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1.  K70Q Adds High-Level Tenofovir Resistance to “Q151M Complex” HIV Reverse Transcriptase through the Enhanced Discrimination Mechanism 
PLoS ONE  2011;6(1):e16242.
HIV-1 carrying the “Q151M complex” reverse transcriptase (RT) mutations (A62V/V75I/F77L/F116Y/Q151M, or Q151Mc) is resistant to many FDA-approved nucleoside RT inhibitors (NRTIs), but has been considered susceptible to tenofovir disoproxil fumarate (TFV-DF or TDF). We have isolated from a TFV-DF-treated HIV patient a Q151Mc-containing clinical isolate with high phenotypic resistance to TFV-DF. Analysis of the genotypic and phenotypic testing over the course of this patient's therapy lead us to hypothesize that TFV-DF resistance emerged upon appearance of the previously unreported K70Q mutation in the Q151Mc background. Virological analysis showed that HIV with only K70Q was not significantly resistant to TFV-DF. However, addition of K70Q to the Q151Mc background significantly enhanced resistance to several approved NRTIs, and also resulted in high-level (10-fold) resistance to TFV-DF. Biochemical experiments established that the increased resistance to tenofovir is not the result of enhanced excision, as K70Q/Q151Mc RT exhibited diminished, rather than enhanced ATP-based primer unblocking activity. Pre-steady state kinetic analysis of the recombinant enzymes demonstrated that addition of the K70Q mutation selectively decreases the binding of tenofovir-diphosphate (TFV-DP), resulting in reduced incorporation of TFV into the nascent DNA chain. Molecular dynamics simulations suggest that changes in the hydrogen bonding pattern in the polymerase active site of K70Q/Q151Mc RT may contribute to the observed changes in binding and incorporation of TFV-DP. The novel pattern of TFV-resistance may help adjust therapeutic strategies for NRTI-experienced patients with multi-drug resistant (MDR) mutations.
doi:10.1371/journal.pone.0016242
PMCID: PMC3020970  PMID: 21249155
2.  N348I in the Connection Domain of HIV-1 Reverse Transcriptase Confers Zidovudine and Nevirapine Resistance 
PLoS Medicine  2007;4(12):e335.
Background
The catalytically active 66-kDa subunit of the human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) consists of DNA polymerase, connection, and ribonuclease H (RNase H) domains. Almost all known RT inhibitor resistance mutations identified to date map to the polymerase domain of the enzyme. However, the connection and RNase H domains are not routinely analysed in clinical samples and none of the genotyping assays available for patient management sequence the entire RT coding region. The British Columbia Centre for Excellence in HIV/AIDS (the Centre) genotypes clinical isolates up to codon 400 in RT, and our retrospective statistical analyses of the Centre's database have identified an N348I mutation in the RT connection domain in treatment-experienced individuals. The objective of this multidisciplinary study was to establish the in vivo relevance of this mutation and its role in drug resistance.
Methods and Findings
The prevalence of N348I in clinical isolates, the time taken for it to emerge under selective drug pressure, and its association with changes in viral load, specific drug treatment, and known drug resistance mutations was analysed from genotypes, viral loads, and treatment histories from the Centre's database. N348I increased in prevalence from below 1% in 368 treatment-naïve individuals to 12.1% in 1,009 treatment-experienced patients (p = 7.7 × 10−12). N348I appeared early in therapy and was highly associated with thymidine analogue mutations (TAMs) M41L and T215Y/F (p < 0.001), the lamivudine resistance mutations M184V/I (p < 0.001), and non-nucleoside RTI (NNRTI) resistance mutations K103N and Y181C/I (p < 0.001). The association with TAMs and NNRTI resistance mutations was consistent with the selection of N348I in patients treated with regimens that included both zidovudine and nevirapine (odds ratio 2.62, 95% confidence interval 1.43–4.81). The appearance of N348I was associated with a significant increase in viral load (p < 0.001), which was as large as the viral load increases observed for any of the TAMs. However, this analysis did not account for the simultaneous selection of other RT or protease inhibitor resistance mutations on viral load. To delineate the role of this mutation in RT inhibitor resistance, N348I was introduced into HIV-1 molecular clones containing different genetic backbones. N348I decreased zidovudine susceptibility 2- to 4-fold in the context of wild-type HIV-1 or when combined with TAMs. N348I also decreased susceptibility to nevirapine (7.4-fold) and efavirenz (2.5-fold) and significantly potentiated resistance to these drugs when combined with K103N. Biochemical analyses of recombinant RT containing N348I provide supporting evidence for the role of this mutation in zidovudine and NNRTI resistance and give some insight into the molecular mechanism of resistance.
Conclusions
This study provides the first in vivo evidence that treatment with RT inhibitors can select a mutation (i.e., N348I) outside the polymerase domain of the HIV-1 RT that confers dual-class resistance. Its emergence, which can happen early during therapy, may significantly impact on a patient's response to antiretroviral therapies containing zidovudine and nevirapine. This study also provides compelling evidence for investigating the role of other mutations in the connection and RNase H domains in virological failure.
Analyzing HIV sequences from a Canadian cohort, Gilda Tachedjian and colleagues identify a common mutation in a little-studied domain of reverse transcriptase that confers resistance to two drug classes.
Editors' Summary
Background.
In the 1980s, infection with the human immunodeficiency virus (HIV), which causes acquired immunodeficiency syndrome (AIDS), was a death sentence. Although the first antiretroviral drugs (compounds that block HIV's life cycle) were developed quickly, single antiretrovirals only transiently suppress HIV infection. HIV rapidly accumulates random changes (mutations) in its genetic material, some of which make it drug resistant. Nowadays, there are many different antiretrovirals. Some inhibit the viral protease, an enzyme used to assemble new viruses. Others block reverse transcriptase (RT), which makes replicates of the genes of the virus. Nucleoside/nucleotide RT inhibitors (NRTIs; for example, zidovudine—also called AZT—and lamivudine) and non-nucleoside RT inhibitors (NNRTIs; for example, nevirapine and efavirenz) interfere with the activity of RT by binding to different sites in its so-called “DNA polymerase domain,” the part of the enzyme that constructs copies of the viral genes. Highly active antiretroviral therapy (HAART), which was introduced in the mid 1990s, combines several antiretrovirals (usually a protease inhibitor and two NRTIs or an NNRTI and two NRTIs) so that the replication of any virus that develops resistance to one drug is inhibited by the other drugs in the mix. When treated with HAART, HIV infection is usually a chronic, stable condition rather than a fatal disease.
Why Was This Study Done?
Unfortunately, HIV that is resistant to drugs still develops in some patients. To improve the prevention and management of drug resistance, a better understanding of the mutations that cause resistance is needed. Resistance to RT inhibitors usually involves mutations in the DNA polymerase domain that reduce the efficacy of NRTIs (including thymidine analogue mutations—also known as TAMs—and lamivudine-resistance mutations) and NNRTIs. Blood tests that detect these resistance mutations (genotype tests) have been used for several years to guide individualized selection of HIV drugs. Recently, however, mutations outside the DNA polymerase domain have also been implicated in resistance to RT inhibitors. In this study, the researchers have used data and samples collected since the mid 1990s by Canada's British Columbia Centre for Excellence in HIV/AIDS to investigate the clinical relevance of a mutation called N348I. This mutation changes an asparagine (a type of amino acid) to an isoleucine in a region of RT known as the connection domain. The researchers have also investigated how this mutation causes resistance to RT inhibitors in laboratory tests.
What Did the Researchers Do and Find?
The researchers analyzed the first two-thirds of the RT gene in viruses isolated from a large number of the Centre's patients. Virus carrying the N348I mutation was present in less than one in 100 patients whose HIV infection had never been treated, but in more than one in 10 treatment-experienced patients. The mutation appeared early in therapy, often in viruses that had TAMs, a lamivudine-resistance mutation called M184V/I, and/or NNRTI resistance mutations. Patients treated with zidovudine and nevirapine were 2.6 times more likely to have the N348I mutation than patients not treated with these drugs. Furthermore, the appearance of the N348I mutation often coincided with an increase in viral load, although other mutations that appeared at a similar time could have contributed to this increase. When the researchers introduced the N348I mutation into HIV growing in the laboratory, they found that it decreased the susceptibility of the virus to zidovudine and to NNRTIs.
What Do These Findings Mean?
These findings show that the treatment of patients with RT inhibitors can select a drug-resistant HIV variant that has a mutation outside the enzyme's DNA polymerase domain. Because this N348I mutation, which is commonly selected in vivo and has also been seen in other studies, confers resistance to two classes of RT inhibitors and can emerge early during therapy, it could have a large impact on patient responses to antiviral regimens that contain zidovudine and nevirapine. Although these findings do not show that the N348I mutation alone causes treatment failure, they may have implications for genotypic and phenotypic resistance testing, which is often used to guide treatment decisions. At present, genotype tests for resistance to RT inhibitors look for mutations only in the DNA polymerase domain of RT. This study is the first to demonstrate that it might be worth looking for the N348I mutation (and for other mutations outside the DNA polymerase domain) to improve the ability of genotypic and phenotypic resistance tests to predict treatment outcomes.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0040335.
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
HIV InSite has comprehensive information on all aspects of HIV/AIDS, including links to fact sheets (in English, French, and Spanish) about antiretrovirals, and chapters explaining antiretroviral resistance testing
NAM, a UK registered charity, provides information about all aspects of HIV and AIDS, including fact sheets on types of HIV drugs, drug resistance, and resistance tests (in English, Spanish, French, Portuguese, and Russian)
The US Centers for Disease Control and Prevention provides information on HIV/AIDS and on treatment (in English and Spanish)
AIDSinfo, a service of the US Department of Health and Human Services provides information for patients on HIV and its treatment
doi:10.1371/journal.pmed.0040335
PMCID: PMC2100143  PMID: 18052601
3.  Proteochemometric Modeling of the Susceptibility of Mutated Variants of the HIV-1 Virus to Reverse Transcriptase Inhibitors 
PLoS ONE  2010;5(12):e14353.
Background
Reverse transcriptase is a major drug target in highly active antiretroviral therapy (HAART) against HIV, which typically comprises two nucleoside/nucleotide analog reverse transcriptase (RT) inhibitors (NRTIs) in combination with a non-nucleoside RT inhibitor or a protease inhibitor. Unfortunately, HIV is capable of escaping the therapy by mutating into drug-resistant variants. Computational models that correlate HIV drug susceptibilities to the virus genotype and to drug molecular properties might facilitate selection of improved combination treatment regimens.
Methodology/Principal Findings
We applied our earlier developed proteochemometric modeling technology to analyze HIV mutant susceptibility to the eight clinically approved NRTIs. The data set used covered 728 virus variants genotyped for 240 sequence residues of the DNA polymerase domain of the RT; 165 of these residues contained mutations; totally the data-set covered susceptibility data for 4,495 inhibitor-RT combinations. Inhibitors and RT sequences were represented numerically by 3D-structural and physicochemical property descriptors, respectively. The two sets of descriptors and their derived cross-terms were correlated to the susceptibility data by partial least-squares projections to latent structures. The model identified more than ten frequently occurring mutations, each conferring more than two-fold loss of susceptibility for one or several NRTIs. The most deleterious mutations were K65R, Q151M, M184V/I, and T215Y/F, each of them decreasing susceptibility to most of the NRTIs. The predictive ability of the model was estimated by cross-validation and by external predictions for new HIV variants; both procedures showed very high correlation between the predicted and actual susceptibility values (Q2 = 0.89 and Q2ext = 0.86). The model is available at www.hivdrc.org as a free web service for the prediction of the susceptibility to any of the clinically used NRTIs for any HIV-1 mutant variant.
Conclusions/Significance
Our results give directions how to develop approaches for selection of genome-based optimum combination therapy for patients harboring mutated HIV variants.
doi:10.1371/journal.pone.0014353
PMCID: PMC3002298  PMID: 21179544
4.  HIV-1 Drug Resistance Emergence among Breastfeeding Infants Born to HIV-Infected Mothers during a Single-Arm Trial of Triple-Antiretroviral Prophylaxis for Prevention of Mother-To-Child Transmission: A Secondary Analysis 
PLoS Medicine  2011;8(3):e1000430.
Analysis of a substudy of the Kisumu breastfeeding trial by Clement Zeh and colleagues reveals the emergence of HIV drug resistance in HIV-positive infants born to HIV-infected mothers treated with antiretroviral drugs.
Background
Nevirapine and lamivudine given to mothers are transmitted to infants via breastfeeding in quantities sufficient to have biologic effects on the virus; this may lead to an increased risk of a breastfed infant's development of resistance to maternal antiretrovirals. The Kisumu Breastfeeding Study (KiBS), a single-arm open-label prevention of mother-to-child HIV transmission (PMTCT) trial, assessed the safety and efficacy of zidovudine, lamivudine, and either nevirapine or nelfinavir given to HIV-infected women from 34 wk gestation through 6 mo of breastfeeding. Here, we present findings from a KiBS trial secondary analysis that evaluated the emergence of maternal ARV-associated resistance among 32 HIV-infected breastfed infants.
Methods and Findings
All infants in the cohort were tested for HIV infection using DNA PCR at multiple study visits during the 24 mo of the study, and plasma RNA viral load for all HIV-PCR–positive infants was evaluated retrospectively. Specimens from mothers and infants with viral load >1,000 copies/ml were tested for HIV drug resistance mutations. Overall, 32 infants were HIV infected by 24 mo of age, and of this group, 24 (75%) infants were HIV infected by 6 mo of age. Of the 24 infants infected by 6 mo, nine were born to mothers on a nelfinavir-based regimen, whereas the remaining 15 were born to mothers on a nevirapine-based regimen. All infants were also given single-dose nevirapine within 48 hours of birth. We detected genotypic resistance mutations in none of eight infants who were HIV-PCR positive by 2 wk of age (specimens from six infants were not amplifiable), for 30% (6/20) at 6 wk, 63% (14/22) positive at 14 wk, and 67% (16/24) at 6 mo post partum. Among the 16 infants with resistance mutations by 6 mo post partum, the common mutations were M184V and K103N, conferring resistance to lamivudine and nevirapine, respectively. Genotypic resistance was detected among 9/9 (100%) and 7/15 (47%) infected infants whose mothers were on nelfinavir and nevirapine, respectively. No mutations were detected among the eight infants infected after the breastfeeding period (age 6 mo).
Conclusions
Emergence of HIV drug resistance mutations in HIV-infected infants occurred between 2 wk and 6 mo post partum, most likely because of exposure to maternal ARV drugs through breast milk. Our findings may impact the choice of regimen for ARV treatment of HIV-infected breastfeeding mothers and their infected infants.
Trial Registration
ClinicalTrials.gov NCT00146380
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Globally, more than 2 million children are infected with the human immunodeficiency virus (HIV) that causes acquired immunodeficiency syndrome (AIDS), and half a million children are newly infected every year. These infections are mainly the result of mother-to-child transmission (MTCT) of HIV during pregnancy, labor and delivery, or through breastfeeding. MTCT can be greatly reduced by treating HIV-positive mothers and their babies with antiretroviral drugs (ARVs). Without ARVs, up to half of babies born to HIV-positive mothers become infected with HIV. This rate of transmission falls to below 5% if a combination of three ARVs is given to the mother throughout pregnancy. Unfortunately, this triple-ARV therapy is too expensive for use in the resource-limited countries where most MTCT occurs. Instead, many such countries have introduced simpler, shorter ARV regimens such as a daily dose of zidovudine (a nucleoside reverse transcriptase inhibitor or NRTI) given to HIV-positive women during late pregnancy coupled with single-dose nevirapine (a non-nucleoside reverse transcriptase inhibitor or NNRTI) at the onset of labor, zidovudine and lamivudine (another NRTI) during labor and delivery, and single-dose nevirapine given to the baby at birth.
Why Was This Study Done?
More than 95% of HIV-exposed children are born in resource-limited settings where breastfeeding is the norm and is crucial for child survival even though it poses a risk of HIV transmission. Consequently, several recent studies have investigated whether MTCT can be further reduced by giving the mother ARVs while she is breastfeeding. In the Kisumu Breastfeeding Study (KiBS), for example, researchers assessed the effects of giving zidovudine, lamivudine, and either nevirapine or nelfinavir (a protease inhibitor) to HIV-infected women from 34 weeks of pregnancy through 6 months of breastfeeding. The results of KiBS indicate that this approach might be a safe, feasible way to reduce MTCT (see the accompanying paper by Thomas and colleagues). However, low amounts of nevirapine and lamivudine are transferred from mother to infant in breast milk and this exposure to ARVs could induce the development of resistance to ARVs among HIV-infected infants. In this KiBS substudy, the researchers investigate whether HIV drug resistance emerged in any of the HIV-positive infants in the parent study.
What Did the Researchers Do and Find?
In KiBS, 32 infants were HIV-positive at 24 months old; 24 were HIV-positive at 6 months old when their mothers stopped taking ARVs and when breastfeeding was supposed to stop. The researchers analyzed blood samples taken from these infants at various ages and from their mothers for the presence of HIV drug resistance mutations (DNA changes that make HIV resistant to killing by ARVs). They detected no resistance mutations in samples taken from 2-week old HIV-positive infants or from the infants who became infected after the age of 6 months. However, they found resistance mutations in a third and two-thirds of samples taken from 6-week and 6-month old HIV-positive infants, respectively. The commonest mutations conferred resistance to lamivudine and nevirapine. Moreover, resistance mutations were present in samples taken from all the HIV-positive infants whose mothers who had received nelfinavir but in only half those taken from infants whose mothers who had received nevirapine. Finally, most of the mothers of HIV-positive infants had no HIV drug resistance mutations, and only one mother-infant pair had an overlapping pattern of HIV drug resistance mutations.
What Do These Findings Mean?
These findings indicate that, in this KiBS substudy, the emergence of HIV drug resistance mutations in HIV-infected infants whose mothers were receiving ARVs occurred between 2 weeks and 6 months after birth. The pattern of mutations suggests that drug resistance most likely arose through exposure of the infants to low levels of ARVs in breast milk rather than through MTCT of drug-resistant virus. These findings need confirming but suggest that infants exposed to ARVs through breast milk—a situation that may become increasingly common given the reduction in MTCT seen in KiBS and other similar trials—should be carefully monitored for HIV infection. Providers should consider the mothers' regimen when choosing treatment for infants who are found to be HIV-infected despite maternal triple drug prophylaxis. Infants exposed to a maternal regimen with NNRTI drugs should receive first-line therapy with lopinavir/ritonavir, a protease inhibitor. The significance of the NRTI mutations such as M184V with regard to response to therapy needs further evaluation. The M184V mutation may result in hypersensitization to other NRTI drugs and delay or reverse zidovudine resistance. Given the limited availability of alternative drugs for infants in resource-limited settings, provision of the standard WHO-recommended first-line NRTI backbone, which includes 3TC, with enhanced monitoring of the infant to ensure virologic suppression, could be considered. Such an approach should reduce both illness and morbidity among infants who become HIV positive through breastfeeding.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/ 10.1371/journal.pmed.1000430.
The accompanying PLoS Medicine Research article by Thomas and colleagues describes the primary findings of the Kisumu Breastfeeding Study
Information is available from the US National Institute of Allergy and Infectious Diseases on HIV infection and AIDS
HIV InSite has comprehensive information on HIV/AIDS
Information is available from Avert, an international AIDS charity, on many aspects of HIV/AIDS, including information on children, HIV, and AIDS and on preventing mother-to-child transmission of HIV (in English and Spanish)
UNICEF also has information about children and HIV and AIDS (in several languages)
The World Health organization has information on mother-to-child transmission of HIV (in several languages), and guidance on the use of ARVs for preventing MTCT
doi:10.1371/journal.pmed.1000430
PMCID: PMC3066134  PMID: 21468304
5.  In Vitro Cross-Resistance Profile of Nucleoside Reverse Transcriptase Inhibitor (NRTI) BMS-986001 against Known NRTI Resistance Mutations 
Antimicrobial Agents and Chemotherapy  2013;57(11):5500-5508.
BMS-986001 is a novel HIV nucleoside reverse transcriptase inhibitor (NRTI). To date, little is known about its resistance profile. In order to examine the cross-resistance profile of BMS-986001 to NRTI mutations, a replicating virus system was used to examine specific amino acid mutations known to confer resistance to various NRTIs. In addition, reverse transcriptases from 19 clinical isolates with various NRTI mutations were examined in the Monogram PhenoSense HIV assay. In the site-directed mutagenesis studies, a virus containing a K65R substitution exhibited a 0.4-fold change in 50% effective concentration (EC50) versus the wild type, while the majority of viruses with the Q151M constellation (without M184V) exhibited changes in EC50 versus wild type of 0.23- to 0.48-fold. Susceptibility to BMS-986001 was also maintained in an L74V-containing virus (0.7-fold change), while an M184V-only-containing virus induced a 2- to 3-fold decrease in susceptibility. Increasing numbers of thymidine analog mutation pattern 1 (TAM-1) pathway mutations correlated with decreases in susceptibility to BMS-986001, while viruses with TAM-2 pathway mutations exhibited a 5- to 8-fold decrease in susceptibility, regardless of the number of TAMs. A 22-fold decrease in susceptibility to BMS-986001 was observed in a site-directed mutant containing the T69 insertion complex. Common non-NRTI (NNRTI) mutations had little impact on susceptibility to BMS-986001. The results from the site-directed mutants correlated well with the more complicated genotypes found in NRTI-resistant clinical isolates. Data from clinical studies are needed to determine the clinically relevant resistance cutoff values for BMS-986001.
doi:10.1128/AAC.01195-13
PMCID: PMC3811251  PMID: 23979732
6.  Emergence of Multiclass Drug–Resistance in HIV-2 in Antiretroviral-Treated Individuals in Senegal: Implications for HIV-2 Treatment in Resouce-Limited West Africa 
Background
The efficacy of various antiretroviral (ARV) therapy regimens for human immunodeficiency virus type 2 (HIV-2) infection remains unclear. HIV-2 is intrinsically resistant to the nonnucleoside reverse-transcriptase inhibitors and to enfuvirtide and may also be less susceptible than HIV-1 to some protease inhibitors (PIs). However, the mutations in HIV-2 that confer ARV resistance are not well characterized.
Methods
Twenty-three patients were studied as part of an ongoing prospective longitudinal cohort study of ARV therapy for HIV-2 infection in Senegal. Patients were treated with nucleoside reverse-transcriptase inhibitor (NRTI)– and PI (indinavir)–based regimens. HIV-2 pol genes from these patients were genotyped, and the mutations predictive of resistance in HIV-2 were assessed. Correlates of ARV resistance were analyzed.
Results
Multiclass drug–resistance mutations (NRTI and PI) were detected in strains in 30% of patients; 52% had evidence of resistance to at least 1 ARV class. The reverse-transcriptase mutations M184V and K65R, which confer high-level resistance to lamivudine and emtricitabine in HIV-2, were found in strains from 43% and 9% of patients, respectively. The Q151M mutation, which confers multinucleoside resistance in HIV-2, emerged in strains from 9% of patients. HIV-1–associated thymidine analogue mutations (M41L, D67N, K70R, L210W, and T215Y/F) were not observed, with the exception of K70R, which was present together with K65R and Q151M in a strain from 1 patient. Eight patients had HIV-2 with PI mutations associated with indinavir resistance, including K7R, I54M, V62A, I82F, L90M, L99F; 4 patients had strains with multiple PI resistance–associated mutations. The duration of ARV therapy was positively associated with the development of drug resistance (P = .02). Nine (82%) of 11 patients with HIV-2 with detectable ARV resistance had undetectable plasma HIV-2 RNA loads (<1.4 log10 copies/mL), compared with 3 (25%) of 12 patients with HIV-2 with detectable ARV resistance (P = .009). Patients with ARV-resistant virus had higher plasma HIV-2 RNA loads, compared with those with non–ARV-resistant virus (median, 1.7 log10 copies/mL [range, <1.4 to 2.6 log10 copies/mL] vs. <1.4 log10 copies/mL [range, <1.4 to 1.6 log10 copies/mL]; P = .003).
Conclusions
HIV-2–infected individuals treated with ARV therapy in Senegal commonly have HIV-2 mutations consistent with multiclass drug resistance. Additional clinical studies are required to improve the efficacy of primary and salvage treatment regimens for treating HIV-2 infection.
doi:10.1086/596504
PMCID: PMC3671065  PMID: 19143530
7.  The K65R Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Exhibits Bidirectional Phenotypic Antagonism with Thymidine Analog Mutations 
Journal of Virology  2006;80(10):4971-4977.
The K65R mutation in human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) is selected in vitro by many d-nucleoside analog RT inhibitors (NRTI) but has been rarely detected in treated patients. In recent clinical trials, the K65R mutation has emerged frequently in patients experiencing virologic failure on antiretroviral combinations that do not include 3′-azidothymidine (AZT). The reason for this change is uncertain. To gain insight, we examined trends in the frequency of K65R in a large genotype database, the association of K65R with thymidine analog mutations (TAMs) and other NRTI mutations, and the viral susceptibility profile of HIV-1 with K65R alone and in combination with TAMs. Among >60,000 clinical samples submitted for genotype analysis that contained one or more NRTI resistance mutations, the frequency of K65R increased from 0.4% in 1998 to 3.6% in 2003. Among samples with K65R, a strong negative association was evident with the TAMs M41L, D67N, L210W, T215Y/F, and K219Q/E (P < 0.005) but not with other NRTI mutations, including the Q151M complex. This suggested that K65R and TAMs are antagonistic. To test this possibility, we generated recombinant HIV-1 encoding K65R in two different TAM backgrounds: M41L/L210W/T215Y and D67N/K70R/T215F/K219Q. K65R reduced AZT resistance from >50-fold to <2.5-fold in both backgrounds. In addition, TAMs antagonized the phenotypic effect of K65R, reducing resistance to tenofovir, abacavir, 2′,3′-dideoxycytidine, dideoxyinosine, and stavudine. In conclusion, K65R and TAMs exhibit bidirectional phenotypic antagonism. This antagonism likely explains the negative association of these mutations in genotype databases, the rare emergence of K65R with antiretroviral therapies that contain AZT, and its more frequent emergence with combinations that exclude AZT.
doi:10.1128/JVI.80.10.4971-4977.2006
PMCID: PMC1472090  PMID: 16641288
8.  The “Connection” Between HIV Drug Resistance and RNase H 
Viruses  2010;2(7):1476-1503.
Currently, nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) are two classes of antiretroviral agents that are approved for treatment of HIV-1 infection. Since both NRTIs and NNRTIs target the polymerase (pol) domain of reverse transcriptase (RT), most genotypic analysis for drug resistance is limited to the first ~300 amino acids of RT. However, recent studies have demonstrated that mutations in the C-terminal domain of RT, specifically the connection subdomain and RNase H domain, can also increase resistance to both NRTIs and NNRTIs. In this review we will present the potential mechanisms by which mutations in the C-terminal domain of RT influence NRTI and NNRTI susceptibility, summarize the prevalence of the mutations in these regions of RT identified to date, and discuss their importance to clinical drug resistance.
doi:10.3390/v2071476
PMCID: PMC2982141  PMID: 21088701
connection subdomain; NRTI; NNRTI; RNase H; drug resistance; HIV
9.  The “Connection” Between HIV Drug Resistance and RNase H 
Viruses  2010;2(7):1476-1503.
Currently, nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) are two classes of antiretroviral agents that are approved for treatment of HIV-1 infection. Since both NRTIs and NNRTIs target the polymerase (pol) domain of reverse transcriptase (RT), most genotypic analysis for drug resistance is limited to the first ∼300 amino acids of RT. However, recent studies have demonstrated that mutations in the C-terminal domain of RT, specifically the connection subdomain and RNase H domain, can also increase resistance to both NRTIs and NNRTIs. In this review we will present the potential mechanisms by which mutations in the C-terminal domain of RT influence NRTI and NNRTI susceptibility, summarize the prevalence of the mutations in these regions of RT identified to date, and discuss their importance to clinical drug resistance.
doi:10.3390/v2071476
PMCID: PMC2982141  PMID: 21088701
connection subdomain; NRTI; NNRTI; RNase H; drug resistance; HIV
10.  Standardized Comparison of the Relative Impacts of HIV-1 Reverse Transcriptase (RT) Mutations on Nucleoside RT Inhibitor Susceptibility 
Determining the phenotypic impacts of reverse transcriptase (RT) mutations on individual nucleoside RT inhibitors (NRTIs) has remained a statistical challenge because clinical NRTI-resistant HIV-1 isolates usually contain multiple mutations, often in complex patterns, complicating the task of determining the relative contribution of each mutation to HIV drug resistance. Furthermore, the NRTIs have highly variable dynamic susceptibility ranges, making it difficult to determine the relative effect of an RT mutation on susceptibility to different NRTIs. In this study, we analyzed 1,273 genotyped HIV-1 isolates for which phenotypic results were obtained using the PhenoSense assay (Monogram, South San Francisco, CA). We used a parsimonious feature selection algorithm, LASSO, to assess the possible contributions of 177 mutations that occurred in 10 or more isolates in our data set. We then used least-squares regression to quantify the impact of each LASSO-selected mutation on each NRTI. Our study provides a comprehensive view of the most common NRTI resistance mutations. Because our results were standardized, the study provides the first analysis that quantifies the relative phenotypic effects of NRTI resistance mutations on each of the NRTIs. In addition, the study contains new findings on the relative impacts of thymidine analog mutations (TAMs) on susceptibility to abacavir and tenofovir; the impacts of several known but incompletely characterized mutations, including E40F, V75T, Y115F, and K219R; and a tentative role in reduced NRTI susceptibility for K64H, a novel NRTI resistance mutation.
doi:10.1128/AAC.05487-11
PMCID: PMC3346663  PMID: 22330916
11.  Frequency of Mutations Conferring Resistance to Nucleoside Reverse Transcriptase Inhibitors in Human Immunodeficiency Virus Type 1-Infected Patients in Korea 
Journal of Clinical Microbiology  2002;40(4):1319-1325.
A nested PCR and direct sequencing methods were used to define human immunodeficiency virus type 1(HIV-1) reverse transcriptase codons 41 to 219 in DNA from 127 peripheral blood mononuclear cell samples obtained from 35 patients treated with nucleoside reverse transcriptase inhibitors (NRTI). The follow-up period after the initiation of NRTI therapy was 61.8 ± 31 months (mean and standard deviation). In addition to NRTI therapy, 32 of 35 patients were simultaneously treated with Korean red ginseng. The annual decrease in the CD4+ T-cell count over 5 years was 13.2/μl. Twenty-eight (80%) of the 35 patients had mutations conferring resistance to NRTI. The frequencies of K70R, T215S/Y/F (i.e., mutation of T at codon 215 to S, Y, or F), D67N/E, K219Q, T69N/S/A, M41L, and L210W mutations conferring resistance to zidovudine were 57.6, 36.4, 36.4, 27.2, 24.2, 21.2, and 12.1%, respectively. Mutations conferring resistance to didanosine and lamivudine were detected in 2 (L74V and M184I; 14.2%) of 11 patients tested and in 4 (M184V; 57%) of 7 patients tested, respectively. In particular, the frequency of T69N/S/A increased sharply after more than 48 months of zidovudine monotherapy. However, Q151M was not detected. As the first report on the frequency of NRTI resistance mutations in Korea, our data suggest that genotypic antiretroviral drug testing should be considered for the design of better drug regimens to improve the management of HIV-1-infected patients.
doi:10.1128/JCM.40.4.1319-1325.2002
PMCID: PMC140365  PMID: 11923351
12.  Efficacy and Safety of Three Antiretroviral Regimens for Initial Treatment of HIV-1: A Randomized Clinical Trial in Diverse Multinational Settings 
PLoS Medicine  2012;9(8):e1001290.
Thomas Campbell and colleagues report findings of a randomized trial conducted in multiple countries regarding the efficacy of antiretroviral regimens with simplified dosing.
Background
Antiretroviral regimens with simplified dosing and better safety are needed to maximize the efficiency of antiretroviral delivery in resource-limited settings. We investigated the efficacy and safety of antiretroviral regimens with once-daily compared to twice-daily dosing in diverse areas of the world.
Methods and Findings
1,571 HIV-1-infected persons (47% women) from nine countries in four continents were assigned with equal probability to open-label antiretroviral therapy with efavirenz plus lamivudine-zidovudine (EFV+3TC-ZDV), atazanavir plus didanosine-EC plus emtricitabine (ATV+DDI+FTC), or efavirenz plus emtricitabine-tenofovir-disoproxil fumarate (DF) (EFV+FTC-TDF). ATV+DDI+FTC and EFV+FTC-TDF were hypothesized to be non-inferior to EFV+3TC-ZDV if the upper one-sided 95% confidence bound for the hazard ratio (HR) was ≤1.35 when 30% of participants had treatment failure.
An independent monitoring board recommended stopping study follow-up prior to accumulation of 472 treatment failures. Comparing EFV+FTC-TDF to EFV+3TC-ZDV, during a median 184 wk of follow-up there were 95 treatment failures (18%) among 526 participants versus 98 failures among 519 participants (19%; HR 0.95, 95% CI 0.72–1.27; p = 0.74). Safety endpoints occurred in 243 (46%) participants assigned to EFV+FTC-TDF versus 313 (60%) assigned to EFV+3TC-ZDV (HR 0.64, CI 0.54–0.76; p<0.001) and there was a significant interaction between sex and regimen safety (HR 0.50, CI 0.39–0.64 for women; HR 0.79, CI 0.62–1.00 for men; p = 0.01). Comparing ATV+DDI+FTC to EFV+3TC-ZDV, during a median follow-up of 81 wk there were 108 failures (21%) among 526 participants assigned to ATV+DDI+FTC and 76 (15%) among 519 participants assigned to EFV+3TC-ZDV (HR 1.51, CI 1.12–2.04; p = 0.007).
Conclusion
EFV+FTC-TDF had similar high efficacy compared to EFV+3TC-ZDV in this trial population, recruited in diverse multinational settings. Superior safety, especially in HIV-1-infected women, and once-daily dosing of EFV+FTC-TDF are advantageous for use of this regimen for initial treatment of HIV-1 infection in resource-limited countries. ATV+DDI+FTC had inferior efficacy and is not recommended as an initial antiretroviral regimen.
Trial Registration
www.ClinicalTrials.gov NCT00084136
Please see later in the article for the Editors' Summary.
Editors' Summary
Background
Despite the enormous gains in reducing HIV-related illness and death over the past decade, there are still considerable challenges to meeting the global goal of universal access to highly active antiretroviral treatment—a combination of effective drugs that attack the HIV virus in various ways—to everyone living with HIV/AIDS who could benefit from treatment. In recognition of the related financial, technical, and system obstacles to providing universal access to HIV treatment, in 2010 the UN agency responsible for HIV/AIDS—UNAIDS—launched an ambitious plan called Treatment 2.0, which aims to simplify the way HIV treatment is currently provided. One of the main focuses of Treatment 2.0 is to simplify drug regimes for the treatment of HIV and to make treatment regimes less toxic. In line with Treatment 2.0, the World Health Organization currently recommends that antiretroviral regimens for the initial treatment of HIV should include two nucleoside reverse transcriptase inhibitors (zidovudine or tenofovir disoproxil fumarate [DF] with lamivudine or emtricitabine) and a non-nucleoside reverse transcriptase inhibitor (efavirenz or nevirapine.)
Why Was This Study Done?
Most of the evidence about the safety and effectiveness of clinical trials come from clinical trials in high-income countries and thus is not generally representative of the majority of people with HIV. So in this study, the researchers conducted a randomized controlled trial in diverse populations in many different settings to investigate whether antiretroviral regimens administered once daily were as effective as twice-daily regimens and also whether a regimen containing the drug atazanavir administered once daily was as safe and effective as a regimen containing efavirenz—data from previous studies have suggested that atazanavir has characteristics, such as its side effect profile, which may make it more suitable for low income settings.
What Did the Researchers Do and Find?
The researchers recruited eligible patients from centers in Brazil, Haiti, India, Malawi, Peru, South Africa, Thailand, the United States, and Zimbabwe—almost half (47%) were women. Then the researchers randomly assigned participants to one of three regimens: efavirenz 600 mg daily plus co-formulated lamivudine-zidovudine 150 mg/300 mg twice daily (EFV+3TC-ZDV); or atazanavir 400 mg once daily, plus didanosine-EC 400 mg once daily, plus emtricitabine 200 mg once daily (ATV+DDI+FTC); or efavirenz 600 mg once daily plus coformulated emtricitabine-tenofovir-DF 200 mg/300 mg once daily (EFV+FTC-TDF). During the study period ATV+DDI+FTC was found to be inferior to EFV+3TC-ZDV, so the Multinational Data Safety Monitoring Board ordered this arm of the trial to stop. Then a year later, due to the low number of treatment failures (deaths, severe HIV disease, or serious opportunistic infections) in the remaining two arms, the board advised the trial to stop early. So the researchers analyzed the data obtained up to this point and pooled the results from all of the centers.
The researchers found that during an average of 184 weeks of follow-up, there were 95 treatment failures (18%) among 526 participants taking EFV+FTC-TDF compared to 98 failures among 519 participants taking EFV+3TC-ZDV. During an average 81 weeks follow-up, there were 108 failures (21%) among 526 participants assigned to ATV+DDI+FTC and 76 (15%) among 519 participants assigned to EFV+3TC-ZDV. As for safety, 243 (46%) participants assigned to EFV+FTC-TDF reached a safety endpoint (grade 3 disease, abnormal lab measurement, or the need to change drug) compared to 313 (60%) in the EFV+3TC-ZDV group. Importantly, the researchers found that there was greater risk of safety events for women assigned to EFV+3TC-ZDV and also that the atazanavir-based regimen had a higher relative efficacy in women compared to men.
What Do These Findings Mean?
These findings suggest that in diverse populations, EFV+FTC-TDF is as effective as EFV+3TC-ZDV but importantly, the once-daily dosing of EFV+FTC-TDF makes this regimen useful for the initial treatment of HIV, especially in low-income countries. Therefore, as per the guidance in Treatment 2.0, EFV+FTC-TDF in a single combination tablet that can be taken once a day is an attractive option. These findings also indicate that as ATV+DDI+FTC was found to be inferior to the other regimens, this combination should not be used in the initial treatment of HIV. These findings also add to the evidence that antiretroviral efficacy and safety can differ between women and men and support further development of sex-specific recommendations for antiretroviral regimen options.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001290.
The UNAIDS website has more information about Treatment 2.0; and the WHO website provides technical information
For an introduction to the treatment of HIV/AIDS see http://www.avert.org/treatment.htm; the AVERT site also has personal stories from women living with HIV/AIDS
AIDSmap provides information for individuals and communities affected by HIV/AIDS
The ACTG website provides information about research to improve treatment of HIV and related complications
doi:10.1371/journal.pmed.1001290
PMCID: PMC3419182  PMID: 22936892
13.  Increased Risk of Q151M and K65R Mutations in Patients Failing Stavudine-Containing First-Line Antiretroviral Therapy in Cambodia 
PLoS ONE  2013;8(8):e73744.
Background
Multi-nucleos(t)ide resistance (MNR) mutations including Q151M, K65R mutations, and insertion at codon 69 of HIV-1 reverse transcriptase coding region may confer resistance to all molecules of nucleos(t)ide reverse transcriptase inhibitors (NRTI). The presence of these mutations is an emerging problem compromising non-nucleoside reverse transcriptase inhibitors and protease inhibitors-based therapies. Furthermore, factors associated with selection of these mutations are still not well defined. The current study aimed to evaluate the frequency and to characterize factors associated with the occurrence of multi-nucleos(t)ide resistance mutations among HIV-1 infected patients failing recommended first-line antiretroviral regimens in Cambodia.
Methodology/Principal Finding
This is a retrospective analysis of HIV-1 drug resistance genotyping of 520 HIV-1 infected patients in virological failure (viral load > 250 copies/mL) while on first-line antiretroviral therapy in Cambodia with at least one reverse transcriptase inhibitor resistance associated mutation. Among these 520 patients, a total of 66 subjects (66/520, 12.7%) presented ≥1 MNR mutation, including Q151M, K65R, and Insert69 for 59 (11.3%), 29 (5.6%), and 2 (0.4%) patients, respectively. In multivariate analysis, both Q151M (p = 0.039) and K65R (p = 0.029) mutations were independently associated with current stavudine- compared to zidovudine-use.
Conclusion
Such selection of mutations by stavudine drastically limits the choice of antiretroviral molecules available for second-line therapy in resource-limited settings. This finding supports the World Health Organization’s recommendation for stavudine phase-out.
doi:10.1371/journal.pone.0073744
PMCID: PMC3756052  PMID: 24015311
14.  Evidence of a Role for the Q151L Mutation and the Viral Background in Development of Multiple Dideoxynucleoside-Resistant Human Immunodeficiency Virus Type 1 
Journal of Virology  2000;74(20):9339-9346.
The majority of human immunodeficiency virus type 1 (HIV-1)-infected patients treated with zidovudine (AZT) plus zalcitabine (ddC) and didanosine (ddI) develop AZT resistance mediated by mutations such as T215Y and M41L. Only a small proportion of patients develop multiple dideoxynucleoside resistance (MDNR) mediated by the Q151M mutation. To gain insight into the factors responsible for the low frequency of selection of Q151M, we evaluated the replication capabilities of recombinant viruses carrying two possible intermediates (151L or 151K) of the Q151M mutation generated in different reverse transcriptase (RT) genetic backgrounds. The 151L and 151K mutations were introduced by site-directed mutagenesis in RTs from two patient-derived HIV-1 isolates that had either wild type (WT) Q or the Q151M (posttreatment isolate) mutation. For comparison, both mutations were also introduced in a laboratory-adapted HIV-1 strain (HIV-1HXB2). Analysis of replication capabilities showed that both 151L and 151K were lethal in RT genetic backgrounds of the WT isolate and in HIV-1HXB2. In contrast, 151L but not 151K allowed virus replication in RT backgrounds of the posttreatment isolate. Three mutations (V35I, S68G, and I178M) were present in the RT background of the posttreatment isolate but not in the WT isolate. Introduction of S68G in the RT of both the WT isolate and HIV-1HXB2 partially restored replication capacity of recombinants carrying the 151L mutation. The S68G mutation alone did not confer a significant replicative disadvantage in WT viruses. Like HIV-1151M, HIV-1151L RT was found to have six- to eightfold resistance to AZT-triphosphate (TP), ddA-TP, and ddC-TP, indicating an MDNR phenotype. However, HIV-1151L was found to be less fit than HIV-1151M, which may explain the preferential selection of HIV-1151M observed in vivo. The demonstrated ability of HIV-1151L/68G to replicate and the associated MDNR suggest that 151L is a potential intermediate of Q151M. The dependence of HIV-1151L on other mutations, such as S68G, for replication may explain the low frequency of the Q151M-mediated pathway of resistance.
PMCID: PMC112361  PMID: 11000201
15.  Effectiveness of antiretroviral therapy and development of drug resistance in HIV-1 infected patients in Mombasa, Kenya 
Access to antiretroviral therapy (ART) is increasing in resource-limited settings (RLS) and can successfully reduce HIV-related morbidity and mortality. However, virologic failure and development of viral drug resistance can result in reduced treatment options and disease progression. Additionally, transmission of resistant virus, and particularly multi-drug resistance, could become a public health concern. This study evaluated treatment success and development of ART drug resistance after short-term treatment among patients attending the Comprehensive HIV Care Centre (CCC) of Coast Province General Hospital, Mombasa, Kenya. One hundred and fifty HIV-infected individuals receiving ART were consecutively recruited to participate in the study. After determination of plasma viral load, patients with detectable viral load levels were subjected to genotypic drug resistance testing. At the time of sampling, 132 of the 150 participants were on ART for more than 6 months (median 21 months, IQR = 12–26). An efficient viral load reduction to below 50 copies/ml was observed in 113 (85.6%) of them. Of the 19 patients with a detectable viral load, sequencing of the protease (PR) and reverse transcriptase (RT) gene was successful in 16. Eleven (11) of these 16 patients were infected with a subtype A1 virus. Major PR mutations were absent, but mutations associated with drug resistance in RT were detected in 14 of the 16 patients (87.5%). High-level resistance against at least 2 drugs of the ART regimen was observed in 9/14 (64.3%). The 3TC mutation M184V and the NNRTI mutation K103N were most frequent but also the multi-drug resistance Q151M and the broad NRTI cross-resistance K65R were observed. The results of this study revealed a high rate of treatment success after short term ART in patients treated at a public provincial hospital in a RLS. Nevertheless, the observed high risk of accumulation of resistance mutations among patients failing treatment and the selection of multi-drug resistance mutations in some, remains of great concern for future treatment options and potential transmission to partners.
doi:10.1186/1742-6405-6-12
PMCID: PMC2704235  PMID: 19531211
16.  EFFECT OF TRANSLOCATION DEFECTIVE REVERSE TRANSCRIPTASE INHIBITORS ON THE ACTIVITY OF N348I, A CONNECTION SUBDOMAIN DRUG RESISTANT HIV-1 REVERSE TRANSCRIPTASE MUTANT 
4′-ethynyl-2-fluoro-2′-deoxyadenosine (EFdA) is a highly potent inhibitor of HIV-1 reverse transcriptase (RT). We have previously shown that its exceptional antiviral activity stems from a unique mechanism of action that is based primarily on blocking translocation of RT; therefore we named EFdA a Translocation Defective RT Inhibitor (TDRTI). The N348I mutation at the connection subdomain (CS) of HIV-1 RT confers clinically significant resistance to both nucleoside (NRTIs) and non-nucleoside RT inhibitors (NNRTIs). In this study we tested EFdA-triphosphate (TP) together with a related compound, ENdA-TP (4′-ethynyl-2-amino-2′-deoxyadenosine triphosphate) against HIV-1 RTs that carry clinically relevant drug resistance mutations: N348I, D67N/K70R/L210Q/T215F, D67N/K70R/L210Q/T215F/N348I, and A62V/V75I/F77L/F116Y/Q151M. We demonstrate that these enzymes remain susceptible to TDRTIs. Similar to WT RT, the N348I RT is inhibited by EFdA mainly at the point of incorporation through decreased translocation. In addition, the N348I substitution decreases the RNase H cleavage of DNA terminated with EFdA-MP (T/PEFdA-MP). Moreover, N348I RT unblocks EFdA-terminated primers with similar efficiency as the WT enzyme, and further enhances EFdA unblocking in the background of AZT-resistance mutations. This study provides biochemical insights into the mechanism of inhibition of N348I RT by TDRTIs and highlights the excellent efficacy of this class of inhibitors against WT and drug-resistant HIV-1 RTs.
PMCID: PMC3551986  PMID: 23273211
EFdA; ENdA; N348I; Translocation Defective Reverse Transcriptase Inhibitors; Reverse Transcriptase; HIV-1; Antivirals
17.  Extended spectrum of HIV-1 reverse transcriptase mutations in patients receiving multiple nucleoside analog inhibitors 
AIDS (London, England)  2003;17(6):791-799.
Objective
To characterize reverse transcriptase (RT) mutations by their association with extent of nucleoside RT inhibitor (NRTI) therapy. To identify mutational clusters in RT sequences from persons receiving multiple NRTI.
Design
A total of 1210 RT sequences from persons with known antiretroviral therapy were analyzed: 641 new sequences were performed at Stanford University Hospital; 569 were previously published.
Methods
Chi-square tests and logistic regression were done to identify associations between mutations and NRTI therapy. Correlation studies were done to identify mutational clusters. The Benjamini-Hochberg procedure was used to correct for multiple comparisons.
Results
Mutations at 26 positions were significantly associated with NRTI including 17 known resistance mutations (positions 41, 44, 62, 65, 67, 69, 70, 74, 75, 77, 116, 118, 151, 184, 210, 215, 219) and nine previously unreported mutations (positions 20, 39, 43, 203, 208, 218, 221, 223, 228). The nine new mutations correlated linearly with number of NRTI; 777 out of 817 (95%) instances occurred with known drug resistance mutations. Positions 203, 208, 218, 221, 223, and 228 were conserved in untreated persons; positions 20, 39, and 43 were polymorphic. Most NRTI-associated mutations clustered into three groups: (i) 62, 65, 75, 77, 115, 116, 151; (ii) 41, 43, 44, 118, 208, 210, 215, 223; (iii) 67, 69, 70, 218, 219, 228.
Conclusions
Mutations at nine previously unreported positions are associated with NRTI therapy. These mutations are probably accessory because they occur almost exclusively with known drug resistance mutations. Most NRTI mutations group into one of three clusters, although several (e.g., M184V) occur in multiple mutational contexts.
doi:10.1097/01.aids.0000050860.71999.23
PMCID: PMC2573403  PMID: 12660525
HIV drug resistance; resistance mutations; reverse transcriptase inhibitors; HIV diagnostic tests; antiretroviral therapy
18.  Anti-Human Immunodeficiency Virus Type 1 Activity and Resistance Profile of 2′,3′-Didehydro-3′-Deoxy-4′-Ethynylthymidine In Vitro 
2′,3′-Didehydro-3′-deoxy-4′-ethynylthymidine (4′-Ed4T) has been identified as a novel nucleoside analog with potent and selective anti-human immunodeficiency virus type 1 (HIV-1) activity and weak cytotoxicity in cell cultures. 4′-Ed4T proved to be 5- to 10-fold more active than its structurally related compound, stavudine (d4T). However, the drug resistance profile of 4′-Ed4T was different from those of d4T and other existing HIV-1 nucleoside reverse transcriptase inhibitors (NRTIs). Approximately 6- to 11-fold decreases in susceptibility to 4′-Ed4T were observed for HIV-1 carrying NRTI-associated mutations (D67N, K70R, T215F, and K219Q) or the lamivudine (3TC)-resistant mutation M184V. In contrast, the susceptibility of the virus carrying the K65R mutation or the multidrug-resistant mutation with the Q151M complex (A62V, V75I, F77L, F116Y, and Q151M) was not altered. Furthermore, the activity of 4′-Ed4T appeared to be enhanced in the presence of K103N, a major nonnucleoside reverse transcriptase inhibitor-resistant mutation. Although 4′-Ed4T was 4.5- to 17.5-fold less active against multidrug-resistant clinical isolates than against a reference strain isolated from a treatment-naïve patient, it was still inhibitory to these isolates at low concentrations. Analysis of 4′-Ed4T-resistant HIV-1 obtained through in vitro selection revealed that the virus was also resistant to 3TC and had two amino acid mutations (P119S and T165A) in addition to the M184V mutation. Since 4′-Ed4T has increased anti-HIV-1 activity, decreased cytotoxicity, and a different resistance profile, it should be considered for further development as a new member of NRTIs.
doi:10.1128/AAC.49.8.3355-3360.2005
PMCID: PMC1196241  PMID: 16048947
19.  Combinations of Mutations in the Connection Domain of Human Immunodeficiency Virus Type 1 Reverse Transcriptase: Assessing the Impact on Nucleoside and Nonnucleoside Reverse Transcriptase Inhibitor Resistance▿  
Recent reports have described the effect of mutations in the connection and RNase H domains of reverse transcriptase (RT) on nucleoside and nonnucleoside reverse transcriptase inhibitor (NRTI and NNRTI, respectively) resistance in the presence of thymidine analog resistance mutations (TAMs) and NNRTI mutations (J. H. Brehm, D. Koontz, J. D. Meteer, V. Pathak, N. Sluis-Cremer, and J. W. Mellors, J. Virol. 81:7852-7859, 2007; K. A. Delviks-Frankenberry, G. N. Nikolenko, R. Barr, and V. K. Pathak, J. Virol. 81:6837-6845, 2007; G. N. Nikolenko, K. A. Delviks-Frankenberry, S. Palmer, F. Maldarelli, M. J. Fivash, Jr., J. M. Coffin, and V. K. Pathak, Proc. Natl. Acad. Sci. U. S. A. 104:317-322, 2007; G. N. Nikolenko, S. Palmer, F. Maldarelli, J. W. Mellors, J. M. Coffin, and V. K. Pathak, Proc. Natl. Acad. Sci. U. S. A. 102:2093-2098, 2005; and S. H. Yap, C. W. Sheen, J. Fahey, M. Zanin, D. Tyssen, V. D. Lima, B. Wynhoven, M. Kuiper, N. Sluis-Cremer, P. R. Harrigan, and G. Tachedjian, PLoS Med. 4:e335, 2007). In the present study, novel mutations in the connection domain of RT (T369I/V), first identified in patient-derived viruses, were characterized, and their effects on NNRTI and NNRTI susceptibility were determined. Furthermore, the effect of N348I on NRTI and NNRTI resistance was confirmed. HIV-1 with either N348I or T369I/V demonstrated reduced susceptibility to nevirapine (NVP), efavirenz (EFV), delaviridine (DLV), and zidovudine (ZDV) compared to wild-type HIV-1. However, HIV-1 with T369I and N348I demonstrated 10- to 60-fold resistance to these same drugs. In clinical samples, these two connection domain RT mutations were predominantly observed in viruses containing TAMs and NNRTI mutations and did not alter the susceptible-resistant classifications of these samples. Introduction of T369I, N348I, or T369I/N348I also reduced replication capacity (RC). These observations suggest that it may be of scientific interest to test these mutations against new NNRTI candidates.
doi:10.1128/AAC.00870-09
PMCID: PMC2863632  PMID: 20194692
20.  Relative Fitness and Replication Capacity of a Multinucleoside Analogue-Resistant Clinical Human Immunodeficiency Virus Type 1 Isolate with a Deletion of Codon 69 in the Reverse Transcriptase Coding Region▿  
Journal of Virology  2007;81(9):4713-4721.
Deletions, insertions, and amino acid substitutions in the β3-β4 hairpin loop-coding region of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been associated with resistance to nucleoside RT inhibitors when appearing in combination with other mutations in the RT-coding region. In this work, we have measured the in vivo fitness of HIV-1 variants containing a deletion of 3 nucleotides affecting codon 69 (Δ69) of the viral RT as well as the replication capacity (RC) ex vivo of a series of recombinant HIV-1 variants carrying an RT bearing the Δ69 deletion or the T69A mutation in a multidrug-resistant (MDR) sequence background, including the Q151M complex and substitutions M184V, K103N, Y181C, and G190A. Patient-derived viral clones having RTs with Δ69 together with S163I showed increased RCs under drug pressure. These data were consistent with the viral population dynamics observed in a long-term-treated HIV-1-infected patient. In the absence of drugs, viral clones containing T69A replicated more efficiently than those having Δ69, but only when patient-derived sequences corresponding to RT residues 248 to 527 were present. These effects could be attributed to a functional interaction between the C-terminal domain of the p66 subunit (RNase H domain) and the DNA polymerase domain of the RT. Finally, recombinant HIV-1 clones bearing RTs with MDR-associated mutations, including deletions at codon 69, showed increased susceptibilities to protease inhibitors in phenotypic assays. These effects correlated with impaired Gag cleavage and could be attributed to delayed maturation and decreased production of active protease in those variants.
doi:10.1128/JVI.02135-06
PMCID: PMC1900151  PMID: 17314158
21.  Multidrug-resistant human immunodeficiency virus type 1 strains resulting from combination antiretroviral therapy. 
Journal of Virology  1996;70(2):1086-1090.
Multidrug-resistant human immunodeficiency virus type 1 (HIV-1) strains with reverse transcriptase (RT) mutations at codons A62-->V, V75-->I, F77-->L, F116-->Y, and Q151-->M have been reported in patients receiving combination therapy with zidovudine (AZT) and didanosine (ddI). Infectious clones with each mutation alone, all five mutations together, and various combinations of mutations were created by site-directed mutagenesis. Mutation Q151-->M conferred partial resistance to AZT, ddI, zalcitibine, and stavudine, whereas a combination of four mutations conferred increased resistance to AZT, ddI, zalcitibine, and stavudine. The positions of residues 75, 77, and 151 in the three-dimensional crystal structure of HIV-1 RT suggest that these residues may affect the ability of the enzyme to discriminate between deoxynucleoside triphosphates and nucleoside analog RT inhibitors. Replication experiments showed that clones with mutation F77-->L but without V75-->I (HIV-1(77), HIV-1(77,151), and HIV-1(77,116,151) had attenuated growth compared with that of the original HIV-1NL4-3 strain and strains containing mutations at both positions 75 and 77 (HIV-1(75,77,151) and HIV-1(75,77,116,15)). Sequence analysis of viral RNA and proviral DNA from several patients indicated that RT mutations developed in a sequential and cumulative pattern over the course of a 2- to 4-year observation period. The present results suggest that drug resistance and viral replicative capacity both may play a role in selection of HIV-1 RT mutations.
PMCID: PMC189915  PMID: 8551567
22.  Comparative Fitness of Multi-Dideoxynucleoside-Resistant Human Immunodeficiency Virus Type 1 (HIV-1) in an In Vitro Competitive HIV-1 Replication Assay 
Journal of Virology  1999;73(7):5356-5363.
We examined whether human immunodeficiency virus type 1 (HIV-1) fitness was altered upon the acquisition of a set or subset of five mutations (A62V, V75I, F77L, F116Y, and Q151M) in the pol gene, which confers resistance to multiple dideoxynucleosides (MDR), as well as the zidovudine resistance-associated mutation T215Y, using a competitive HIV-1 replication assay in a setting of an HXB2D genetic background. Target H9 cells were exposed to a 50:50 mixture of paired infectious molecular clones, and HIV-1 in the culture supernatant was transmitted to new cultures every 7 to 10 days. The polymerase-encoding region of the virus was sequenced at various time points, and the relative proportion of the two viral populations was determined. In the absence of drugs, the comparative order for replicative fitness was HIV-162/75/77/116/151 > HIV-177/116/151 > HIV-1151 > wild-type HIV-1 (HIV-1wt) > HIV-175/77/116/151 > HIV-1151/215 > HIV-1215. In the presence of zidovudine or didanosine, the order was HIV-162/75/77/116/151 > HIV-177/116/151 > HIV-175/77/116/151 > HIV-1151 > HIV-1215. HIV-1215S(TCC), a putative intermediate infectious clone for HIV-1215, replicated comparably to HIV-1wt, while two putative intermediates for HIV-1151 [HIV-1151L(CTG) and HIV-1151K(AAG)] replicated much less efficiently than HIV-1wt and HIV-1151, suggesting that for HIV-1151 to develop, two base substitutions are likely to occur concurrently or within a short interval. These data may illustrate the molecular basis by which HIV-1151 emerges much less frequently than HIV-1215. The present data also demonstrate that several MDR HIV-1 variants are more fit than HIV-1wt in the absence of drugs and that resistance-associated mutations and drug pressure are critical variates for HIV-1 fitness.
PMCID: PMC112591  PMID: 10364282
23.  In Vitro Antiretroviral Activity and In Vitro Toxicity Profile of SPD754, a New Deoxycytidine Nucleoside Reverse Transcriptase Inhibitor for Treatment of Human Immunodeficiency Virus Infection 
SPD754 (AVX754) is a deoxycytidine analogue nucleotide reverse transcriptase inhibitor (NRTI) in clinical development. These studies characterized the in vitro activity of SPD754 against NRTI-resistant human immunodeficiency virus type 1 (HIV-1) and non-clade B HIV-1 isolates, its activity in combination with other antiretrovirals, and its potential myelotoxicity and mitochondrial toxicity. SPD754 was tested against 50 clinical HIV-1 isolates (5 wild-type isolates and 45 NRTI-resistant isolates) in MT-4 cells using the Antivirogram assay. SPD754 susceptibility was reduced 1.2- to 2.2-fold against isolates resistant to zidovudine (M41L, T215Y/F, plus a median of three additional nucleoside analogue mutations [NAMs]) and/or lamivudine (M184V) and was reduced 1.3- to 2.8-fold against isolates resistant to abacavir (L74V, Y115F, and M184V plus one other NAM) or stavudine (V75T/M, M41L, T215F/Y, and four other NAMs). Insertions at amino acid position 69 and Q151M mutations (with or without M184V) reduced SPD754 susceptibility 5.2-fold and 14- to 16-fold, respectively (these changes gave values comparable to or less than the corresponding values for zidovudine, lamivudine, abacavir, and didanosine). SPD754 showed similar activity against isolates of group M HIV-1 clades, including A/G, B, C, D, A(E), D/F, F, and H. SPD754 showed additive effects in combination with other NRTIs, tenofovir, nevirapine, or saquinavir. SPD754 had no significant effects on cell viability or mitochondrial DNA in HepG2 or MT-4 cells during 28-day exposure at concentrations up to 200 μM. SPD754 showed a low potential for myelotoxicity against human bone marrow. In vitro, SPD754 retained activity against most NRTI-resistant HIV-1 clinical isolates and showed a low propensity to cause myelotoxicity and mitochondrial toxicity.
doi:10.1128/AAC.50.2.625-631.2006
PMCID: PMC1366874  PMID: 16436719
24.  HIV-1 Antiretroviral Resistance 
Drugs  2012;72(9):e1-e25.
The efficacy of an antiretroviral (ARV) treatment regimen depends on the activity of the regimen’s individual ARV drugs and the number of HIV-1 mutations required for the development of resistance to each ARV — the genetic barrier to resistance. ARV resistance impairs the response to therapy in patients with transmitted resistance, unsuccessful initial ARV therapy and multiple virological failures. Genotypic resistance testing is used to identify transmitted drug resistance, provide insight into the reasons for virological failure in treated patients, and help guide second-line and salvage therapies. In patients with transmitted drug resistance, the virological response to a regimen selected on the basis of standard genotypic testing approaches the responses observed in patients with wild-type viruses. However, because such patients are at a higher risk of harbouring minority drug-resistant variants, initial ARV therapy in this population should contain a boosted protease inhibitor (PI) — the drug class with the highest genetic barrier to resistance.
In patients receiving an initial ARV regimen with a high genetic barrier to resistance, the most common reasons for virological failure are nonadherence and, potentially, pharmacokinetic factors or minority transmitted drug-resistant variants. Among patients in whom first-line ARVs have failed, the patterns of drug-resistance mutations and cross-resistance are often predictable. However, the extent of drug resistance correlates with the duration of uncontrolled virological replication. Second-line therapy should include the continued use of a dual nucleoside/nucleotide reverse transcriptase inhibitor (NRTI)-containing backbone, together with a change in the non-NRTI component, most often to an ARV belonging to a new drug class.
The number of available fully active ARVs is often diminished with each successive treatment failure. Therefore, a salvage regimen is likely to be more complicated in that it may require multiple ARVs with partial residual activity and compromised genetic barriers of resistance to attain complete virological suppression. A thorough examination of the patient’s ARV history and prior resistance tests should be performed because genotypic and/or phenotypic susceptibility testing is often not sufficient to identify drug-resistant variants that emerged during past therapies and may still pose a threat to a new regimen. Phenotypic testing is also often helpful in this subset of patients. ARVs used for salvage therapy can be placed into the following hierarchy: (i) ARVs belonging to a previously unused drug class; (ii) ARVs belonging to a previously used drug class that maintain significant residual antiviral activity; (iii) NRTI combinations, as these often appear to retain in vivo virological activity, even in the presence of reduced in vitro NRTI susceptibility; and rarely (iv) ARVs associated with previous virological failure and drug resistance that appear to have possibly regained their activity as a result of viral reversion to wild type. Understanding the basic principles of HIV drug resistance is helpful in guiding individual clinical decisions and the development of ARV treatment guidelines.
doi:10.2165/11633630-000000000-00000
PMCID: PMC3689909  PMID: 22686620
25.  HIV-1 mutational pathways under multidrug therapy 
Background
Genotype-derived drug resistance profiles are a valuable asset in HIV-1 therapy decisions. Therapy decisions could be further improved, both in terms of predicting length of current therapy success and in preserving followup therapy options, through better knowledge of mutational pathways- here defined as specific locations on the viral genome which, when mutant, alter the risk that additional specific mutations arise. We limit the search to locations in the reverse transcriptase region of the HIV-1 genome which host resistance mutations to nucleoside (NRTI) and non-nucleoside (NNRTI) reverse transcriptase inhibitors (as listed in the 2008 International AIDS Society report), or which were mutant at therapy start in 5% or more of the therapies studied.
Methods
A Cox proportional hazards model was fit to each location with the hazard of a mutation at that location during therapy proportional to the presence/absence of mutations at the remaining locations at therapy start. A pathway from preexisting to occurring mutation was indicated if the covariate was both selected as important via smoothly clipped absolute deviation (a form of regularized regression) and had a small p-value. The Cox model also allowed controlling for non-genetic parameters and potential nuisance factors such as viral resistance and number of previous therapies. Results were based on 1981 therapies given to 1495 distinct patients drawn from the EuResist database.
Results
The strongest influence on the hazard of developing NRTI resistance was having more than four previous therapies, not any one existing resistance mutation. Known NRTI resistance pathways were shown, and previously speculated inhibition between the thymidine analog pathways was evidenced. Evidence was found for a number of specific pathways between NRTI and NNRTI resistance sites. A number of common mutations were shown to increase the hazard of developing both NRTI and NNRTI resistance. Viral resistance to the therapy compounds did not materially effect the hazard of mutation in our model.
Conclusions
The accuracy of therapy outcome prediction tools may be increased by including the number of previous treatments, and by considering locations in the HIV genome which increase the hazard of developing resistance mutations.
doi:10.1186/1742-6405-8-26
PMCID: PMC3162516  PMID: 21794106

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