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

• Published in: Retrovirology 2010-10, Vol.7 (1), p.80-80, Article 80
• Main Authors: Xu, Hong-Tao, Quan, Yudong, Asahchop, Eugene, Oliveira, Maureen, Moisi, Daniella, Wainberg, Mark A
• Format: Article
• Language: English
• Subjects: Cells, Cultured; Drug resistance; Enzymes; Escherichia coli; HIV; HIV Infections - virology; HIV Reverse Transcriptase - antagonists & inhibitors; HIV Reverse Transcriptase - metabolism; HIV-1 - classification; HIV-1 - drug effects; HIV-1 - enzymology; Human immunodeficiency virus; Human immunodeficiency virus 1; Humans; Medical research; Molecular weight; Plasmids; Recombinant Proteins - antagonists & inhibitors; Recombinant Proteins - metabolism; Reverse Transcriptase Inhibitors - pharmacology; Ribonuclease H - metabolism; RNA-Directed DNA Polymerase - metabolism
• ISSN: 1742-4690; 1742-4690
• DOI: 10.1186/1742-4690-7-80

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. 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. 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. 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.