Abstract
BackgroundHIV-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.MethodsRecombinant 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 tRNA3Lys 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.ResultsThe 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.ConclusionsOverall 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.
Highlights
Human immunodeficiency virus type 1 (HIV-1) genetic diversity is reflected by the existence of three groups (M, N, and O), of which group M is responsible for greater than 90% of HIV-1 infections
Purification of recombinant HIV-1 reverse transcriptase (RT) from subtype B and subtype C The subtype C HIV-1 RT sequence used in this study differs from consensus subtype B RT by 6.96% of amino acids
This level of variation is in agreement with previous reports showing that HIV-1 RT subtypes differ from one another by ≈ 5%-6% of amino acids [16]
Summary
Human immunodeficiency virus type 1 (HIV-1) genetic diversity is reflected by the existence of three groups (M, N, and O), of which group M is responsible for greater than 90% of HIV-1 infections. Eight NRTIs and four non-nucleoside reverse transcriptase inhibitor (NNRTIs) are approved for treatment of HIV-1 infection. The former are activated by host enzymes to their active triphosphate forms (diphosphate for tenofovir), which bind to the active site of RT, acting as competitive inhibitors of RT and interfering with the addition of incoming nucleosides to growing viral DNA chains. 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
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