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 p 66 and p 15 RNase H regions of HIV-1 reverse transcriptase was compared using a polyclonal rabbit immune serum raised against a synthetic peptide from the RNase H region of reverse transcriptase (aa 511-527) and six monoclonal antibodies binding to discontinuous epitopes in the RNase H region of p 66. The antigens used in Western blot analysis included recombinantly expressed homodimeric p 66 digested with the HIV-1 protease for generation of the p 51 and p 15 polypeptides and two different length RNase H domains expressed as Trp E fusion proteins (aa 410-560 and aa 441-560). The polyclonal rabbit antibody binding to a continuous epitope recognized both the Trp E-fusion proteins and also the polypeptides p 66 and p 15 generated by processing of homodimeric p 66 with the viral protease. Two additional cleavage products with estimated molecular weights of 9 and 11 kDa were also detected. The anti-RNase H MAbs binding to discontinuous epitopes recognized only the RNase H domain of the p 66 polypeptide and the Trp E-RNase H fusion protein when this was expressed together with the C-terminal part of the polymerase domain. The results indicate conformational differences between the RNase H domain of the p 66 subunit and the RNase H p 15 polypeptide.