CHARACTERIZATION OF HIV-1 REVERSE-TRANSCRIPTASE WITH ANTIBODIES INDICATES CONFORMATIONAL DIFFERENCES BETWEEN THE RNASE-H DOMAINS OF P 66 AND P 15

Antibody binding to the p66 and p15 RNaseH regions of HIV-1 reverse tr anscriptase was compared using a polyclonal rabbit immune serum raised against a synthetic peptide from the RNaseH region of reverse transcr iptase (aa 511-527) and six monoclonal antibodies binding to discontin uous epitopes in the RNaseH region of p66. The antigens used in Wester n blot analysis included recombinantly expressed homodimeric p66 diges ted with the HIV-1 protease for generation of the p51 and p15 polypept ides and two different length RNaseH domains expressed as TrpE fusion proteins (aa 410-560 and aa 441-560). The polyclonal rabbit antibody b inding to a continuous epitope recognized both the TrpE-fusion protein s and also the polypeptides p66 and p15 generated by processing of hom odimeric p66 with the viral protease. Two additional cleavage products with estimated molecular weights of 9 and 11 kDa were also detected. The anti-RNaseH MAbs binding to discontinuous epitopes recognized only the RNaseH domain of the p66 polypeptide and the TrpE-RNaseH fusion p rotein when this was expressed together with the C-terminal part of th e polymerase domain. The results indicate conformational differences b etween the RNaseH domain of the p66 subunit and the RNaseH p15 polypep tide.