title: Mechanisms that prevent template inactivation by HIV-1 reverse transcriptase RNase H cleavages.
abstract: The RNase H activity of human immunodeficiency virus, type 1 (HIV-1) reverse transcriptase (RT) cleaves the viral genome concomitant with minus strand synthesis. We previously analyzed RT-mediated pausing and RNase H cleavage on a hairpin-containing RNA template system and reported that RT generated 3' end-directed primary and secondary cuts while paused at the base of the hairpin during synthesis. Here, we report that all of the prominent cleavage products observed during primer extension on this template correlated with pause induced cuts. Products that persisted throughout the reaction corresponded to secondary cuts, about eight nucleotides in from the DNA primer terminus. This distance allows little overlap of intact template with the primer terminus. We considered whether secondary cuts could inactivate further synthesis by promoting dissociation of the primer from the template. As anticipated, 3' end-directed secondary cuts decreased primer extendibility. This provides a plausible mechanism to explain the persistence of secondary cut products in our hairpin template system. Improving the efficiency of synthesis by increasing the concentration of dNTPs or addition of nucleocapsid protein (NC) reduced pausing and the generation of pause related secondary cuts on this template. Further studies reveal that 3' end-directed primary and secondary cleavages were also generated when synthesis was stalled by the presence of 3'-azido-3'-deoxythymidine at the primer terminus, possibly contributing to 3'-azido-3'-deoxythymidine inhibition. Considered together, the data reveal a role for NC and other factors that enhance DNA synthesis in the prevention of RNase H cleavages that could be detrimental to viral replication.
authors.affiliation: Department of Biochemistry and Biophysics, University of Rochester, Rochester, New York 14642, USA.
authors.forename: Vandana
authors.initials: V
authors.lastname: Purohit
authors.forename: Bernard P
authors.initials: BP
authors.lastname: Roques
authors.forename: Baek
authors.initials: B
authors.lastname: Kim
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bambara
mesh_terms: D024509:3' Flanking Region; D017931:DNA Primers / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004279:DNA, Viral / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D003854:Deoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D016679:Genome, Viral; D054303:HIV Reverse Transcriptase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D003854:Deoxyribonucleotides; D019590:Nucleocapsid Proteins; D054303:HIV Reverse Transcriptase; D016914:Ribonuclease H