Mapping of a neutralizing epitope in the surface envelope protein of porcine endogenous retrovirus subgroup B

Abstract

Pigs are thought to be the most suitable donor animal for xenotransplantation. However, pigs harbour potentially hazardous infectious agents, termed porcine endogenous retroviruses (PERVs), in their genome. In this study, we generated a mAb against PERV-B surface (SU) envelope protein (Env), designated KRT1. KRT1 binding was detected by an indirect immunofluorescence assay and flow cytometric analysis on cells infected with PERV-B. KRT1 neutralized PERV-B pseudotype virus and specifically recognized PERV-B SU Env, but not PERV-A SU Env by immunoblotting analysis. The peptide-ELISA revealed that KRT1 recognized a linear peptide sequence (ALEPPHNLPVP) residing in a proline-rich region that is one of the subdomains of SU Env. In conclusion, the KRT1 antibody will serve as a useful tool for the study of PERV-B and, more importantly, it may provide new protective strategies against PERV-B infection in xenotransplantation.Xenotransplantation is a solution to the shortage of human donors, and pigs are considered as the most suitable donor animal (Blusch et al., 2002). Although specific-pathogenfree pigs will be used for xenotransplantation, all pigs possess potentially hazardous infectious viral agents, named porcine endogenous retroviruses (PERVs), in their genome (Blusch et al., 2002). PERVs are members of the genus Gammaretrovirus and are divided into three subgroups (termed PERV-A, PERV-B and PERV-C) based on their different receptor usages (Le Tissier et al., 1997;Patience et al., 1997;Denner, 2008). PERV-A and PERV-B may transmit from pigs to humans, because humans have functional receptors for both PERV-A and PERV-B (Takeuchi et al., 1998;Denner, 2008). Although the risks caused by the infection with PERVs have not been fully assessed at present, they may potentially cause leukaemia, anaemia, immunodeficiency and cancers like other gammaretroviruses (Kawakami et al., 1980;Wilson et al., 2000;Denner, 2008). Gammaretroviral envelope (Env) consists of surface (SU) and transmembrane (TM) subunits, which interact via a disulfide bond between SU CX 2 C-and TM CX 7 C-motifs (Wallin et al., 2004). PERV SU Env consists of four subdomains, known as variable regions A and B (VRA and VRB), proline-rich region (PRR) and the C-terminal region (CR). Of these domains, VRA, VRB and PRR are divergent between subgroups (Le Tissier et al., 1997;Watanabe et al., 2005). In ecotropic and amphotropic murine leukaemia viruses, the receptor-binding domain needed for binding with host receptors spans VRA and VRB. In PERV-A and PERV-B, PRR might be involved in the proper binding to their receptors together with VRA and VRB (Watanabe et al., 2005). Recently, it was revealed that CR was also important for the infectivities of PERV-A and PERV-C (Gemeniano et al., 2006;Argaw et al., 2008).The information about the antigenicity of PERV Env is quite limited (Chiang et al., 2007) and the antigenic property of PERV-B Env has not yet been reported. In this study, we generated a neutralizing mAb against PERV-B and determined the neutralizing epitop...

Methods and Tools for Detection and Evaluation of the Risks of Porcine Endogenous Retrovirus in Porcine to Human Xenotransplantation

“…More recent developments in this field include new monoclonal antibodies with neutralizing activity. Nakaya, et al, have described a monoclonal antibody that potently neutralizes PERV-B, but not PERV-A (Nakaya, Hoshino et al 2011). Interestingly, the epitope for this neutralizing antibody maps to the proline-rich region, a finding that corresponds to previously described observations that correlate human cell infection with determinants within the PRR (see section 2.2.3).…”

“…A monoclonal antibody specific to the PERV gag protien was shown useful for detecting infection (Chiang, Chang et al 2005) . Recently, a monoclonal antibody directed against the PERV-B envelope protein detected a specific peptide sequence in the proline rich region of SU as an epitope for PERV infection (Nakaya, Hoshino et al 2011) . These monoclonal antibodies may prove useful for screening for PERV antigen, although to date no xenotransplantation studies have used the antibody on porcine or human xenotransplant recipient samples.…”

Detailed Mapping of Determinants within the Porcine Endogenous Retrovirus Envelope Surface Unit Identifies Critical Residues for Human Cell Infection within the Proline-Rich Region

“…Of note, insertion of tags tolerated in other gammaretroviruses into the PERV PRR renders vectors carrying the tagged envelopes noninfectious (C. Wilson, data not shown), further supporting the distinct structural impact of the PRR on envelope function. Recently, it was reported that a neutralizing antibody to PERV-B recognizes a peptide within the PRR (15), suggesting that the region is exposed to antibodies. Interestingly, the particular epitope identified is absent from both PERV-A and PERV-C (15).…”

“…Recently, it was reported that a neutralizing antibody to PERV-B recognizes a peptide within the PRR (15), suggesting that the region is exposed to antibodies. Interestingly, the particular epitope identified is absent from both PERV-A and PERV-C (15).…”

Dysfunction of Bovine Endogenous Retrovirus K2 Envelope Glycoprotein Is Related to Unsuccessful Intracellular Trafficking

“…IFA was applied as previously described, with slight modifications (26). Briefly, cells transfected with indicated plasmids were fixed with 4% PFA on ice for 30 min, followed by blocking in blocking buffer at room temperature (RT) for 1 h, reaction with anti-FLAG M2 diluted at 1:500 in antibody dilution buffer (PBS containing 1% BSA) at RT for 2 h, staining with anti-mouse IgG conjugated with Alexa Fluor 594 (Invitrogen) diluted at 1:1,500 in antibody dilution buffer at RT for 1 h, and staining with 1 ng/ml 4=,6-diamidino-2-phenylindole (DAPI) solution at RT for 5 min.…”