Comparison of replication-competent molecular clones of porcine endogenous retrovirus class A and class B derived from pig and human cells

Vertically transmitted endogenous retroviruses pose an infectious risk in the course of pig-to-human transplantation of cells, tissues, and organs. Two classes of polytropic type C porcine endogenous retroviruses (PERV) which are infectious for human cells in vitro are known. Recently, we described the cloning and characterization of replication-competent PERV-B sequences from productively infected human cells (F. Czauderna, N. Fischer, K. Boller, R. Kurth, and R. R. Tönjes, J. Virol. 74:4028–4038, 2000). Here, we report the isolation of infectious molecular PERV-A and PERV-B clones from pig cells and compare these proviruses with clones derived from infected human 293 cells. In addition to clone PERV-A(42) derived from 293 cells, four “native” full-length proviral PERV sequences derived from a genomic library of the porcine cell line PK15 were isolated. Three identical class A clones, designated PK15-PERV-A(42), PK15-PERV-A(45), and PK15-PERV-A(58), and one class B clone, PK15-PERV-B(213), were characterized. PK15-PERV-B(213) is highly homologous but distinct from the previously described clone PERV-B(43). PK15-PERV-A(58) demonstrates close homology to PERV-A(42) in env and to PERV-C in long terminal repeat, gag, and pro/pol sequences. All three PERV clones described here were replication competent upon infection of susceptible cell lines. The findings suggest that the pig genome harbors a limited number of infectious PERV-A and -B sequences.A better understanding of the cellular and molecular basis of transplant rejection and the generation of transgenic donor animals bearing genes that mediate protection towards rejection (3, 24, 25) have stimulated approaches to use xenotransplantation, i.e., the therapeutic use of animal cells, tissues, and organs, to overcome the shortage of allogeneic transplants (7). Pigs are preferred as donors for xenotransplants (10).Major concerns have been raised about the possibility of introducing new microbial agents from the animal into the recipient, leading to xenozoonosis (2, 11, 18, 27). Viruses that are germ line transmitted, i.e., porcine endogenous retroviruses (PERV) (21), and DNA viruses that can persist without symptoms in their natural host and are transmitted via intrauterine or transplacentar pathways, e.g., herpesviruses (8), are of particular interest.Approximately 50 integration sites of PERV exist in the genomes of different pig breeds (1, 14, 21), and at least three classes are known (14, 28). Those classes, named PERV-A, -B, and -C (PERV-C is also known as PERV-MSL), display high sequence homology in the genes for group-specific antigens (gag) and polymerase (pol) but differ in the envelope (env) genes which determine the host range. In addition, the existence of multiple other PERV sequences in domestic pigs and their phylogenetic relatives has been described. However, only classes A, B, and C appear to be infectious (22).PERV that are released from different pig cell lines are able to infect human cells in vitro (15, 32, 33). PERV-C (1) is ecotropic compared to PERV-A and PERV-B, which are polytropic as deduced from pseudotype experiments utilizing the corresponding env genes (28).A retrospective investigation of 160 patients who had been treated with porcine cells and tissues showed no evidence for transmission of PERV (20); however, no long-term transplantation of a whole vascularized organ has been attempted so far. In contrast, a recent study utilizing NOD/SCID mice revealed PERV infection in several tissue compartments after transplantation of pig pancreatic islets, indicating the xenozoonotic potential of those retroviruses (31).Recently, we have reported the isolation of replication-competent PERV-B molecular clones derived from human embryonic kidney cells infected with PERV (293 PERV-PK) (5). In this communication, we describe the cloning and characterization of PERV-A and PERV-B proviral sequences derived from the porcine kidney cell line PK15 as well as the characterization of the molecular clone PERV-A(42); isolated from 293 PERV-PK cells (5). [Hereafter, clones derived from cell line 293 PERV-PK will be designated 293-PERV-B(33), 293-PERV-B(43), and 293-PERV-A(42); clones derived from cell line PK15 will be designated PK15-PERV-A(58), and so on.] Three proviruses, one PERV-B and two PERV-A clones, produce infectious and replication-competent particles upon transfection of susceptible cells and subsequent infection of different human cell lines. Thus, this study provides the first functional PERV-A and PERV-B clones isolated directly from the pig genome and allows the comparison of proviral PERV sequences from different origins at the molecular and cellular level.