RESUMO
BACKGROUND: HIV-1 subtype B is the most prevalent in developed countries and, consequently, it has been extensively studied. On the other hand, subtype C is the most prevalent worldwide and therefore is a reasonable target for future studies. Here we evaluate the acquisition of resistance and the viability of HIV-1 subtype B and C RT clones from different isolates that were subjected to in vitro selection pressure with zidovudine (ZDV) and lamivudine (3TC). METHODS/PRINCIPAL FINDINGS: MT4 cells were infected with chimeric virus pseudotyped with RT from subtype B and C clones, which were previously subjected to serial passage with increasing concentrations of ZDV and 3TC. The samples collected after each passage were analyzed for the presence of resistance mutations and VL. No differences were found between subtypes B and C in viral load and resistance mutations when these viruses were selected with 3TC. However, the route of mutations and the time to rebound of subtype B and C virus were different when subjected to ZDV treatment. In order to confirm the role of the mutations detected, other clones were generated and subjected to in vitro selection. RT subtype B virus isolates tended to acquire different ZDV resistance mutations (Q151M and D67N or T215Y, D67D/N and F214L) compared to subtype C (D67N, K70R, T215I or T215F). CONCLUSIONS/SIGNIFICANCE: This study suggests that different subtypes have a tendency to react differently to antiretroviral drug selection in vitro. Consequently, the acquisition of resistance in patients undergoing antiretroviral therapy can be dependent on the subtypes composing the viral population.
Assuntos
Fármacos Anti-HIV/farmacologia , Farmacorresistência Viral/genética , Transcriptase Reversa do HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Linhagem Celular , HIV-1/genética , Humanos , Lamivudina/farmacologia , Mutação , Zidovudina/farmacologiaRESUMO
OBJECTIVES: Non-B human immunodeficiency virus (HIV)-1 subtypes possess several amino acid signatures in the viral protease that distinguish them from subtype B, some of which are reported as secondary drug-related mutations. We have previously shown a strong statistical interdependency of residues 71, 89 and 90 in subtype G, but the impact of substitutions on protease inhibitor (PI) resistance is unknown. PATIENTS AND METHODS: We selected subtype G viruses from patients with diverse amino acid combinations at codons 71 (A/T), 74 (T/S), 89 (I/L/M/V) and 90 (L/M). Viral protease genes were inserted into an HIV molecular clone (HXB2). PI drug susceptibilities of chimeric viruses were determined. RESULTS: In isolates displaying 89I/V in combination with A71 or T74, a reversal to subtype G wild-type 89M was observed after growth in the absence of PI. The presence of 71T in one isolate and 74S in another allowed the persistence of 89I. Mutation 90M conferred intermediate but significant degrees of drug resistance to ritonavir and nelfinavir in subtype G viruses. The combination of 71T or 74S, 89I and 90M resulted in higher levels of resistance to those PIs. CONCLUSIONS: Our results point to the hypothesis that 71T or 74S stabilizes 89I in the protease of subtype G, whose association was previously seen by Bayesian network analyses. The association of 89I with 90M may further increase the PI resistance of subtype G viruses when compared with 90M alone, highlighting novel mutational profiles for drug resistance in this non-B subtype.
Assuntos
Substituição de Aminoácidos/genética , Farmacorresistência Viral/genética , Inibidores da Protease de HIV/farmacologia , Protease de HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/genética , Genótipo , Infecções por HIV/virologia , HIV-1/classificação , HIV-1/isolamento & purificação , Humanos , Testes de Sensibilidade Microbiana , Mutação , Análise de Sequência de DNARESUMO
Human immunodeficiency virus type 1 subtype C isolates belong to one of the most prevalent strains circulating worldwide and are responsible for the majority of new infections in the sub-Saharan region and other highly populated areas of the globe. In this work, the impact of drug-resistance mutations in the protease gene of subtype C viruses was analysed and compared with that of subtype B counterparts. A series of recombinant subtype C and B viruses was constructed carrying indinavir (IDV)-resistance mutations (M46V, I54V, V82A and L90M) and their susceptibility to six FDA-approved protease inhibitor compounds (amprenavir, indinavir, lopinavir, ritonavir, saquinavir and nelfinavir) was determined. A different impact of these mutations was found when nelfinavir and lopinavir were tested. The IDV drug-resistance mutations in the subtype C protease backbone were retained for a long period in culture without selective pressure when compared with those in subtype B counterparts in washout experiments.
Assuntos
Inibidores da Protease de HIV/farmacologia , Protease de HIV/genética , HIV-1/efeitos dos fármacos , Indinavir/farmacologia , Farmacorresistência Viral/genética , HIV-1/classificação , HIV-1/fisiologia , Lopinavir , Testes de Sensibilidade Microbiana , Mutação , Nelfinavir/farmacologia , Pirimidinonas/farmacologia , Especificidade da Espécie , Replicação ViralRESUMO
Human immunodeficiency virus type 1 subtype B and C proteases were manipulated to contain 90M, 88D, or 89L, and their in vitro biological properties were studied. We showed that D30N has significantly more impact in subtype C than in subtype B counterparts, accounting for the reported low prevalence of this mutation in patients failing nelfinavir-based regimens.
Assuntos
Inibidores da Protease de HIV/farmacologia , Protease de HIV/genética , HIV-1/efeitos dos fármacos , HIV-1/enzimologia , Mutação/genética , Nelfinavir/farmacologia , Linhagem Celular , Clonagem Molecular , Primers do DNA , Farmacorresistência Viral , Infecções por HIV/virologia , HIV-1/genética , Humanos , Isoenzimas/genética , Fenótipo , RNA Viral/genética , Proteínas Recombinantes de Fusão/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Replicação Viral/efeitos dos fármacosRESUMO
In order to characterize the impact of genetic polymorphisms on the susceptibility of subtype C strains of human immunodeficiency virus type 1 to protease inhibitors (PIs), a subtype B protease that originated from an infectious clone was modified through site-directed mutagenesis to include the amino acid residue signatures of subtype C viruses (I15V, M36I, R41K, H69K, L89 M) with (clone C6) or without (clone C5) an I93L polymorphism present as a molecular signature of the worldwide subtype C protease. Their susceptibilities to commercially available PIs were measured by a recombinant virus phenotyping assay. We could not detect any differences in the 50% inhibitory concentration (IC(50)s) of amprenavir, indinavir, ritonavir, saquinavir, and nelfinavir for the clones analyzed. However, we did observe hypersusceptibility to lopinavir solely in clone C6, which includes the I93L substitution (a 2.6-fold decrease in the IC(50) compared to that for the subtype B reference strain). The same phenotypic behavior was observed for 11 Brazilian and South African clinical isolates tested, in which only subtype C isolates carrying the I93L mutation presented significant hypersusceptibility to lopinavir.
