Comparative biochemical analysis of recombinant reverse transcriptase enzymes of HIV-1 subtype B and subtype C

Retrovirology. 2010 Oct 7:7:80. doi: 10.1186/1742-4690-7-80.

Abstract

Background: HIV-1 subtype C infections account for over half of global HIV infections, yet the vast focus of HIV-1 research has been on subtype B viruses which represent less than 12% of the global pandemic. Since HIV-1 reverse transcriptase (RT) is a major target of antiviral therapy, and since differential drug resistance pathways have been observed among different HIV subtypes, it is important to study and compare the enzymatic activities of HIV-1 RT derived from each of subtypes B and C as well as to determine the susceptibilities of these enzymes to various RT inhibitors in biochemical assays.

Methods: Recombinant subtype B and C HIV-1 RTs in heterodimeric form were purified from Escherichia coli and enzyme activities were compared in cell-free assays. The efficiency of (-) ssDNA synthesis was measured using gel-based assays with HIV-1 PBS RNA template and tRNA3(Lys) as primer. Processivity was assayed under single-cycle conditions using both homopolymeric and heteropolymeric RNA templates. Intrinsic RNase H activity was compared using 5'-end labeled RNA template annealed to 3'-end recessed DNA primer in a time course study in the presence and absence of a heparin trap. A mis-incorporation assay was used to assess the fidelity of the two RT enzymes. Drug susceptibility assays were performed both in cell-free assays using recombinant enzymes and in cell culture phenotyping assays.

Results: The comparative biochemical analyses of recombinant subtype B and subtype C HIV-1 reverse transcriptase indicate that the two enzymes are very similar biochemically in efficiency of tRNA-primed (-) ssDNA synthesis, processivity, fidelity and RNase H activity, and that both enzymes show similar susceptibilities to commonly used NRTIs and NNRTIs. Cell culture phenotyping assays confirmed these results.

Conclusions: Overall enzyme activity and drug susceptibility of HIV-1 subtype C RT are comparable to those of subtype B RT. The use of RT inhibitors (RTIs) against these two HIV-1 enzymes should have comparable effects.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cells, Cultured
  • HIV Infections / virology
  • HIV Reverse Transcriptase / antagonists & inhibitors*
  • HIV Reverse Transcriptase / metabolism*
  • HIV-1 / classification
  • HIV-1 / drug effects
  • HIV-1 / enzymology*
  • Humans
  • RNA-Directed DNA Polymerase / metabolism
  • Recombinant Proteins / antagonists & inhibitors
  • Recombinant Proteins / metabolism
  • Reverse Transcriptase Inhibitors / pharmacology*
  • Ribonuclease H / metabolism

Substances

  • Recombinant Proteins
  • Reverse Transcriptase Inhibitors
  • reverse transcriptase, Human immunodeficiency virus 1
  • HIV Reverse Transcriptase
  • RNA-Directed DNA Polymerase
  • Ribonuclease H