title: Effects of mutations in the G tract of the human immunodeficiency virus type 1 polypurine tract on virus replication and RNase H cleavage.
abstract: The RNase H cleavages that generate and remove the polypurine tract (PPT) primer during retroviral reverse transcription must be specific in order to create a linear viral DNA that is suitable for integration. Lentiviruses contain a highly conserved sequence consisting of six guanine residues at the 3' end of the PPT (hereafter referred to as the G tract). We introduced mutations into the G tract of a human immunodeficiency virus type 1-based vector and determined the effects on the virus titer and RNase H cleavage specificity. Most mutations in the G tract had little or no effect on the virus titer. Mutations at the second and fifth positions of the G tract increased the proportion of two-long-terminal-repeat (2-LTR) circle junctions with one or two nucleotide insertions. The second and fifth positions of the G tract make specific contacts with amino acids in the RNase H domain that are important for RNase H cleavage specificity. These complementary data define protein-nucleic acid interactions that help control the specificity of RNase H cleavage. When the G-tract mutants were analyzed in a viral background that was deficient in integrase, in most cases the proportion of consensus 2-LTR circle junctions increased. However, in the case of a mutant with Ts at the second and fifth positions of the G tract, the proportion of 2-LTR circle junctions containing the one-nucleotide insertion increased, suggesting that linear viral DNAs containing an extra base are substrates for integration. This result is consistent with the idea that the 3' end-processing reactions of retroviral integrases may help to generate defined ends from a heterogenous population of linear viral DNAs.
authors.affiliation: HIV Drug Resistance Program, NCI-Frederick, P.O. Box B, Building 539, Room 130A, Frederick, MD 21702-1201, USA.
authors.forename: John G
authors.initials: JG
authors.lastname: Julias
authors.forename: Mary Jane
authors.initials: MJ
authors.lastname: McWilliams
authors.forename: Stefan G
authors.initials: SG
authors.lastname: Sarafianos
authors.forename: W Gregory
authors.initials: WG
authors.lastname: Alvord
authors.forename: Eddy
authors.initials: E
authors.lastname: Arnold
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Hughes
mesh_terms: D001483:Base Sequence; D017931:DNA Primers / Q000737:chemistry*; D015497:HIV-1 / Q000235:genetics / Q000502:physiology*; D008969:Molecular Sequence Data; D009154:Mutation; D016914:Ribonuclease H / Q000494:pharmacology*; D020079:Terminal Repeat Sequences; D014779:Virus Replication
chemical_list: D017931:DNA Primers; D016914:Ribonuclease H