HIV-1 Naturally-Occurring BF Intersubtype Recombination in Gag-Protease is Associated to Reduction in Viral Replication Capacity

ESPADA CONSTANZA ELEONORA; MELO F; ZANOTTO PM; MARTÍNEZ PERALTA LILIANA; CAROBENE MAURICIO GUILLERMO

Barcelona

Congreso; AIDS Vaccine 2013; 2013

Background: HIV-1 epidemic is moving towards an increasing complexity and prevalence of recombinant forms. In addition, HIV-1 recombinants might exhibit variable biological behaviors, and different responses to immunologic and therapeutic interventions. gag and pol are closely related genes since both polyproteins precursors are cleaved by the viral protease (PR). In addition, the gag region is not only the target of the host cellular immunologic response but also mutations in this region may affect the response to protease inhibitors. So far, there are no in vitro studies evaluating the replication capacity (RC) of HIV-1 non-B subtypes. In the present study, we compare Gag-Protease-associated RC between pure subtype B vs. BF recombinants variants highly represented in South America. Methods: HIV-1 CRF12_BF-like and CRF28_BF-like gag-protease sequences from two subjects from Buenos Aires (S1 and S2, respectively) with chronic infection were used to construct a chimeric NL4-3 strain. This NL4-3 strain was modified using the unique restriction sites BssHII (nucleotide [nt] 703) and BstBI (nt 2548) to generate 3 variants: a) pNL-Bst (subtype B), where a BstBI restriction site has been introduced, b) pNL-CRF12_BF-like (CRF12_BF-like), carrying the proviral gag-protease sequence derived from S1 and c) pNL-CRF28_BF-like (CRF28_BF-like), carrying the gag-protease sequence derived from S2. The resulting chimeras were sequenced to ensure that BF recombinant gag-protease sequences and restriction sites were intact. Viruses were generated by transfection of HEK293T cells using Lipofectamine 2000 (Invitrogen). MT2 cell line was infected at a MOI of 0.1 and viral replication capacity was examined by quantifying p24 antigen (ag) in cell culture supernatants 24, 48, 72 and 96 h post-infection (Murex, Abbot). Results: At all time points, except at 24 h, infection with NL4-3 wt resulted in a significantly higher p24 ag concentration in culture supernatant when compared with that of the NL-Bst counterpart (0.27, 3.52, 296 and 2354 vs. 0.55, 1.7, 41 and 1672 ng/ml, respectively, p