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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 Downloaded from www.microbiologyresearch.org by 027011 G 2011 SGM IP: 78.47.27.170 On: Thu, 13 Oct 2016 17:45:03 Printed in Great Britain 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 Downloaded from www.microbiologyresearch.org by IP: 78.47.27.170 On: Thu, 13 Oct 2016 17:45:03 583 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 584 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, Downloaded from www.microbiologyresearch.org by IP: 78.47.27.170 On: Thu, 13 Oct 2016 17:45:03 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. Holzschu, D. L., Delaney, M. A., Renshaw, R. W. & Casey, J. W. (1998). The nucleotide sequence and spliced pol mRNA levels of the Herchenröder, O., Turek, R., Neumann-Haefelin, D., Rethwilm, A. & Schneider, J. (1995). Infectious proviral clones of chimpanzee foamy virus (SFVcpz) generated by long PCR reveal close functional relatedness to human foamy virus. Virology 214, 685–689. Ho, S. Y., Phillips, M. J., Drummond, A. J. & Cooper, A. (2005). Accuracy of rate estimation using relaxed-clock models with a critical focus on the early metazoan radiation. Mol Biol Evol 22, 1355–1363. nonprimate spumavirus bovine foamy virus. J Virol 72, 2177–2182. Kang, Y. & Cullen, B. R. (1998). Derivation and functional characterization of a consensus DNA binding sequence for the tas transcriptional activator of simian foamy virus type 1. J Virol 72, 5502–5509. Kang, Y., Blair, W. S. & Cullen, B. R. (1998). Identification and 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. functional characterization of a high-affinity Bel-1 DNA binding site located in the human foamy virus internal promoter. J Virol 72, 504– 511. Kupiec, J. J., Kay, A., Hayat, M., Ravier, R., Peries, J. & Galibert, F. (1991). Sequence analysis of the simian foamy virus type 1 genome. Gene 101, 185–194. Leendertz, F. H., Zirkel, F., Couacy-Hymann, E., Ellerbrok, H., Morozov, V. A., Pauli, G., Hedemann, C., Formenty, P., Jensen, S. A. & other authors (2008). Interspecies transmission of simian foamy References virus in a natural predator-prey system. J Virol 82, 7741–7744. Bieniasz, P. D., Rethwilm, A., Pitman, R., Daniel, M. D., Chrystie, I. & McClure, M. O. (1995). A comparative study of higher primate foamy viruses, including a new virus from a gorilla. Virology 207, 217–228. Calattini, S., Nerrienet, E., Mauclere, P., Georges-Courbot, M. C., Saib, A. & Gessain, A. (2004). Natural simian foamy virus infection in wild-caught gorillas, mandrills and drills from Cameroon and Gabon. J Gen Virol 85, 3313–3317. Calattini, S., Betsem, E. B., Froment, A., Mauclere, P., Tortevoye, P., Schmitt, C., Njouom, R., Saib, A. & Gessain, A. (2007). Simian foamy virus transmission from apes to humans, rural Cameroon. Emerg Infect Dis 13, 1314–1320. Liu, W., Worobey, M., Li, Y., Keele, B. F., Bibollet-Ruche, F., Guo, Y., Goepfert, P. A., Santiago, M. L., Ndjango, J. B. & other authors (2008). Molecular ecology and natural history of simian foamy virus infection in wild-living chimpanzees. PLoS Pathog 4, e1000097. Löchelt, M., Muranyi, W. & Flügel, R. M. (1993). Human foamy virus genome possesses an internal, Bel-1-dependent and functional promoter. Proc Natl Acad Sci U S A 90, 7317–7321. Mannigel, I., Stange, A., Zentgraf, H. & Lindemann, D. (2007). Correct capsid assembly mediated by a conserved YXXLGL motif in prototype foamy virus Gag is essential for infectivity and reverse transcription of the viral genome. J Virol 81, 3317–3326. Charleston, M. A. & Robertson, D. L. (2002). Preferential host Murray, S. M., Picker, S. L., Axthelm, M. K., Hudkins, K., Alpers, C. E. & Linial, M. L. (2008). Replication in a superficial epithelial cell niche switching by primate lentiviruses can account for phylogenetic similarity with the primate phylogeny. Syst Biol 51, 528–535. explains the lack of pathogenicity of primate foamy virus infections. J Virol 82, 5981–5985. BEAST: Bayesian evolutionary analysis by sampling trees. BMC Evol Biol 7, 214. Pacheco, B., Finzi, A., McGee-Estrada, K. & Sodroski, J. (2010). Species- Drummond, A. J. & Rambaut, A. (2007). Duda, A., Stange, A., Luftenegger, D., Stanke, N., Westphal, D., Pietschmann, T., Eastman, S. W., Linial, M. L., Rethwilm, A. & Lindemann, D. (2004). Prototype foamy virus envelope glycoprotein leader peptide processing is mediated by a furin-like cellular protease, but cleavage is not essential for viral infectivity. J Virol 78, 13865– 13870. Eastman, S. W. & Linial, M. L. (2001). Identification of a conserved residue of foamy virus Gag required for intracellular capsid assembly. J Virol 75, 6857–6864. Epstein, M. A. (2004). Simian retroviral infections in human beings. Lancet 364, 138–139, author reply 139–140. specific inhibition of foamy viruses from South American monkeys by New World Monkey TRIM5a proteins. J Virol 84, 4095–4099. Peters, K., Barg, N., Gartner, K. & Rethwilm, A. (2008). Complex effects of foamy virus central purine-rich regions on viral replication. Virology 373, 51–60. Pfrepper, K. I., Rackwitz, H. R., Schnölzer, M., Heid, H., Löchelt, M. & Flügel, R. M. (1998). Molecular characterization of proteolytic processing of the Pol proteins of human foamy virus reveals novel features of the viral protease. J Virol 72, 7648–7652. Prasad, A. B., Allard, M. W. & Green, E. D. (2008). Confirming the phylogeny of mammals by use of large comparative sequence data sets. Mol Biol Evol 25, 1795–1808. Heneine, W., Switzer, W. M., Sandstrom, P., Brown, J., Vedapuri, S., Schable, C. A., Khan, A. S., Lerche, N. W., Schweizer, M. & other authors (1998). Identification of a human population infected with Renne, R., Friedl, E., Schweizer, M., Fleps, U., Turek, R. & NeumannHaefelin, D. (1992). Genomic organization and expression of simian simian foamy viruses. Nat Med 4, 403–407. Rethwilm, A., Darai, G., Rosen, A., Maurer, B. & Flügel, R. M. (1987). Herchenröder, O., Renne, R., Loncar, D., Cobb, E. K., Murthy, K. K., Schneider, J., Mergia, A. & Luciw, P. A. (1994). Isolation, cloning, and Molecular cloning of the genome of human spumaretrovirus. Gene 59, 19–28. http://vir.sgmjournals.org foamy virus type 3 (SFV-3). Virology 186, 597–608. Downloaded from www.microbiologyresearch.org by IP: 78.47.27.170 On: Thu, 13 Oct 2016 17:45:03 585 A. Schulze and others Switzer, W. M., Bhullar, V., Shanmugam, V., Cong, M. E., Parekh, B., Lerche, N. W., Yee, J. L., Ely, J. J., Boneva, R. & other authors (2004). Frequent simian foamy virus infection in persons occupationally exposed to nonhuman primates. J Virol 78, 2780–2789. Thümer, L., Rethwilm, A., Holmes, E. & Bodem, J. (2007). The complete nucleotide sequence of a New World simian foamy virus. Virology 369, 191–197. Tobaly-Tapiero, J., Bittoun, P., Neves, M., Guillemin, M. C., Lecellier, C. H., Puvion-Dutilleul, F., Gicquel, B., Zientara, S., Giron, M. L. & other authors (2000). Isolation and characterization of an equine foamy virus. J Virol 74, 4064–4073. Verschoor, E. J., Langenhuijzen, S., van den Engel, S., Niphuis, H., Warren, K. S. & Heeney, J. L. (2003). Structural and evolution- ary analysis of an orangutan foamy virus. J Virol 77, 8584– 8587. 586 Wang, G. & Mulligan, M. J. (1999). Comparative sequence analysis and predictions for the envelope glycoproteins of foamy viruses. J Gen Virol 80, 245–254. Wattel, E., Vartanian, J. P., Pannetier, C. & Wain-Hobson, S. (1995). Clonal expansion of human T-cell leukemia virus type I-infected cells in asymptomatic and symptomatic carriers without malignancy. J Virol 69, 2863–2868. Winkler, I., Bodem, J., Haas, L., Zemba, M., Delius, H., Flower, R., Flügel, R. & Löchelt, M. (1997). Characterization of the genome of feline foamy virus and its proteins shows distinct features different from those of primate spumaviruses. J Virol 71, 6727–6741. Wolfe, N. D., Switzer, W. M., Carr, J. K., Bhullar, V. B., Shanmugam, V., Tamoufe, U., Prosser, A. T., Torimiro, J. N., Wright, A. & other authors (2004). Naturally acquired simian retrovirus infections in central African hunters. Lancet 363, 932–937. Downloaded from www.microbiologyresearch.org by IP: 78.47.27.170 On: Thu, 13 Oct 2016 17:45:03 Journal of General Virology 92