Comparison of two commercial assays for the detection of insertion mutations of HIV-1 reverse transcriptase.

Insertions in the beta3-beta4 fingers subdomain of HIV-1 reverse transcriptase (RT) confer cross-resistance to various nucleoside analogs. The detection of these rearrangements in the region of codons 67-70 of RT is of primary importance for adapting and optimizing combination treatment regimen. Recent reports suggest that some genotyping techniques based on the hybridization of oligonucleotide probes may fail to detect insertion mutants of HIV-1 RT. In the present study, we have evaluated the efficiency of two commercial kits TruGene (based on Dye Primer sequencing) and Viroseq (Big Dye Terminator technique) for the detection of insertion mutations. The data were compared with an in-house dRhodamine sequencing method. Overall, all these cycle sequencing techniques were operative in the detection of insertion mutants. The best peak homogeneity in the electrophoregrams was observed with the Dye primer technique. However, specific compression artifacts were frequently encountered with this technique, rendering ambiguous the interpretation of the electrophoregrams in several regions of the sequence. This shortcoming did not occur with dRhodamine Dye terminator or Bigdye terminator cycle sequencing. In any case, a manual inspection of the electrophoregrams is highly recommended, for all types of cycle sequencing techniques, especially for detecting new mutational patterns of the RT and protease genes. Finally, some specific problems were encountered with the softwares provided with both Trugene and Viroseq kits.

Use of drug resistance sequence data for the systematic detection of non-B human immunodeficiency virus type 1 (HIV-1) subtypes: how to create a sentinel site for monitoring the genetic diversity of HIV-1 at a country scale.

To assess the molecular epidemiology of human immunodeficiency virus type 1 (HIV-1), a screening method was developed for identification of non-B subtypes from sequence data obtained for resistance testing. The method is based on the evaluation of the percentage of divergence of a given sequence from the reference B subtype HXB2. Analysis of 1720 reverse-transcriptase (RT) and 1824 protease sequences stored in a database allowed for the determination of a threshold level of divergence from HXB2 above which a non-B subtype could be unambiguously characterized regardless of the pattern of resistance mutations (>8.6% for RT; >10.8% for protease). This conclusion was validated by phylogenetic analysis of RT, protease, and env genes. Overall, 72 (4.2%) and 73 (4.0%) non-B sequences were identified in the RT and protease coding regions, respectively. This method allows for the rapid detection of non-B subtypes among retrospective, recent, and future RT and/or protease sequence databases.

Mutation L210W of HIV-1 reverse transcriptase in patients receiving combination therapy. Incidence, association with other mutations, and effects on the structure of mutated reverse transcriptase.

Mutation L210W of HIV-1 reverse transcriptase (RT) is one of the six main mutations that confer in vivo resistance to zidovudine. Surprisingly, this mutation has received scant appraisal and its contribution to the genotypic resistance to nucleoside analogs is not well understood. The aim of this study was: (1) to study the frequency of mutation L210W in a large collection of HIV-1 sequences (2,049 samples, including 395 DNA and 1,654 RNA sequences) from patients receiving combination therapy, and (2) to analyze its association with the other mutations that confer resistance to zidovudine. A mutation at codon 210 (mainly L210W) was found in 647 (32%) of the 2,049 sequences analyzed. Only 43 (<7%) of these 647 genomes were also mutated at codon 70 (p < 10(-5)). In contrast, 98% of these 647 sequences were also mutated at codon 215 (essentially T215Y/F), and 94% at codon 41 (mainly M41L). These data showing a close association between L210W, T215Y/F, and M41L, and a mutual exclusion between K70R and L210W, were confirmed by analyzing the sequences stored in the HIV-1 sequences available through the Stanford HIV RT and Protease Database. Follow-up studies demonstrated that L210W appeared always after T215Y/F. This observation is consistent with crystallographic studies which suggested that the aromatic side chain of Trp 210 could stabilize the interaction of Phe/Tyr215 with the dNTP-binding pocket. This molecular cross-talk between amino acid chains occurs nearby the conserved Asp113 residue. Since the lateral chain of Arg70 may also interact with Asp113, this is likely to create a sterical hindrance around this residue. Thus, the R-->K reversion of codon 70 may represent a compensatory mechanism allowing a functional rearrangement of the dNTP-binding pocket in the mutated RT.

Mutation patterns of the reverse transcriptase and protease genes in human immunodeficiency virus type 1-infected patients undergoing combination therapy: survey of 787 sequences.

The aim of the present study was to evaluate the resistance-associated mutations in 302 human immunodeficiency virus type 1 (HIV-1)-infected patients receiving combination therapy and monitored in Marseille, France, hospitals from January 1997 to June 1998. In the reverse transcriptase (RT) gene, the most frequent mutations were found at codons 215 (53%), 41 (34%), and 67, 70, 184, and 210 (>20%). One deletion and two insertions in the beta3-beta4 hairpin loop of the finger subdomain (codon 69) were detected. Interesting associations and/or exclusions of specific mutations were observed. In 96% of RT genes, a mutation at codon 70 (most frequently, K70R) was associated with a wild-type genotype at position 210 (P < 10(-5)). Similarly, a mutation at codon 210 (most frequently, L210W) was generally associated with mutations at codons 41 (92%) and 215 (96%) but not at codon 219 (16%) or codon 70 (4%) (P < 10(-5)). In the protease gene, the most prevalent mutations were at codons 63 (84%), followed by codons 10, 36, 71, 77, and 93 (ca. 20%). As for RT, pairwise associations of mutations were observed. Analysis of the mutation patterns for patients with undetectable HIV-1 loads revealed a high proportion (65%) of wild-type RT genotypes but only 18% wild-type protease genotypes. For patients with high viral loads (>100,000 copies/ml), more than 50% of the RT and protease genes displayed three or more mutations. The significant correlation between the level of viremia in plasma and the number of resistance mutations in the protease (P = 0.007) and RT (P = 0.00078) genes strengthens the importance of defining the genotype of the predominant HIV-1 quasispecies before initiating antiretroviral therapy.