Establishment and characterization of molecular clones of porcine endogenous retroviruses replicating on human cells

The use of pig xenografts is being considered to alleviate the shortage of allogeneic organs for transplantation. Ln addition to the problems overcomi ng immunological and physiological barriers, the existence of numerous porc ine microorganisms poses the risk of initiating a xenozoonosis. Recently, d ifferent classes of type C porcine endogenous retoviruses (PERV) which are infectious for human cells in vitro have been partially described. We there fore examined whether completely intact proviruses exist that produce infec tious and replication-competent virions. Several proviral PERV sequences we re cloned and characterized, One molecular PERV class B clone, PERV-B(43), generated infectious particles after transfection into human 293 cells. A s econd clone, PERV-B(33), which was highly homologous to PERV-B(43), showed a G-to-ii mutation in the first start codon (Met to Ile) of the env gene, p reventing this provirus from replicating. However, a genetic recombinant, P ERV-B(33)/ATG, carrying a restored env start codon, became infectious and c ould be serially passaged on 293 cells similar to virus clone PERV-B(43), P ERV protein expression was detected 24 to 48 h posttransfection (p.t.) usin g cross-reacting antiserum, and reverse transcriptase activity was found at 12 to 14 days p.t. The transcriptional start and stop sites as well as the splice donor and splice acceptor sites of PERV mRNA were mapped, yielding a subgenomic cnv transcript of 3.1 kb, PERV-B(33 and PERV-B(43) differ in t he number of copies of a 39-bp segment in the U3 region of the long termina l repeat. Strategies to identify and to specifically suppress or eliminate those proviruses from the pig genome might help in the production of PERV-f ree animals.