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

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Methods</p> <p>Recombinant subtype B and C HIV-1 RTs in heterodimeric form were purified from <it>Escherichia coli </it>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^Lysas 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.</p> <p>Results</p> <p>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.</p> <p>Conclusions</p> <p>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.</p

Development of an Allele-Specific PCR for Detection of the K65R Resistance Mutation in Patients Infected with Subtype C Human Immunodeficiency Virus Type 1 ▿ †

The selection of drug-resistant variants of human immunodeficiency virus type 1 (HIV-1) is an impediment to the efficiency of antiretroviral (ARV) therapy. We have developed an allele-specific real-time PCR assay to explore the presence of K65R minority species among treated HIV-1 subtype B and C infections. Thirty HIV-1 subtype C- and 26 subtype B-infected patients lacking K65R as determined by conventional sequencing methods were studied, and viral minority species were found in four HIV-1 subtype C samples

High Prevalence of the K65R Mutation in Human Immunodeficiency Virus Type 1 Subtype C Isolates from Infected Patients in Botswana Treated with Didanosine-Based Regimens

We analyzed the reverse transcriptase genotypes of human immunodeficiency virus type 1 subtype C viruses isolated from 23 patients in Botswana treated with didanosine-based regimens. The K65R mutation was selected either alone or together with the Q151M, S68G, or F116Y substitution in viruses from seven such individuals. The results of in vitro passage experiments were consistent with an apparent increased propensity of subtype C viruses to develop the K65R substitution

Impact of Human Immunodeficiency Virus Type 1 Subtype C on Drug Resistance Mutations in Patients from Botswana Failing a Nelfinavir-Containing Regimen

Among 16 human immunodeficiency virus-infected (subtype C) Batswana patients who failed nelfinavir (NFV)-containing regimens, the most prevalent mutation observed was D30N (54%), followed by L90M (31%). L89I, K20T/I, and E35D polymorphic changes were also identified. These findings suggest that subtype C viruses in Botswana may develop resistance to NFV via subtype-specific pathways