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 chara cterization of full-length expression-competent HIV-1 env genes direct ly from patient samples. Design: HIV exists as a quasispecies which ca n be disturbed by in vitro culture, in which numerous members of the p opulation 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 prod ucts play major roles in the initiation and spread of infection we nee d to study genes with open reading frames. Methods: A nested polymeras e chain reaction (PCR) approach has been used to rescue intact (2.6 kb ) env genes, which are cloned into a T7-promoter-containing vector. Ex pression of gp160 in CV-1 cells is detected by Western blot. Expressio n-competent clones are sequenced and resulting sequences used for phyl ogenetic studies. Translation products are analysed in relation to the known immunogenic structure of gp160. Results: From random patient sa mples 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 s ubtypes A, D and an A/D recombinant were recovered. Conclusion: An eff ective procedure is described for the isolation of HIV-1 env genes dir ectly from patient samples, which has worked for A, B and D subtypes t o date. The PCR primers can be utilized with other subtypes with the p ossible 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 produc ts generated may be more relevant to disease progression in vivo and v accine formulations than those obtained from viruses selected in long- term culture.