Identification and characterization of HIV-specific RNase H by monoclonal antibody.

Abstract

Human immune deficiency virus (HIV) replicates by conversion of the RNA genome into the double‐stranded DNA provirus. The reverse transcriptase is not the only enzymatic function crucial in DNA‐provirus synthesis. A viral‐coded RNase H activity which specifically degrades RNA in RNA‐DNA hybrids has been shown to be essential as well. Here we demonstrate that the HIV‐reverse transcriptase which consists of a two‐polypeptide complex, p66 and p51, copurifies with an RNase H activity which exhibits properties of a processive exonuclease. Only the p66 molecule, not p51, is active as polymerase as evidenced by activated gel analysis. p66 exhibits RNase H activity when precipitated as immune complex by a monoclonal antibody raised against a bacterially expressed carboxy‐terminal portion of p66. The monoclonal antibody which does not interfere with enzyme activity also precipitates a second population of molecules with RNase H activity which is of low mol. wt, p15. This RNase H appears therefore to be derived from the carboxy terminus of p66 during processing to the p51 polypeptide. It exhibits low template‐binding ability and is of a non‐processing mode of action which may be due to the absence of the reverse transcriptase domain. These results lend experimental support to the hypothesis that the RNase H gene maps at the carboxy terminus of the reverse transcriptase. Since both RNase H populations are virus‐coded they may be essential for retrovirus replication in general and useful targets for chemotherapeutic agents.

Immunologic and proteolytic analysis of HIV-1 reverse transcriptase structure

“…In addition, a 15-kDa fragment was not detected in disrupted virions by immunoblotting. Hansen et al (1988) have reported detecting a 15-kDa RNase H activity in immunoprecipitates from extracts of HIV-l virions. We are in the process of investigating this apparent discrepancy.…”

Dynamic binding orientations direct activity of HIV reverse transcriptase

“…First, RT uses the viral RNA genome as a template and a host-cell transfer RNA as a primer to synthesize a minus-strand DNA, producing an RNA-DNA hybrid [5][6][7] . This duplex becomes the substrate of the RNase H domain of RT, which cleaves the RNA strand at numerous points, leaving behind short RNA segments hybridized to the nascent DNA [8][9][10] . Among these RNAs, two specific purine-rich sequences, known as the polypurine tracts (PPTs), serve as unique primers to initiate the synthesis of plus-strand DNA [11][12][13] , thereby creating the double-stranded DNA viral genome.…”

Human immunodeficiency virus‐1 reverse transcriptase heterodimer stability

“…The native enzyme has DNA polymerase and RNase H activities and exists as a heterodimer containing polypeptides of approximately 66 and 5 1 kDa (DiMarzo-Veronese et al, 1986; Lightfoote et al, 1986). The p66 subunit contains both the DNA polymerase and RNase H functional domains, whereas the smaller polypeptide, p51, lacks the carboxyl-terminal RNase H domain (Hansen et al, 1988). RT and the viral DNA integration protein are synthesized as part of the gag-pol precursor polyprotein (for review see, *This paper is dedicated to the memory of Dean L. Richard.…”