MURDOCH RESEARCH REPOSITORY

http://researchrepository.murdoch.edu.au

This is the author's final version of the work, as accepted for publication following peer review but without the publisher's layout or pagination.

Groenewoud, M.J., Fagrouch, Z., van Gessel, S., Niphuis, H., Bulavaite, A., Warren, K.S. , Heeney, J. L. and Verschoor, E. J. (2010) Characterization of novel polyomaviruses from Bornean and Sumatran orang-

utans. Journal of General Virology, 91 (3). pp. 653-658.

http://researchrepository.murdoch.edu.au/3637

Copyright © 2010 SGM It is posted here for your personal use. No further distribution is permitted.

1

Characterization of novel polyomaviruses from Bornean and 1

Sumatran orangutans 2

3

Marlous J. Groenewoud1,4, Zahra Fagrouch1, Sabine van Gessel1,5, Henk Niphuis1, 4

Kestutis Sasnauskas2, Kristin S. Warren3, Jonathan L. Heeney1,6, and Ernst J. 5

Verschoor1,* 6

7

1 Department of Virology, Biomedical Primate Research Centre, Rijswijk, 8

The Netherlands 9

2 Institute of Biotechnology, Vilnius, Lithuania 10

3 School of Veterinary and Biomedical Sciences, Murdoch University, Perth, 11

Western Australia 12

4 Present address: Department of Physiological Chemistry and Centre for Biomedical 13

Genetics, University Medical Center, Utrecht, The Netherlands 14

5 Present address: Department of Human Genetics, Radboud University Nijmegen 15

Medical Centre, Nijmegen, The Netherlands 16

6 Present address: Department of Veterinary Medicine, University of Cambridge, 17

Cambridge, United Kingdom 18

19

*Corresponding author. 20

E. J. Verschoor 21

Department of Virology 22

Biomedical Primate Research Centre 23

Lange Kleiweg 139 24

2288GJ Rijswijk 25

JGV Papers in Press. Published November 18, 2009 as doi:10.1099/vir.0.017673-0

2

The Netherlands 1

2

Tel: +31-15-2842661 3

Fax: +31-15-2842601 4

E-mail address: verschoor@bprc.nl 5

6

Running title: novel polyomaviruses from orangutans 7

8

Word count Summary: 124 9

Word count Text (including legends): 1799 10

11

Number of figures: 3 12

13

Number of supplementary figures: 314

3

ABSTRACT 1

2

Serological screening of sera from orangutans demonstrated a high percentage 3

of sera that cross-reacted with SV40 polyomavirus antigens. Analysis of archival 4

DNA samples from seventy-one Bornean and eight Sumatran orangutans with a 5

broad-spectrum PCR assay resulted in the detection of polyomavirus infections in 6

eleven animals from both species. Sequence analysis of the amplicons revealed 7

considerable differences between the polyomaviruses from Bornean and Sumatran 8

orangutans. The genome from two polyomaviruses, one from each species, was 9

therefore amplified and sequenced. Both viral genomes revealed a characteristic 10

polyomavirus architecture, but lacked an obvious agnogene. Neighbor-joining 11

analysis positioned the viruses in a larger cluster together with viruses from bats, 12

bovines, rodents, and several primate polyomaviruses from chimpanzees, African 13

green monkeys, squirrel monkeys, and the human Merkel cell polyomavirus.14

4

Polyomaviruses (PyV) are dsDNA viruses with a relatively small circular 1

genome of approximately 5 kb. In humans, five different PyV have been described 2

until now. The two best-studied human viruses, JCV and BKV, are associated with 3

serious diseases in immunosuppressed persons (Jiang et al., 2009). The others, 4

WUPyV, KIPyV , and Merkel cell PyV (McPyV), have been found in patients with 5

respiratory tract infections (WU and KI), or in patients with Merkel cell carcinoma 6

(MCC), a rare aggressive skin cancer, respectively(Allander et al., 2007; Feng et al., 7

2008; Gaynor et al., 2007). 8

Another polyomavirus, simian virus 40 (SV40), has also been detected in 9

humans, but these data remain controversial (Martini et al., 2007). Its natural hosts are 10

Asian macaques (Koliaskina, 1963). SV40 may have entered the human population as 11

a contaminant of polio vaccine batches that were prepared on primary macaque 12

kidney cells (Sweet & Hilleman, 1960). It causes cell transformation in vitro, but also 13

induces tumors in rodents. In its natural host SV40 infection generally goes unnoticed, 14

but when the host is immunosuppressed SV40 can induce disease symptoms 15

comparable to JCV-induced progressive multifocal leukoencephalopathy (PML) in 16

humans (Axthelm et al., 2004; Chretien et al., 2000; Horvath et al., 1992; Simon et 17

al., 1992; Simon et al., 1999). 18

In addition to SV40, other polyomaviruses have been described in nonhuman 19

primates. African green monkeys (LPV or AgmPyV), chacma baboons (SA12), 20

squirrel monkeys (SquiPyV), and chimpanzees (ChPyV) are all naturally infected 21

with PyV, but no association with any disease in the healthy, immunocompetent host 22

has been reported until now (Johne et al., 2005; Valis et al., 1977; Verschoor et al., 23

2008a; zur Hausen & Gissmann, 1979). The finding of polyomaviruses in apes 24

(chimpanzee), Old World monkeys (baboons, African green monkeys), and New 25

5

World monkeys (squirrel monkey) suggests an extensive spread of PyV in primates. 1

As part of an ongoing project with the aim to investigate polyomavirus infections in 2

nonhuman primates, we here report the detection and genetic characterization of 3

polyomaviruses from Bornean and Sumatran orangutans. 4

A panel of 95 sera from orangutans was screened in an ELISA using virus-5

like particles derived from SV40 VP1, which is the major capsid protein(Verschoor et 6

al., 2008b). A substantial number of orangutan sera were positive for antibodies that 7

reacted with this protein, indicating that orangutans can be infected with SV40, or a 8

related polyomavirus. In total, 43% of the sera (41 out of 95) reacted positively in the 9

test. 10

Next, archival DNA specimens which had been extracted from frozen blood 11

were examined for evidence of polyomavirus infections. All blood samples from 12

Bornean orangutans (Pongo pygmaeus) had previously been acquired from wild-13

caught animals, or animals housed at the Wanariset orangutan rehabilitation centre in 14

Kalimantan, Indonesia, while those from the Sumatran species (Pongo abelii) had 15

been collected from wild-caught and zoo animals. With a broad-spectrum PCR assay, 16

targeted specifically to the VP1 gene(Johne et al., 2005), we analyzed 79 DNA 17

samples obtained from 71 Bornean orangutans and eight Sumatran orangutans. PyV-18

like sequences of approximately 250 bp were detected in eight Bornean orangutans 19

(11.4%), and in three Sumatran animals (37.5%). The PCR fragments were gel-20

purified using the Zymoclean™ Gel DNA Recovery Kit (Zymo Research Corp, 21

Orange, USA), and sequence analysis was performed directly on the purified 22

amplicons (Baseclear BV, Leiden, The Netherlands). Alignment of the sequences 23

revealed a clear distinction between viruses from both species. While the intraspecies 24

nucleotide identity in the Sumatran and Bornean apes appeared limited, the sequences 25

6

derived from each species differed considerably (59-61% sequence identity) 1

(Supplementary figure 1). This finding was confirmed by the amplification and 2

sequence analysis of five full-length VP1 sequences derived from two Sumatran 3

orangutans (SU77 and Pi), and three Bornean individuals (Cl, Ku, and Bo). Viral VP1 4

genes from the Sumatran animals differed 1 nucleotide on a total length of 1,143 nt 5

(0.17%), while two VP1 variants were found in the Bornean orangutan, differing 10 nt 6

from each other (0.9%). The deduced protein sequences were 100 % similar within 7

each orangutan species. The direct comparison of the Sumatran and Bornean VP1 8

genes again showed substantial differences. Alignment analysis revealed 63% 9

nucleotide identity, while the inferred protein sequences were 27% different 10

(Supplementary figures 2 and 3). Sequences have been submitted to EMBL Database 11

under accession numbers FN356900 – FN356910. 12

From two animals representing each species, P. pygmaeus Ora-Bo and P. 13

abelii Ora-Pi, the circular dsDNA genomes were amplified, essentially as described 14

previously (Verschoor et al., 2008a). OraPyV-Bor was amplified with outer primer 15

pair Bora-Fout 5’- AATCCTTACCCAGTTACTTCTTTGCTG-3’ and Bora-Rout 5’-16

TCTGTATTTCATGCTTCCATCATTAG, and the inner set Bora-Fin 17

5’ATGATGCCACCTGTACAGGGACAAC-3’ and Bora-Rin 5’-18

CCACTATGTCACATGCTGACACATAC-3’. To amplify OraPyV-Sum we used to 19

outer primer set Pora-Fout 5’-AATGTCAGAAATCCATACCCTGTAACCTCC-3’ 20

and Pora-Rout 5’-ATACCTTGGCAGACCTCTATATTGCTTAG-3’, and the inner 21

primers Pora-Fin 5’-AGTCTTATGCCTAAAATGCAAGGTCAGCC-3’ and Pora-22

Rin 5’-CTGCACAGCTTAGAAAAAGTCCATCACC-3’. The 5 kb subgenome 23

fragments were cloned in the pJET1.2 vector (CloneJET™ PCR Cloning Kit, 24

Fermentas, Germany), and sequenced by using a primer-walking strategy. Finally, the 25

7

VP1 sequences and the 5 kb PCR fragments were combined to construct complete 1

PyV genomes. 2

The viral genomes differed considerably in length. The virus from Ora-Bo 3

(OraPyV-Bor) measured 5,168 bp, while the Sumatran isolate (OraPyV-Sum) was 4

5,358 bp in length, which is one of the longest primate polyomavirus genomes 5

identified thus far. The genome architecture of the viruses is characteristic of that of 6

the Polyomaviridae with an early region encoding the small T (stAg) and large T 7

antigens (LTAg), and a late region with genes for the VP1, VP2, and VP3 structural 8

proteins. Both viruses lack an agnogene open reading frame (orf) located 5' to the 9

VP2 orf, which has been reported for several primate PyV (Khalili et al., 2005; 10

Verschoor et al., 2008a). 11

The stAg and LTAg are transcribed from a single messenger RNA (mRNA). 12

The stAg orf is relatively short and encodes proteins of 197 and 194 aa. in length for 13

OraPyV-Bor and -Sum, respectively. The LTAgs are transcribed from a spliced 14

mRNA. Putative splice-donor and -acceptor sites were calculated using SpliceView 15

(http://zeus2.itb.cnr.it/~webgene/wwwspliceview.html) (Rogozin & Milanesi, 1997) 16

and GeneSplicer programs 17

(http://www.cbcb.umd.edu/software/GeneSplicer/gene_spl.shtml) (Pertea et al., 18

2001), and the most likely sites were determined on the basis of comparison with 19

known PyV LTAgs. The putative LTags vary significantly in size (693 and 735 aa. for 20

Bor and Sum, respectively), and contain multiple domains common to other PyV 21

LTAgs that are involved in viral DNA replication and host cell transformation (Figure 22

1.) (Sullivan et al., 2000). Both LTAgs possess the conserved J domain (HPDKGG), 23

which is important for efficient DNA replication and transformation. A pRB tumor 24

suppressor-binding motif (LXCXE), which is critical for DNA replication is also 25

8

present, but in case of OraPyV-Sum the leucine (L) residue has been changed into an 1

isoleucine (I). A zinc- binding motif (CX2CX7HX3HX2H) and an ATP-binding motif 2

(GX4GK) are also found in both LTags. Like in other polyomavirus LTags, a 3

conserved binding motif similar to the conserved region 1 of the adenovirus E1A 4

protein ((E/D)X3LX(E/D)LX2(L/I)) is found in the N-terminal part of the LTags 5

(Pipas, 1992). As for the pRB motif, this domain is fully conserved in OraPyV-Bor, 6

but in -Sum there are a glutamine (Q) and a valine (V) instead of a glutamic acid (E) 7

or an aspartic acid (D). Finally, putative nuclear localization sequences (NLS) are also 8

present in both proteins. 9

In contrast to most polyomaviruses, except for the mouse and squirrel monkey 10

PyV, the VP2/3 orfs terminate immediately after the nuclear transport signal (NLS), 11

resulting in relatively short proteins. The VP2 and VP3 antigens of OraPyV-Bor are 12

311 and 190 aa. in length, and those of -Sum 317 and 202 aa. Alignment of the VP1 13

proteins with other published polyomavirus VP1 proteins revealed various regions of 14

relative conservation, alternated by highly variable regions (Figure 2). Most variation 15

is located in the BC-, DE-, EF-, and HI-loops that protrude from the VP1 protein 16

structure and that mediate receptor-binding, and that contain principal antigenic 17

domains (Li et al., 2000; Liddington et al., 1991; Murata et al., 2008; Neu et al., 18

2008; Stehle et al., 1996; Stehle & Harrison, 1997). 19

The excessive variation in the nucleotide sequences of VP1 precludes accurate 20

alignment and phyogenetic analysis. We therefore used the protein alignment to 21

evaluate the genetic relationships of the orangutan polyomaviruses with the other PyV 22

(Figure 3). The orangutan viruses cluster with a heterogeneous group of viruses that 23

have been isolated from bats, bovines, and rodents, and a variety of primate PyV from 24

chimpanzees, African green monkeys, New World squirrel monkeys and humans 25

9

(bootstrap value of 76%). Viruses in this group are well-separated from the avian 1

PyVs, the WU and KI polyomaviruses, and a tight cluster formed by JCV, BKV, 2

SA12 and SV40. 3

Orangutans from the islands of Borneo and Sumatra have diverged 4

approximately 1.1 million years ago, while they have been physically separated for 5

10.000 to 15.000 years ago (Warren et al., 2001). From their position in the tree it 6

becomes less evident that the Bornean and Sumatran PyV have evolved from the 7

same ancestor virus. Instead, a scenario describing two independent transmissions 8

from as yet unknown hosts becomes more likely. In view of this it is interesting to 9

note that PyV have been characterized from rodents and bats. Both groups of animals 10

are found worldwide, together representing over 60% of the mammal species, and are 11

notorious for their role as vectors for zoonotic transmissions of viruses. However, as 12

our current knowledge about polyomaviruses from primates, but also other mammals, 13

is far from complete, and more data concerning the prevalence of polyomaviruses in 14

other species will be needed to properly address this issue. 15

16

ACKNOWLEDGEMENTS 17

This study was supported by the European Community Research Infrastructures 18

Program grant RII3-CT-2006-026155 'European Primate Network: Specialized 19

Infrastructures and Procedures for Biological and Biomedical Research (EUPRIM-20

NET). 21

22

10

References 1

Legends to Figures 2

3

Figure 1 4

Alignment of large T antigens of OraPyV-Bor and OraPyV-Sum. Grey shaded boxes 5

indicate sequence similarities and identities between the proteins. The open boxes 6

designate conserved domains. 7

8

Figure 2 9

Comparison of VP1 proteins from orangutans with VP1 from other mammalian and 10

avian polyomaviruses. Grey-shaded boxes indicate the areas of more than 70% amino 11

acid identity. Underlined regions designate loops in the VP1 proteins. The GenBank 12

accession numbers of the viruses used are: NC_001515 (MuPyV), NC_001663 13

(HaPyV), NC_010277 (McPyV), M30540 (LPV), AY691168 (ChPyV), NC_001442 14

(BoPyV), NC_009951 (SquiPyV), NC_011310 (MyoPyV), NC_007922 (CrPyV), 15

NC_004800 (GoPyV), NC_007923 (FiPyV), AB453166 (BFDPyV), NC_001669 16

(SV40), NC_001699 (JCV), AY614708 (SA12), NC_001538 (BKV), EF127906 17

(KIPyV), and EF444549 (WUPyV). OraPyV-Bor and OraPyV-Sum genomes have 18

been deposited under accession numbers FN356900 and FN356901. 19

20

Figure 3 21

Phylogenetic tree constructed using the VP1 protein of avian and mammalian 22

polyomaviruses. Orangutan PyV are in bold. Mammalian viruses are indicated by 23

grey shading. Sequence alignments were made by using MacVector version 10.6. The 24

GapStreeze program (Los Alamos HIV Sequence Database; http://www.hiv. 25

11

lanl.gov/content/hiv-db/GAPSTREEZE/gap.html) was used to remove columns with a 1

gap tolerance of 20%. Phylogenetic analysis was performed by the neighbor-joining 2

method using the JTT matrix model as implemented in MEGA version 4 (Tamura et 3

al., 2007). Bootstrap values (as % of 1000 re-samplings) are indicated. Bar, 0.2 amino 4

acid residue replacements per site. 5

6 7

Allander, T., Andreasson, K., Gupta, S., Bjerkner, A., Bogdanovic, G., Persson, 8

M. A. A., Dalianis, T., Ramqvist, T. & Andersson, B. (2007). Identification 9

of a third human polyomavirus. Journal of virology 81, 4130-4136. 10

Axthelm, M. K., Koralnik, I. J., Dang, X., Wuthrich, C., Rohne, D., Stillman, I. 11

E. & Letvin, N. L. (2004). Meningoencephalitis and demyelination are 12

pathologic manifestations of primary polyomavirus infection in 13

immunosuppressed rhesus monkeys. Journal of neuropathology and 14

experimental neurology 63, 750-758. 15

Chretien, F., Boche, D., Lorin de la Grandmaison, G., Ereau, T., Mikol, J., 16

Hurtrel, M., Hurtrel, B. & Gray, F. (2000). Progressive multifocal 17

leukoencephalopathy and oligodendroglioma in a monkey co-infected by 18

simian immunodeficiency virus and simian virus 40. Acta neuropathologica 19

100, 332-336. 20

Feng, H., Shuda, M., Chang, Y. & Moore, P. S. (2008). Clonal integration of a 21

polyomavirus in human Merkel cell carcinoma. Science 319, 1096-1100. 22

Gaynor, A. M., Nissen, M. D., Whiley, D. M., Mackay, I. M., Lambert, S. B., Wu, 23

G., Brennan, D. C., Storch, G. A., Sloots, T. P. & Wang, D. (2007). 24

Identification of a novel polyomavirus from patients with acute respiratory 25

tract infections. PLoS pathogens 3, e64. 26

12

Horvath, C. J., Simon, M. A., Bergsagel, D. J., Pauley, D. R., King, N. W., 1

Garcea, R. L. & Ringler, D. J. (1992). Simian virus 40-induced disease in 2

rhesus monkeys with simian acquired immunodeficiency syndrome. The 3

American journal of pathology 140, 1431-1440. 4

Jiang, M., Abend, J. R., Johnson, S. F. & Imperiale, M. J. (2009). The role of 5

polyomaviruses in human disease. Virology 384, 266-273. 6

Johne, R., Enderlein, D., Nieper, H. & Muller, H. (2005). Novel polyomavirus 7

detected in the feces of a chimpanzee by nested broad-spectrum PCR. Journal 8

of virology 79, 3883-3887. 9

Khalili, K., White, M. K., Sawa, H., Nagashima, K. & Safak, M. (2005). The 10

agnoprotein of polyomaviruses: a multifunctional auxiliary protein. J Cell 11

Physiol 204, 1-7. 12

Koliaskina, G. I. (1963). [Virological and Serological Data on Latent Infection 13

Caused by Ob40 (Sv40) Virus in Macaca Rhesus Monkeys.]. Voprosy 14

virusologii 29, 450-452. 15

Li, P. P., Nakanishi, A., Tran, M. A., Salazar, A. M., Liddington, R. C. & 16

Kasamatsu, H. (2000). Role of simian virus 40 Vp1 cysteines in virion 17

infectivity. Journal of virology 74, 11388-11393. 18

Liddington, R. C., Yan, Y., Moulai, J., Sahli, R., Benjamin, T. L. & Harrison, S. 19

C. (1991). Structure of simian virus 40 at 3.8-A resolution. Nature 354, 278-20

284. 21

Martini, F., Corallini, A., Balatti, V., Sabbioni, S., Pancaldi, C. & Tognon, M. 22

(2007). Simian virus 40 in humans. Infectious agents and cancer 2, 13. 23

13

Murata, H., Teferedegne, B., Sheng, L., Lewis, A. M., Jr. & Peden, K. (2008). 1

Identification of a neutralization epitope in the VP1 capsid protein of SV40. 2

Virology 381, 116-122. 3

Neu, U., Woellner, K., Gauglitz, G. & Stehle, T. (2008). Structural basis of GM1 4

ganglioside recognition by simian virus 40. Proc Natl Acad Sci U S A 105, 5

5219-5224. 6

Pertea, M., Lin, X. & Salzberg, S. L. (2001). GeneSplicer: a new computational 7

method for splice site prediction. Nucleic Acids Res 29, 1185-1190. 8

Pipas, J. M. (1992). Common and unique features of T antigens encoded by the 9

polyomavirus group. Journal of virology 66, 3979-3985. 10

Rogozin, I. B. & Milanesi, L. (1997). Analysis of donor splice sites in different 11

eukaryotic organisms. Journal of molecular evolution 45, 50-59. 12

Simon, M. A., Chalifoux, L. V. & Ringler, D. J. (1992). Pathologic features of SIV-13

induced disease and the association of macrophage infection with disease 14

evolution. AIDS Res Hum Retroviruses 8, 327-337. 15

Simon, M. A., Ilyinskii, P. O., Baskin, G. B., Knight, H. Y., Pauley, D. R. & 16

Lackner, A. A. (1999). Association of simian virus 40 with a central nervous 17

system lesion distinct from progressive multifocal leukoencephalopathy in 18

macaques with AIDS. The American journal of pathology 154, 437-446. 19

Stehle, T., Gamblin, S. J., Yan, Y. & Harrison, S. C. (1996). The structure of 20

simian virus 40 refined at 3.1 A resolution. Structure 4, 165-182. 21

Stehle, T. & Harrison, S. C. (1997). High-resolution structure of a polyomavirus 22

VP1-oligosaccharide complex: implications for assembly and receptor 23

binding. Embo J 16, 5139-5148. 24

14

Sullivan, C. S., Cantalupo, P. & Pipas, J. M. (2000). The molecular chaperone 1

activity of simian virus 40 large T antigen is required to disrupt Rb-E2F 2

family complexes by an ATP-dependent mechanism. Mol Cell Biol 20, 6233-3

6243. 4

Sweet, B. H. & Hilleman, M. R. (1960). The vacuolating virus, S.V. 40. Proc Soc 5

Exp Biol Med 105, 420-427. 6

Tamura, K., Dudley, J., Nei, M. & Kumar, S. (2007). MEGA4: Molecular 7

Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular 8

biology and evolution 24, 1596-1599. 9

Valis, J. D., Newell, N., Reissig, M., Malherbe, H., Kaschula, V. R. & Shah, K. V. 10

(1977). Characterization of SA12 as a simian virus 40-related papovavirus of 11

chacma baboons. Infect Immun 18, 247-252. 12

Verschoor, E. J., Groenewoud, M. J., Fagrouch, Z., Kewalapat, A., van Gessel, 13

S., Kik, M. J. & Heeney, J. L. (2008a). Molecular characterization of the first 14

polyomavirus from a New World primate: squirrel monkey polyomavirus. J 15

Gen Virol 89, 130-137. 16

Verschoor, E. J., Niphuis, H., Fagrouch, Z., Christian, P., Sasnauskas, K., 17

Pizarro, M. C. & Heeney, J. L. (2008b). Seroprevalence of SV40-like 18

polyomavirus infections in captive and free-ranging macaque species. J Med 19

Primatol 37, 196-201. 20

Warren, K. S., Verschoor, E. J., Langenhuijzen, S., Heriyanto, Swan, R. A., 21

Vigilant, L. & Heeney, J. L. (2001). Speciation and intrasubspecific variation 22

of Bornean orangutans, Pongo pygmaeus pygmaeus. Molecular biology and 23

evolution 18, 472-480. 24

15

zur Hausen, H. & Gissmann, L. (1979). Lymphotropic papovaviruses isolated from 1

African green monkey and human cells. Med Microbiol Immunol 167, 137-2

153. 3

4

5

J domain

pRb-binding motif

ATP-binding motif

Zinc-binding domain

E1A-binding motif

NLS

OraPyV -BorOraPyV -S umChPyVL PVS quiPyVMePyVS V 4 0S A 1 2BK VJ CVK I PyVWU PyVMuPyVHaPyVMyoPyVBoPyVFiPyVBFDPyVCrPyVGoPyV

20 40 60 80 100 120 140M A P K R K G E G C R - - - - - - - - - K T - - C P - - - - - - - - Q P K P V P K L I V K G N V Q V L G L K - T G P D S V T Q I E C F L N P R M G E N D - - - - - - E H K G W D M D T V K K L L L L T A - W Q - - - - M T T Y R K E I P T Y S T A R I P L P M L N E D L T C N T L M M W E A V S V K T E V V G V S T LM S C K R K R G G D S K T K C T P C P T R K K - C P - - - - - - - - T P A P V P K L I V K G G V E V L D I R - T G P D S I T T I E A F L N P R M G F N - - - - - - - Q S S E Q N Y G F S D K I T T A T S R S E - - - - D K P K Q N T L P C Y S C A R I A L P L L N E D L T C A S L Q M W E A V S V K T E V V G I S S FM A P P R K R A R C V S T P T K V K - C V P K K C P - - - - - - - - V P T P V P K L L V K G G V E V L N I I - T G P D S T T E I E L Y L E P R M G I N S P - T G - - D K K E W - Y G Y S E V I H H A D G - Y D - - - - N N L L N I Q M P Q Y S C A R V Q L P M L N T D M T C D T L M M W E A V S C K T E I V G I G S LM A P Q R K R Q D G - - - - - - - - A - C K K T C P - - - - - - - - I P A P V P R L L V K G G V E V L E V R - T G P D A I T Q I E A Y L N P R M G N N - - - - - - - I P S E D L Y G Y S N S I N T A F S K A S - - - - D T P N K G T L P C Y S V A V I K L P L L N E D M T C D T I L M W E A V S V K T E V V G I S S LM P L K K R S A S A - - - - - - - - - - - - - - - - - - - P R K - - T P Q E V P R L L I S G G V E V L G L K - T G P D S I T E V E A F L N P R M G L E - - - - - - - - N T D N H Y G Y S E N V T V A K K K D D - - - - D K P L K N Q L P C Y S V A K I E L P M L N E D L T N D T I L M W E C I S V K T E V V G I N T LM A P K R K A S S T C K T P K R Q C I P K P G C C P - - - - - - - - N V A S V P K L L V K G G V E V L S V V - T G E D S I T Q I E L Y L N P R M G V N - - - S P D L P T T S N W Y T Y T Y D L Q P K G S - S P - - - - D Q P I K E N L P A Y S V A R V S L P M L N E D I T C D T L Q M W E A I S V K T E V V G I S S LM A P T K R K G S - - - - - - - - - - - - - - - C P G A A P K K P K E P V Q V P K L V I K G G I E V L G V K - T G V D S F T E V E C F L N P Q M G N P - - - - - - - - - D E H Q K G L S K S L A A E K Q F T D - - - - D S P D K E Q L P C Y S V A R I P L P N L N E D L T C G N I L M W E A V T V K T E V I G V T A MM P P A K R K G E - - - - - - - - - - - - - - - C P G A A P K K P K D P V R V P K L L I R G G V E V L E V K - T G V D S I T E V E C F L T P E M G D A - - - - - - - - - N E H L R G Y S Q R V T C D V T F E N - - - - D A P Q K K T L P C Y S T A R I P L P N L N E D L T C G N I L M W E A V T V K T E V L G V T S MM A P T K R K G E - - - - - - - - - - - - - - - C P G A A P K K P K E P V Q V P K L L I K G G V E V L E V K - T G V D A I T E V E C F L N P E M G D P - - - - - - - - - D E N L R G F S L K L S A E N D F S S - - - - D S P E R K M L P C Y S T A R I P L P N L N E D L T C G N L L M W E A V T V Q T E V I G I T S MM A P T K R K G E R - - - - - - - - - - - - - - - - - - - - - - - K D P V Q V P K L L I R G G V E V L E V K - T G V D S I T E V E C F L T P E M G D P - - - - - - - - - D E H L R G F S K S I S I S D T F E S - - - - D S P N R D M L P C Y S V A R I P L P N L N E D L T C G N I L M W E A V T L K T E V I G V T S LM S C T P C R P Q K R L T - - - - R P R S Q - - - - - - - - - - V P R V Q T L A T E V K K G G V E V L A A V P L S E E T E F K V E L F V K P V I G N T T A A Q D G R E P T P H Y W S I S S A I H D K E S G S S I K V E E T P D A D T T V C Y S L A E I A P P D I P N Q V S E C D M K V W E L Y R M E T E L L V V - P LM A C T A K P A C T A K P G R - - S P R S Q - - - - - - - - - - P T R V Q S L P K Q V R K G G V D V L A A V P L S E E T E F K V E L F V K P V I G N A - - - - E G - - T T P H Y W S I S S P L K T A E A A N V - - - - - T P D A D T T V C Y S L S Q V A P P D I P N Q V S E C D M L I W E L Y R M E T E V L V L - P VM A P K R K S G V S - - - K C E T - K C T K A - C P - - - - - - - - R P A P V P K L L I K G G M E V L D L V - T G P D S V T E I E A F L N P R M G Q P P T - P E S L T E G G Q Y Y G W S R G I N L A T S D T E - - - - D S P G N N T L P T W S M A K L Q L P M L N E D L T C D T L Q M W E A V S V K T E V V G S G S LM A P K R K S G A S S - - R C A N - P C G K P - C P - - - - - - - - K P A N V P K L I M R G G V G V L D L V - T G E D S I T Q I E A Y L N P R M G Q N - K - P G T G T D G - Q Y Y G F S Q S I K V N S S L T A - - - - D E V K A N Q L P Y Y S M A K I Q L P T L N E D L T C D T L Q M W E A V S V K T E V V G V G S LM A P K K R S S - - - - - - - - - - - - - - - - - - - - - - - - - - A P S E V P K L I I S G G I E V L S V R - T G P D S I T T I E A Y L N P R M G Q P - - - - - - - - P E S D F Y G F S D N I T V S Q S F E A - - - - D Q P Q I K E I P C Y S M A K I N L P L L N E D I T C D T I L M W E A I S V K T E V V G I S S LM S R M R K N M N - - - - - - - - - - - - - - - - P - P K K G L K G Q P S P V P K L I I K G G I E V L G L R - T G P D S T T T I E L F L N P R M G Q S - - - - - - - - T E S E Y Y G F S D N Q R G S T S R T D - - - - E D L I S A E L P R Y S L G V V Q L P L L N E K L T D D V L L M W E A V S C K T E V V G V N T LM A P K K G N G S - - - - - - - - - - - - - - - C P - - - - - - - - R P Q Q V P K L I V K G G I E V L D V K - T G P D S T T T I E A Y L N P R V G Q N - - - - - - - - - - - - - W G F S T E I T V A S N G Y N - - - - D A P H L T E I P C Y S C A R I S L P L L N E D I T C P T L L M W E A V S V K T E V V G I S S MM S - Q K G K G S - - - - - - - - - - - - - - - C P - - - - - - - - R P Q Q V P R L L V K G G I E V L D V K - S G P D S I T T I E A Y L Q P R P G Q K - - - - - - - - - - - - - N G Y S T V I T V Q A E G Y Q - - - - D A P H S T E V P C Y S C A R I P L P T I N D D I T C P T L L M W E A V S V K T E V V G V S S IM G P K R P R A A - - - - - - - - - - - - - - - - - - - - - - - - - G P S P V P R L L I K G G I E V L E V K - T G P D S F T T I E A Y L N P R M G L D - - - - - - - - - - - - - Y G F S E A I T V A H T L N P - - - - D V P P K K Q L P T Y S C A R I G L P E L N D D M T K L Q I L M W E A V S C K T E V V G I T S LM A P K V K R P R N - - - - - - - - - - - - - - - - - - - - - - - - G P V P V P R L L V K G G I E V L G V R - T G P D S T T T I E A Y L N P R M G T D - - - - - - - - - - - - - N G F S Q A V T V A T S L N P - - - - D V P P K A E L P C Y S C A R I G L P M L N E D M T T P E I L M W E A V S V K T E V V G V T T M

OraPyV -BorOraPyV -S umChPyVL PVS quiPyVMePyVS V 4 0S A 1 2BK VJ CVK I PyVWU PyVMuPyVHaPyVMyoPyVBoPyVFiPyVBFDPyVCrPyVGoPyV

160 180 200 220 240 260 280 300V N C H M P S K R M Y D D Q - - - G I G F P I E G M N F H M F A V G G E P L D L Q F I T S N Y K T E Y - - - - - - - K D K Y V G P D G K S S T Q - - - - Q A L N T A Y K S K L L K D G A F P V E C W C P D P F K N E N S R Y Y G S Y T G G Q S T P P V M Q F T N T V T T V L L D E N W V V G P L C K G D S L Y V S A CV N V H S A A K K E T D S Q - - - G P A L P I E G L N Y H M F A V G G E P L E L Q G L V M N Y E A K Y N E N S - - - S T V I S I K K V T N A D M T A K N Q V L D M A A K A T L D S D A L Y P V E M W C P D P S K N E N T R Y F G S Y T G G L T T P P V L Q F T N T V T T V L L D E N G - V G P L C K G D G L F L S C AI S V H L L E A K M A A K E G G D G P S Q P I E G M N Y H M F A V G G E P L D L Q G I E S N A L T K Y A S A I P P K T I H P N D I A K L A E E E K P Q L Q G L V P K A K A R L D K D G F Y P I E E W S P G P S R N E N S R Y F G S F V G G L N T P P N L Q F T N A V T T V L L D E N G - V G P L C K G D G L F V S A A

V N L H Q G G K Y I Y G S S - - - S G C V P V Q G T T Y H M F A V G G E P L E L Q G L V A S S T A T Y - - - - - - - P D D V V A I K N M K P G N - - - - Q G L D P K A K A L L D K D G K Y P V E V W C P D P S K N E N T R Y Y G S F T G G A T T P P V M Q F T N S V T T V L L D E N G - V G P L C K G D K L F L S C AV N T H S Y G K R D G E - - - - - S P S M P I V G L N Y H F F A V G G E P I E M Q Y I V Q N F Q C D Y - - - - - - - P V G V V A M K P S P L S T - - - - Q V L D P K Q K S K L T G D G I F P I E C W A P D P S K N E N A R Y F A T Y T G G L N T P P V L N I T N T V T T I L L N E N G - V G P L C K G D Q L H I A A DI N V H Y W D M K R V H D Y - - - G A G I P V S G V N Y H M F A I G G E P L D L Q G L V L D Y Q T E Y P K T T N G G P I T I E T V L G R K M T P - - K N Q G L D P Q A K A K L D K D G N Y P I E V W C P D P S K N E N S R Y Y G S I Q T G S Q T P T V L Q F S N T L T T V L L D E N G - V G P L C K G D G L F I S C AL N L H S G T Q K T H E N - - - - G A G K P I Q G S N F H F F A V G G E P L E L Q G V L A N Y R T K Y - - - - - - - P A Q T V T P K N A T V D S - - - - Q Q M N T D H K A V L D K D N A Y P V E C W V P D P S K N E N T R Y F G T Y T G G E N V P P V L H I T N T A T T V L L D E Q G - V G P L C K A D S L Y V S A VL N L H A E A Q K V H D N - - - - G A G R P V Q G S N F H F F S V G G E P L E L Q G V L H N Y R T T Y - - - - - - - P E G T I A P K N P T A E S - - - - Q V M N T E H K A Y L D K T G A Y P V E C W V P D P S R N E N T R Y F G T Y T G G E N V P P V L H V T N T A T T V L L D E Q G - V G P L C K A D S L Y V S A VL N L H A G S Q K V H E H - - - - G G G K P I Q G S N F H F F A V G G E P L E M Q G V L M N Y R S K Y - - - - - - - P D G T I T P K N P T A Q S - - - - Q V M N T D H K A Y L D K N N A Y P V E C W V P D P S R N E N A R Y F G T F T G G E N V P P V L H V T N T A T T V L L D E Q G - V G P L C K A D S L Y V S A AM N V H S N G Q A T H D N - - - - G A G K P V Q G T S F H F F S V G G E A L E L Q G V L F N Y R T K Y - - - - - - - P D G T I F P K N A T V Q S - - - - Q V M N T E H K A Y L D K N K A Y P V E C W V P D P T R N E N T R Y F G T L T G G E N V P P V L H I T N T A T T V L L D E F G - V G P L C K G D N L Y L S A VV N A L G N T N - - - - - - - - - G V V H G L A G T Q L Y F W A V G G Q P L D V V G V T P T D K Y K G - - - - - - - P T T Y T I N P P G D P R T L H V - Y N S N - T P K A K V T S E - R Y S V E S W A P D P S R N D N C R Y F G R V V G G A A T P P V V S Y G N N S T I P L L D E N G - I G I L C L Q G R L Y I T C AL N A G I L T T - - - - - - - - - G G V G G I A G P Q L Y F W A V G G Q P L D V L G L A P T E K Y K G - - - - - - - P A Q Y T V N P K T N G T V P H V - Y S S S E T P R A R V T N E - K Y S I E S W V A D P S R N D N C R Y F G R M V G G A A T P P V V S F S N N S T I P L L D E N G - I G I L C L Q G R L Y I T C AL D V H G F N K P T D T V N T - K G I S T P V E G S Q Y H V F A V G G E P L D L Q G L V T D A R T K Y K E E G - - - V V T I K T I T K K D M V N - - K D Q V L N P I S K A K L D K D G M Y P V E I W H P D P A K N E N T R Y F G N Y T G G T T T P P V L Q F T N T L T T V L L D E N G - V G P L C K G E G L Y L S C VL N V H G Y G S R S E T K D - - I G I S K P V E G T T Y H M F A V G G E P L D L Q G L V Q N Y N A N Y - E A A - - - I V S I K T V T G K A M T S - - T N Q V L D P T A K A K L D K D G R Y P I E I W G P D P S K N E N S R Y Y G N F T G G T G T P P V M Q F T N T L T T V L L D E N G - V G P L C K G D G L Y L S A AC N V H S A V H R E F E N E - - - G A G F P V Q G L N F H F F A V G G E P L E L Q M V V D N H K C R Y - - - - - - - P A G L A A L Q Q A P K T A - - - - Q V L D P Q L K A N L I K D G T F P V E V W C P D P A K N E N S R Y Y G S Y T G G L T T P P V L Q F T N T V T T I L L N E N G - V G P L C K G D G L F L S C AT T C H G Y K K R Y S P S A G - Q G S A M P I E G I N Y H F F A V G G E P L E I Q F I C E D F K A P Y H - - - - - - P T E T I V P P K D K L S N - - K S Q V L D P T L K G I L D K D G V Y P V E C W C P D P S K N E N T R Y F G T Y T G G V S T P P V L Q F T N T V T T I L L D E N G - V G P L C K A D K L Y I T A AL N M H S Y G L R A F G G Y - - - G G G Y T I E G S H I H F F S V G G E P L D L Q G L M Q N H S T Q Y - - - - - - - P S P L V G P K K P D G T T D D S A Q V L N P I Y K A K L D K D A T Y P I E C W C P D P S R N E N S R Y F G S Y T G G V E T P P V L S F T N T S T T I L L D E N G - V G P L C K G D G L Y L S S AL N M H S G A F R A F N G Y - - - G G G F T I C G P R I H F F S V G G E P L D L Q A C M Q N S K T V Y - - - - - - - P A P L I G P G E G E P R E T A - - Q V L D T G Y K A R L D K D G L Y P I E C W C P D P A K N E N T R Y Y G N L T G G P E T P P V L A F T N T T T T I L L D E N G - V G P L C K G D G L F L S A AT N L H S G M K R T V S G Y - - - G A G I P I E G L N L H F F A I G G E P L D L Q G L V Q N Y T A T Y - - - - - - - P E G V I A P K L A D A K A - - - - Q I L D P T L K A K L D K D G T Y P V E C W S P D P S R N E N T R Y F G S Y T G G T Q T P P V L P I G N S V T T V L L D E N G - I G P L C K G D G L Y I S A VC N V H S A S I R M N G G Y - - - G V G R P I E G L N C H M F A V G G E P L E L Q G C V Q N W S T T Y - - - - - - - P S G V V A P P L K D A K A - - - - Q V L D P G L K A R L D K D G A Y P V E C W C P D P S R N E N T R Y F G T Y T G G Q Q T P P V L P F T N T V T T V L L D E N G - V G P L C K G D G L Y L S C V

OraPyV -BorOraPyV -S umChPyVL PVS quiPyVMePyVS V 4 0S A 1 2BK VJ CVK I PyVWU PyVMuPyVHaPyVMyoPyVBoPyVFiPyVBFDPyVCrPyVGoPyV

320 340 360 380 400 420 440 460D I V G F Y V - A N D G S M K Y R G L P R Y F D I T L R K R I V K N P Y P V T S L L N N L F T G M M P P V Q G Q P M D S R L F T Q V E E V R V Y D G N R K - - - D F Q E N P D M V R Y I D Q F - G Q D Q T - - - - - - R P P I R M H - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -D I C G F F T N A D - G S K Q Y R G L P R Y F N V T L R K R N V R N P Y P V T S L L S S L F S S L M P K M Q G Q P M E G - R D A Q V E E V R V Y E G - T E - - - S V T A D P D M R R F I D K F - G Q K Q T - - - - - - D V P Q - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -D I C G V M V K A D N E A I R Y R G L S R Y F K V T L R K R A V K N P Y P I T S L L G S L F T G L M P K M D G Q P M T G - P D A Q V E E V R I Y Q G - K E - - - G L P A D P D M K R Y I D Q F - G Q E Q T - - - - - - P T P T P A A P A A - - - - - - - - - - - - - - - - - - - - - - - - - - -D I A G V H T N Y S - E T Q S W R G L P R Y F N V T L R K R I V K N P Y P V S S L L N S F F S G L M P Q I Q G Q P M E G - V S G Q V E E V R I F E G - T E - - - G L P G D P D L N R Y V D K F - C Q N Q T - - - - - - V L P V S N D M - - - - - - - - - - - - - - - - - - - - - - - - - - - - -D I C G F H I E E D - N K Y K Y R G L P R Y F K L V L R K R V V K N P Y P V S T L L N T L F T Q M S P N V Q G Q D M T - - - - K Q V E E V K I Y E G - T E - - - K L P G D P D M I R F R N Q F - G Q E E T - - - - - - V I P V I T N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -H I V G F L F K T S - G K M A L H G L P R Y F N V T L R K I W V K N P Y P V V N L I N S L F S N L M P K V S G Q P M E G - K D N Q V E E V R I Y E G - S E - - - Q L P G N P D I V R F L D K F - G Q E K T V Y P K P S V A P A A V T F Q S N Q Q D K G K A P L K G P Q K A S Q K E S Q T Q Q L LD I C G L F T N T S - G T Q Q W K G L P R Y F K I T L R K R S V K N P Y P I S F L L S D L I N R R T Q R V D G Q P M I G - M S S Q V E E V R V Y E D - T E - - - E L P G D P D M I R Y I D E F - G Q T T T - - - - - - R M Q - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -D I C G L F T N S S - G T Q Q W R G L S R Y F K I S L R K R S V K N P Y P I S F L L S D L I N R R T T R V Q G Q P M Y G - M N A Q V E E V R V Y D G - T E - - - E L P G D P D M M R Y I D K Y - G Q H Q T - - - - - - K M L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -D I C G L F T N S S - G T Q Q W R G L A R Y F K I R L R K R S V K N P Y P I S F L L S D L I N R R T Q R V D G Q P M Y G - M E S Q V E E V R V F D G - T E - - - R L P G D P D M I R Y I D K Q - G Q L Q T - - - - - - K M L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -D V C G M F T N R S - G S Q Q W R G L S R Y F K V Q L R K R R V K N P Y P I S F L L T D L I N R R T P R V D G Q P M Y G - M D A Q V E E V R V F E G - T E - - - E L P G D P D M M R Y V D K Y - G Q L Q T - - - - - - K M L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -D M L G T A N - - - - - S R I H T P M A R F F R L H F R Q R R V K N P F T M N V L Y K Q V F N R P T E T V D A Q V G V T - E V T M V E E I G P L P P S I Q - - T T L P T S V N L T Q L P R T V T L Q S Q A - - - - - - P L L N T Q Q N S K - - - - - - - - - - - - - - - - - - - - - - - - - - -D L L G V N K - - - - - N R V H T G L S R F F R L H F R Q R R V R N P Y T I N L L Y K Q V F N K P A D D I S G Q L Q V T - E V T M T E E T G P L P P T V E G N V G V P T T S N L S H L P A T V T L Q A T G - - - - - - P I L N T Q G - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -D I M G W R V T R N Y D V H H W R G L P R Y F K I T L R K R W V K N P Y P M A S L I S S L F N N M L P Q V Q G Q P M E G - E N T Q V E E V R V Y D G - T E - - - P V P G D P D M T R Y V D R F - G K T K T - - - - - - V F P G N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -D V M G W Y I E Y N S A G W H W R G L P R Y F N V T L R K R W V K N P Y P V T S L L A S L Y N N M L P T I E G Q P M E G - E A A Q V E E V R I Y E G - T E - - - A V P G D P D V N R F I D K Y - G Q Q H T K P P - - - A K P A N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -D I C G F R T Q D N - K K M Q F R G L P R Y F N V T L R K R L V K N P Y P V S S L L N T L F S D M M P K V Q G Q V M E G - D K A Q V K E V R V Y E G - V E - - - R L P G D P D M I R Y R D K Y - G Q E K T - - - - - - K I P A V A N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -D I C G F L T Q P N - D Q Q Q F R G L P R Y M S V T L R K R L V K N P Y P I A S I L T S L F T N S L P P V T S Q E M D - - - - K Q V E E V R I Y Q G - V E - - - G L P G D P D M V R Y I N K F - G Q E E T - - - - - - C I P K - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -D V A G T F V Q Q T S Q K Q Y W R G L P R Y F N I T L R K R A V K N P Y P V T G L L T S L F N G L M P R M Q G Q S M S G - P T A Q V E E V R V Y E G - M E - - - G L P G D P T M E R H L D Q Q - G Q S H T - - - - - - - N P A Q D - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -D V A G T Y V D Q R G - R Q Y W R G L P R Y F S I Q L R K R N V R N P Y P V S G L L N S L F N D L M P R M T G Q S M Q G - S D A Q V E E V R V Y E G - M E - - - G L A - - P E I D - - - - - - - - M P P K - - - - - - - A P R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -D I V G L Y T A Q Y D E T Q Y F R G L P R Y F S V T L R K R V V K N P Y P I S S L L T S L F G N I L P R M D G Q P M Q G - K D G Q V E E V R I Y Q G - V E - - - P L P G D P T L T R T V D R Y - G Q N E P - - - - - - V L P H R E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -D I C G F Y S E Q Y S Q K Q H F R G L P R Y F S V S L R K R L V R N P Y P V S S L L T S L F T S I I P K M Q G Q P M V G - D N S Q V E E V R V Y E G - V E - - - P L P G D P D I S R F V D R F - G E Q H T - - - - - - H M P S N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

BC-loop

EF-loop

HI-loop

DE-loop

MuPyV

HaPyV

McPyV

LPV

OraPyV-Sum

ChPyV

OraPyV-Bor

BoPyV

SquiPyV

MyoPyV

CrPyV

GoPyV

FiPyV

BFDPyV

SV40

JCV

SA12

BKV

KIPyV

WUPyV

0.2

97

76

71

69

99

9988

100

100

78

Mouse

Hamster

African green monkey

Sumatran orangutan

Western common chimpanzee

Bat

Human

Bornean orangutan

Bovine

Squirrel monkey

Crow

Goose

Finch

Parakeet

Human

Chacma baboon

Human

Human

Human

Macaque