Download Complete nucleotide sequence and evolutionary analysis of a

Journal of General Virology (2011), 92, 582–586
Short
Communication
DOI 10.1099/vir.0.027011-0
Complete nucleotide sequence and evolutionary
analysis of a Gorilla foamy virus
Andrea Schulze,13 Philippe Lemey,23 Jörg Schubert,1 Myra O. McClure,3
Axel Rethwilm1 and Jochen Bodem1
Correspondence
1
Jochen Bodem
2
Jochen.Bodem@vim.
uni-wuerzburg.de
Received 7 September 2010
Accepted 21 November 2010
Institute of Virology and Immunobiology, University of Würzburg, Germany
Department of Microbiology and Immunology, K. U., Leuven, Belgium
3
Division of Medicine, Imperial College London, UK
To shed light on primate foamy virus (FV) evolution, we determined the complete nucleotide
sequence of the gorilla simian foamy virus (SFVgor). Starting from a conserved region in the
integrase (IN) domain of the pol gene we cloned the viral genome to the 59 and 39 LTR into
plasmid vectors and elucidated its nucleotide sequence. The sequences of both LTRs were
determined by nucleotide sequencing of separate PCR products from the primer-binding site or
the bel region and LTRs. All protein motifs conserved among the primate FV were identified in
SFVgor. Using phylogenetic analysis of the Gag, Pol and Env amino acid sequences, we
demonstrate that SFVgor consistently clusters in accordance with a scenario of virus–host
co-divergence.
Foamy viruses (FVs) form a subfamily of retroviruses.
Although FV cause a persistent infection, they are harmless,
implying that the virus may be a valuable tool for viral
vector development. FV have been isolated and cloned
from a variety of primate (for references see below) and
non-primate hosts (Holzschu et al., 1998; Tobaly-Tapiero
et al., 2000; Winkler et al., 1997). Evolutionary studies
would benefit from the characterization of the FV genomic
diversity, but to date, only a few primate FVs from
macaques (SFVmac) (Kupiec et al., 1991), African green
monkey (SFVagm) (Renne et al., 1992), orang-utan
(SFVora) (Verschoor et al., 2003), chimpanzees (SFVcpz)
(Herchenröder et al., 1994, 1995), spider monkey
(SFVspm) (Thümer et al., 2007), marmoset and squirrel
monkey (Pacheco et al., 2010) have been cloned and
completely sequenced. Although the gorilla simian FV
(SFVgor) has been previously isolated and partially
sequenced (Bieniasz et al., 1995; Calattini et al., 2004,
2007; Wolfe et al., 2004), the complete sequence of the
SFVgor represents the last missing piece of the FV genomic
puzzle within the great apes. Since gorillas are the closest
related primates to humans, next to chimpanzees, the
complete sequence of SFVgor has the potential to provide
additional insights into virus and primate evolution, which
are known to be related by a history co-speciation (Switzer
et al., 2004). Facilitated by only small differences between
3These authors contributed equally to this paper.
The GenBank/EMBL/DDBJ accession number for the sequence
reported in this paper is HM245790.
Supplementary material is available with the online version of this paper.
582
cellular restriction factors and between the immune system
of gorillas and humans, gorilla-derived retroviruses can be
transmitted to humans. This could result in a potential
threat even with the virus being non-pathogenic in its
natural host. These potential dangers are highlighted by the
fact that, at least for SFVcpz and SFVora, such inter-species
transmissions are not rare events and were even observed
for SFVgor as well (Calattini et al., 2007; Heneine et al.,
1998; Switzer et al., 2004). In this regard it should be noted
that the prototype foamy virus (PFV), which was supposed
to be a specific human isolate, is most probably a
transmission from chimpanzees (Epstein, 2004).
To broaden our knowledge of FV evolution and genomic
diversity, we have cloned and determined the sequence of
the complete SFVgor. The SFVgor isolate, derived from a
western lowland gorilla and described by Bieniasz et al.
(1995), was propagated in baby hamster kidney cells
(BHK-21) in a co-culture system, as previously described,
in order to yield high infection rates (Bieniasz et al., 1995).
Total DNA was isolated from the infected cells and served
as template in the PCRs. The viral genome was amplified in
two segments starting from the 59 LTR to the integrase
(IN) region and from the IN to the 39 LTR. The PCR
products were cloned into the PCR-XL-Topo (Invitrogen)
vector in accordance with the manufacturer’s manual and
three independent clones of each fragment were isolated
and completely sequenced by primer walking. Sequence
assembly was performed with DNASTAR Lasergene Software.
Since some of the isolated clones contained frame-shift
mutations in the pol region, we reamplified, cloned and
resequenced this part of the genome. These sequences
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SFVgor nucleotide sequence and evolutionary analysis
showed no frame-shifts or unexpected stop codons. To
determine the complete sequences of both LTRs, and to
verify the obtained sequence a primer located at the start of
the 59 LTR was used to amplify the genome up to the gag
gene, whereas the 39 end was amplified from the bel region
until the 39 end of the LTR. All sequencing primers used in
this study and a detailed clone description is available upon
request.
Sequence similarity analysis revealed the overall length of
the LTR to be 1283 nt (Supplementary Table S1, available
in JGV Online). Based on this analysis the U3, R, U5
regions were defined as being 944, 181 and 158 bp,
respectively. A TATA box motif was identified from 226
to 221 in the LTR. Sequence comparison with the PFV Tas
responsive element (TRE) (Rethwilm et al., 1987) revealed
a highly conserved sub-sequence from 247 to 258. A
splice-donor sequence was found in the R region, defining
exon one as 51 nt. The primer-binding site is located, as
expected, downstream of the LTR. A central polypurinetract is located at 6490–6501 (Peters et al., 2008).
The position of the internal promoter (IP) was determined
by similarity to PFV at nt position 9329 (Löchelt et al.,
1993). A TATA box sequence at 220 to 215 was found
upstream of the potential IP. In addition, due to the high
similarity of the PFV and SFVgor sequences, we could
determine a potential TRE at 2151 to 2133 of the IP
(Kang & Cullen, 1998). This TRE shows a sequence identity
of 18 of the 19 nt to its PFV counterpart. The 39
polypurine-tract was determined directly before the 39
LTR at 10949–10972. A poly(A) signal was identified from
12072 to 12077 in the 39 LTR and it can be assumed that
the poly(A) addition takes place at nt 12097 of the proviral
DNA. This would define a genomic transcript with
11153 nt. The nucleotide sequence of SFVgor has been
deposited to GenBank (accession no. HM245790).
The Gag ORF is 644 aa in length (Supplementary Table
S1). A cytoplasmic targeting and retention signal was
determined from aa 43 to 66 (Kang et al., 1998). Two late
domain PSAP motifs (at aa 214–217 and 280–283) and
three YXXL motifs (at 109–112, 200–203 and 463–466)
were identified. The latter were shown to be essential for
particle assembly of PFV (Eastman & Linial, 2001). Gag
encodes three glycine–arginine-rich boxes (502–507, 547–
553 and 594–598), which contribute to the nuclear
localization of Gag and are involved in the binding of
nucleic acids. The potential cleavage site (CS) of SFVgor at
the carboxyl terminus of Gag (aa position 616, HTVN/
TVTQ) was found to display high similarity to the PFV CS
[RAVN/TVTQ (Mannigel et al., 2007)].
The Pol ORF consists of 1141 aa (Supplementary Table
S1). The catalytic centre of the protease (DSGA) is located
at aa 24–27. There is only one efficiently used protease CS
in the FV Pol, which is located between reverse transcriptase (RT) and IN. This CS is well conserved with the
amino acid sequence YVVN/NNDK to PFV (YVVN/
CNTK) (Pfrepper et al., 1998) and located after aa 752.
http://vir.sgmjournals.org
This protein processing would give rise to an IN protein of
389 aa. The catalytic centre (YVDD) of the RT encompasses the aa residues 312–315. The zinc-finger motif of the
IN was found at H814–H818–C848–C851 and the (DD35E)
catalytic centre motif at D880–D937–E973.
The env gene encodes a 987 aa protein (Supplementary
Table S1). The FV Env precursor is cleaved in contrast to
the ortho-retroviral Env by cellular furin-like proteases in a
particle-associated leader peptide (LP), the surface (SU)
and transmembrane (TM) proteins. The LP/SU cleavage is
likely to occur at aa 126 (RIAR/ALRA) (Pfrepper et al.,
1998), while the SU/TM cleavage site was determined by
similarity at aa 568 (NQRR/KREVN) (Duda et al., 2004).
The Tas ORF was defined from 9577 to 10476 giving rise to
a protein of 299 aa (Supplementary Table S1). This protein
shows a strikingly high similarity in the DNA-binding
domain to the Tas sequence of PFV (Supplementary Tables
S2 and S3, available in JGV Online), which is reflected by a
high conservation of the TREs in both promoters.
The Bet ORF encompasses the 59 Tas reading frame from
nt 9577 to 9838 (Supplementary Table S1). The latter is
joined by splicing with nt 10140, resulting in a Bet ORF of
1443 nt and 480 aa, respectively. The Bet amino acid
sequence, with 80 % similarity, is highly homologous to the
PFV counterpart.
To determine the evolutionary relationship between
SFVgor and other simian or non-primate FV, we
reconstructed the evolutionary history of the concatenated
gag, pol and env amino acid sequences for all cloned FV and
compared this to the host evolutionary relationships
inferred from their mitochondrial DNA sequences. We
employed Bayesian Markov chain Monte Carlo (MCMC)
analysis implemented in BEAST (Wang & Mulligan, 1999) to
estimate posterior distributions of time-measured phylogenetic trees (Drummond & Rambaut, 2007). To calibrate
both the host and viral phylogenies under the assumption
of ancient co-speciation, we identified two host calibration
dates available from the fossil record that could be applied
to nodes with consistent relative divergence times
(Supplementary Material, available in JGV Online). The
host and viral evolutionary histories are summarized on
the same timescale in Fig. 1. All primate FV sequences
clustered according to their host species, which would
argue for a long-lasting co-evolutionary process. However,
the relationships among the feline (F), bovine (B) and
equine (E) FV, with FFV basal to BFV and EFV, did not
mirror the host phylogeny, which places the horse lineage
basal to the bovine and feline cluster. It has, however,
been shown that relationships among these hosts are not
consistent among different genomic segments (Drummond
& Rambaut, 2007).
The matching virus and primate host trees are a
prerequisite to the assumption of co-speciation, but also
the ages of the corresponding common ancestors are
expected to be concordant. If this was not the case then the
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583
A. Schulze and others
Fig. 1. Host (black) and foamy virus (grey)
evolutionary history on an absolute timescale.
We represent the viral lineage in the human
host as a dotted line because this is the result
of an interspecies transmission and humans
are not a natural host for FV. Numbers along
the branches depict the posterior probability
support for the respective clusters; host and
virus posterior probabilities are shown above
and below the branches, respectively. Bars at
the nodes represent the 95 % credible intervals for the node ages; host and virus node
bars are shown as white opaque and black
opaque bars, respectively. Data from fossil
records of the human–orang-utan split (12–16
million years, Myr) and the human–gorilla split
(6.5–8 Myr) (Ho et al., 2005) were used for
calibration.
matching phylogenies could also be explained by preferential host switching (Prasad et al., 2008), which postulates
that viruses are more likely to jump between related host
species. With a few exceptions, there is generally a wide
overlap among the credible intervals for the virus and host
divergence times (Fig. 1). The PFV-SFVcpz divergence is
for example younger than the respective host divergence
times, but this can be expected as the divergence is the
result of viral evolution within chimpanzee species that
forms geographical isolated clusters. PFV is in fact the
result of a cross-species transmission from chimpanzees to
humans rather than co-speciation (Charleston &
Robertson, 2002). Retroviruses are actively crossing into
humans exposed to non-human primates, from chimpanzees as well as other non-human primates (Switzer et al.,
2004; Wolfe et al., 2004). Furthermore, there are also many
examples of FV cross-species transmission among nonhuman primates, e.g. from cercopithecus monkeys to
chimpanzees (Epstein, 2004) and from colobus monkeys to
chimpanzees (Liu et al., 2008). Although this complicates
the history of FV, not all cross-species transmission will
lead to successful onwards transmission in the new host
and co-speciation may still hold as a long-term evolutionary process.
Assuming ancient co-speciation in primate hosts, the viral
rate of amino acid substitution was estimated to be
1.3161028 (1.146102821.4861028) substitutions per
site per year. This is a remarkable low rate of evolution for
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a virus with RT that is as error-prone as other retroviruses.
It has been suggested that FV may replicate through clonal
expansion, analogous to primate T-lymphotropic viruses
(Leendertz et al., 2008; Verschoor et al., 2003), thereby
avoiding the introduction of many RT-induced mutations
over short timescales. However, although most tissues
harbour latent provirus in naturally infected rhesus
macaques, viral replication does take place in oral mucosa
(Murray et al., 2008), which complicates an explanation of
low evolutionary rates by clonal expansion.
We decided to analyse further the evolution of SFVgor by
using sequences derived from primary isolates, since
differences from the complete SFVgor sequence to the
isolates would argue for an adaptation to the cell culture
conditions. We determined sequence variations up to 5 %
in the 425 bp IN fragment. The complete SFVgor sequence
showed a high homology to previously published wild-type
isolates within a range of 95.5 % nt identity. The published
sequences of the 425 bp IN fragment of SFVcpz were
aligned as control and showed divergence in the same
range. In addition, differences in the same order were
found previously for SFVcpz (Wattel et al., 1995).
Several studies have suggested that FV sequence diversity
can be used as a marker for host evolution. The sequence
data presented here appear to confirm this assumption.
However, comparing our trees to the trees previously
published for the 425 bp fragment in the IN coding region,
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Journal of General Virology 92
SFVgor nucleotide sequence and evolutionary analysis
an obvious difference in the grouping of the SFVgor can be
observed. Bieniasz et al. (1995) grouped this fragment in
the branch near to SFVmac and SFVagm, in contrast to
whole Pol sequence, which is grouped in the SFVcpz
branch. This indicates that small sequence fragments may
lead to different phylogenetic trees, which would argue for
the use of complete sequences in order to get reliable
results.
sequencing of simian foamy viruses from chimpanzees (SFVcpz): high
homology to human foamy virus (HFV). Virology 201, 187–199.
In summary, this report underlines that FVs are, indeed,
old viruses, which display a long co-evolution with their
respective hosts. Differences between infected animals
account for less than 5 % sequence deviation. The reason
why FV evolution is such a slow process remains to be
discovered.
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Acknowledgements
We would like to thank Tanja Schied for technical support and Sylvia
Geubig for help with the preparation of the manuscript. This work
was supported by the Deutsche Forschungsgemeinschaft (B03006/1-2
and SFB 479). P. L. was supported by a postdoctoral fellowship from
the Fund for Scientific Research (FWO) Flanders.
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