Genetic divergence of human immunodeficiency virus type 1 Ethiopian clade C reverse transcriptase (RT) and rapid development of resistance against nonnucleoside inhibitors of RT

Abstract

We sequenced and phylogenetically analyzed the reverse transcriptase (RT) region of five human immunodeficiency virus type 1 isolates from treatment-naive Ethiopian émigrés to Israel. Heteroduplex mobility assays were performed to confirm the clade C status of env genomic regions. The RT sequences showed that the strains clustered phylogenetically with clade C viruses, and a KVEQ-specific motif of silent mutations (amino acids 65, 106, 138, and 161, respectively) at resistance sites was present in the polymerase region of all studied Ethiopian isolates and subtype C reference strains. In addition, many other silent mutations were observed in the clade C viruses at various resistance sites. In general, the Ethiopian isolates were more closely related genotypically to a clade C reference strain from Botswana (southern Africa) than to previously sequenced Ethiopian reference strains. Genotypic analysis showed that two Ethiopian isolates naturally harbored the mutations K70R and G190A associated with resistance to ZDV and nonnucleoside reverse transcriptase inhibitors, respectively. Phenotypic assays revealed that the K70R substitution in this context did not reduce susceptibility to ZDV, whereas the G190A substitution resulted in high-level resistance to nevirapine (NVP). Moreover, variants resistant to NVP, delavirdine (DLV), and efavirenz (EFV) were more rapidly selected at lower drug doses culture with clade C than with clade B wild-type isolates. In the case of subtype C, selection with NVP and/or EFV led to the appearance of several previously unseen mutations in RT, i.e., V106M and S98I, as well as other mutations that have been previously reported (e.g., K103N, V106A, V108I, and Y181C). After selection with DLV, a polymorphism, A62A, initially observed in the Ethiopian isolate 4762, mutated to A62V; the latter is a secondary substitution associated with multidrug resistance against nucleoside RT inhibitors. Phenotypic analysis of clade C mutants selected against NVP, DLV, and EFV revealed broad cross-resistance, particularly in regard to NVP and DLV. These findings suggest that RT genotypic diversity may influence the emergence of drug resistance.

FIG. 1.

Phylogenetic profiles of clinical isolates from five drug-naive Ethiopian individuals. (A) HMA of isolates from five Ethiopian drug-naive patients. HMAs were performed as described in Materials and Methods. Heterocomplexes were formed by mixing the amplified DNA from the viral isolates with the PCR-amplified env sequences of reference strains. A more rapid migration on acrylamide gels indicates the relative degree of similarity between the unknown isolate and the reference strain sequences. For comparative purposes, the results with a wild-type clade B virus (i.e., 4746 virus) is included. (B) Phylogenetic analysis of reverse transcriptase sequences from HIV-1 Ethiopian isolates. Phylogenetic analysis comparing the RT regions of HIV-1 pol genes from five Ethiopian clinical isolates and 20 different reference strains. Tree topology was inferred from the neighbor-joining method and was based on an alignment of 397 nucleotides from which columns containing gaps were deleted. The subtype O prototype isolate was treated as an outgroup.

FIG. 1.

Phylogenetic profiles of clinical isolates from five drug-naive Ethiopian individuals. (A) HMA of isolates from five Ethiopian drug-naive patients. HMAs were performed as described in Materials and Methods. Heterocomplexes were formed by mixing the amplified DNA from the viral isolates with the PCR-amplified env sequences of reference strains. A more rapid migration on acrylamide gels indicates the relative degree of similarity between the unknown isolate and the reference strain sequences. For comparative purposes, the results with a wild-type clade B virus (i.e., 4746 virus) is included. (B) Phylogenetic analysis of reverse transcriptase sequences from HIV-1 Ethiopian isolates. Phylogenetic analysis comparing the RT regions of HIV-1 pol genes from five Ethiopian clinical isolates and 20 different reference strains. Tree topology was inferred from the neighbor-joining method and was based on an alignment of 397 nucleotides from which columns containing gaps were deleted. The subtype O prototype isolate was treated as an outgroup.

FIG. 2.

Progression to resistance against NVP in cell culture. Ethiopian clade C and clade B control viruses were selected for resistance against NVP by growing cells in the presence of increasing concentrations of drugs. Concentrations of NVP selecting for primary resistance mutations and the amount of time required (in weeks) are shown.