title: Structural determinants of slippage-mediated mutations by human immunodeficiency virus type 1 reverse transcriptase.
abstract: Single-base deletions at nucleotide runs or -1 frameshifting by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) result from template slippage during polymerization. In crystal structures of HIV-1 RT complexed with DNA-DNA template-primer, the palm subdomain in the template cleft contacts the template backbone near the proposed site of slippage via the Glu(89) side chain. We investigated the role of Glu(89) in frameshifting by perturbing this interaction. Substitutions with Asp, Gly, Ala, Val, Ser, Thr, Asn, or Lys were created in recombinant HIV RT, and frameshift frequencies of the resulting mutant RTs were measured. All substitutions led to reduced -1 frameshifting by HIV-1 RT (2-40-fold). Interestingly, the suppression of -1 frameshifting frequently coincided with an enhancement of +1 frameshifting (3-47-fold) suggesting that Glu(89) can influence the slippage of both strands. Glu(89) substitutions also led to reduced rates of dNTP misincorporation that paralleled reductions in -1 frameshifting, suggesting a common structural mechanism for both classes of RT error. Our results reveal a major influence of Glu(89) on slippage-mediated errors and dNTP incorporation fidelity. The crystal structure of HIV-1 RT reveals a salt bridge between Glu(89) and Lys(154), which may facilitate -1 frameshifting; this concept is supported by the observed reduction in -1 frameshifting for K154A and K154R mutants.
authors.affiliation: Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA.
authors.forename: Monica E
authors.initials: ME
authors.lastname: Hamburgh
authors.forename: Kenneth A
authors.initials: KA
authors.lastname: Curr
authors.forename: Melissa
authors.initials: M
authors.lastname: Monaghan
authors.forename: Vasudev R
authors.initials: VR
authors.lastname: Rao
authors.forename: Snehlata
authors.initials: S
authors.lastname: Tripathi
authors.forename: Bradley D
authors.initials: BD
authors.lastname: Preston
authors.forename: Stefan
authors.initials: S
authors.lastname: Sarafianos
authors.forename: Eddy
authors.initials: E
authors.lastname: Arnold
authors.forename: Thomas
authors.initials: T
authors.lastname: Darden
authors.forename: Vinayaka R
authors.initials: VR
authors.lastname: Prasad
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry; D017931:DNA Primers / Q000737:chemistry; D004279:DNA, Viral / Q000235:genetics; D024882:Drug Resistance, Viral; D016368:Frameshift Mutation; D017353:Gene Deletion; D005814:Genes, Viral; D018698:Glutamic Acid / Q000737:chemistry; D054303:HIV Reverse Transcriptase / Q000235:genetics*; D015497:HIV-1 / Q000378:metabolism; D007763:Lac Operon; D008239:Lysine / Q000737:chemistry; D008957:Models, Genetic; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009841:Oligonucleotides / Q000737:chemistry; D011487:Protein Conformation; D012194:RNA-Directed DNA Polymerase; D011994:Recombinant Proteins / Q000737:chemistry; D013698:Templates, Genetic; D014779:Virus Replication
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D009841:Oligonucleotides; D011994:Recombinant Proteins; D018698:Glutamic Acid; D004247:DNA; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D008239:Lysine