent finding of
HBZ mRNA as the sole viral gene product expressed in
ATL patients also suggests a role of HBZ mRNA in the sur-
vival of leukemic cells in vivo and in HTLV-1-associated
oncogenesis [47]. Although originally reported to be
exclusive to PTLV-1 [28], we previously reported that HBZ
is conserved among PTLV-1, -2, and -3 [31]. More
recently, others have demonstrated that an HTLV-
3(Pyl43) molecular clone expressed an antisense mRNA
[48]. Although these results confirm the predicted HBZ
gene region in this virus [34], additional studies are
required to evaluate the functionality of the HTLV-3 HBZ
protein. We now show by sequence analysis that an HBZ
homolog is also present in HTLV-4 emphasizing the
potential importance of this protein and mRNA in viral
replication, persistence, and leukemogenesis [28,46]. The
carboxyl terminus of the HBZ ORF contains a 21 aa
arginine rich region that is relatively conserved in PTLV
and known cellular bZIP transcription factors, followed
by a less conserved leucine zipper region that possesses
five or four highly conserved leucine heptads in HTLV-1
and all other PTLVs, respectively (Fig. 12). HTLV-1 has five
leucine heptads similar to that found in mammalian bZIP
proteins, while all other PTLVs, including PTLV-4, have
four leucine heptads followed by leucine octet (Fig. 12).
In PTLVs, the first residue in the initial leucine heptad is a
nonpolar amino acid other than leucine (Fig. 12). This
single amino acid substitution has not affected the func-
tionality of the leucine zipper in HTLV-1 but requires fur-
ther study of its affect in other PTLV HBZs [25,41]. As
reported previously, HTLV-2(MoT) is the only PTLV-2
strain that does not have the full complement of leucine
heptads a result of a single nucleotide deletion at position
6823 that causes a frameshift in the predicted HBZ
sequence [31].

Discussion
Here we report the first complete nucleotide sequence and
genomic characterization of the recently discovered
HTLV-4. We show that the genome of this novel human
virus is genetically equidistant from HTLV-1, HTLV-2, and
HTLV-3. Robust phylogenetic and molecular clock analy-
sis confirms that HTLV-4 clearly falls outside the diversity
of PTLV-1, PTLV-2, and PTLV-3, demonstrating that
HTLV-4 is the only known member of a distinct PTLV
group we call PTLV-4. Combined, these results strongly

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C (D -0 U)
a, cz~ C

D c: C\1 C\
-)+ + + +
2- +
n

m AD


eg c-Myb
supershift


c-Myb -0

non-sp -0


HTLV-4
c-myb
site probe


1 2 3 4 5 6


Figure 8
EMSA using a 32P-labeled probe representing the c-
Myb binding site within the HTLV-4 LTR (lane I)
incubated with Jurkat nuclear extract (lanes 2-6). A
100-fold excess of unlabeled probe sequence (specific com-
petitor, lane 3) or an unlabeled oligonucleotide containing
mutations within the c-Myb binding site (non-specific com-
petitor, lane 4) were added as indicated. Non-specific (lane
5) and Myb-specific (lane 6) antibodies were added and the
supershifted band is indicated on the right panel, which is a
longer exposure of the left panel.



support the HTLV-4/PTLV-4 nomenclature proposed for
this virus [6]. The phylogenetic stability seen across HTLV-
4 and other PTLV genomes also demonstrates the absence
of major recombination events occurring in PTLV despite
evidence of dual infections in humans and primates
[9,49]. Furthermore, these results support the distinct evo-
lutionary history of HTLV-4 and other PTLVs demonstrat-
ing that they are not recent genetic recombinants from
pre-existing viral genomes. This finding contrasts with
other retroviruses like HIV in which frequent recombina-
tion contributes substantially to genetic diversity [50].

Bayesian MCMC statistical methods have recently been
developed to accurately infer dates of evolutionary events,


to investigate the origin ofviral epidemics, and to estimate
historical population dynamics [32,51]. Molecular dating
of the HTLV-4 predecessor using these robust methods
suggests that this novel PTLV lineage originated almost
200 millennia ago, which predates the inferred origin of
the ancestors of HTLV-1, HTLV-2, and HTLV-3 by about
76,000 191,000 ya [31]. Two equally parsimonious
hypotheses on the origin of HTLV-4 can thus be proposed
by the inferred ancient existence of the PTLV-4 lineage.
First, it is possible that HTLV-4(1863LE) is a current
descendent of the ancestral PTLV-4 that infected humans
as they evolved in Africa and represents a strain circulating
within humans living in this geographic region. Interest-
ingly, the inferred date of the HTLV-4 ancestor also coin-
cides with the appearance of Homo sapiens sapiens,
estimated to have occurred around 200 400 K ya, sug-
gesting the emergent human lineage may have been a suit-
able host for the ancestral PTLV-4. If this is not just an
evolutionary historical coincidence of both virus and
host, then HTLV-4 may indeed be the oldest human del-
taretrovirus as inferred from the molecular dating of all
four HTLV groups. Alternatively, HTLV-4(1863LE) could
also be the result of a more recent zoonotic infection with
a very divergent STLV present in NHPs in the forests of
Cameroon. Additional information on the diversity of
HTLV-4 and its likely simian counterpart will be needed to
determine whether HTLV-4(1863LE) truly originated as
H. sapiens sapiens evolved, and persists in humans today,
or represents a more recent zoonotic transmission from
an NHP. As of yet, a simian counterpart of HTLV-4 has not
been identified in Cameroon or elsewhere despite the
identification of other novel STLVs in this region
[9,10,22]. Nonetheless, the inability to find "STLV-4" may
be due to sampling and screening biases in the selection
of NHP species and the geographic locations examined
[9,32].

The inference of an ancient split of HTLV-4 (1863 LE) from
the PTLV-2 lineage, combined with the wide geographic
distribution of STLVs and a history of STLVs crossing into
humans [2,8-10,18-21], all imply that HTLV-4 infection
may be more prevalent. Repeated and historical cross-spe-
cies infections of humans with various STLV-1 strains led
to the emergence and dissemination of several HTLV-1
subtypes in West-Central Africa [2,4-6]. Similar evidence
suggests that the newly identified HTLV-3 infections also
potentially arose from multiple, independent past or con-
temporary introductions of different STLV-3 strains into
humans [6,8,31]. Given that both HTLV-1 and HTLV-2
followed human population migrations out of Africa and
across the globe as humans evolved, HTLV-4 and HTLV-3
may also have spread globally. A more precise determina-
tion of the origin and distribution of HTLV-4 infection
will require further studies, such as expanded surveillance
in both humans and NHPs. However, serosurveys for
HTLV-4 may be complicated by the inability to discrimi-

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IT"G Length = 42
TG'c Energy = -17.7 kcal/mole



C-G C
T
iT /




/ Rex core
G-C
G-C

C-G
425 466

Figure 9
Plot of predicted RNA stem loop secondary struc-
ture of HTLV-4(1863LE) LTR region. Position of the
Rex responsive element (RexRE) core is indicated.


nate this infection from HTLV-2 since they both show
similar WB profiles and the sensitivity of serological
assays for identifying HTLV-4 is currently unknown
[6,35]. Thus, additional diagnostic tools are required to
determine the level of HTLV-4 penetration into the gen-
eral population and to search for the potential primate
origin of HTLV-4(1863LE). Screening for HTLV-4 will be
facilitated by the development and application of sero-
logic and molecular assays based on the sequences
reported here. For example, since the HTLV-4 Gag matrix
and nucleocapsid and the envelope surface proteins are
divergent from PTLV-1, PTLV-2, and PTLV-3 it may be pos-
sible to use them in serologic assays to differentiate the
four PTLV groups.

Virus classification is a topic of ongoing discussion and
suggestions for nomenclature are typically based on
lumping or splitting of taxa into distinct groups. Deltaret-
rovirus species are classified by the International Commit-
tee on Taxonomy of Viruses (ICTV) by differences in
genome sequence and viral oncogenes, antigenic proper-
ties, natural host range, and pathogenicity. For example,
HTLV-1 and HTLV-2 are distinguished mostly by phyloge-
netic diversity and variable disease outcomes of each
virus. Recently, a new deltaretrovirus species, STLV-5, was
proposed based on limited analyses of small tax/rex
sequences from a Macaca arctoides (strain MarB43) that
was originally classified as STLV-1 [4,10]. Herein, we
show by using robust phylogenetic analysis of major cod-
ing regions and complete viral genomes that expansion of
the current PTLV nomenclature from four to six putative
major taxonomic species or groups should be considered.
Our natural classification of PTLV groups is based on rig-


orous phylogenetic inference that demonstrates with high
confidence the formation of very distinctive mono-
phyletic lineages outside the diversity of all known viral
groups, combined with genetic distances demonstrating
the putative new lineage is nearly equidistant from all pre-
viously characterized groups, and the placement of the
new PTLV groups near the root of the PTLV phylogeny.
The first four PTLV phylogroups consist of HTLV-1/STLV-
1, HTLV-2, HTLV-3/STLV-3, and HTLV-4. We confirm the
existence of the putative STLV-5(MarB43) lineage, while
the sixth group consists of the STLV-2(PanP) and STLV-
2(PP1664) viruses. However, for simplicity we suggest
maintaining the STLV-2 nomenclature historically used
for this particular viral group. Each proposed new viral
group clearly falls outside the diversity of their nearest
PTLV relatives (PTLV-1 and HTLV-2, respectively), is
monophyletic with strong bootstrap support and poste-
rior probabilities, and are all roughly genetically equidis-
tant from other PTLVs, and hence should all be classified
as distinct viral species. As with all viral nomenclature,
PTLV classification as proposed here will require approval
of ICTV.

In addition to understanding viral evolutionary history,
analysis of full-length genomes can also provide basic
information on the replication and pathogenic potential
of new viruses. Thus, we examined in detail the genetic
structure and sequence of HTLV-4 to determine if impor-
tant functional motifs involved in viral expression and
HTLV-induced leukemogenesis are preserved [26-30,44].
All enzymatic, regulatory, and structural proteins are well
conserved in HTLV-4(1863LE), including conserved func-
tional motifs in Tax that are important for viral gene
expression and T-cell proliferation, suggesting HTLV-4 is
replication competent. We also observed several impor-
tant molecular features of the HTLV-4 genome involved in
viral expression and pathogenicity that are either similar
or distinct from other HTLVs. For example, the absence of
a PDZ domain in the Tax protein of HTLV-4(1863LE),
known to be important in cellular signal transduction and
T-cell transformation [29-31], is similar to what is seen in
HTLV-2 but not in HTLV-1 and HTLV-3 [27]. The absence
of PDZ suggests that the HTLV-4 Tax may be more pheno-
typically similar to the HTLV-2 than the HTLV-1 Tax. Fur-
thermore, the high amino acid identity of the Tax4 and
Tax2 proteins also suggests that Tax4 may function simi-
larly to Tax2 [27]. However, whether the absence of a PDZ
domain in HTLV-4 is associated with an absence of spe-
cific cellular and/or clinical outcomes like HTLV-2 will
require further investigation.

We also identified unique putative c-Myb and Pbx-1 tran-
scription factor binding sites in the U3 region of the LTR
of HTLV-4(1863LE). c-Myb is a proto-oncogene that is
expressed in T cells induced by mitogen or antigenic stim-
ulation and is involved in cell cycle progression and pro-

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M1 Nuclear Localization Signal CBP/P300 binding
HTLV-4 (1863LE) MPfHPGFGQSLLYGYPVYVFGDCVQADWCPISGGLCSPRLHRHALLA NPEHQITWDPIDGRVVGSPLQYLIPRLPSFPI2RTSKTLKVLTPPT PVTPK
HTLV-2 (MoT) ....................... .... ... .. ........ ....L. .......... ............... ....R ............ ..
HTLV-2 (Efe) ................... ....... .. ..... T .......... ..... ......... S .............. .R ............ ..
STLV-2 (PP16644 ) ................... ........ V ......T ......... ..... ........................................... S
HTLV-1 (ATK) ..... .... ............ G ........... A ......... ............... I..A .. ....................... I HT ..N
HTLV-3(2026ND) . .................................. .............. .. S.A ........... T..... . .. . AA ...
STLV-3 (TGE2117) .. ................... .............. .......... .............. S. ............... T .. ..... .AT...
M22 (NF-kB transactivation) Nuclear Export Signal
HTLV-4 (1863LE) VPPSFFQSVRRHSPYRNGCLETTLGEQLP 3IFPEPGLRPQNVYTIWGKTIVCLYIYQLSPPMTWPLIPHVIFCNPRQLGAFLSNVPPKRLEELLYKLYL
HTLV-2(MoT) ...A....M.K.T........ .. ..D.......... .. T .. V.. ............. . ........TK..I.........MF.
HTLV-2 (Efe) .. .A .... M.K.T... .... ....D... ...... .. I. T .. .. V.... LF. ....... ... H.K..... TK.. .........MF.
STLV-2 (PP1664) I..A....M.KL....... .YP. ..D..... D .......I..T..R.V... .L........ ........ .H.K.. .T..T... ........ .IF
HTLV-1(ATK) I....L.AM.KY..F.. .YM.P.. .QH.. .3..D....... L..L..GSV..M.L.. I... .L.. ..... H.G ..... T..Y I.. ...... IS.
HTLV-3 (2026ND) I.....HA .KK .T.F..N ..L....... 3..D .......I..M ..SSV .. .L. ..................H.E......TR..T. ...... IF.
STLV-3(TGE2117) ......HA..K.T.F..N. ..L....... ..D .......... M..SSV ....L........ .......... .H.E......TR . ........ IF
NES
HTLV-4 (1863LE) HTAIIILPEDALPTTLFQPVRAPCVQTTWNTGLLPYQPNLTTPGLIWTFNDGSPMISGPCPKAGQPSLVVQSSLLIFERFQTKAYHPSYLLSHQLIQYS
HTLV-2 (MoT) ...TV.V ... ....M........ I..A.C......HSI ........ ........... ..Y.. ...... ........ K.E...F . ...
HTLV-2 (Efe) .. V .V .... D ....M ........ I..A .C ...... HSI ....................................... K .....F . ....
STLV-2 (PP1664) .... ....T ...... ........ A .D......HSLI ......................P....... .... K.....F .. .....
HTLV-1 (ATK) L ...... C....... A.. .VTL.A.QN ....FHST ..........T..T .........D......L. .. SF. .HK..... ... .... G ....
HTLV-3 (2026ND) S......... NCF .......T ...A ..AP.H..... C.KEIA........T ............E. .......... TF..QQ..... S..AF ... .K..H..
STLV-3(TGE2117) S ... .....NCF ....... T...AI.AP.H ..... C.KEI .... V...T............ E ...... TF..QQ.....N..AF ... K..H..
M47 (CREB/ATF transactivation)
HTLV-4 (1863LE) SFHHLYLLFDEYTIPFE LKEKEGDDR-DNDPLPGATASPQGQN---- 345
HTLV-2(MoT) ...N.H......N..V.I..NKE.A..NG.--- ---------- 331
HTLV-2 (Efe) ...N.H.......N..V.I..NKE.A..NG.QP.G.----AA.DESSA----- 344
STLV-2(PP1664) ...N.H...E..N..X.Y..N.. .A..S-.S..G.SNLGAA..ESSA----- 347
HTLV-1(ATK) ... S.H...E.. N..I...N...A.. NDHEPQISPGGLE.PSEKHFRK E 353
HTLV-3(2026ND) ...S.H...E...........N..GANVDD.EPRDGS---Q.PARGQIAEPV 35
STLV-3 (TGE2117) .. S.H... E....V...... N.. GANVND.EPQDEP---Q.PTRGQIAER 350
CR2 binding PDZ

Figure 10
Comparison of predicted Tax amino acid sequences of selected prototypical primate T-cell lymphotropic
viruses. Shown in boxes are known functional motifs: NLS, nuclear localization signal; (CBP)/P300, cAMP response element
(CREB) binding protein; NES, nuclear export signal; CR2, C-terminal transcriptional activating domain; PDZ, PDZ binding
motif; M I, M22, and M47 are motifs important for Tax transactivation and NF-kp activation (38).


liberation of T lymphocytes, such that continuous
deregulation of cell cycling may play a role in leukemo-
genesis [52]. c-Myb has been shown to bind to the HTLV-
1 and feline leukemia virus LTRs to increase viral tran-
scription [53,54]. Like c-Myb, dysregulation of the home-
oprotein Pbx-1 can also increase leukemogenesis by
disturbing hematopoiesis [55]. We demonstrate here that
the potential c-Myb binding site in the HTLV-4 LTR specif-
ically binds c-Myb, suggesting that it may also promote
LTR-mediated viral expression and which may help over-
come the loss of the distal 21-bp repeat element observed
in the HTLV-4 LTR. For example, Pbx-1 has been demon-
strated to up-regulate transcription of another retrovirus,
murine leukemia virus (MuLV), by binding to conserved
Pbx-1 transcription factor sites present in MuLV LTRs [56].
The presence of putative c-Myb and Pbx-1 binding sites in
the HTLV-4 LTR may provide novel mechanisms of tran-
scriptional control at both the viral and cellular levels not
previously known for HTLV. Nevertheless, involvement of
the putative novel binding sites in viral transcription and
leukemogenesis will require additional studies.

Although originally reported to be exclusive to HTLV-1
[28], we now provide additional evidence for a putative
HBZ region among all PTLVs, including HTLV-4(1863LE).


Despite the absence of canonical bZIP domains, prelimi-
nary experiments show that proteins are transcribed from
the HTLV-3, and -4 antisense mRNAs and all were potent
inhibitors of Tax induction of HTLV LTR activity with sim-
ilar cellular localizations like that of the HTLV-1 HBZ
(unpublished data). These results not only confirm the
predicted HBZ sequences and proteins in these viruses but
also demonstrate the potential importance of HBZ in
PTLV replication. The finding of a potential bZIP region
on the antisense strand of all PTLV genomes also indicates
that the nomenclature for this protein should be renamed
from HBZ to AEP for antisense encoding protein as sug-
gested [48]. The potential role of AEP in HTLV-induced
oncogenesis may be less clear since HTLV-1 and HTLV-2
infection result in different clinical outcomes, while
pathologies for HTLV-3 and HTLV-4 have not yet been
reported. Additional studies are required to confirm the
potential effect of the predicted AEP transcripts and pro-
teins on HTLV-4 and PTLV expression and any role they
may have on leukemogenesis.

Conclusions
The novel HTLV-4 genome independently evolved from
an ancient deltaretrovirus lineage and contains many of
the functional motifs important for viral expression and


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% sim HTLV-4 (ORFIV)
75% HTLV-1 (pl3I)
72% STLV-2(ORFII)
67% HTLV-3(ORFIV)
75% HTLV-2 (p28xII)
HTLV-4(ORFV)

HTLV-4 (ORFIV)
HTLV-1 (pl311)
STLV-2(ORFII)
HTLV-3 (ORFIV)
HTLV-2 (p28"II)
HTLV-4(ORFV)


Active region
--------------- -------------------------
MLIISPLP VWTESSFRIP RVWRLCTRR VPHLWGTMFG?PTS
MPFFSPLSRFRTEPPLWIPSLRVWRLCSSRLVSRFRWALFH PAP
SNLQKFYSLVSPFERFRTEPSLRVPRFRLCTGVPHFWGALFSATP
Mitochondrial Targeting Sequence MFH PTS

Highly Conserved Region
-----MDHLGPHRWTSCRLASPIPYPSPPLLPH TNLQDPQSPYPTNHSCHPQGSTLLLSVRAEA6
SRPTGHLSRAS. ..... .RY TV .. ...... .EM.----------------------------
ACPSRHLPGAPA ... ... R...S...... ..A ... ...E..... G.HSPY.. .L. .D..R.FPIH.--
PCPTGHVPRTS.Y ......R.Q.S.T..... T......E.YPH..GSH.P..C.D..D.SI..PR.--- 1
TCPPGHLSRAPT.. ......R.Q.S ............. E. K...G ... SH.. L..... .C. IN.K. 7


HTLV-4(ORFIV) QPLPQRMS
HTLV-1 (pl3) --------
STLV-2(ORFII) --------
HTLV-3 (ORFIV) --------
HTLV-2 (p28xII) H... KW.PGTNPRGSAPLPRLPRTWPPSPKHLHHLGKNRSMPIPIPAFPTHDMATYTPCHILPPQTIRSL
HTLV-4 (ORFV) --------------------------------------------M.V. S.. L. PHS ..... Q..AAWRF


HTLV-4 (ORFIV)
HTLV-1 (pl3I)
STLV-2(ORFII)
HTLV-3 (ORFIV)
48% HTLV-2 (p28xII)
HTLV-4(ORFV)


PHQGASKTIRRTSIQNVPTHRDSHSPPGGRPTHHNVPTREGSLYPDCLVYRTSPLSLHLNNPRSNMDLQ
SK.C.PQA....PL.TLS...RHNH.A.R..AY.PISACSSTLC.NYLEH....IPAKPDY.WPD...-


Figure II
Comparison of predicted accessory protein sequences of selected primate T-cell lymphotropic viruses. Upper
alignment, HTLV-4(1863LE) open reading frame (ORF) IV compared to HTLV-I (pl 3"), STLV-2 ORFII, HTLV-3(2026ND)
ORFIV, and HTLV-2 p28XI. Location of conserved mitochondrial targeting sequence in the HTLV-I p 3" protein and highly
conserved amino acid region are boxed. Lower alignment, HTLV-4(1863LE) ORFV compared to HTLV-2 ORFII (p28XI). % Sim,
percent amino acid similarity of HTLV-4 ORFs to other PTLV ORFs.


possibly oncogenesis, including two novel transcription
factor binding sites in the LTR. More studies are needed to
further characterize the unique molecular features of
HTLV-4 identified here, and to determine whether HTLV-
4 is endemic and pathogenic in humans to better under-
stand the public health importance of this novel human
virus.

Methods
DNA preparation and PCR-based genome walking
DNA was prepared from uncultured PBMCs available
from person 1863LE identified in the original PTLV sur-
veillance study in Cameroon reported in detail elsewhere
[6]. DNA integrity was confirmed by 3-actin polymerase
chain reaction (PCR) as previously described [6]. All DNA
preparation and PCR assays were performed in a labora-
tory where only human specimens are processed and
tested according to recommended precautions to prevent
contamination. To obtain the full-length genomic
sequence of HTLV-4 we first PCR-amplified small regions
of each major coding region by using nested PCR and
degenerate PTLV primers (Fig. 1). The tax (730-bp),
polymerase (pol) (662-bp), and envelope (env) (319-bp)
sequences were amplified by using primers and condi-


tions provided elsewhere [6,31]. An additional short
HTLV-4 sequence, 440-bp in length, that overlaps the end
of tax and the beginning of the 3'LTR was amplified using
standard PCR conditions and 45 C annealing with the
external primers PGTAXF7a 5'TGATGGIWSICCIATGATT-
TCCGG 3', PGTAXF7b 5'TGATGGGTCT CCTATG ATIT
CCGG3' and PGTATA1+2R1 5'TCCTGAA CYGTCYY
YRCGCTITTATAG3' and the internal primers PGTAXF8
5'TGCCCIAARIMIGGICAGCCATCTIT3' and PGTAT
A1+2R1.

HTLV-4(1863LE)-specific primers were then designed
from sequences obtained in each of the four viral regions
described above and were used in nested, long-template
PCRs (Expand High Fidelity kit containing both Taq and
Tgo DNA polymerases (Roche)) to fill in the gaps in the
genome as depicted in Fig. 1. The external and internal
primer sequences for the LTR-pol fragment are 1863LF2
5'CCAAGGACAAAACTAGCAGGGACT3' and 1863PR4
5'GGGGATGGTAAAGGCGAAGTAGGG3', and 1863LF3
5'CGTCCCAGCCCAGCCTCAAAACCA 3'and 1863PR5
5'GGGAATCTGGAAGAAAGCGTCCGT3', respectively.
The external and internal primer sequences for the pol-env
fragment are 1863PF3 5'GTCCTCTCATGGTCTCCCAGTT-



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atfl
c-fos
c-jun
Crebl
HTLV-1(ATK)
HTLV-1(Mel)
STLV-1(Tan)
STLV-3(TGE2117)
STLV-3(PH969)
STLV-3(CT0604)
STLV-3(NG409)
STLV-3(PPAF3)
HTLV-3(2026ND)
HTLV-2 (Efe)
HTLV-2(G12)
HTLV-2(Gab)
HTLV-2(Kay)
HTLV-2 (MoT)
STLV-2(PanP)
STLV-2(PP1664)
HTLV-4 (1863LE)


EIRLMKNREAARECRRKKKE- -YVKC---
RIRRERNKMAAAKCRNRRRE- -LTDT----
ERKRLRNRIAASKCRKRKLE--RISR-----
EVRLMKNREAARECRRKKKE--YVKC-----
ERKWRQGAEKAKQHSARKEK ----MQELG
ERKWRQTEQGAKQRSARKEK ----MTELG
ERKWRQGTEKAKRHSARKEK ----MQGLG
ERRKRRREELEERKRRKKERR QLDCIDMLG
ERRKRRREELEERKRQKKERR QLDCIDMLG
EHRKRRREELEERKRRKKERR QLDCIDMLG
EHRKRRREELEERRRRKKERR QLDCIDMLG
ERRKRRREELEERKRRKKER QQLDCIDALG
ERRRRCREELEERKRQKKERR QLDCIDMLG
RRRKERLREKDQKRREREERE QLECIGILG
RRRKERLREKDQKRREREERE QLECIGLLG
RRRKERLQEKDQKRREREERE QLECIGLLG
RRRKERQREKDQKRREREERE QLECIGLLG
RRRKERLREKDQKRREREERE QLECIGLLG
QRRRERLREKEEKRREREKRE QLECIGLLG
QRRRERLREKEEKRREREKRE QLECIGLLG
ARRRERLQERERKRKEREARE QQRKCMDLLG
Arginine rich
DNA-binding domain


ENRVAI
2AETD(
EEKVK
ENRVAI
)GYTR(
)GYAR(
)GYTR(
EGFCE]
EGFCE1
EGFCG]
EGFCD]
EGFCD]
EGFCD]
)GFCD]
)GFCD]
)GFCD]
)GFCD]
)GFCD]
EGYCE1
EGYCE1
EGYCD]


Figure 12
Comparison of predicted amino acid sequences of primate T-cell lymphotropic viruses and cellular basic leu-
cine zipper (bZIP) transcription factors. Conserved arginine rich and potential leucine zipper regions of the bZIP proteins
are boxed. Alternate amino acid sequence resulting from frameshift mutation in HTLV-2(MoT) leucine zipper region is shown
in italics.


TCCCAG 3' and 1863ER 5'GCTGGAGTGG
TAGGAGGAGATAC3', and 1863PF5 5'CACTTCCTG
GGCCAAATCATACATCCAGATC3' and 1863ER3
5'GGCTGGCCTGAA GTACTGGGATGCC3', respectively.
The external and internal primer sequences for the env-tax
fragment are 1863EF1 5'CCTGCC AAAACCT GATCACC
TATTC3' and 1863TR1 5'CGACAAC TCGTCCATC
GATGG3' and 1863EF2 5'CCCTGTATCTCTTCCCACACT-
GGGTA C3' and 1863TR2 5'GGG GAGCATAATC
CACCGGAGATGG3', respectively. The remaining 3' end
of the genome was obtained by using the primers
1863pXF1 5'AACTCCGCCAATACACCCAACAGG3' and
1863LR1 5'GGAGGGGTITGAGTACAGCGGGCT3' in a
single round of PCR amplification.

PCR products were purified with a Qiaquick PCR purifica-
tion kit (Qiagen), and sequenced in both directions with
a BigDye terminator cycle kit and automated sequencers
(Applied Biosystems). Selected PCR products were also
cloned into the pCR4-TOPO vector using the TOPO TA
Cloning kit (Invitrogen) and recombinant plasmid DNA
was prepared using the Qiagen plasmid purification kit
prior to automated sequencing.

Sequence analysis
Percent nucleotide divergence was calculated using the
GAP program in the Genetic Computer Group's (GCG)
Wisconsin package [57]. Examination of functional


genetic motifs involved in viral expression, regulation,
and HTLV-induced oncogenesis was done by detailed
comparison of the HTLV-4 genome with full-length PTLV
sequences [26-29,31,44]. Identification of potential tran-
scription factor binding sites in the HTLV-4 genome was
performed using the program TESS (Transcription Ele-
ment Search System) [58]. Secondary structure of the LTR
RNA was determined using the program RNAstructure
v4.2 program [59]. Comparison of full-length PTLV
genomes available at GenBank and determination of
genetic recombination was done using HTLV-4(1863LE)
as the query sequence and the F84 (maximum likelihood)
model and a transition/transversion ratio of 2.28 imple-
mented in the program SimPlot [60]. Prediction of splice
acceptor (sa) and splice donor (sd) sites was done using
an artificial neural network implemented in the
NetGene2 program [61] and with the Spliceview program
[62].

Nucleotide substitution saturation was evaluated using
pair-wise transition and transversion versus divergence
plots using the DAMBE program [63]. Unequal nucleotide
composition was measured by using the TREE-PUZZLE
program [64]. Phylogenetic trees were inferred with the
parameters estimated from the Clustal W [65] sequence
alignments of each gene and the full-length genome after
removing indels by using Modeltestv3.7 [66] and Neigh-
bor-Joining (NJ) methods in the MEGAv4.0 [67] program


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ENQNKT IEELK I
LDEKSA LTEIAlL
KSQNTE LSTASI I
ENQNKT IEELK I1
LGEVES EAERRII
LSEAES EAERGI L
LDEVES LAERR L
LGYVDF RENQR I
SGYIDF ERENQ I1
LGYIDF EQENQQ11
LGYIDF EQENQ I1
LGYIDF EQENQ I1
LGYIDF ERESQ I1
LGYIDV EEEKK I
LGYVDF EEEKK I
LGYVDF EEEKK I
LGYVDF EGEKKV I
LGYVDF KGKRRM
GYIGV EGERTCI
GYISV EGERTCI
QGYIDV EKERK 11
Leucine Zipper


CDLYSN
jKEKEI
ZEQVAQt
DLYCHE
4QEKEDI
4QENDDI
JQEKED
tADCEES
IADCEES
tADCEGS
tAGCEES
tAGCEES
tAGCEES
9EECEQG
9EECERG
9EECERG
9EECERG
GRNVSGC
MGECERE
9EECEQE
EEECNHN


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and maximum-likelihood (ML) analysis in PAUP* [68],
TREE-PUZZLE [64], and PhyML [69]. The reliability of the
inferred tree topology was tested with 100 (PAUP*) to
1000 bootstrap replicates (NJ and PhyML) or 100,000
puzzling steps (TREE-PUZZLE). Trees were viewed and
edited using FigTree v1.1.2 [70].

PTLV evolutionary rates and divergence times
In order to estimate a reliable divergence time for the
cenancestor (most recent common ancestor) of the HTLV-
4(1863LE) lineage, we generated separate alignments of
gag, pol, env, and tax genes from all full-length PTLV
genomes available at GenBank by using Clustal W.
Sequence gaps and 3rd codon positions were removed,
and minor adjustments in the alignment were made man-
ually. The best fitting evolutionary model for the aligned
sequences was determined using a hierarchical likelihood
ratio test as described elsewhere [68]. A variant of the GTR
model, allowing four different substitution rate categories
(rAOc = rAOT = rc,CT = 1, rAOc = 9.35, rc.G = 0.67, rc4T =
5.79), with gamma-distributed rate heterogeneity (a =
0.694) and an estimated proportion of invariable sites
(0.185), was determined to best fit the data.

The molecular clock hypothesis, or constant rate of evolu-
tion, for the PTLV tree was tested with the likelihood ratio
test [71]. Likelihoods were calculated using the best fitting
nucleotide substitution model either with or without the
enforcement of the global clock constraint with the pro-
gram PAML [72]. The PTLV evolutionary rate assuming
the global molecular clock model was estimated by using
the divergence time of 40,000 60,000 years ago (ya) for
the Melanesian HTLV-1 lineage (HTLV-lmel) and
12,000-30,000 ya for the most recent common ancestor
of HTLV-2a/HTLV-2b native American strains according
to the formula: evolutionary rate (r) = branch length (bl)/
divergence time (t) [23]. Such divergence dates were based
on well-established genetic and archaeological evidence
suggesting that ancestors of indigenous Melanesians and
Australians migrated from Southeast Asia or the introduc-
tion of ancestral indigenous Indians into North America
via the Bering Straight during those times [3,4,32]. The
evolutionary rate was also estimated by employing a Baye-
sian Markov Chain Monte Carlo (MCMC) molecular
clock method, allowing for either a strict or a relaxed
molecular clock [51], implemented in the BEAST software
package [73]. For each analysis, we used the calibration
dates discussed above as a strong prior for the time of the
most recent common ancestor (tMRCA) of the HTLV-
1Mel/HTLV-la,b and HTLV-2a,b lineages, respectively. In
practice, the upper and lower divergence times estimated
from anthropological data were used to define the interval
of a strong uniform prior distribution from which the
MCMC sampler would sample possible divergence times
for the corresponding node in the tree. For each model,


the Bayesian calculation consisted of three independent
100,000,000 generations MCMC with sampling every
1,000th generation. Convergence of the MCMC was
assessed by calculating the effective sampling size (ESS) of
the runs using the program Tracer [74]. All parameter esti-
mates showed significant ESSs (>150). The tree with the
maximum product of the posterior clade probabilities
(maximum clade credibility tree) was chosen from the
posterior distribution of 5,000 sampled trees (after burn-
ing in the first 5001 sampled trees) with the program Tree-
Annotator version 1.4.6 included in the BEAST software
package [73]. Both the constant coalescent and Yule Proc-
ess were used as tree priors and gave identical results.

DNA transfection
Approximately 1 million 293 cells were seeded on a 100
mm dish and incubated for 24 h at 37C. Cells were then
transfected with a c-Myb expression vector using Lipo-
fectamine-PLUS (Invitrogen). Cells were lysed 48 hours
using 1 x passive lysis buffer (Promega). Whole cell extract
was stored at-80 C.

Electrophoretic mobility shift assay (EMSA)
The double-stranded oligonucleotide probe representing
the c-Myb binding site within the HTLV-4 LTR (sense, 5'-
TCGAGAAAGGTCAACTGTCTCACACAAAC-3'; anti-
sense, 3'-TCGAGTITGTGTGAGACAGTTGACCTITC-5')
was end-labeled with [a-32P]dCTP using Klenow enzyme
(Invitrogen). The DNA-binding reaction was incubated
for 1 h at room temperature using 5 ng of labeled probe
and binding buffer (10 mM Tris [pH 7.9], 50 mM NaC1, 1
mM EDTA 10 mM dithiothreitol, 0.5% non-fat dry milk,
5% glycerol) supplemented with 2 ug of sheared salmon
sperm DNA, 1 ug poly-dI-dC (Sigma St. Louis, MO), and
5 ug 293 cell extract in a final volume of 15 ul. The super-
shift was performed by adding 1 ug of anti-c-Myb mono-
clonal antibody (Upstate Biotechnology, Charlottesville,
VA) or non-specific PC10 monoclonal antibody (Santa
Cruz Biotechnology, Santa Cruz, CA) to the binding reac-
tion for 1 h at room temperature. Unlabeled double-
stranded (sense, 5'-TCGAGAAAGGTCGTATGTCT-
CACACAAAC-3'; antisense, 3'-TCGAGTITGTGTGAGA-
CATACGACCTITC-5') non-specific oligonucleotide
contained mutations at three positions (underlined)
within the predicted c-Myb binding site. Specific and non-
specific competitors were added in a 100-fold excess over
labeled probe. DNA-protein complexes were resolved on
a 4% non-denaturing polyacrylamide gel in 0.5x Tris-
borate-EDTA at 150 V for 2.5 h.

Nucleotide sequence accession numbers
The complete HTLV-4(1863LE) proviral sequence has
been deposited in GenBank with accession number
EF488483. GenBank accession numbers for the complete
PTLV genomes used in this paper are [HTLV-1(ATK) =


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1020291, [HTLV-1(ATL-YS)= H199491, [HTLV-1(Mel5) =
L025341, [HTLV-1 (Boi) = L36905], [STLV-1(TE4) =
Z46900], [STLV-1(Tan90) = AF074966], [HTLV-2(MoT) =
M10060], [HTLV-2(Kay96) = AF356584], [HTLV-2(Gab)
= Y13051], [HTLV-2(SP-WV)= AF139382], [HTLV-2(G2)
= L11456], [HTLV-2(G12)= L11456], [HTLV-2(Efe) =
Y14365], [STLV-2(Pan-p) = U90557], [STLV-2(ppl664) =
Y14570], [HTLV-3(2026ND) = D0093792], [HTLV-
3(Pyl43) = D0462191], [STLV-3(CT0604)
NC 003323], [STLV-3(Ph969) = Y07616], [STLV-
3(TGE2117) = AY217650], [STLV-3(NG409)
AY222339], [STLV-3(Ppaf3) = AF517775], [STLV-
5(MarB43)= AY590142].

Competing interests
Some authors (WMS, NDW, DSB, TMF, WH) have applied
for a patent for the discovery of HTLV-4.

Authors' contributions
WMS conceived, designed and coordinated the study,
analyzed, acquired and interpreted the data, and wrote the
manuscript. MS, RRG, and AK helped design the study,
performed detailed phylogenetic analysis of the
sequences, and helped write the manuscript. SHQ and HJ
together obtained the full-length genome of HTLV-4, ana-
lyzed the sequences, and participated in writing the man-
uscript. SJM and KNP helped characterize the LTR
regulatory elements and participated in writing the manu-
script. NDW, DSB, TMF, and WH helped design the study,
assisted in analysis of the data, and participated in writing
the manuscript. All authors read and approved the final
manuscript.

Additional material


Additional file 1
Supplementary figures. Figure S1. Pair-wise transition (s; blue line) and
transversion (v, green line) versus divergence plots in ,i. ... HTLV-4
(1863LE) genes using 1st + 2nd or 3rd codon positions (cdp). Genetic
distances were calculated with the Tamura and Nei 1993 (TN93) model
and plotted against the estimated number of transitions and transversions
for each pair-wise comparison using the DAMBE program. Figure S2.
Evolutionary relationship of major genes and the entire genome of HTLV-
4(1863LE) to other PTLVs by using either Neighbor-Joining (NJ; a-f) or
maximum likelihood (ML, g-j) methods. The percentage of replicate trees
in which the associated taxa clustered together in the bootstrap test (100-
1000 replicates) is shown at the branch nodes. Branch lengths are drawn
to scale and only bootstrap values greater than 70% are shown. Branches
leading to PTLV-1, HTLV-2, and PTLV-3 sequences are drawn in red,
blue, and green, respectively. The branches leading to HTLV-4(1863LE),
STLV-2, and to the divergent STLV-5(MarB43) strain are drawn in
magenta, purple, and yellow, respectively.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1742-
4690-6-9-Sl.ppt]


Acknowledgements
N.D.W. is supported by a National Institutes of Health (NIH) Director's
Pioneer Award Program (grant number DPI-OD000370) and an Interna-
tional Research Scientist Development Award from the NIH Fogarty Inter-
national Center (KO I TW00003- I). This research was supported in part by
the Global Viral Forecasting Initiative. Use of trade names is for identifica-
tion only and does not imply endorsement by the U.S. Department of
Health and Human Services, the Public Health Service, or the Centers for
Disease Control and Prevention. The findings and conclusions in this report
are those of the authors and do not necessarily represent the views of the
Centers for Disease Control and Prevention. K.N.P. was supported by NIH
grant #R25 M 69234, and work in the S.J.M.laboratory was supported by
NIH grant #R21 A1078307.

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