Structural determinants of slippage-mediated mutations by human immunodeficiency virus type 1 reverse transcriptase

Monica E. Hamburgh, Kenneth A. Curr, Melissa Monaghan, Vasudev R. Rao, Snehlata Tripathi, Bradley D. Preston, Stefan Sarafianos, Eddy Arnold, Thomas Darden, Vinayaka R. Prasad

Research output: Contribution to journalArticle

16 Citations (Scopus)

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 Glu89 side chain. We investigated the role of Glu89 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 Glu89 can influence the slippage of both strands. Glu89 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 Glu89 on slippage-mediated errors and dNTP incorporation fidelity. The crystal structure of HIV-1 RT reveals a salt bridge between Glu89 and Lys154, which may facilitate -1 frameshifting; this concept is supported by the observed reduction in -1 frameshifting for K154A and K154R mutants.

Original languageEnglish (US)
Pages (from-to)7421-7428
Number of pages8
JournalJournal of Biological Chemistry
Volume281
Issue number11
DOIs
StatePublished - Mar 17 2006

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HIV-1
Mutation
Substitution reactions
Crystal structure
HIV Reverse Transcriptase
DNA Primers
RNA-Directed DNA Polymerase
DNA
Viperidae
Polymerization
Nucleotides
Salts
Human immunodeficiency virus 1 reverse transcriptase

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structural determinants of slippage-mediated mutations by human immunodeficiency virus type 1 reverse transcriptase. / Hamburgh, Monica E.; Curr, Kenneth A.; Monaghan, Melissa; Rao, Vasudev R.; Tripathi, Snehlata; Preston, Bradley D.; Sarafianos, Stefan; Arnold, Eddy; Darden, Thomas; Prasad, Vinayaka R.

In: Journal of Biological Chemistry, Vol. 281, No. 11, 17.03.2006, p. 7421-7428.

Research output: Contribution to journalArticle

Hamburgh, ME, Curr, KA, Monaghan, M, Rao, VR, Tripathi, S, Preston, BD, Sarafianos, S, Arnold, E, Darden, T & Prasad, VR 2006, 'Structural determinants of slippage-mediated mutations by human immunodeficiency virus type 1 reverse transcriptase', Journal of Biological Chemistry, vol. 281, no. 11, pp. 7421-7428. https://doi.org/10.1074/jbc.M511380200
Hamburgh, Monica E. ; Curr, Kenneth A. ; Monaghan, Melissa ; Rao, Vasudev R. ; Tripathi, Snehlata ; Preston, Bradley D. ; Sarafianos, Stefan ; Arnold, Eddy ; Darden, Thomas ; Prasad, Vinayaka R. / Structural determinants of slippage-mediated mutations by human immunodeficiency virus type 1 reverse transcriptase. In: Journal of Biological Chemistry. 2006 ; Vol. 281, No. 11. pp. 7421-7428.
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