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• W1968675487 abstract "We followed genotypic changes in HIV-1 gp41 during enfuvirtide therapy in a group of antiretroviral-experienced patients. The study confirmed the development of a resistance mutation (N43D), but revealed an interesting compensatory mutation (E137K), which was either present in the pretreatment virus or developed during therapy. In a functional assay of viral envelope fusogenicity we found that the 43D genotype had a 1.4 log10 decrease in infectivity, which could be restored by the introduction of 137K. During treatment of HIV-1 the occurrence of drug-resistant variants is a major obstacle for the long-term success of therapy. It has been established that genotypic resistance mutations are a strong independent predictor of protease inhibitor treatment outcome [1]. Studies have tried to determine the clinical importance of one or more resistance mutations [2]. An inverse correlation between the number of resistance mutations in genes targeted by antiretroviral drugs and viral fitness has been reported [3]. This suggests that continued treatment despite resistance may confer an advantage in patients with limited options in treatment [2]. Enfuvirtide is the first drug to target the HIV-1 viral envelope (Env) protein [4]. The Env protein consists of two functional subunits; the surface subunit gp120 and the transmembrane subunit gp41. Upon receptor attachment, large conformational changes in the gp41 subunit initiate and complete the membrane fusion step. Gp41 contains well-conserved heptad repeat domains (HR1 and HR2) [5]. During fusion, HR1 helices form a trimeric coiled-coil structure upon which the HR2 helices fold back, thereby forcing the membranes into close proximity. Enfuvirtide blocks this step by competitively mimicking the viral interactions between HR1 and HR2 [6]. Genotypic changes in HR1, especially positions 36V/D, 38A/S and 43D/S, have a pronounced impact on enfuvirtide susceptibility [7–9]. To assay the fitness of enfuvirtide-resistant variants we sought to detect the genotypic changes in HR1 as well as HR2. Nine highly antiretroviral-experienced patients were enrolled in the study. One missed follow-up and was withdrawn. From eight patients HIV-1 plasma RNA was isolated and amplified by reverse transcriptase–polymerase chain reaction. Of these, we obtained a sequence from five patients. These patients were sampled at the subsequent visits to the outpatient clinic. Informed consent was obtained from all patients. The study was approved by the Ethics Committee at the County of Aarhus, Denmark. All five patients in the study experienced a temporary decline in plasma HIV-RNA when enfuvirtide was introduced to the antiretroviral regimen. Patient 1 had a viral load decrease of 1.8 log10 initially but rebounded at day 21. Simultaneously, 43D was detected and remained throughout the observation period. At day 48, a 137K mutation appeared in conjunction with 43D. Sequencing of HIV-RNA from patient 2 revealed a N43S mutation after more than 23 months of enfuvirtide therapy. Patients 6 and 7 harboured resistance mutations V38A (day 14) and G36D (day 66), respectively. Patient 9 experienced a decrease (2.5 log10) in viral load upon enfuvirtide initiation, but at day 101 a complete rebound had occurred. Sequence analysis confirmed the interesting mutation constellation 43D–137K. Of interest is the presence of 137K as a natural viral polymorphism in patients 7 and 9 supported by the Los Alamos sequence database [10]. The enfuvirtide-resistant genotype 43D, the single HR2 mutant 137K, and the double mutant 43D–137K were generated in a bicistronic HIV-1 envelope vector (M. Tolstrup, in preparation). MLV HIV-1 envelope pseudotyped viruses were generated by transient transfection of 293 T cells. Titre was measured using flow cytometry. The wild type, the single 137K, and the double mutant 43D–137K had comparable infectivity with 5.3 (± 0.13), 5.2 (± 0.08) and 5.1 (± 0.15) log10 IU/ml, respectively. The 43D single mutants had an infectivity of 3.9 (± 0.01) log10 IU/ml, which was found to be significantly less than wild type (P < 0.0001, Student's t-test). Figure 1 (a) shows the relative infectivity. The decreased infectivity (8% of wild type) of the 43D mutant corresponds to a fitness reduction of 92%, whereas the double mutant 43D–137K and single 137K display infectivity comparable to wild type (87 and 98%, respectively).Fig. 1: Relative infectivity of the four genotypes and inhibitory effect of enfuvirtide. (a) Infectivity of the four genotypes as measured in a single-round assay is displayed as the percentage compared with wild type; the average of four independent experiments. The infectivity of the 43D genotype was statistically significant from all others at P < 0.001. No statistically significant difference was found between any of the remaining three genotypes. (b) Enfuvirtide inhibition of the four genotypes depicted as percentage infectivity compared with no enfuvirtide. Enfuvirtide IC50 values were obtained by fitting a linear regression line to the inhibitory curve. Student's t-test was applied to detect difference relative to wild type. IC50 values; wild type (0.066 μg/ml), 137K (0.085 μg/ml), 43D (1.85 μg/ml), 43D–137K (2.1 μg/ml); the average of three independent experiments.Wild type;43D;137K;43D–137K.The inhibitory effect of enfuvirtide on the four genotypes is depicted in Fig. 1(b). The IC50 values for the single 43D and double 43D–137K genotypes are significantly larger than wild type, with 1.85 and 2.1 μg/ml (P < 0.001, Student's t-test), respectively. This corresponds to an approximately 30-fold increase in resistance. To investigate further the cause of the observed infectivity differences between the genotypes, we investigated the level of HIV-1 Env in the producer cells. First, we verified an equal amount of transcribed RNA between the genotypes. Second, we measured the cell surface expression of the Env protein by flow cytometry. Both assays revealed identical levels across the four genotypes. In this study we found that in patients treated with enfuvirtide the occurrence of 43D in HIV-1 Env gp41 results in an approximately 30-fold increased resistance and reduces the viral fitness by 92%. Importantly, a complete rescue of viral fitness was caused by the 137K genotype that either developed during treatment or was present before treatment. This results in a dominant viral population with wild-type fitness and low susceptibility to enfuvirtide. These data suggest that the negatively charged aspartate is unfavourable in the formation of the coiled-coil structure and structural rearrangements during fusion. This is the first report to test the 43D single mutant because other studies have used primary isolates that have been obscured by the compensatory mutation 137K [7,11]. Considering the clear effect on fusion competence of introducing 137K on top of 43D presented here, we therefore hypothesize that the constraint on forming the fusion-competent coiled-coil lies in the charge repulsion observed in the 43D–137E genotype. Structure modeling predictions recently indicated that mutations in residues 135–145 in HR2 could improve the stability of the coiled-coil [12]. Our finding of a compensatory mutation in HR2 substantiates this. It is therefore highly plausible that other enfuvirtide-resistant variants with mutations at position 36V/E and 38A/E could interact with residues in HR2 in an analogous manner. This furthers the speculation on the clinical implications for the presence of the 137K/Q polymorphism. It is evident that viruses with 137K will have a shorter evolutionary pathway to develop an enfuvirtide-resistant phenotype combined with wild-type fitness. We summarized 63 enfuvirtide patients reported in seven studies [7,8,11,13–15]. The results indicated a disproportion of the 43D resistance mutation based on HR2 genotype [74% (14/19) of 43D carry mutation at 137; P = 0.014 Fisher's exact test]. Along this line it has previously been suggested that the pre-existence of baseline fitness compensatory mutations could influence virological response because of rapid resistance evolution [3]. It will therefore be of interest to investigate the time to virological failure in enfuvirtide-treated patients depending on the HR2 genotypic profile. Sponsorship: M.T. is a scholar at the Research School in Genetic Medicine. This work was supported by the Scandinavian Society for Antimicrobial Chemotherapy." @default.
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• W1968675487 date "2007-02-19" @default.
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• W1968675487 title "Full fusion competence rescue of the enfuvirtide resistant HIV-1 gp41 genotype (43D) by a prevalent polymorphism (137K)" @default.
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• W1968675487 doi "https://doi.org/10.1097/qad.0b013e3280187558" @default.
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