Altered strand transfer activity of a multiple-drug-resistant human immunodeficiency virus type 1 reverse transcriptase mutant with a dipeptide fingers domain insertion

Laura A. Nguyen, Waaqo Daddacha, Sean Rigby, Robert A. Bambara, Baek Kim

Research output: Contribution to journalArticle

5 Scopus citations

Abstract

Prolonged highly active anti-retroviral therapy with multiple nucleoside reverse transcriptase inhibitors for the treatment of patients infected with human immunodeficiency virus type 1 (HIV-1) can induce the development of an HIV-1 reverse transcriptase (RT) harboring a dipeptide insertion at the RT fingers domain with a background thymidine analog mutation. This mutation renders viral resistance to multiple nucleoside reverse transcriptase inhibitors. We investigated the effect of the dipeptide fingers domain insertion mutation on strand transfer activity using two clinical RT variants isolated during the pre-treatment and post-treatment of an infected patient, termed pre-drug RT without dipeptide insertion and post-drug RT with Ser-Gly insertion, respectively. First, the post-drug RT displayed elevated strand transfer activity compared to the pre-drug RT, with two different RNA templates. Second, the post-drug RT exhibited less RNA template degradation than the pre-drug RT but higher polymerization-dependent RNase H activity. Third, the post-drug RT had a faster association rate (kon) for template binding and a lower equilibrium binding constant Kd for the template, leading to a template binding affinity tighter than that of the pre-drug RT. The k off values for the pre-drug RT and the post-drug RT were similar. Finally, the removal of the dipeptide insertion from the post-drug RT abolished the elevated strand transfer activity and RNase H activity, in addition to the loss of azidothymidine resistance. These biochemical data suggest that the dipeptide insertion elevates strand transfer activity by increasing the interaction of the RT with the RNA donor template, promoting cleavage that generates more invasion sites for the acceptor template during DNA synthesis.

Original languageEnglish (US)
Pages (from-to)248-262
Number of pages15
JournalJournal of Molecular Biology
Volume415
Issue number2
DOIs
StatePublished - Jan 13 2012
Externally publishedYes

Keywords

  • HIV-1
  • fingers domain insertion
  • multiple drug resistance
  • reverse transcriptase
  • strand transfer

ASJC Scopus subject areas

  • Molecular Biology

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