An efficient method for the rescue and analysis of functional HIV-1 env genes: Evidence for recombination in the vicinity of the tat/rev splice site

Objective: To establish a robust procedure for the isolation and characterization of full-length expression-competent HIV-1 env genes directly from patient samples. Design: HIV exists as a quasispecies which can be disturbed by in vitro culture, in which numerous members of the population are likely to be defective due to the high error rate of the viral reverse transcriptase. Defective viruses are unlikely to play a dominant role in disease progression. Since env gene translation products play major roles in the initiation and spread of infection we need to study genes with open reading frames. Methods: A nested polymerase chain reaction (PCR) approach has been used to rescue intact (2.6 kb) env genes, which are cloned into a T7-promoter-containing vector. Expression of gp160 in CV-1 cells is detected by Western blot. Expression-competent clones are sequenced and resulting sequences used for phylogenetic studies. Translation products are analysed in relation to the known immunogenic structure of gp160. Results: From random patient samples collected in London clinics, only HIV-1 subtype B was found. Two of the samples contained viruses with an additional pair of cysteine residues in their V1 regions. For samples collected in Uganda, HIV-1 subtypes A, D and an A/D recombinant were recovered. Conclusion: An effective procedure is described for the isolation of HIV-1 env genes directly from patient samples, which has worked for A, B and D subtypes to date. The PCR primers can be utilized with other subtypes with the possible exception of subtype O viruses. Phylogenetic analyses revealed the potential importance of a G/C-rich region near the tat/rev splice site as a site of recombination. The sequences and translation products generated may be more relevant to disease progression in vivo and vaccine formulations than those obtained from viruses selected in long-term culture.