Objective: We have shown that HIV-1 clade C variants contain a valine codon 106 polymorphism (GTG) that facilitates a V106M transition (GTG←ATG) after selection with efavirenz (EFV). This study evaluates the prevalence of V106 (GTG) and 106M (ATG) codons in clinical isolates as well as the effects of V106M on resistance to non-nucleoside reverse transcriptase inhibitors (NNRTI).
Methods: Genotypic analysis ascertained sequence diversity at codon 106, including both valine polymorphisms (GTA and GTG) and the V106A (GCA) and V106M (ATG) resistance-conferring mutations in B (n = 440) and non-B (n = 84) clinical isolates. Cell-based phenotypic assays were performed to determine the effects of V106M and V106A on levels of resistance to EFV, nevirapine and delavirdine.
Results: Most subtype B isolates harbored GTA (valine) at codon 106 (97% of cases) while the GTG (valine) polymorphism was generally present in clade C viruses (94% of cases). Under conditions of EFV but not nevirapine or delavirdine pressure (n = 8) in tissue culture, clade C isolates developed the V106M mutation (GTG←ATG), conferring high-level (100–1000-fold) cross-resistance to all NNRTI. Generation of V106M recombinant viruses by site-directed mutagenesis confirmed the ability of V106M to confer NNRTI cross-resistance. This mutation also developed in three of six EFV-treated patients harboring clade C infections. In current genotypic interpretative reports (including 15 algorithmic databases), V106A is listed as an nevirapine-specific mutation while V106M is not recognized.
Conclusions: V106M may be a signature mutation in clade C patients treated with EFV and may have the potential to confer high-level multi-NNRTI resistance.
From the McGill University AIDS Centre, Jewish General Hospital, Montreal, Quebec, Canada, aHarvard School of Public Health, Boston, Massachusetts, USA, bBotswana-Harvard Laboratory, Gaborone, Botswana, the cDepartment of Medicine, Stanford University, Palo Alto, California, USA, and the dDépartement de Microbiologie et d’'Immunologie, Montreal, Quebec, Canada.
Correspondence to B. Brenner or M. Wainberg, McGill AIDS Centre, Jewish General Hospital, 3755 Cote Ste Catherine Road, Montreal, Quebec, Canada H3T 1E2.
Received: 30 September 2002; revised: 24 October 2002; accepted: 24 October 2002.
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.
1.Pallela FJ, Delaney KM, Moorman AC, Loveless MO, Fuhrer J, Satten GA, et al
. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. N Engl J Med
2.Loemba H, Brenner B, Parniak MA, Ma'ayan S, Spira B, Moisi D, et al
. Genetic divergence of HIV-1 Ethiopian clade C reverse transcriptase (RT) and rapid development of resistance against non-nucleoside inhibitors of RT. Antimicrob Agents Chemother
3.Loemba H, Wainberg MA, Brenner BG. Effects of HIV-1 clade diversity on HIV-1 virulence and antiretroviral drug sensitivity. Recent Rev Devel Virol
4.Pillay D, Walker AS, Gibb DM, de Rossi S, Kaye M, Ait-Khaled M, et al
. Impact of human immunodeficiency virus type 1 subtypes on virologic response and emergence of drug resistance among children in the Paediatric European Network for Treatment of AIDS (PENTA) 5 Trial. J Infect Dis
5.Staszewski S, Morales-Ramirez J, Tashima KT, Rachlis A, Skiest D, Stanford J, et al
. Efavirenz plus zidovudine and lamivudine, efavirenz plus indinavir, and indinavir plus zidovudine and lamivudine in the treatment of HIV-1 infection in adults. N Engl J Med
6.Robbins G, Shafer R, Smeaton L, de Grottola V, Pettinelli C, Synder S, et al
. Antiretroviral strategies in naive HIV+ subjects: comparisons of sequential 3-drug regimens (ACTG 384). Program and abstracts of the XIV International Conference on AIDS.
Barcelona, July 7–12, 2002 [abstract LbOr20A].
7.Loemba H, Brenner B, Parniak MA, Ma'ayan S, Spira B, Moisi D, et al. Polymorphisms of cytotoxic T-lymphocyte (CTL) and T-helper epitopes within reverse transcriptase (RT) of HIV-1 subtype C from Ethiopia and Botswana following selection of drug resistance. Antiviral Res
8.Shafer RW, Gonzales MJ, Brun-Vezinet F. Online comparison of HIV-1 drug resistance algorithms identifies rates and causes of discordant interpretations. Antiviral Ther
9.Gu Z, Gao Q, Li X, Parniak MA, Wainberg MA. Novel mutation in the human immunodeficiency virus type 1 reverse transcriptase gene that encodes cross-resistance to 2′,3′-dideoxyinosine and 2′,3′-dideoxycytidine. J Virol
10.Salomon H, Wainberg MA, Brenner BG, Quan Y, Rouleau D, Cote P, et al
. Prevalence of HIV-1 viruses resistant to antiretroviral drugs in 81 individuals newly infected by sexual contact or intravenous drug use. AIDS
11.Hammond J, Larder BA, Schinasi RF, Mellors JW. Mutations in retroviral genes associated with drug resistance. http://www.hiv
13.Beerenwinkel N, Schmidt B, Walter H, Kaiser R, Lengauer T, Hoffmann D, et al
. Diversity and complexity of HIV-1 drug resistance: A bioinformatics approach to predicting phenotype from genotype. Proc Natl Acad Sci, USA
14.D'Aquila RT, Schapiro JM, Brun-Vezinet F, Clotet B, Conway B, Demeter LM, et al
. Drug resistance mutations in HIV-1. Topics in HIV Med
15.Frater AJ, Beardall A, Ariyoshi K, Churchill D, Galpin S, Clarke JR, et al
. Impact of baseline polymorphisms in RT and protease on outcome of highly active antiretroviral therapy in HIV-1-infected African patients. AIDS
16.Palmer S, Alaeus A, Albert J, Cox S. Drug susceptibility of subtypes A, B, C, D, and E human immunodeficiency virus type 1 isolates. AIDS Res Hum Retroviruses
17.Caride E, Hertogs K, Larder B, Dehertogh P, Brindeiro R, Machado E, et al. Genotypic and phenotypic evidence of different drug-resistance mutation patterns between B and non-B subtype isolates of human immunodeficiency virus type 1 found in individuals failing HAART. Virus Genes
18.Caride E, Brindeiro R, Hertogs K, Larder B, Dehertogh P, Machado E, et al. Drug-resistant reverse transcriptase genotyping and phenotyping of B and non-B subtypes (F and A) of human immunodeficiency virus type I found in Brazilian patients failing HAART. Virology
19.Loveday C, van Hooff F, Johnson M. Inferior virological response to highly active antiretroviral therapy in patients with HIV-1 subtype C infection: a case controlled study. Antiviral Ther
20.Archer RH, Dykes C, Gerondelis P, Lloyd A, Fay P, Reichman RC, et al. Mutants of human immunodeficiency virus type (HIV-1) reverse transcriptase resistant to nonnucleoside reverse transcriptase inhibitors demonstrate altered rates of RnaseH cleavage that correlate with HIV-1 replication fitness in cell culture. J Virol
21.Bacheler LT, Anton ED, Kudish, P, Baker D, Bunville J, Krakowski K, et al
. Human immunodeficiency virus type 1 mutations selected in patients failing efavirenz combination therapy. Antimicrob Agents Chemother