Determinants of high titre in recombinant porcine endogenous retroviruses
Pigs are a potential organ source for xenotransplantation, however the production of replication competent virus from endogenous loci possess a threat of a zoonotic infection. Two classes of human tropic replication competent (HTRC) virus are produced PERV-A and -B, while one ecotropic class is also expressed, PERV-C. Miniature swine have been bred to remove all the HTRC loci but human tropic virus was still produced and identified as PERV-A/C recombinants. Some of these AJC recombinants replicate on human cells with increased infectivity compared to PERV- A, one such is PERV-BTI which is approximately 500-fold more infectious. In this study we have confirmed the increased infectivity of PERV-BTI and shown that particles are produced from chronically infected cell lines at similar rate. Comparison of the LTR activity and gag-pol found a 3 to 8-fold increase in PERV- BTI infectivity while comparison of MLV particles pseudotyped with the PERV-Env identified this as the major determinant of the increased infectivity. More detailed investigation identified a substitution of isoleucine to valine at position 140 near the VRC in the RBD and the Proline Rich Region (PRR) as causing the increased Env infectivity. The recombination event resulting in PERV-BTI has produced a novel juxtaposition of the two epitopes, each of which is derived from a different parental sequence, which has resulted in a more infectious virus. Investigations of the biological consequence of the recombination have found that the PERV-C derived PRR increases Env incorporation into budded particles. PERV-BTI was found to have an extended host-range as it can infect cells expressing the human PERV-A receptor (HuPAR-1) whereas PERV-A is effectively restricted to infection on the HuPAR-2 receptor. No difference was identified with the membrane fusion ability of either Env. It is postulated that the BTI Env has increased affinity for the HuPAR receptors, specifically HuPAR-1, caused by the modification of the tertiary structure of the receptor binding domain. And that this is mediated by the PRR, a binding assay will be developed to investigate this. Finally, a DNA shuffling system has been developed to evaluate other AJC recombinants and confirm the relevance of position 140 and the PRR in increased infectivity. This will also facilitate a study on the potential and consequences of the production of a PERV-B/C recombinant.