Effects of mutations in the G tract of the human immunodeficiency virus type 1 polypurine tract on virus replication and RNase H cleavage

John G. Julias, Mary Jane McWilliams, Stefan G. Sarafianos, W. Gregory Alvord, Eddy Arnold, Stephen H. Hughes

Research output: Contribution to journalArticlepeer-review

29 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)13315-13324
Number of pages10
JournalJournal of virology
Volume78
Issue number23
DOIs
StatePublished - Dec 2004

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Immunology
  • Insect Science
  • Virology

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