Highly active antiretroviral therapy with the three classes of antiretroviral drugs, namely nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTI and NNRTI) and protease inhibitors (PI) has helped to stabilize disease progression of predominantly subtype B infections in Western countries . However, global epidemics with group M (non-B, A through J) and O clades in resource-poor nations are expanding and it is essential to develop effective management strategies for non-B clade infections [2,3]. Most commercialized drugs have been studied against clade B viruses and clade B reverse transcriptase (RT) and protease. To date, limited information is available on other subtypes in regard to virological or treatment response [3,4].
Efavirenz (EFV) given once daily in combination with two NRTI possesses potent antiviral activity . Recent studies, including preliminary analysis of the ACTG 384 study suggest that a first-line regimen of choice is zidovudine (ZDV)/lamivudine (3TC)/EFV . We have observed that resistance to EFV in clade C viruses can develop in vitro as a result of a V106M mutation that arises due to a natural GTG polymorphism at codon 106 in clade C variants, that is distinct from the nevirapine (NVP)-associated mutation at position 106 (i.e., V106A) commonly seen in clade B viruses [2,7]. The GTG polymorphism is largely limited to clade C viruses, yielding V106M in EFV-selected cells, while the naturally occurring codon in clade B viruses is GTA. We now show the prevalence of this GTG polymorphism in clinical isolates, the in vitro and in vivo evolution of EFV resistance in isolates carrying this polymorphism, and the consequences of V106M on responses to other NNRTI. We have also confirmed the significance of the V106M mutation by site-directed mutagenesis.
Subjects and methods
Study subjects and viral isolates
The clinical isolates in this study include subtype A (n = 29), B (n = 440), C (n = 39), D (n = 4), A/E (n = 3) and G (n = 9) variants. The RT region of plasma viral RNA from clinical samples was genotyped to ascertain viral subtype (clade) and to determine the nucleotide sequence at codon 106 . Viruses were isolated from peripheral blood mononuclear cells (PBMC) of the three EFV-experienced patients who harbored the V106M mutation.
To ascertain the effect of the valine codon 106 polymorphism (GTG) on evolution of NNRTI drug resistance, viruses were amplified from clade C clinical isolates obtained from five treatment-naive subjects from Botswana and three treatment-naive Ethiopians . Using serial passage in increasing concentrations of drugs, HIV-1 variants resistant to EFV and NVP were selected . Recombinant clones carrying V106A or V106M were also constructed by cloning the BH10 polymerase gene into the pGEM-5Zf (+) vector (GIBCO-BRL Inc., Montreal, Canada) as described previously .
Genotypic and phenotypic resistance testing
Sequencing of extracted DNA was performed by Visible Genetics TruGene or by ABI technology to determine genotypic changes associated with drug resistance . Scoring the presence and drug susceptibility of V106M viruses was performed using the Visible Genetics Inc., Virco Inc., Stanford, ABL, and geno2pheno algorithms [8,11–14].
Cell culture-based phenotypic assays were performed in cord blood mononuclear cells infected with recombinant, EFV-selected, or clinical clade C isolates to determine the extent to which antiretroviral drugs inhibited in vitro HIV-1 replication, as described previously [2,7]. The 50% drug inhibitory concentrations (IC50) for EFV, NVP, and delavirdine (DLV) were ascertained both pre- and post-selection with antiviral drugs.
Prevalence of codon 106 substitutions in different clades
We determined the prevalence of the GTG polymorphism and V106M mutation in clinical isolates from the Montreal area. Whereas clade B isolates overwhelmingly expressed the valine GTA at codon 106 (97% of cases), clade C isolates carried the valine GTG (94% of cases) (Fig. 1). Subtypes, other than for clade C, generally expressed the GTA nucleotide sequence at codon 106, although one case each of subtypes D and G also expressed GTG (Fig. 1). Whereas, the V106A or V106I mutations rarely developed in clade B infections, the V106M mutation was observed in three of six clade C persons treated with EFV/d4T/3TC or EFV/ZDV/3TC. In one female, elevated viremia (3000 copies/ml) was associated with V106M and M184V; in the other two females with viremia of 10 241 and 19 363 copies/ml, 106M and K103N were present alone or with T215Y.
Emergence of V106M in isolates with the GTG polymorphism
To establish the consequences of the GTG substitution and V106M mutation on NNRTI resistance profiles, clade C clinical isolates from eight treatment-naive persons were selected for NVP and EFV resistance in tissue culture (Table 1). In all selections with EFV, V106M was observed to arise. In contrast, V106M never arose when the same clade C isolates were subjected to NVP. Similarly, V106M never arose under pressure with DLV . In the case of the clade C viruses, the presence of V106M was followed by Y188H or Y188C if selection pressure with EFV was continued.
Phenotypic susceptibility of V106M viral isolates
The drug susceptibility of three of the in vitro EFV-selected isolates harboring V106M is shown in Table 2. All viruses harboring V106M following selection with EFV showed high-level cross-resistance to all approved NNRTI. This included two EFV-selected isolates in which V106M was the only NNRTI mutation present (Table 2).
Viruses isolated from the PBMC of the three EFV-treated individuals harboring V106M were also evaluated for NNRTI drug susceptibility. As depicted in Fig. 2, the patient harboring V106M as the only NNRTI mutation showed resistance to EFV, NVP, and DLV. These data confirm that V106M confers high-level cross-resistance to all NNRTI. The remaining two patients also showed NNRTI resistance although these isolates also harbored the K103N mutation (Fig. 2). The cross-resistance observed with V106M variants selected either in vitro or in vivo, is in the range observed for K103N and Y188C/H/L in clade B or C infections (Table 2, Fig. 2).
Site-directed mutagenesis was performed to introduce the V106M mutation into the subtype B infectious recombinant virus, BH10. This virus possessed a phenotypic pattern consistent with high-level cross-resistance to all approved NNRTI (Table 2). In contrast, the V106A mutation introduced into BH10 yielded resistance only to NVP, consistent with previous observations (Table 2, Fig. 2) [8,11].
Sequences of viral variants harboring V106M as a sole NNRTI resistance-associated mutation were entered into a number of sequence databases. Three clinical isolates sequenced by ABI and sent for Virco Virtual phenotypic analysis did not score the V106M mutation. While the Visible Genetics interpretative system did report V106M as an unexpected mutation, the Trugene algorithm predicted sensitivity to all NNRTI. In five of six Stanford algorithms, the five ABL databases, the Los Alamos and IAS-USA databases, the V106M mutation was not scored [8,11–14]. The Stanford Specialty and geno2pheno algorithms scored V106M as resistance to NVP with sensitivity to both EFV and DLV [8,13]. Thus, no currently available algorithmic database recognizes V106M as a mutation conferring NNRTI cross-resistance.
The rapid expansion of new HIV-1 subtypes poses new challenges in HIV-1 disease management. To date, limited information is available on the efficacy of antiretroviral drugs and the emergence of drug resistance among genotypically diverse non-B viral subtypes. Our study has identified a novel multi-NNRTI resistance mutation that may develop in clade C infections after EFV treatment. The valine polymorphism at codon 106 (GTG) in clade C variants favors occurrence of a V106M (GTG←ATG) mutation following either in vitro or in vivo exposure to EFV. This GTG polymorphism that is present in 94% of clade C isolates was observed rarely in clade B variants (1.5% of cases) (Fig. 1). V106M arose in three of six persons with clade C infections who were treated with EFV, but was seen at an incidence of < 0.6 % in NNRTI-treated clade B infections.
Several studies have suggested that viral subtype does not generally influence antiretroviral response to NRTI or PI [3,4,15–19], but that non-B clade viruses may respond to NNRTI differently than do clade B variants . Both HIV-2 viruses and clade O viruses show innate resistance to all NNRTI due to the presence of naturally occurring Y181I and Y181C in RT, respectively . It is interesting that V106M only arose with EFV whereas Y181C was the favored mutation selected with NVP. It has been reported that Y181C is more replicatively fit than V106A in clade B isolates . Further studies are required to determine the relative drug susceptibility and fitness of NNRTI mutations in different clade backbones.
The principal resistance mutations associated with multi-NNRTI resistance in clade B infections are Y188C and K103N [20,21]. When clade C viruses were serially passaged in the presence of high doses of EFV, Y188H developed in addition to V106M. The Y188H mutation has been previously observed for subtype B viruses exposed to two experimental NNRTI, loviride and TIBO R82913 . In addition to V106M, the K103N resistance mutation arose in two of three individuals with clade C viruses who were treated with EFV.
V106M has not been reported as a mutation conferring resistance to NNRTI by any resistance database. While V106A/I has been described in clade B infections, resistance profiles predict resistance to NVP but sensitivity to DLV and EFV. These findings underscore the limitations of genotypic interpretative reports that fail to include polymorphisms or clade designation. Although these considerations may add complexity to reports, they will also allow easier retrospective analysis of resistance mutations that may arise in non-B HIV infections. At present, only Visible Genetics, ABL and geno2pheno software provide detailed analyses of polymorphisms, and while the Virco Virtual Phenotype provides clade designation, reports from Visible Genetics do not.
Inexpensive treatment strategies for developing countries are urgently required. Both NVP and EFV are drugs of choice given their efficacy and potential for single daily dosing. The emergence of V106M in patients harboring clade C infections will be a matter of concern if cross-resistance to all approved NNRTI will be shown to occur as a result of this mutation. Physicians should be aware of the V106M mutation and its potential impact on virological responsiveness.
Sponsorship: This work was sponsored by the Canadian Institutes for Health Research (CIHR), the Canadian Foundation for AIDS research (CANFAR), Fonds de la Recherche en Santé du Québec (FRSQ), and by the Secure the Future Programme of Bristol-Myers Squibb, Inc.
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