HIV-1 reverse transcriptase: polymerization properties of the p51 homodimer compared to the p66/p51 heterodimer.

The polymerase activity of the p51 homodimeric form of HIV reverse transcriptase was characterized by activity gel analysis, steady-state kinetic measurements, and processivity assays, and the activity was shown to be highly similar to that for the p66/p51 heterodimer. Recombinant 51- and 66-kDa reverse transcriptase proteins were individually expressed from an HIV-1 Pol gene having an accumulation of natural amino acid mutations compared to the BH10 clone (Ratner et al., 1985). The preparation of an active p51 homodimer critically depended on low temperature during its expression in bacterial cultures. Activity gel analysis demonstrates that refolded p51 protein derived from denatured p66/p51 heterodimer yields an active polymerase. The p51 homodimer has approximately one-half the activity and processivity of the heterodimer, while both enzymes have similar thermostability. Steady-state measurements reveal no significant differences in apparent affinities for substrate or homopolymeric template-primer, suggesting that the subunits in both enzyme forms have similar conformations. Template challenge experiments show that the off-rates for template-primer are lower, but as indicated by primer extension analyses, processivity is less for p51 homodimer. These results show that the RNase H domain is not essential for the assembly of the functional polymerase, but suggest that it enhances processivity.

Two proteins with reverse transcriptase activities associated with hepatitis B virus-like particles.

Recent studies suggest that hepatitis B virus (HBV), despite being a DNA virus, replicates via an RNA intermediate (R. H. Miller, P. L. Marion, and S. W. Robinson, Virology 139:64-72, 1984; J. Summers and W. S. Mason, Cell 29:403-415, 1982). The HBV life cycle is therefore a permuted version of the RNA retroviral life cycle. Sequence homology between retroviral reverse transcriptase and the putative HBV polymerase gene product suggests the presence of an HBV reverse transcriptase (H. Toh, H. Hajashida, and T. Miyata, Nature (London) 305:827-829, 1983). As yet, there has been no direct evidence that reverse transcriptase activity is present in the viral particle. We used activity gel analysis to detect the in situ catalytic activities of DNA polymerases after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our studies demonstrated that HBV-like particles secreted by a differentiated human hepatoma cell line transfected with genomic HBV DNA contain two major polymerase activities which migrate as approximately 90- and approximately 70-kilodalton (kDa) proteins. This demonstrated, for the first time, that HBV-like particles contain a novel DNA polymerase-reverse transcriptase activity. Furthermore, we propose that the 70-kDa reverse transcriptase may be produced by proteolytic self-cleavage of the 90-kDa precursor protein.