A comparison of computational models with and without genotyping for prediction of response to second-line HIV therapy.

OBJECTIVES: We compared the use of computational models developed with and without HIV genotype vs. genotyping itself to predict effective regimens for patients experiencing first-line virological failure. METHODS: Two sets of models predicted virological response for 99 three-drug regimens for patients on a failing regimen of two nucleoside/nucleotide reverse transcriptase inhibitors and one nonnucleoside reverse transcriptase inhibitor in the Second-Line study. One set used viral load, CD4 count, genotype, plus treatment history and time to follow-up to make its predictions; the second set did not include genotype. Genotypic sensitivity scores were derived and the ranking of the alternative regimens compared with those of the models. The accuracy of the models and that of genotyping as predictors of the virological responses to second-line regimens were compared. RESULTS: The rankings of alternative regimens by the two sets of models were significantly correlated in 60-69% of cases, and the rankings by the models that use a genotype and genotyping itself were significantly correlated in 60% of cases. The two sets of models identified alternative regimens that were predicted to be effective in 97% and 100% of cases, respectively. The area under the receiver-operating curve was 0.72 and 0.74 for the two sets of models, respectively, and significantly lower at 0.55 for genotyping. CONCLUSIONS: The two sets of models performed comparably well and significantly outperformed genotyping as predictors of response. The models identified alternative regimens predicted to be effective in almost all cases. It is encouraging that models that do not require a genotype were able to predict responses to common second-line therapies in settings where genotyping is unavailable.

Computational models can predict response to HIV therapy without a genotype and may reduce treatment failure in different resource-limited settings.

OBJECTIVES: Genotypic HIV drug-resistance testing is typically 60%-65% predictive of response to combination antiretroviral therapy (ART) and is valuable for guiding treatment changes. Genotyping is unavailable in many resource-limited settings (RLSs). We aimed to develop models that can predict response to ART without a genotype and evaluated their potential as a treatment support tool in RLSs. METHODS: Random forest models were trained to predict the probability of response to ART (≤400 copies HIV RNA/mL) using the following data from 14 891 treatment change episodes (TCEs) after virological failure, from well-resourced countries: viral load and CD4 count prior to treatment change, treatment history, drugs in the new regimen, time to follow-up and follow-up viral load. Models were assessed by cross-validation during development, with an independent set of 800 cases from well-resourced countries, plus 231 cases from Southern Africa, 206 from India and 375 from Romania. The area under the receiver operating characteristic curve (AUC) was the main outcome measure. RESULTS: The models achieved an AUC of 0.74-0.81 during cross-validation and 0.76-0.77 with the 800 test TCEs. They achieved AUCs of 0.58-0.65 (Southern Africa), 0.63 (India) and 0.70 (Romania). Models were more accurate for data from the well-resourced countries than for cases from Southern Africa and India (P < 0.001), but not Romania. The models identified alternative, available drug regimens predicted to result in virological response for 94% of virological failures in Southern Africa, 99% of those in India and 93% of those in Romania. CONCLUSIONS: We developed computational models that predict virological response to ART without a genotype with comparable accuracy to genotyping with rule-based interpretation. These models have the potential to help optimize antiretroviral therapy for patients in RLSs where genotyping is not generally available.

Modelling response to HIV therapy without a genotype: an argument for viral load monitoring in resource-limited settings.

In the absence of widespread access to individualized laboratory monitoring, which forms an integral part of HIV patient management in resource-rich settings, the roll-out of highly active antiretroviral therapy (HAART) in resource-limited settings has adopted a public health approach based on standard HAART protocols and clinical/immunological definitions of therapy failure. The cost-effectiveness of HIV-1 viral load monitoring at the individual level in such settings has been debated, and questions remain over the long-term and population-level impact of managing HAART without it. Computational models that accurately predict virological response to HAART using baseline data including CD4 count, viral load and genotypic resistance profile, as developed by the Resistance Database Initiative, have significant potential as an aid to treatment selection and optimization. Recently developed models have shown good predictive performance without the need for genotypic data, with viral load emerging as by far the most important variable. This finding provides further, indirect support for the use of viral load monitoring for the long-term optimization of HAART in resource-limited settings.

Multiple mutations in HIV-1 reverse transcriptase confer high-level resistance to zidovudine (AZT).

Human immunodeficiency virus (HIV) isolates with reduced sensitivity to zidovudine (3'-azido-3'-deoxythymidine, AZT) from individuals with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex were studied to determine the genetic basis of their resistance. Most were sequential isolates obtained at the initiation of and during therapy. Comparative nucleotide sequence analysis of the reverse transcriptase (RT) coding region from five pairs of sensitive and resistant isolates identified three predicted amino acid substitutions common to all the resistant strains (Asp67----Asn, Lys70----Arg, Thr215----Phe or Tyr) plus a fourth in three isolates (Lys219----Gln). Partially resistant isolates had combinations of these four changes. An infectious molecular clone constructed with these four mutations in RT yielded highly resistant HIV after transfection of T cells. The reproducible nature of these mutations should make it possible to develop rapid assays to predict zidovudine resistance by performing polymerase chain reaction amplification of nucleic acid from peripheral blood lymphocytes, thereby circumventing current lengthy HIV isolation and sensitivity testing.

HIV with reduced sensitivity to zidovudine (AZT) isolated during prolonged therapy.

The drug sensitivities of human immunodeficiency virus (HIV) isolates from a group of patients with acquired immunodeficiency syndrome (AIDS) or AIDS-related complex (ARC) who were receiving zidovudine (3'-azido-3'-deoythymidine, AZT) therapy were tested by means of a newly developed plaque assay in CD4+ HeLa cells. Fifty percent inhibitory dose (ID50) values of 18 isolates from untreated individuals ranged between 0.01 microM and 0.05 microM. In contrast, most isolates from patients who had received zidovudine for 6 months or more exhibited decreased sensitivity characterized by changes in ID50 or ID95 values (or both), with isolates from several patients (5/15) showing 100-fold increases in ID50. The latter isolates were also insensitive to 3'-azido-2',3'-dideoxyuridine; however, the isolates were still sensitive to 2',3'-dideoxycytidine, 2',3'-dideoxy-2',3'-didehydrothymidine, or phosphonoformate. It cannot be determined from this small sample of patients whether development of a less sensitive virus phenotype results in clinical resistance. Appearance of such variants was not associated with a consistent increase in viral p24 concentrations in patient plasma and did not herald any sudden deterioration in clinical status. More extensive studies are required to determine the clinical significance. Thus, it would be premature to alter any treatment protocols for HIV-infected individuals at present.

Potential mechanism for sustained antiretroviral efficacy of AZT-3TC combination therapy.

Combinations of antiretroviral drugs that prevent or delay the appearance of drug-resistant human immunodeficiency virus-type 1 (HIV-1) mutants are urgently required. Mutants resistant to 3'-azidothymidine (AZT, zidovudine) became phenotypically sensitive in vitro by mutation of residue 184 of viral reverse transcriptase to valine, which also induced resistance to (-)2'-deoxy-3'-thiacytidine (3TC). Furthermore, AZT-3TC coresistance was not observed during extensive in vitro selection with both drugs. In vivo AZT-3TC combination therapy resulted in a markedly greater decreased in serum HIV-1 RNA concentrations than treatment with AZT alone, even though valine-184 mutants rapidly emerged. Most samples assessed from the combination group remained AZT sensitive at 24 weeks of therapy, consistent with in vitro mutation studies.

Resistance to ddI and sensitivity to AZT induced by a mutation in HIV-1 reverse transcriptase.

Serial human immunodeficiency virus type-1 (HIV-1) isolates were obtained from five individuals with acquired immunodeficiency syndrome (AIDS) who changed therapy to 2',3'-dideoxyinosine (ddI) after at least 12 months of treatment with 3'-azido-3'-deoxythymidine (zidovudine, AZT). The in vitro sensitivity to ddI decreased during the 12 months following ddI initiation, whereas AZT sensitivity increased. Analysis of the reverse transcriptase coding region revealed a mutation associated with reduced sensitivity to ddI. When this mutation was present in the same genome as a mutation known to confer AZT resistance, the isolates showed increased sensitivity to AZT. Analysis of HIV-1 variants confirmed that the ddI resistance mutation alone conferred ddI and 2',3'-dideoxycytidine resistance, and suppressed the effect of the AZT resistance mutation. The use of combination therapy for HIV-1 disease may prevent drug-resistant isolates from emerging.

Zidovudine resistance predicted by direct detection of mutations in DNA from HIV-infected lymphocytes.

Zidovudine-resistant strains of HIV have recently been isolated from individuals during prolonged treatment. Analysis of the HIV reverse transcriptase (RT) gene from clinical isolates revealed that resistance was due to multiple nucleotide changes conferring specific amino acid substitutions in this enzyme. In order to correlate the degree of resistance with these amino acid changes, we constructed a series of infectious HIV variants with specific combinations of mutations in the RT gene and assessed their sensitivity to zidovudine. The reproducible nature of the mutations seen in clinical isolates has enabled the polymerase chain reaction to be used to identify lesions associated with resistance. This procedure was validated by analysis of sensitive and resistant clinical isolates with RT genes of known DNA sequence. Using a 'double' amplification procedure, zidovudine sensitivity was assessed by direct detection of specific mutations in DNA from peripheral-blood lymphocyte samples. This should make it possible to test large numbers of individuals receiving zidovudine therapy, with the aim of establishing the clinical significance of the resistant isolates.

Phenotypic and genotypic analysis of clinical HIV-1 isolates reveals extensive protease inhibitor cross-resistance: a survey of over 6000 samples.

OBJECTIVE: To evaluate in HIV-1 the extent of phenotypic and genotypic antiretroviral drug resistance and cross-resistance towards the protease inhibitors (PIs) saquinavir, ritonavir, indinavir and nelfinavir among a set of patient samples originating from European and US routine clinical practice and submitted for phenotypic drug resistance testing and/or genotypic analysis. The mutational pattern(s) underlying both resistance and cross-resistance to PIs was investigated. METHOD: Over 6000 patient isolates with plasma viral load greater than 1000 copies/ml plasma were analysed. Phenotypic resistance was evaluated by a recombinant virus assay. Phenotypic resistance is expressed as the fold-increase of the 50% inhibitory concentration (IC50) value of a compound for a patient-derived recombinant virus isolate compared with that for a wild-type laboratory virus. Genotypic analysis is reported as amino acid changes at positions in the HIV-1 protease compared to a wild-type reference. RESULTS: Phenotypic resistance to any single PI was observed in 17 to 25% of the clinical isolates investigated. Phenotypic cross-resistance among PIs (> 10-fold increase in IC50 value) was detected in 59 to 80% of the samples resistant (> 10-fold increase in IC50 value) to at least one PI. The prevalent mutations in PI-resistant isolates involved substitutions at codons 10, 36, 46, 54, 71, 77, 82 and 90. The most frequent mutational pattern in samples with PI cross-resistance involved combined substitutions at positions 10 and 90, extended with substitutions at positions 54, 71, 77, 82 or 84. CONCLUSIONS: Extensive use of first-generation PIs leads to the emergence of HIV-1 isolates possessing cross-resistance to all members of this class. Identification of particular mutational profiles among these isolates may assist in the design of new generation inhibitors with specific activity against protease-mutant HIV strains.

HIV-1 biological phenotype and the development of zidovudine resistance in relation to disease progression in asymptomatic individuals during treatment.

OBJECTIVE: To determine which parameters are associated with clinical progression during zidovudine treatment of asymptomatic HIV-1-infected individuals. METHODS: Twenty-four initially asymptomatic HIV-1-infected individuals were treated with zidovudine and followed until the development of AIDS or for approximately 3 years. HIV-1 phenotype was determined by cocultivation of patient cells with donor lymphocytes, and by a new assay of direct cocultivation with MT-2 cells. Specific mutations in the HIV-1 reverse transcriptase (RT) gene conferring resistance to zidovudine were detected using a selective polymerase chain reaction. RESULTS: Progression to AIDS was more rapid in individuals harbouring syncytium-inducing (SI) viral isolates or showing a conversion from non-syncytium-inducing (NSI) to SI viral isolates. One out of 20 patients who spent a total of 559 months harbouring an NSI phenotype progressed to AIDS, whereas eight out of 12 patients who spent a total of 223 months harbouring an SI phenotype progressed to AIDS (P < 0.001). There was no significant difference between SI and non-SI isolates in the frequency of five mutations causing zidovudine resistance. However, all SI isolates obtained after 2 years of treatment contained mutations in codons 41 and 215 of the RT gene, whereas only five out of 11 (45%) NSI isolates obtained at that time had this combination of mutations. CONCLUSIONS: Conversion to the SI phenotype cannot be prevented by zidovudine treatment. The presence or appearance of an SI virus heralded disease progression in zidovudine-treated individuals. Further research is required to investigate the relationship between virus phenotype and development of zidovudine resistance.

Tipranavir inhibits broadly protease inhibitor-resistant HIV-1 clinical samples.

OBJECTIVE: Although the use of HIV-1 protease inhibitors (PI) has substantially benefited HIV-1-infected individuals, new PI are urgently needed, as broad PI resistance and therapy failure is common. METHODS: The antiviral activity of tipranavir (TPV), a non-peptidic PI, was assessed in in vitro culture for 134 clinical isolates with a wide range of resistance to currently available peptidomimetic PI. The susceptibility of all 134 variants was then re-tested with the four PI simultaneously with TPV, using the Antivirogram assay. RESULTS: Of 105 viruses with more than tenfold resistance to three or four PI and an average of 6.1 PI mutations per sample, 95 (90%) were susceptible to TPV; eight (8%) had four- to tenfold resistance to TPV and only two (2%) had more than tenfold resistance. CONCLUSIONS: The substantial lack of PI cross-resistance to TPV shown by highly PI-resistant clinical isolates makes TPV an attractive new-generation HIV inhibitor.

World-wide variation in HIV-1 phenotypic susceptibility in untreated individuals: biologically relevant values for resistance testing.

OBJECTIVES: To examine the natural phenotypic variability in drug susceptibility among recombinant HIV-1 isolates from a large number of untreated HIV-positive individuals from wide-ranging geographic locations, and to use this information to establish biologically relevant cut-off values for phenotypic antiretroviral susceptibility testing. METHODS: Phenotypic susceptibility to 14 antiretroviral agents was determined for HIV-1 samples from > 1000 treatment-naive individuals in seven clinical trials. Samples were from the USA (n = 351), Germany (n = 306), Canada (n = 265), and South Africa (n = 358). Geometric mean fold-resistance and confidence intervals were determined relative to a standard laboratory wild-type virus. RESULTS: Baseline fold-resistance was approximately log-normally distributed for all antiretroviral agents examined. There was no evidence of large geographical differences in average antiviral susceptibility. Geometric mean fold-resistance for each of 14 antiviral agents was similar (+/- 0.5-fold) for samples derived from the USA, Canada, Germany, or South Africa. The non-nucleoside reverse transcriptase inhibitors (NNRTI) exhibited the broadest distribution of susceptibility; approximately 97.5% of all isolates had < 2.5-4.0, < 3.0-4.5, and < 5-10 fold-decrease in susceptibility to five protease inhibitors, six nucleoside analogues, and three NNRTI, respectively. No consistent geographic pattern or clade effect (B versus C) in either the mean or the distribution of baseline antiretroviral susceptibility was observed. CONCLUSIONS: Phenotypic drug susceptibility of HIV-1 in untreated individuals varies markedly from drug to drug, with broadly similar patterns world-wide. These results have important implications in defining the 'normal range' of phenotypic susceptibility to antiretroviral agents and establish biologically relevant cut-off values for this phenotypic drug susceptibility test.

The M184V mutation in HIV-1 reverse transcriptase (RT) conferring lamivudine resistance does not result in broad cross-resistance to nucleoside analogue RT inhibitors.

OBJECTIVE: To investigate the prevalence and magnitude of M184V-mediated changes in susceptibility to zalcitabine, didanosine, stavudine and abacavir (1592U89 succinate) in a cohort of lamivudine-treated patients. DESIGN AND METHODS: A total of 255 samples from patients treated with lamivudine and zidovudine with or without other nucleoside reverse transcriptase inhibitors (NRTI) were analysed for susceptibility to zidovudine, lamivudine, zalcitabine, didanosine and stavudine using a recombinant virus assay. Seventy-three samples originated from patients exposed to zidovudine and lamivudine only. A subset of 27 samples was investigated for cross-resistance to abacavir. Resistance was defined as a change in median inhibitory concentration more than fivefold compared with wild-type (high-level resistance, > 10-fold). A genotypic analysis of plasma-derived reverse transcriptase coding regions was carried out in samples with cross-resistance. RESULTS: The majority of samples displayed wild-type or greater than wild-type sensitivity to zalcitabine, didanosine and stavudine: resistance was seen in 17.2, 9 and 6.3% of the total sample population, respectively. Of these, 1.2, 2.7 and 2.4%, respectively, showed high-level resistance. The prevalence of resistance to a particular NRTI was lower in samples from patients not pretreated with that NRTI and in samples from patients exposed to zidovudine-lamivudine only. Cross-resistance was more prevalent in samples with high ZDV resistance. There was no obvious correlation between cross-resistance and genotype; all but two samples were mutant at codon 184. There were no consistent changes at positions associated with zidovudine resistance. The majority of samples from a subset (n=27) were four- to eightfold less sensitive to abacavir. There were no other genotypic changes in addition to M184V known to be associated with abacavir resistance. CONCLUSIONS: Cross-resistance was not commonly observed in this lamivudine-treated cohort. M184V per se is not expected to compromise subsequent treatment with NRTI such as didanosine-stavudine or combinations containing abacavir.

Mutational analysis of two conserved sequence motifs in HIV-1 reverse transcriptase.

Two conserved sequence motifs, occurring in HIV-1 reverse transcriptase at residues 110-116 and 183-190, have been studied using site-directed mutagenesis of the cloned gene. In particular, aspartates at positions 185 and 186 have each been mutated to either asparagine or glutamate. The resulting mutant proteins were catalytically inactive but still able to bind the template-primer complex, poly rA-oligo dT. Other mutations in these regions resulted in reduced reverse trascriptase activity but the mutation of tyrosine-183 to serine caused a significant increase in the Km for dTTP and the Ki for inhibition by 3'-azidothymidine-triphosphate, 2',3'-dideoxythymidine-triphosphate and phosphonoformic acid.

Mutational patterns in the HIV genome and cross-resistance following nucleoside and nucleotide analogue drug exposure.

A variety of key mutations in HIV reverse transcriptase (RT) have been associated with nucleoside reverse transcriptase inhibitor (NRTI) exposure, which give rise to a diverse range of effects in terms of altered drug susceptibilities, viral replicative capacity and RT biochemistry. There are three basic mechanisms of resistance conferred by specific mutations in the coding region of RT. The first is drug discrimination, whereby a particular drug or drugs are either selectively excluded from uptake or from the RT-primer-template catalytic complex. Drug discrimination is, for the most part, relatively specific for individual drugs. Repositioning of the template-primer to prevent a catalytically competent complex in the presence of a bound drug molecule has also been observed in some instances, and forms a second mechanism. The third, and potentially most significant for long-term efficacy of the NRTIs, is pyrophosphorolysis, the primary mode of resistance to zidovudine. Mutations selected by this drug or stavudine serve to elevate the natural rate of the reverse reaction for RT. Pyrophosphorolysis uncouples the last nucleoside monophosphate added to the proviral transcript, and attaches it to either a free pyrophosphate (regenerating a deoxynucleoside triphosphate) or to a nucleoside di- or triphosphate (usually ATP). Uncoupling a chain-terminating NRTI residue therefore rescues reverse transcription and reduces drug susceptibility across the class, since the process is not specific for the selecting drug. Of all the nucleoside-associated mutations, the best known and most studied are the six associated with thymidine analogue exposure. These six mutations (M41L, D67N, K70R, L210W, T215Y/F, K219Q) enhance RT pyrophosphorolysis to confer high-level viral resistance to zidovudine, and clinically significant loss of response to stavudine and didanosine. They have also been found to confer reduced susceptibility to lamivudine and abacavir, particularly when present alongside other NRTI-induced changes. Other key mutations generally confer more limited resistance to specific agents, although the primary lamivudine- and abacavir-associated M184V substitution generates a broad spectrum of drug-dependent phenotypes, and uncommon mutational complexes conferring resistance across the entire class are well known. In addition to 'classical' multi-nucleoside-resistant genotypes, database-driven 'virtual phenotyping' for accumulations of NRTI-associated mutations around a core of thymidine analogue-induced changes predicts drug susceptibilities below wild-type across the entire NRTI class, even in the absence of key mutations associated with individual agents. When the natural range of drug susceptibilities for treatment-naive isolates is used as the basis for defining resistance, retrospective analysis of clinical isolates in the Virco database shows a significantly increased incidence of reduced susceptibility for the dideoxy NRTIs (didanosine, stavudine and zalcitabine) that was undetected in previous assays. These data imply a cumulative degradation of response to

Isolation and characterization of acyclovir-resistant strains of herpes simplex virus.

A number of clinical studies have documented herpes simplex infections which appear to be resistant to nucleoside analogs; these include idoxuridine [1,2] and acyclovir [3]. Few, if any of the viruses isolated from such patients have yet been thoroughly characterized. We have isolated a number of acyclovir-resistant mutants by selection for resistance in tissue culture. The study of the biochemical and biological properties of these mutants has given some insight into the likely nature of resistant clinical strains. We have devised a number of simple tests to allow classification of laboratory mutants. We also draw attention to some of the difficulties the clinical virologist may encounter when analyzing putative resistant virus isolated from treated patients.

Structural studies of the acquired immunodeficiency syndrome virus reverse transcriptase.

The clinical success of zidovudine has established the human immunodeficiency virus (HIV) reverse transcriptase (RT) as a valid target for the design of drugs to treat acquired immunodeficiency syndrome. In order to facilitate structural studies of this enzyme, expression systems in Escherichia coli, which allow the production of large amounts of RT, have been established. Using this recombinant material the RT has been purified and crystallized. Crystallographic studies currently underway are aimed at elucidating the three-dimensional structure of HIV RT. The availability of a bacterial expression system has enabled structural/functional studies of the RT by site-directed mutagenesis. These studies have identified amino acid residues that are essential for activity of the enzyme and might be involved in substrate binding. It is hoped that structural information of this nature will allow the rational design of HIV RT inhibitors.

The rabbit study: ritonavir and saquinavir in combination in saquinavir-experienced and previously untreated patients.

Thirteen protease inhibitor-naive patients with HIV-1 infection, and 12 patients with a median of 58 months prior treatment with saquinavir (SQV) monotherapy, were treated with SQV (400 mg twice daily) and ritonavir (RIT, 500 mg twice daily) in a study designed to assess the effect of prior treatment with SQV monotherapy on the antiretroviral activity of RIT-SQV combination therapy. Median baseline viral load and CD4+ cell counts were 155,000 and 262,000 copies/ml and 333 and 225 cells/mm3 in the naive and experienced groups, respectively. Mean viral load changes at 24 weeks were -1.63 and -0.27 log copies/ml in the naive and SQV-experienced groups, respectively (intent-to-treat analysis). Baseline genotype by point mutation assay and sequencing in the SQV-experienced group was highly predictive of virological response. Eight of 11 SQV-experienced patients had evidence of phenotypic resistance to RIT at baseline, despite previous treatment with SQV only. There was strong correlation between phenotypic resistance to RIT and the presence of the L90M mutation. We conclude that prolonged prior treatment with saquinavir monotherapy may produce cross-resistance to ritonavir and reduce the subsequent response to ritonavir-saquinavir in combination. In this study, both phenotypic resistance to ritonavir and presence of the L90M mutation predicted the viral load response to ritonavir-saquinavir.

3'-Azido-3'-deoxythymidine resistance suppressed by a mutation conferring human immunodeficiency virus type 1 resistance to nonnucleoside reverse transcriptase inhibitors.

Nonnucleoside reverse transcriptase (NNRT) inhibitors (R82913; (+)-S-4,5,6,7-tetrahydro-9-chloro-5-methyl-6-(3-methyl-2-butenyl)- imidazo[4,5,1-jk][1,4]-benzodiazepin-2(1H)-thione; Cl-TIBO; and BI-RG-587, nevirapine) were used to select resistant human immunodeficiency virus type 1 (HIV-1) variants by passage in cell cultures of wild-type or 3'-azido-3'-deoxythymidine (zidovudine; AZT)-resistant strains. Similar to other NNRT inhibitors, Cl-TIBO induced a single mutation (Y181 to C) in reverse transcriptase (RT) that accounted for the resistance. BI-RG-587 induced a different mutation (V106-->A) in AZT resistance backgrounds. A series of viable HIV-1 variants was constructed by site-directed mutagenesis of the RT, which harbored multiple drug resistance mutations, including Y181 to C. HIV-1 that was co-resistant to NNRT inhibitors and 2',3'-dideoxyinosine resulted when a 2',3'-dideoxyinosine resistance mutation (L74 to V) was also present in RT. By contrast, however, the Y181 to C mutation in an AZT resistance background significantly suppressed resistance to AZT, while it conferred resistance to NNRT inhibitors. However, the V106-->A substitution did not cause suppression of preexisting AZT resistance. Since certain combinations of nucleoside analogs and NNRT inhibitors might result in the development of co-resistance, careful analysis of clinical isolates obtained during combination therapy will be needed to determine the potential significance of these observations.