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.
Szilvay
A M
AM
National Centre for Research in Virology, University of Bergen, Norway.
Nornes
S
S
Kannapiran
A
A
Haukanes
B I
BI
Endresen
C
C
Helland
D E
DE
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D001666:Binding Sites, Antibody; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology; D016914:Ribonuclease H / Q000737:chemistry* / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H