Porcine endogenous retroviruses (PERV) represent a major safety concern in pig-to-human xenotransplantation. To date, no PERV infection of a xenograft recipient has been recorded; however, PERVs are transmissible to human cells in vitro. Some recombinants of the A and C PERV subgroups are particularly efficient in infection and replication in human cells. Transcription of PERVs has been described in most pig cells, but their sequence and insertion polymorphism in the pig genome impede identification of transcriptionally active or silenced proviral copies. Furthermore, little is known about the epigenetic regulation of PERV transcription. Here, we report on the transcriptional suppression of PERV by DNA methylation in vitro and describe heavy methylation in the majority of PERV 5' long terminal repeats (LTR) in porcine tissues. In contrast, we have detected sparsely methylated or nonmethylated proviruses in the porcine PK15 cells, which express human cell-tropic PERVs. We also demonstrate the resistance of PERV DNA methylation to inhibitors of methylation and deacetylation. Finally, we show that the high permissiveness of various human cell lines to PERV infection coincides with the inability to efficiently silence the PERV proviruses by 5'LTR methylation. In conclusion, we suggest that DNA methylation is involved in PERV regulation, and that only a minor fraction of proviruses are responsible for the PERV RNA expression and porcine cell infectivity.

Department of Cellular and Viral Genetics Institute of Molecular Genetics Academy of Sciences of the Czech Republic Prague Czech Republic

Zobrazit více v PubMed

Tisato V, Cozzi E. 2012. Xenotransplantation: an overview of the field. Methods Mol. Biol. 885:1–16 PubMed

Ekser B, Ezzelarab M, Hara H, van der Windt DJ, Wijkstrom M, Bottino R, Trucco M, Cooper DK. 2012. Clinical xenotransplantation: the next medical revolution? Lancet 379:672–683 PubMed

Scobie L, Takeuchi Y. 2009. Porcine endogenous retrovirus and other viruses in xenotransplantation. Curr. Opin. Organ Transplant. 14:175–179 PubMed

Mueller NJ, Takeuchi Y, Mattiuzzo G, Scobie L. 2011. Microbial safety in xenotransplantation. Curr. Opin. Organ Transplant. 16:201–206 PubMed

Denner J. 2011. Infectious risk in xenotransplantation–what post-transplant screening for the human recipient? Xenotransplantation 18:151–157 PubMed

Denner J, Tönjes RR. 2012. Infection barriers to successful xenotransplantation focusing on porcine endogenous retroviruses. Clin. Microbiol. Rev. 25:318–343 PubMed PMC

Mattiuzzo G, Takeuchi Y, Scobie L. 2012. Potential zoonotic infection of porcine endogenous retroviruses in xenotransplantation. Methods Mol. Biol. 885:263–279 PubMed

Patience C, Takeuchi Y, Weiss RA. 1997. Infection of human cells by an endogenous retrovirus of pigs. Nat. Med. 3:282–286 PubMed

Krach U, Fischer N, Czauderna F, Tönjes RR. 2001. Comparison of replication-competent molecular clones of porcine endogenous retroviruses class A and class B derived from pig and human cells. J. Virol. 75:5465–5472 PubMed PMC

Wilson CA. 2008. Porcine endogenous retroviruses and xenotransplantation. Cell. Mol. Life Sci. 65:3399–3412 PubMed PMC

Bartosch B, Stefanidis D, Myers R, Weiss R, Patience C, Takeuchi Y. 2004. Evidence and consequence of porcine endogenous retrovirus recombination. J. Virol. 78:13880–13890 PubMed PMC

Oldmixon BA, Wood JC, Ericsson TA, Wilson CA, White-Scharf ME, Andersson G, Greenstein JL, Schuurman HJ, Patience C. 2002. Porcine endogenous retrovirus transmission characteristics of an inbred herd of miniature swine. J. Virol. 76:3045–3048 PubMed PMC

Harrison I, Takeuchi Y, Bartosch B, Stoye JP. 2004. Determinants of high titer in recombinant porcine endogenous retroviruses. J. Virol. 78:13871–13879 PubMed PMC

Wood JC, Quinn G, Suling KM, Oldmixon BA, Van Tine BA, Cina R, Arn S, Huang CA, Scobie L, Onions DE, Sachs DH, Schuurman HJ, Fishman JA, Patience C. 2004. Identification of exogenous forms of human-tropic porcine endogenous retrovirus in miniature Swine. J. Virol. 78:2494–2501 PubMed PMC

Patience C, Switzer WM, Takeuchi Y, Griffiths DJ, Goward ME, Heneine W, Stoye JP, Weiss RA. 2001. Multiple groups of novel retroviral genomes in pigs and related species. J. Virol. 75:2771–2775 PubMed PMC

Le Tissier P, Stoye JP, Takeuchi Y, Patience C, Weiss R. 1997. Two sets of human-tropic pig retrovirus. Nature 389:681–682 PubMed

Niebert M, Tönjes RR. 2003. Analyses of prevalence and polymorphisms of six replication-competent and chromosomally assigned porcine endogenous retroviruses in individual pigs and pig subspecies. Virology 313:427–434 PubMed

Groenen MA, Archibald AL, Uenishi H, Tuggle CK, Takeuchi Y, Rothschild MF, Rogel-Gaillard C, Park C, Milan D, Megens H-J, Li S, Larkin DM, Kim H, Frantz LAF, Caccamo M, Ahn H, Aken BL, Anselmo A, Anthon C, Auvil L, Badaoui B, Beattie CW, Bendixen C, Berman D, Blecha F, Blomberg J, Bolund L, Bosse M, Botti S, Buije Z, Bystrom M, Capitanu B, Carvalho-Silva D, Chardon P, Chen C, Cheng R, Choi SH, Chow W, Clark RC, Clee C, Crooijmans RP, Dawson HD, Dehais P, De Sapio F, Dibbits B, Drou N, Du ZQ, Eversole K, Fadista J, Fairley S, Faraut T, Faulkner GJ, Fowler KE, Fredholm M, Fritz E, Gilbert JG, Giuffra E, Gorodkin J, Griffin DK, Harrow JL, Hayward A, Howe K, Hu ZL, Humphray SJ, Hunt T, Hornshøj H, Jeon JT, Jern P, Jones M, Jurka J, Kanamori H, Kapetanovic R, Kim J, Kim JH, Kim KW, Kim TH, Larson G, Lee K, Lee KT, Leggett R, Lewin HA, Li Y, Liu W, Loveland JE, Lu Y, Lunney JK, Ma J, Madsen O, Mann K, Matthews L, McLaren S, Morozumi T, Murtaugh MP, Narayan J, Nguyen DT, Ni P, Oh SJ, Onteru S, Panitz F, Park EW, Park HS, Pascal G, Paudel Y, Perez-Enciso M, Ramirez-Gonzalez R, Reecy JM, Rodriguez-Zas S, Rohrer GA, Rund L, Sang Y, Schachtschneider K, Schraiber JG, Schwartz J, Scobie L, Scott C, Searle S, Servin B, Southey BR, Sperber G, Stadler P, Sweedler JV, Tafer H, Thomsen B, Wali R, Wang J, Wang J, White S, Xu X, Yerle M, Zhang G, Zhang J, Zhang J, Zhao S, Rogers J, Churcher C, Schook LB. 2012. Analyses of pig genomes provide insight into porcine demography and evolution. Nature 491:393–398 PubMed PMC

Karlas A, Irgang M, Votteler J, Specke V, Ozel M, Kurth R, Denner J. 2010. Characterization of a human cell-adapted porcine endogenous retrovirus PERV-A/C. Ann. Transplant. 15:45–54 PubMed

Dieckhoff B, Kessler B, Jobst D, Kues W, Petersen B, Pfeifer A, Kurth R, Niemann H, Wolf E, Denner J. 2009. Distribution and expression of porcine endogenous retroviruses in multi-transgenic pigs generated for xenotransplantation. Xenotransplantation 16:64–73 PubMed

Bittmann I, Mihica D, Plesker R, Denner J. 2012. Expression of porcine endogenous retroviruses (PERV) in different organs of a pig. Virology 433:329–336 PubMed

Scheef G, Fischer N, Krach U, Tönjes RR. 2001. The number of a U3 repeat box acting as an enhancer in long terminal repeats of polytropic replication-competent porcine endogenous retroviruses dynamically fluctuates during serial virus passages in human cells. J. Virol. 75:6933–6940 PubMed PMC

Quinn G, Langford G. 2001. The porcine endogenous retrovirus long terminal repeat contains a single nucleotide polymorphism that confers distinct differences in estrogen receptor binding affinity between PERV A and PERV B/C subtypes. Virology 286:83–90 PubMed

Denner J, Specke V, Thiesen U, Karlas A, Kurth R. 2003. Genetic alterations of the long terminal repeat of an ecotropic porcine endogenous retrovirus during passage in human cells. Virology 314:125–133 PubMed

Scheef G, Fischer N, Flory E, Schmitt I, Tönjes RR. 2002. Transcriptional regulation of porcine endogenous retroviruses released from porcine and infected human cells by heterotrimeric protein complex NF-Y and impact of immunosuppressive drugs. J. Virol. 76:12553–12563 PubMed PMC

Wilson CA, Laeeq S, Ritzhaupt A, Colon-Moran W, Yoshimura FK. 2003. Sequence analysis of porcine endogenous retrovirus long terminal repeats and identification of transcriptional regulatory regions. J. Virol. 77:142–149 PubMed PMC

Lee YJ, Park SH, Bae EH, Jung YT. 2012. Characterization of molecular clones of porcine endogenous retrovirus-A containing different numbers of U3 repeat boxes in the long terminal repeat region. J. Virol. Methods 181:103–108 PubMed

Hejnar J, Plachý J, Geryk J, Machon O, Trejbalová K, Guntaka RV, Svoboda J. 1999. Inhibition of the Rous sarcoma virus long terminal repeat-driven transcription by in vitro methylation: different sensitivity in permissive chicken cells versus mammalian cells. Virology 255:171–181 PubMed

Blazkova J, Trejbalova K, Gondois-Rey F, Halfon P, Philibert P, Guiguen A, Verdin E, Olive D, Van Lint C, Hejnar J, Hirsch I. 2009. CpG methylation controls reactivation of HIV from latency. PLoS Pathog. 5:e1000554.10.1371/journal.ppat.1000554 PubMed DOI PMC

Kauder SE, Bosque A, Lindqvist A, Planelles V, Verdin E. 2009. Epigenetic regulation of HIV-1 latency by cytosine methylation. PLoS Pathog. 5:e1000495.10.1371/journal.ppat.1000495 PubMed DOI PMC

Walsh CP, Chaillet JR, Bestor TH. 1998. Transcription of IAP endogenous retroviruses is constrained by cytosine methylation. Nat. Genet. 20:116–117 PubMed

Lavie L, Kitova M, Maldener E, Meese E, Mayer J. 2005. CpG methylation directly regulates transcriptional activity of the human endogenous retrovirus family HERV-K (HML-2). J. Virol. 79:876–883 PubMed PMC

Matouskova M, Blazkova J, Pajer P, Pavlicek A, Hejnar J. 2006. CpG methylation suppresses transcriptional activity of human syncytin-1 in non-placental tissues. Exp. Cell Res. 312:1011–1020 PubMed

Gimenez J, Montgiraud C, Pichon JP, Bonnaud B, Arsac M, Ruel K, Bouton O, Mallet F. 2010. Custom human endogenous retroviruses dedicated microarray identifies self-induced HERV-W family elements reactivated in testicular cancer upon methylation control. Nucleic Acids Res. 38:2229–2246 PubMed PMC

Leung DC, Dong KB, Maksakova IA, Goyal P, Appanah R, Lee S, Tachibana M, Shinkai Y, Lehnertz B, Mager DL, Rossi F, Lorincz MC. 2011. Lysine methyltransferase G9a is required for de novo DNA methylation and the establishment, but not the maintenance, of proviral silencing. Proc. Natl. Acad. Sci. U. S. A. 108:5718–5723 PubMed PMC

Trejbalova K, Blazkova J, Matouskova M, Kucerova D, Pecnova L, Vernerova Z, Heracek J, Hirsch I, Hejnar J. 2011. Epigenetic regulation of transcription and splicing of syncytins, fusogenic glycoproteins of retroviral origin. Nucleic Acids Res. 39:8728–8739 PubMed PMC

Wolf G, Nielsen AL, Mikkelsen JG, Pedersen FS. 2013. Epigenetic marking and repression of porcine endogenous retroviruses. J. Gen. Virol. 94:960–970 PubMed

Rowe HM, Trono D. 2011. Dynamic control of endogenous retroviruses during development. Virology 411:273–287 PubMed

Park SJ, Huh JW, Kim DS, Ha HS, Jung YD, Ahn K, Oh KB, Park EW, Chang KT, Kim HS. 2010. Analysis of the molecular and regulatory properties of active porcine endogenous retrovirus gamma-1 long terminal repeats in kidney tissues of the NIH-Miniature pig. Mol. Cells 30:319–325 PubMed

Bartosch B, Weiss RA, Takeuchi Y. 2002. PCR-based cloning and immunocytological titration of infectious porcine endogenous retrovirus subgroup A and B. J. Gen. Virol. 83:2231–2240 PubMed

Gustafson KS. 2008. Locked nucleic acids can enhance the analytical performance of quantitative methylation-specific polymerase chain reaction. J. Mol. Diagn. 10:33–42 PubMed PMC

Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−delta delta CT) method. Methods 25:402–408 PubMed

Morandi L, Franceschi E, de Biase D, Marucci G, Tosoni A, Ermani M, Pession A, Tallini G, Brandes A. 2010. Promoter methylation analysis of O6-methylguanine-DNA methyltransferase in glioblastoma: detection by locked nucleic acid based quantitative PCR using an imprinted gene (SNURF) as a reference. BMC Cancer 10:48.10.1186/1471-2407-10-48 PubMed DOI PMC

Kalina J, Senigl F, Micakova A, Mucksova J, Blazkova J, Yan H, Poplstejn M, Hejnar J, Trefil P. 2007. Retrovirus-mediated in vitro gene transfer into chicken male germ line cells. Reproduction 134:445–453 PubMed

Mattiuzzo G, Matouskova M, Takeuchi Y. 2007. Differential resistance to cell entry by porcine endogenous retrovirus subgroup A in rodent species. Retrovirology 4:93. PubMed PMC

Scobie L, Hector R, Matouskova M, Ribes J, Mattiuzzo G, Crossan C, Hejnar J, Takeuchi Y. 2011. PERV-C integration, expression and transmission of PERV to human cells. Xenotransplantation 18:279–280

Parker MD, Chambers PA, Lodge JP, Pratt JR. 2008. Ischemia-reperfusion injury and its influence on the epigenetic modification of the donor kidney genome. Transplantation 86:1818–1823 PubMed

Jonsson SR, LaRue RS, Stenglein MD, Fahrenkrug SC, Andresdottir V, Harris RS. 2007. The restriction of zoonotic PERV transmission by human APOBEC3G. PLoS One 2:e893.10.1371/journal.pone.0000893 PubMed DOI PMC

Dieckhoff B, Petersen B, Kues WA, Kurth R, Niemann H, Denner J. 2008. Knockdown of porcine endogenous retrovirus (PERV) expression by PERV-specific shRNA in transgenic pigs. Xenotransplantation 15:36–45 PubMed

Mattiuzzo G, Ivol S, Takeuchi Y. 2010. Regulation of porcine endogenous retrovirus release by porcine and human tetherins. J. Virol. 84:2618–2622 PubMed PMC

Senigl F, Auxt M, Hejnar J. 2012. Transcriptional provirus silencing as a crosstalk of de novo DNA methylation and epigenomic features at the integration site. Nucleic Acids Res. 40:5298–5312 PubMed PMC

Takeuchi Y, Patience C, Magre S, Weiss RA, Banerjee PT, Le Tissier P, Stoye JP. 1998. Host range and interference studies of three classes of pig endogenous retrovirus. J. Virol. 72:9986–9991 PubMed PMC

Lavillette D, Kabat D. 2004. Porcine endogenous retroviruses infect cells lacking cognate receptors by an alternative pathway: implications for retrovirus evolution and xenotransplantation. J. Virol. 78:8868–8877 PubMed PMC

Akiyoshi DE, Denaro M, Zhu H, Greenstein JL, Banerjee P, Fishman JA. 1998. Identification of a full-length cDNA for an endogenous retrovirus of miniature swine. J. Virol. 72:4503–4507 PubMed PMC

Li E. 2002. Chromatin modification and epigenetic reprogramming in mammalian development. Nat. Rev. Genet. 3:662–673 PubMed

Okahara G, Matsubara S, Oda T, Sugimoto J, Jinno Y, Kanaya F. 2004. Expression analyses of human endogenous retroviruses (HERVs): tissue-specific and developmental stage-dependent expression of HERVs. Genomics 84:982–990 PubMed

Muir A, Lever A, Moffett A. 2004. Expression and functions of human endogenous retroviruses in the placenta: an update. Placenta 25(Suppl A):S16–S25 PubMed

Seifarth W, Frank O, Zeilfelder U, Spiess B, Greenwood AD, Hehlmann R, Leib-Mösch C. 2005. Comprehensive analysis of human endogenous retrovirus transcriptional activity in human tissues with a retrovirus-specific microarray. J. Virol. 79:341–352 PubMed PMC

Cho K, Lee YK, Greenhalgh DG. 2008. Endogenous retroviruses in systemic response to stress signals. Shock 30:105–116 PubMed

Kwon DN, Greenhalgh DG, Cho K. 2009. Cloning and characterization of endogenous retroviruses associated with postinjury stress signals in lymphoid tissues. Shock 32:80–88 PubMed

Tucker AW, Scobie L. 2006. Retroviraemia and low health status in pigs. Vet. Rec. 159:367–368 PubMed

Schmidt P, Forsman A, Andersson G, Blomberg J, Korsgren O. 2005. Pig islet xenotransplantation: activation of porcine endogenous retroviruses in the immediate post-transplantation period. Xenotransplantation 12:450–456 PubMed

Paradis K, Langford G, Long Z, Heneine W, Sandstrom P, Switzer WM, Chapman LE, Lockey C, Onions D, Otto E. 1999. Search for cross-species transmission of porcine endogenous retrovirus in patients treated with living pig tissue. The XEN 111 Study Group. Science 285:1236–1241 PubMed

Di Nicuolo G, D'Alessandro A, Andria B, Scuderi V, Scognamiglio M, Tammaro A, Mancini A, Cozzolino S, Di Florio E, Bracco A, Calise F, Chamuleau RA. 2010. Long-term absence of porcine endogenous retrovirus infection in chronically immunosuppressed patients after treatment with the porcine cell-based Academic Medical Center bioartificial liver. Xenotransplantation 17:431–439 PubMed

Di Nicuolo G, van de Kerkhove MP, Hoekstra R, Beld MG, Amoroso P, Battisti S, Starace M, di Florio E, Scuderi V, Scala S, Bracco A, Mancini A, Chamuleau RA, Calise F. 2005. No evidence of in vitro and in vivo porcine endogenous retrovirus infection after plasmapheresis through the AMC-bioartificial liver. Xenotransplantation 12:286–292 PubMed

Patience C, Patton GS, Takeuchi Y, Weiss RA, McClure MO, Rydberg L, Breimer ME. 1998. No evidence of pig DNA or retroviral infection in patients with short-term extracorporeal connection to pig kidneys. Lancet 352:699–701 PubMed

Levy MF, Argaw T, Wilson CA, Brooks J, Sandstrom P, Merks H, Logan J, Klintmalm G. 2007. No evidence of PERV infection in healthcare workers exposed to transgenic porcine liver extracorporeal support. Xenotransplantation 14:309–315 PubMed

Scobie L, Padler-Karavani V, Le Bas-Bernardet S, Crossan C, Blaha J, Matouskova M, Hector RD, Cozzi E, Vanhove B, Charreau B, Blancho G, Bourdais L, Tallacchini M, Ribes JM, Yu H, Chen X, Kracikova J, Broz L, Hejnar J, Vesely P, Takeuchi Y, Varki A, Soulillou JP. 2013. Long-term IgG response to porcine Neu5Gc antigens without transmission of PERV in burn patients treated with porcine skin xenografts. J. Immunol. [Epub ahead of print.]10.4049/jimmunol.1301195 PubMed DOI PMC

Senigl F, Plachy J, Hejnar J. 2008. The core element of a CpG island protects avian sarcoma and leukosis virus-derived vectors from transcriptional silencing. J. Virol. 82:7818–7827 PubMed PMC

He J, Yang Q, Chang LJ. 2005. Dynamic DNA methylation and histone modifications contribute to lentiviral transgene silencing in murine embryonic carcinoma cells. J. Virol. 79:13497–13508 PubMed PMC

Santoni FA, Hartley O, Luban J. 2010. Deciphering the code for retroviral integration target site selection. PLoS Comput. Biol. 6:e1001008.10.1371/journal.pcbi.1001008 PubMed DOI PMC

Specke V, Rubant S, Denner J. 2001. Productive infection of human primary cells and cell lines with porcine endogenous retroviruses. Virology 285:177–180 PubMed