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Objectives Dolutegravir is an integrase strand-transfer inhibitor that has shown unprecedented robustness against the emergence of HIV drug-resistant strains in treatment-naive individuals. The R263K substitution in integrase was identified through culture selection as a resistance-associated substitution for dolutegravir and was recently detected in two treatment-experienced participants in the SAILING clinical trial, who experienced dolutegravir-based treatment failure, one of whom was infected by a subtype C virus. The objective of this study was to characterize the R263K substitution in HIV-1 subtype C integrase. Design and methods We used cell-free strand transfer assays and tissue culture experiments to characterize the R263K substitution in HIV-1 subtype C integrase in comparison with subtype B. Results Cell-free biochemical assays showed that the R263K substitution diminished subtype C integrase strand-transfer activity by decreasing the affinity of integrase for target DNA. Similarly, both viral infectiousness and replication capacity were reduced by the R263K substitution in tissue culture. Decrease in enzyme activity and viral infectiousness exceeded 35 and 50%, respectively - significantly more than in HIV-1 subtype B. R263K in HIV-1 subtype C also conferred low levels of resistance against dolutegravir and high levels of cross-resistance against elvitegravir, but not raltegravir. Conclusions The R263K substitution is more deleterious to integrase strand-transfer activity and viral infectiousness in HIV-1 subtype C than in subtype B. Our results suggest that cross-resistance may prevent treatment-experienced individuals who are experiencing treatment failure with dolutegravir from being subsequently treated with elvitegravir.

OBJECTIVE Interest in the role of antibody-dependent cellular cytotoxicity (ADCC) in protection from HIV infection has grown since analyses of the RV144 HIV vaccine trial results found ADCC correlated with protection. Natural killer (NK) cells are among the effector cells that mediate ADCC. The level of antibody-induced NK cell activation depends on NK cell education through inhibitory NK cell receptor human leukocyte antigen (HLA) ligand interactions. Here, we investigated the impact of NK cell education on the delivery of Granzyme B (GzB) to target cells. DESIGN Lymphocytes from 50 HIV-uninfected [30 Bw4 (Bw4) and 20 Bw4 (Bw6)] KIR3DL1 homozygote persons were used as effectors and cocultured with gp120-coated target cells in the presence of a single source of anti-HIV gp120 antibody to ascertain whether NK cell education status influenced the level of GzB delivered to target cells. METHODS The GTL assay assessed the frequency of GzB-positive (%GzB) CEM.NKr.CCR5 target cells generated by effectors from each individual. The frequency of CD107a, interferon (IFN)-γ and CCL4 NK cells was assessed as a measure of antibody-induced NK cell activation. RESULTS KIR3DL1 NK cells from the Bw4 group were more functional than KIR3DL1 NK cells. Despite this, the %GzB target cells generated in the GTL assay did not differ according to the KIR3DL1-HLA-B genotype of the effector cells. The %GzB cells positively correlated with the frequency of CD16KIR3DL1 NK cells in the effector population. CONCLUSION ADCC potency does not depend on NK cell education.

BACKGROUND HIV-1 group O (HIV-O) is a rare variant that is characterized by a high number of natural polymorphisms in the integrase coding region that may impact on susceptibility to integrase strand transfer inhibitors (INSTIs) and on the emergence of resistance substitutions. We previously reported that HIV-O is more susceptible to RAL than HIV-1 group M (HIV-M). METHODS The aim of this study was to assess pathways of resistance to INSTIs in group 0 variants. Accordingly, we selected for resistance to each of raltegravir (RAL), elvitegravir (EVG), and dolutegravir (DTG) in cord blood mononuclear cells using HIV group O subtypes A and B, an HIV-O divergent isolate, and HIV-1 group M (subtype B, which served as a reference). Site-directed mutagenesis was performed on the pCOM2.5 HIV group 0 infectious clone to ascertain the impact of INSTI resistance substitutions at positions Q148R, N155H, and R263K within integrase on susceptibility to INSTIs and infectiousness. RESULTS Cell culture selections of group O variants yielded similar patterns of resistance to RAL, EVG, and DTG as observed for subtype B. In the DTG selections, subtype B yielded S153Y, whereas a natural S153A polymorphism sometimes led to A153V in group O. The pCMO2.5/Q148R and pCMO2.5/N155H variants displayed far higher levels of resistance to DTG (>1000 FC) than was seen for group M viruses. CONCLUSIONS HIV-O harboring Q148R and N155H shows higher resistance to DTG compared with HIV-M subtype B.

HIV resistance to current anti-HIV drugs and drug toxicity have created a need for new anti-HIV agents. We have examined and characterized a synthetic resveratrol analog, termed 3,3',4,4',5,5'-hexahydroxy-trans-stilbene (M8), for potential anti-HIV activity. Here, we demonstrate that M8 possesses potent anti-HIV activity against several HIV variants with EC50 values in the low μM range. M8 was shown to act at a very early step of HIV entry prior to fusion to host cells. These results demonstrate that this novel resveratrol derivative possesses potent anti-HIV-1 activity and may have a mechanism of action that is different from current anti-HIV-1 drugs including entry inhibitors. Further structure-guided design might lead to the development of newer improved resveratrol derivatives that could have value either in therapy or as microbicides to prevent the sexual transmission of HIV-1.

International audience; Background: Current therapies have succeeded in controlling AIDS pandemic. However, there is a continuing need for new drugs, in particular those acting through new and as yet unexplored mechanisms of action to achieve HIV infection cure. We took advantage of the unique feature of proviral genome to require both activation and inhibition of splicing of viral transcripts to develop molecules capable of achieving long lasting effect on viral replication in humanized mouse models through inhibition of Rev-mediated viral RNA biogenesis. Results: Current HIV therapies reduce viral load during treatment but titers rebound after treatment is discontinued. We devised a new drug that has a long lasting effect after viral load reduction. We demonstrate here that ABX464 compromises HIV replication of clinical isolates of different subtypes without selecting for drug resistance in PBMCs or macrophages. ABX464 alone, also efficiently compromised viral proliferation in two humanized mouse models infected with HIV that require a combination of 3TC, Raltegravir and Tenofovir (HAART) to achieve viral inhibition in current protocols. Crucially, while viral load increased dramatically just one week after stopping HAART treatment, only slight rebound was observed following treatment cessation with ABX464 and the magnitude of the rebound was maintained below to that of HAART for two months after stopping the treatment. Using a system to visualize single HIV RNA molecules in living cells, we show that ABX464 inhibits viral replication by preventing Rev-mediated export of unspliced HIV-1 transcripts to the cytoplasm and by interacting with the Cap Binding Complex (CBC). Deep sequencing of viral RNA from treated cells established that retained viral RNA is massively spliced but importantly, normal cellular splicing is unaffected by the drug. Consistently ABX464 is non-toxic in humans and therefore represents a promising complement to current HIV therapies. Conclusions: ABX464 represents a novel class of anti-HIV molecules with unique properties. ABX464 has a long lasting effect in humanized mice and neutralizes the expression of HIV-1 proviral genome of infected immune cells including reservoirs and it is therefore a promising drug toward a functional cure of HIV.

Dolutegravir (DTG) is a second-generation integrase strand transfer inhibitor (INSTI) and INSTIs are the latest class of potent anti-HIV drugs. Compared to the first generation INSTIs, raltegravir (RAL) and elvitegravir (EVG), DTG shows a limited cross-resistance profile. More interestingly, clinical resistance mutations to DTG in treatment-naive patents have not been observed to this date. This review summarizes recent studies on resistance mutations to DTG and on our understanding of the mechanisms of resistance to DTG as well as future directions for research.

Three limonoids, trichiconin A (1) possessing a new carbon skeleton of a rearranged A,B-ring system and trichiconins B (2) and C (3) featuring an unprecedented A,B,D-seco skeleton, were isolated from the twigs of Trichilia connaroides. The carbon scaffold of trichiconin A is designated as trichiconane. Their structures with absolute stereochemistry were determined by spectroscopic data, X-ray crystallography, and CD analysis. Compounds 2 and 3 showed modest anti-HIV activities.

Compound A is a novel nucleotide-competing HIV-1 reverse transcriptase (RT) inhibitor (NcRTI) that selects for a unique W153L substitution that confers hypersusceptibility to tenofovir, while the K65R substitution in RT confers resistance against tenofovir and enhances susceptibility to NcRTIs. Although the K65R substitution is more common in subtype C viruses, the impact of subtype variability on NcRTI susceptibility has not been studied. In the present study, we performed experiments with compound A by using purified recombinant RT enzymes and viruses of subtypes B and C and circulating recombinant form CRF_A/G. We confirmed the hypersusceptibility of K65R substitution-containing RTs to compound A for subtype C, CRF_A/G, and subtype B. Steady-state kinetic analysis showed that K65R RTs enhanced the susceptibility to compound A by increasing binding of the inhibitor to the nucleotide binding site of RT in a subtype-independent manner, without significantly discriminating against the natural nucleotide substrate. These data highlight the potential utility of NcRTIs, such as compound A, for treatment of infections with K65R substitution-containing viruses, regardless of HIV-1 subtype.

In a global clinical trial conducted in 9 countries and 4 continents, pretreatment drug resistance and HIV-1 subtype were independently associated with virologic failure, suggesting that pretreatment genotyping should be considered whenever feasible.

Drug resistance represents a key aspect of human immunodeficiency virus (HIV) treatment failure. It is important to develop nonhuman primate models for studying issues of drug resistance and the persistence and transmission of drug-resistant viruses. However, relatively little work has been conducted using either simian immunodeficiency virus (SIV) or SIV/HIV recombinant viruses for studying resistance against integrase strand transfer inhibitors (INSTIs). Here, we used a T-cell-tropic SIV/HIV recombinant virus in which the capsid and vif regions of HIV-1 were replaced with their SIV counterparts (simian-tropic HIV-1 [stHIV-1] (SCA,SVIF) ) to study the impact of a number of drug resistance substitutions in the integrase coding region at positions E92Q, G118R, E138K, Y143R, S153Y, N155H, and R263K on drug resistance, viral infectivity, and viral replication capacity. Our results show that each of these substitutions exerted effects that were similar to their effects in HIV-1. Substitutions associated with primary resistance against dolutegravir were more detrimental to stHIV-1 (SCA,SVIF) infectiousness than were resistance substitutions associated with raltegravir and elvitegravir, consistent with data that have been reported for HIV-1. These findings support the role of stHIV-1 (SCA,SVIF) as a useful model with which to evaluate the role of INSTI resistance substitutions on viral persistence, transmissibility, and pathogenesis in a nonhuman primate model.

Background Antiretroviral preexposure prophylaxis has been shown to reduce the risk of human immunodeficiency virus type 1 (HIV-1) infection in some studies, but conflicting results have been reported among studies, probably due to challenges of adherence to a daily regimen. Methods We conducted a double-blind, randomized trial of antiretroviral therapy for preexposure HIV-1 prophylaxis among men who have unprotected anal sex with men. Participants were randomly assigned to take a combination of tenofovir disoproxil fumarate (TDF) and emtricitabine (FTC) or placebo before and after sexual activity. All participants received risk-reduction counseling and condoms and were regularly tested for HIV-1 and HIV-2 and other sexually transmitted infections. Results Of the 414 participants who underwent randomization, 400 who did not have HIV infection were enrolled (199 in the TDF-FTC group and 201 in the placebo group). All participants were followed for a median of 9.3 months (interquartile range, 4.9 to 20.6). A total of 16 HIV-1 infections occurred during follow-up, 2 in the TDF-FTC group (incidence, 0.91 per 100 person-years) and 14 in the placebo group (incidence, 6.60 per 100 person-years), a relative reduction in the TDF-FTC group of 86% (95% confidence interval, 40 to 98; P=0.002). Participants took a median of 15 pills of TDF-FTC or placebo per month (P=0.57). The rates of serious adverse events were similar in the two study groups. In the TDF-FTC group, as compared with the placebo group, there were higher rates of gastrointestinal adverse events (14% vs. 5%, P=0.002) and renal adverse events (18% vs. 10%, P=0.03). Conclusions The use of TDF-FTC before and after sexual activity provided protection against HIV-1 infection in men who have sex with men. The treatment was associated with increased rates of gastrointestinal and renal adverse events. (Funded by the National Agency of Research on AIDS and Viral Hepatitis [ ANRS] and others; ClinicalTrials.gov number, NCT01473472.)

Understanding the HIV integrase protein and mechanisms of resistance to HIV integrase inhibitors is complicated by the lack of a full length HIV integrase crystal structure. Moreover, a lentiviral integrase structure with co-crystallised DNA has not been described. For these reasons, we have developed a structural method that utilizes free software to create quaternary HIV integrase homology models, based partially on available full-length prototype foamy virus integrase structures as well as several structures of truncated HIV integrase. We have tested the utility of these models in screening of small anti-integrase compounds using randomly selected molecules from the ZINC database as well as a well characterized IN:DNA binding inhibitor, FZ41, and a putative IN:DNA binding inhibitor, HDS1. Docking studies showed that the ZINC compounds that had the best binding energies bound at the IN:IN dimer interface and that the FZ41 and HDS1 compounds docked at approximately the same location in integrase, i.e. behind the DNA binding domain, although there is some overlap with the IN:IN dimer interface to which the ZINC compounds bind. Thus, we have revealed two possible locations in integrase that could potentially be targeted by allosteric integrase inhibitors, that are distinct from the binding sites of other allosteric molecules such as LEDGF inhibitors. Virological and biochemical studies confirmed that HDS1 and FZ41 share a similar activity profile and that both can inhibit each of integrase and reverse transcriptase activities. The inhibitory mechanism of HDS1 for HIV integrase seems to be at the DNA binding step and not at either of the strand transfer or 3' processing steps of the integrase reaction. Furthermore, HDS1 does not directly interact with DNA. The modeling and docking methodology described here will be useful for future screening of integrase inhibitors as well as for the generation of models for the study of integrase drug resistance.

The increasing prevalence of acquired and transmitted HIV-1 drug resistance is an obstacle to successful antiretroviral therapy (ART) in the low- and middle-income countries (LMICs) hardest hit by the HIV-1 pandemic. Genotypic drug resistance testing could facilitate the choice of initial ART in areas with rising transmitted drug resistance (TDR) and enable care-providers to determine which individuals with virological failure (VF) on a first- or second-line ART regimen require a change in treatment. An inexpensive near point-of-care (POC) genotypic resistance test would be useful in settings where the resources, capacity, and infrastructure to perform standard genotypic drug resistance testing are limited. Such a test would be particularly useful in conjunction with the POC HIV-1 viral load tests that are currently being introduced in LMICs. A POC genotypic resistance test is likely to involve the use of allele-specific point mutation assays for detecting drug-resistance mutations (DRMs). This study proposes that two major nucleoside reverse transcriptase inhibitor (NRTI)-associated DRMs (M184V and K65R) and four major NNRTI-associated DRMs (K103N, Y181C, G190A, and V106M) would be the most useful for POC genotypic resistance testing in LMIC settings. One or more of these six DRMs was present in 61.2% of analyzed virus sequences from ART-naïve individuals with intermediate or high-level TDR and 98.8% of analyzed virus sequences from individuals on a first-line NRTI/NNRTI-containing regimen with intermediate or high-level acquired drug resistance. The detection of one or more of these DRMs in an ART-naïve individual or in a individual with VF on a first-line NRTI/NNRTI-containing regimen may be considered an indication for a protease inhibitor (PI)-containing regimen or closer virological monitoring based on cost-effectiveness or country policy.

Drug resistance has been observed in regard to all anti-retroviral drugs that have been studied until now. The most recent drug to be approved for use in HIV therapy is dolutegravir, an HIV integrase inhibitor, that was approved for therapy by the Food and Drug Administration in the United States in 2013. Dolutegravir is the only HIV drug that has not selected for resistance mutations in the clinic when used as part of first-line therapy. We have hypothesized that this is due to the long binding time of dolutegravir to the integrase enzyme as well as greatly diminished replication capacity on the part of viruses that might become resistant to dolutegravir and that are unable to successfully replicate in infected individuals.

Mutations that confer resistance to nucleoside analog reverse transcriptase inhibitors of HIV-1 can be divided into two major classes: thymidine analog mutations (TAMs) and TAM suppressors. M184V, K65R and L74V are TAM suppressors that emerge under the selective pressure of non-thymidine analogs. Each of the three TAM suppressors have been shown to decrease the level of resistance to 3´-azido-3´-deoxythymidine against a background of certain combinations of TAMs. L74V and M184V have also been associated with decreased phenotypic susceptibility to tenofovir disoproxil fumarate in vitro. In this review, the effects associated with the L74V mutation, which confer resistance to didanosine and abacavir, are discussed. The clinical significance of this mutation and the underlying biochemical mechanisms of inhibition, resistance and resensitization are also discussed in the context of drug regimens containing didanosine and/or abacavir, in combination with 3´-azido -3´-deoxythymidine and/or tenofovir disoproxil fumarate.

Among 16 human immunodeficiency virus-infected (subtype C) Batswana patients who failed nelfinavir (NFV)-containing regimens, the most prevalent mutation observed was D30N (54%), followed by L90M (31%). L89I, K20T/I, and E35D polymorphic changes were also identified. These findings suggest that subtype C viruses in Botswana may develop resistance to NFV via subtype-specific pathways.

Large-cohort studies in North America, Europe, and Thailand have shown that zidovudine/azidothymidine (AZT) monotherapy, given at the late stages of pregnancy, is of proven benefit in reducing mother-to-infant HIV transmission by 51% to 68%. AZT monotherapy will not be of long-term benefit for mothers because no single drug can counteract viral infection; benefits to babies will be short-lived if HIV-1 is acquired through breastfeeding after birth. Unfortunately, ongoing mutation of HIV under conditions of drug pressure allows for the evolution and selection of AZT-resistant viruses. Emergence of AZT-resistant variants in pregnant mothers (7-29%) and their infected offspring (5-21%) has been described in several studies. Drug resistance arises more frequently in those mothers who received AZT therapy before pregnancy. Recent advances in combination chemotherapy may provide alternative strategies in prevention of vertical transmission and drug resistance. Genotypic screening of the HIV-1 isolated from pregnant mothers may provide rational modifications in antiretroviral (ARV) strategies to circumvent vertical HIV transmission. This may be of advantage for resource-rich nations but not for underdeveloped nations with limited access to ARVs. Public health programs are vital to have an impact on the tragic pandemic of pediatric AIDS.

The capsid (CA) sequence of human immunodeficiency virus type 1 (HIV-1) Gag protein consists of two independently folded domains named the N-terminal domain (NTD) and C-terminal domain (CTD) that are connected by a flexible linker. Most of the CTD sequence adopts rigid structure except for the last 11 amino acids (positions 354 to 364) that are disordered even in the context of the downstream SP1 and nucleocapsid (NC) sequence. Although disordered, this short peptide region plays a crucial role in HIV-1 replication. In this study, we identified three second-site mutations within Gag named A238T, G358S, and N373K that rescued a deleterious mutation R362A located at the C-terminus of CA. A238T is located within the NTD of CA, G358S and N373K are positioned proximal to R362A. One of the mechanisms underlying this compensation event is correction of reduced packaging of viral RNA into the R362A mutated viruses, as shown by the results of RNase protection assays, native Northern blots experiments as well as filter-binding assays. These data suggest that one potential function for the C-terminal disordered sequence of CA in HIV-1 replication is to regulate HIV-1 RNA packaging.

The L74V and M184V mutations in the reverse transcriptase (RT) gene of human immunodeficiency virus type 1 (HIV-1) are frequently associated with resistance to the nucleoside reverse transcriptase inhibitors abacavir, didanosine, and lamivudine. Yet viruses containing any of these mutations often display hypersusceptibility to zidovudine (ZDV). Two distinct mechanisms have been described to explain HIV-1 drug resistance. One of these involves diminished rates of incorporation of the nucleotide analogue by mutated RT, while the other mechanism involves increased rates of phosphorolytic excision of the drug-terminated primer. To understand the biochemical mechanisms responsible for the hypersensitization of L74V-containing viruses to ZDV, we studied the efficiency of excision of ZDV-monophosphate (ZDV-MP)-terminated primers by recombinant wild-type and mutated HIV-1 RTs in cell-free assays. We observed that the L74V mutation in RT caused reductions in ATP-dependent removal of ZDV-MP from newly synthesized viral DNA. In addition, we determined that the L74V and M184V mutations did not affect the ratio between the populations of RT-DNA/DNA complexes found at pre- and posttranslocational stages; however, they might have affected proper alignment between incorporated chain terminator and pyrophosphate donor, substrate orientation, affinity for ATP, and/or primer-template substrate. Finally, we confirmed previous findings that L74V-containing viruses display diminished replication capacity and that this is associated with reduced levels of synthesis of early reverse-transcribed viral DNA molecules.

Background. Resistance testing is considered standard of care in HIV medicine, but there is no standard interpretation system for genotype tests. We sought to determine how much agreement exists within a group of experts in the interpretation of complex genotypes. Methods. Genotypes from clinical specimens were sent to an international panel of 12 resistance experts. Phenotypic susceptibility testing of these clinical isolates was performed with antivirogram. Experts predicted phenotype fold change category ( 4.0- to 7.0-fold change, > 7.0- to 10-fold change, > 10- to 20-fold change, or > 20-fold change) and predicted expected drug activity for each of 16 antiretroviral drugs. Experts were also asked to make treatment recommendations on the basis of the genotype. Results. The experts predicted the exact phenotype fold change category correctly 44% of the time, but they varied widely by antiretroviral drug (range, 25%-74%). The highest accuracy was observed for lamivudine (74%) and the nonnucleoside reverse transcriptase inhibitors (66%-69%). Experts generally predicted higher levels of resistance to the remaining nucleoside reverse transcriptase inhibitors than what was found by phenotypic testing. Agreement among experts in predicting phenotype fold change category ranged widely depending on the drug (median agreement, 42% [range, 28%-74%]); the same pattern was observed in predicting expected drug activity (median agreement, 45% [range, 32%-87%]). Experts agreed on treatment recommendations in a median of 79% of instances, and recommendations were consistent over time, with blinded retesting. Conclusions. Although their ability to predict phenotype from a genotype varied for individual antiretroviral drugs, this expert panel had a high degree of agreement in deriving treatment recommendations from the genotype.

Combination antiretroviral therapy (ART) has dramatically decreased morbidity and mortality among human immunodeficiency virus (HIV) type 1 (HIV-1)-infected patients through the durable suppression of viral replication to undetectable levels (22). Although ART has changed significantly in the decades since the advent of monotherapy, nucleoside and nucleotide reverse transcriptase (RT) inhibitors (NRTIs and NtRTIs, respectively) remain important foundations of therapy. The current guidelines for the treatment of HIV infection in the United States and the United Kingdom recommend dual NRTI and NtRTI therapy in combination with a protease inhibitor (PI) or a nonnucleoside reverse transcriptase inhibitor (NNRTI) for most patients (1, 3). New NRTI and NtRTI options for HIV therapy include two new fixed-dose once-daily combinations, abacavir (ABC) plus lamivudine (3TC) and tenofovir disoproxil fumarate (TDF) plus emtricitabine (FTC).

HIV-1 uses to prime reverse transcription of its viral RNA. In this process, the 3′-end of must be annealed to the primer binding site of HIV-1 genomic RNA, and the two molecules together form a complex structure. During annealing, the nucleocapsid (NC) protein enhances the unwinding of tertiary structures within both RNA molecules. Moreover, the packaging of occurs prior to viral budding at a time when NC is still part of the Pr55Gag polyprotein. In contrast, Pr55Gag is able to produce virus-like particles on its own. We have recently shown that an N-terminal extended form of NC (mGag), containing all of the minimal elements required for virus-like particle formation, possesses greater affinity for HIV-1 genomic RNA than does NC alone. We have now studied the -annealing properties of mGag in comparison to those of NC and report that the former is more efficient in this regard than the latter. We have also tested each of a mutant version of mGag, an extended form of mGag, and an almost full-length form of Gag, and showed that all of these possessed greater tRNA-annealing capacity than did the viral NC protein. Yet, surprisingly, multimerization of Gag-related proteins did not abrogate this annealing process but rather resulted in dramatically reduced levels of reverse transcriptase processivity. These results suggest that the initial stages of reverse transcription may be regulated by the multimerization of Pr55Gag polyprotein at times prior to the cleavage of NC.

The Gag protein of human immunodeficiency virus type 1 contains a 14-amino-acid region, termed SP1, between the capsid and downstream nucleocapsid sequences. Although SP1 is known to be indispensable for virus production, the mechanisms involved are mostly unclear. In this study, we demonstrate that an M368A mutation within SP1 severely diminished the ability of Gag to associate with cellular membranes. Although wild-type levels of membrane binding were restored to the M368A Gag by a second-site L20K mutation within matrix, the resultant Gag mutant L20K-M368A remained defective in virus production. This latter deficit was partially consequent to the binding of L20K-M368A Gag to nonraft membranes as opposed to raft association seen for wild-type Gag. Further analysis revealed that the majority of membrane-bound M368A Gag proteins were small oligomers, indicating a multimerization defect. In support of this observation, purified recombinant Gag derivatives containing the M368A mutation formed much lower amounts of high-molecular-weight complexes that were pelletable at 21,000 × g than did wild-type Gag. Based on the myristyl switch model, we propose that the M368A mutation inhibits Gag multimerization and, as a result, restricts the binding of Gag to cellular membranes.

There is a need for new antiretroviral drugs with activity against HIV isolates resistant to currently available agents and improved short and long-term tolerability profiles. Clinical trial designs for nucleotide and nucleoside reverse transcriptase inhibitors (NRTIs) are restricted by the characteristics of these agents (for example, their cross-resistance, resistance threshold and interaction profiles), the ethical need to ensure that patients are not maintained on suboptimal regimens, and regulatory requirements (for example, with regards to trial designs and patient populations). For example, consideration of cross-resistance profiles must influence the way in which an NRTI in development is sequenced to minimize any impact on future treatment options. The resistance threshold is determined by the number of mutations required to diminish sensitivity to a given drug. Pharmacokinetic or pharmacodynamic interactions restrict how NRTIs may be combined during clinical development. Doses may be selected on the basis of results from short-term monotherapy studies in treatment-naive patients, but such studies cannot establish the long-term efficacy or tolerability of new agents used in combination regimens. Confirmatory studies in treatment-naive populations do not meet the medical and regulatory needs for clinical data in treatment-experienced populations, while studies in treatment-experienced populations are subject to numerous clinical and logistical difficulties. Intensification, switch and hybrid study designs all offer suitable approaches to the evaluation of NRTIs with novel resistance profiles. Switch studies are particularly useful for agents with resistance profiles that suggest a specific sequencing approach in treatment and for those with the potential, based on pharmacokinetic data, for interactions with other agents. The successful development of new NRTIs will depend upon a thorough appreciation of these many and complex issues, not only among those involved in the design of clinical studies, but also those contributing to their review and conduct.

This study compared the incidence of HIV-1 variants harboring mutations conferring resistance to thymidine analogues, ie, thymidine analogue mutations (TAMs), nonnucleoside reverse transcriptase (RT) inhibitors (NNMs), lamivudine (3TC) (ie, M184V), and protease inhibitors (PIs) acquired in primary HIV infection (PHI) (n = 59) to their observed prevalence in a corresponding potential transmitter (PT) population of persons harboring resistant infections (n = 380). Both of these populations in the context of this cohort analysis possessed similar demographics. Whereas the frequencies of observed TAMs, NNMs, M184V, and protease-associated mutations (PRAMs) were similar in the PT groups, the prevalence of M184V and major PI mutations were significantly lower in the PHI group (PHI/PT ratios of 0.14 and 0.39, respectively). There was a decreased prevalence in the PHI population of resistant viruses co-expressing NNMs or TAMs with M184V compared with viruses that harbored NNMs or TAMs in the absence of M184V (P < 0.0001). It was also observed that individuals in the PT subgroups who harbored RT mutations or PRAMs with M184V had lower levels of plasma viremia than individuals who lacked M184V (P < 0.05). These findings suggest that both decreased viremia and viral fitness in the case of M184V-containing HIV-1 variants may impact on viral transmissibility.

The M184V substitution in human immunodeficiency virus type 1 reverse transcriptase (RT) is rapidly selected in tissue culture following serial passage of wild-type virus in the presence of increasing concentrations of lamivudine (3TC). M184V is also associated with several alterations of RT enzymatic function in vitro that may adversely affect viral fitness or replication capacity, which creates a potential rationale for its maintenance once it has been selected by antiviral chemotherapy. However, the relative effectiveness of nucleoside RT inhibitors that are structurally unrelated to 3TC in selecting and/or maintaining M184V has not been investigated. In the present study, we have studied the abilities of a variety of drugs, i.e., zalcitabine (ddC), didanosine (ddI), abacavir (ABC), and the novel nucleoside SPD754, in addition to 3TC, to maintain the presence of M184V in tissue culture and have shown that SPD754, ABC, and 3TC are able to preserve M184V in mixed dual infections consisting of wild-type viruses and clinical isolates which contained the M184V mutation. Moreover, M184V could also be maintained in these cultures when a subtherapeutic concentration of 3TC (i.e., 0.05 μM) was used. In contrast, neither ddI nor ddC was able to maintain M184V to the same extent as the other drugs after 10 weeks of tissue culture in mixtures of wild-type viruses and isolates containing M184V in different proportions.

L’émergence et la transmission de souches résistantes du virus de l’immunodéficience humaine (VIH), qui se traduisent par une perte significative de l’efficacité du traitement anti-VIH, s’imposent aujourd’hui comme un problème de santé publique dans la plupart des pays du Sud. À cet égard, il est urgent que l’accès aux antirétroviraux dans les pays à faibles revenus s’accompagne de la mise en place de programmes régionaux et nationaux visant à améliorer l’adhésion des patients à la thérapie, afin de limiter le développement des résistances. La qualité des systèmes de santé, les particularités épidémiologiques du VIH/sida ainsi que le choix limité dans l’éventail des médicaments disponibles obligent les pays du Sud à définir la meilleure association thérapeutique initiale., HIV drug resistance has been associated with treatment failure in Western countries but the lessons learned can be useful in optimization of highly active antiretroviral treatment (HAART) in resource-poor settings. There is a need to improve access to HAART in such regions, but appropriate strategies must be rapidly implemented, such as adapted programs to facilitate adherence to therapy, rational use of genotypic drug resistance monitoring in specific situations, and use of alternative treatment regimens. The implications of HIV genetic diversity must also be considered in management of drug resistance.

The nucleoside and nucleotide reverse transcriptase inhibitors (NRTIs/NtRTIs) remain an enduring feature of combination therapy. As NRTI/NtRTI options continue to expand, questions arise about how best to combine these agents to create effective dual NRTI/NtRTI backbones in antiretroviral regimens while avoiding treatment-emergent drug resistance. Clinicians must consider how NRTIs/NtRTIs such as tenofovir DF (TDF), abacavir (ABC), and emtricitabine (FTC), as well as new once-daily and coformulated NRTIs/NtRTIs, interact with older agents when combined in novel regimens and how sequencing the new NRTIs can preserve future treatment options. Resistance data from clinical trials have revealed important information on the patterns, prevalence, and effects of resistance seen among patients experiencing virologic failure. In recent years, the prevalence of some mutations such as M184V and Q151M has remained relatively constant, while the L74V mutation, the 69 insertions, and thymidine analogue mutations have decreased in prevalence. Other mutations such as K65R and Y115F, while still relatively uncommon, are increasing in prevalence. This increase may be due to the use of new treatment combinations that select for these mutations at a higher rate. Clinical trials suggest that new regimens containing TDF or ABC select for K65R and that this mutation is observed more frequently with TDF; in contrast, L74V is observed more frequently in ABC-containing regimens but is not commonly selected by TDF-containing regimens. Several lines of evidence are converging to suggest that the presence of zidovudine may decrease the risk of L74V and K65R in ABC- or TDF-containing regimens. This review summarizes the clinical implications of resistance profiles associated with new NRTI/NtRTI regimens in current use and in advanced clinical studies.

To detect and characterize polymerase gene ( pol) polymorphisms and mutation patterns in HIV-1C-infected Batswana patients treated with reverse transcriptase inhibitors, samples from AIDS patients treated with highly active antiretroviral therapy (HAART) were sequenced for the region encompassing the entire HIV-1 protease (PR) and the first 335 amino acids of reverse transcriptase (RT). Amongst the 16 patients treated with antiretroviral (ARV) drugs, eight started HAART regimens containing didanosine, stavudine and nevirapine (ddI/d4T/NVP) or efavirenz (EFV) (arm A) while the others started with zidovudine (AZT) and lamivudine (3TC) given together as combivir (CBV) with either NVP or EFV as arm B. Arm B is the first line regimen currently provided by the Botswana ARV national programme. Greater efficacy, in terms of treatment duration, was observed in patients in arm B (14 months) as compared with patients in arm A (9 months); P10 RNA copies/ml) and a significantly improved immunological parameter (ΔCD4=207.0 +48.1 cells/μl; P

We studied the effects of recombinant human growth hormone (r-hGH) on human immunodeficiency virus type 1 replication by growing both wild-type and drug-resistant variants of virus in the presence of various concentrations of eight different antiretroviral drugs. r-hGH had no significant effect on either viral replication or the 50% inhibitory concentrations of these compounds.

The 11th Conference on Retroviruses and Opportunistic Infections was held on February 8–11, 2004 in San Francisco, CA and presented a wealth of new information on viral resistance and on new agents in preclinical and clinical development. In this Perspective, highlights from the conference and their clinical implications will be discussed.

Genetic diversity is a hallmark of HIV-1 infection with regard to the expansion of distinct viral subtypes (clades A, B, C, D, E, F, G, K, and O) in different geographical regions. Here, we discuss the issues of HIV-1 sensitivity to antiretroviral drugs and drug resistance in the context of HIV-1 subtype diversity. Virtually all available evidence suggests that all subtypes of HIV display similar sensitivity to antiviral drugs, but viruses from some subtypes or geographical regions may occasionally have a greater propensity to develop resistance against certain drugs than other viral variants. In some situations, the types of mutations associated with resistance may vary, as a result of subtle differences among subtypes with regard to the genetic code. This consideration notwithstanding, drug resistance is unlikely to become a more serious issue in developing than developed countries, and there is an urgency to make anti-HIV drugs available to all who are in need.

The plight of millions of HIV-infected individuals without access to antiretroviral (ARV) medications constitutes an enormous problem. Religious values can influence policymakers in public and personal health issues. This article posits that Jewish religious law mandates the broadest possible access to ARV medications for HIV-infected individuals, and argues that wealthy countries must assist poorer ones to facilitate access.

Nevirapine and other members of the non-nucleoside reverse transcriptase inhibitor (NNRTI) family of anti-HIV-1 drugs are essential components of antiretroviral treatment regimens. Unfortunately, drug resistance has become an important issue with respect to all therapeutic targets in HIV-1. This paper summarizes current knowledge about the mutations in the reverse transcriptase gene of HIV-1 that are responsible for drug resistance and the mechanisms whereby drug resistance develops.

The M184V mutation in HIV reverse transcriptase (RT) is associated with high-level resistance against the nucleoside inhibitor lamivudine as well as diminished viral replication capacity. We have previously demonstrated that HIV variants containing the M184V mutation were relatively unable to successfully undergo compensatory mutagenesis following deletion of an A-rich loop located upstream of the primer binding site (PBS). To understand the mechanisms involved, we synthesized viral RNA templates containing different compensatory mutations that were emergent during the long-term culture of the A-rich loop-deleted viruses. These templates were then used in cell-free reverse transcription initiation assays and in tRNA primer placement assays performed with either recombinant wild-type RT or recombinant RT containing the M184V substitution. The results showed that the RNA template that contained the A-rich loop deletion was impaired in ability to initiate reverse transcription and that the presence of the M184V substitution in RT amplified this effect. Clearance from pausing at position +3 during synthesis of viral DNA was identified as a sensitive step in this reaction that could not be efficiently bypassed with the M184V mutant enzyme. Increased efficiency of initiation was seen with the deleted RNA templates that also contained mutations identified in the revertant viruses, provided that these mutations facilitated formation of a competent binary tRNA/RNA complex. These findings provide biochemical evidence that initiation of tRNALys3-primed DNA synthesis is an important rate-limiting step in reverse transcription that correlates with viral replication fitness.

Although protease inhibitors (PIs) have dramatically improved outcomes in HIV-infected patients, half still fail treatment with PI-based combination therapy. Genetic pressure from incomplete viral suppression rapidly selects for HIV variants with protease gene mutations that confer reduced susceptibility to PI drugs. A number of specific amino acid substitutions have been associated with PI resistance. However, high-level resistance to individual PIs requires the accumulation of several primary and secondary mutations, developing along drug-specific, step-wise pathways. HIV variants resistant to saquinavir and ritonavir usually contain L90M and V82A substitutions, respectively. Indinavir resistance may be linked to substitutions at positions 46 or 82. Resistance to nelfinavir is primarily associated with D30N but may alternatively be found with L90M. Resistance during exposure to amprenavir can follow development of I50V, which also may confer resistance to lopinavir. Failure during treatment with atazanavir is closely linked to I50L. The overlapping of these pathways can lead to multiple-PI resistance, limiting therapeutic options in antiretroviral-experienced patients. Reduced susceptibility to more than one PI is most likely to be associated with amino acid substitutions at six positions: 10, 46, 54, 82, 84 and 90. Other mutations (D30N, G48V, I50V or I50L) are relatively specific for particular PIs and are less likely to produce cross resistance. Certain resistance mutations selected by exposure to one PI may actually increase susceptibility to others. Patients newly diagnosed with HIV infection are increasingly found to harbour virus that is resistant to the more commonly used drugs. Newer PIs may select for mutations that result in less cross resistance with older agents.

The Tat protein of human immunodeficiency virus type 1 (HIV-1) has been shown to restrict premature reverse transcription at late stages of virus infection and to thus ensure the integrity of the viral RNA genome for packaging. To gain further insights into the roles of Tat in HIV-1 reverse transcription, we have assessed its effects on the first-strand transfer during the synthesis of minus-strand DNA through use of a reconstituted cell-free system. The results demonstrated that a form of Tat, containing only the first exon (Tat72), was able to enhance the first-strand transfer as efficiently as did the viral nucleocapsid protein. Coincidentally, this form of Tat was unable to inhibit the production of minus-strand strong-stop DNA. Further studies with various mutated forms of Tat showed that its Cys-rich region, rather than the core and Arg-rich domains, was essential for this strand transfer activity. Moreover, this activity of Tat is largely independent of the TAR RNA structure. Although full-length Tat protein (Tat86) was also able to promote strand transfer, this activity was limited by a strong overall inhibition of reverse transcription because of the presence of the second Tat exon. Other nucleic-acid-binding proteins (e.g., single-strand DNA-binding protein) were employed as negative controls and were unable to promote strand transfer in these assays. We propose that Tat possesses nucleic acid chaperone activity and can promote the first-strand transfer during HIV-1 reverse transcription; however, these activities are restricted by the second Tat exon, and the roles of these Tat activities in viral replication remain to be elucidated.

Human immunodeficiency viruses (HIVs) and the related bovine lentiviruses bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV) utilize the viral Tat protein to activate viral transcription. The arginine-rich RNA-binding domains of the Tat proteins bind to their cognate transactivation response element (TAR) RNA hairpins located at the 5′ ends of the viral mRNAs, resulting in enhanced processivity of RNA polymerase II. It has previously been shown that HIV type 1 (HIV-1) Tat requires the cellular cyclin T1 protein for high-affinity RNA binding whereas BIV Tat and JDV Tat bind with high affinity on their own and adopt distinct β-hairpin conformations when complexed to RNA. Here we have engineered the BIV and JDV Tat-TAR interactions into HIV-1 and show that the heterologous interactions support viral replication, correlating well with their RNA-binding affinities. Viruses engineered with a variant TAR able to bind all three Tat proteins replicate efficiently with any of the proteins. In one virus containing a noncognate Tat-TAR pair that neither interacts nor efficiently replicates (HIV-1 TAR and BIV Tat), viral revertants were isolated in which TAR had become mutated to generate a functional BIV Tat binding site. Our results support the view that incremental changes to TAR structure can provide routes for evolving new Tat-TAR complexes while maintaining active viral replication.

The M184V mutation in human immunodeficiency virus (HIV) reverse transcriptase is associated with high-level resistance to both (−)2′,3′-dideoxy-3′-thiacytidine (3TC) and (−)2′,3′-dideoxy-5-fluoro-3′-thiacytidine as well as low-level resistance to 2′,3′-dideoxyinosine, 2′,3′-dideoxycytidine, and abacavir. This mutation is also associated with diminished HIV replicative fitness as well as several functional changes in enzyme activity, including diminutions in polymerase processivity, pyrophosphorylysis, and nucleotide primer unblocking. Despite the fact that M184V encodes up to 1,000-fold resistance to 3TC, we asked whether this drug might still display some antiviral effect in regard to viruses containing this mutation. Cell-free assays revealed that high concentrations of 3TC triphosphate (i.e., >100 μM) could affect chain termination and/or inhibit purified reverse transcriptase containing the M184V substitution. This effect became more pronounced with elongation of reverse transcriptase products. In newly infected cells (i.e., peripheral blood mononuclear cells), we found that the amount of full-length reverse transcriptase product was diminished in the presence of 2 to 10 μM 3TC, although no decrease in the first product of the reverse transcriptase reaction, i.e., minus strong-stop DNA, was observed. In the presence of two other HIV inhibitors, e.g., nevirapine and indinavir, 3TC exerted additive effects in tissue culture at concentrations only marginally higher than the 50% inhibitory concentration (IC50). Reverse transcriptases cloned from clinical isolates harboring M184V in the context of multidrug resistance had similar IC50values for 3TC triphosphate compared to reverse transcriptase containing only the M184V mutation. These results suggest that viruses containing M184V can retain a higher degree of sensitivity to 3TC than previously assumed.

HIV-1 infection is characterized by genetic diversity wherein distinct viral subtypes (clades A, B, C, D, E, F, G, K and O) are expanding in different geographical regions. This article deals with the topic of HIV-1 subtype diversity in the context of sensitivity to antiretroviral drugs, drug resistance and viral fitness. Increasing evidence suggests that all clades of HIV probably display similar sensitivity to antiviral drugs. However, viruses from some subtypes and/or geographical regions may have a greater propensity to develop resistance against certain drugs than do other viral variants. In addition, differences in regard to replication capacity or fitness may exist among various HIV subtypes and differences in this regard may potentially become magnified under conditions of drug resistance. Immunological pressures may also play an important role in the evolution of viral subtypes that may impact on ultimate drug resistance profiles.

None of the available antiretroviral drugs that are currently used in the clinic to treat infection with HIV-1 is directed against the RNase H active site of the reverse transcriptase. Here we developed a nonradioactive, 96-well plate assay designed to be used for high-throughput screening of compounds capable of inhibiting the RNase H activity of HIV-1 reverse transcriptase. We employed a tRNA as substrate that was labeled with digoxygenin-modified reporter residues. The labeled tRNA was prehybridized with a DNA oligonucleotide that contained a single biotinylated residue at its 5′-terminus to ensure its attachment to streptavidin-coated microplates. The uncleaved, immobilized DNA/tRNA substrate was detected through the use of established ELISA protocols. Incubation with purified HIV-1 reverse transcriptase initiated RNase H degradation and caused a signal reduction to negligible background levels. In contrast, the signal intensity remained unaffected when using an RNase H deficient mutant enzyme. The assay was validated using the hydrazone derivative BBNH that was previously shown to inhibit RNase H degradation below concentrations of 10 μM.

Although advances in highly active antiretroviral therapy (HAART) have made long-term suppression of HIV an achievable goal of therapy, a substantial proportion of first-line regimens will eventually fail. Successful long-term treatment requires consideration of downstream treatment options at the time of initiating or changing regimens. An understanding of the patterns and interactions of resistance mutations, and the appropriate use of genotypic and phenotypic testing is an important component of successful drug sequencing. Resistance to multiple nucleoside reverse transcriptase inhibitors (NRTIs) may result from several genotypically distinct pathways, including the Q151M (151 complex), the 69 insertion complex, two distinct thymidine analogue mutational pathways and the K65R mutation. Knowledge of the clinical implications of these and other resistance pathways, as well as the antagonism or synergy between mutations, helps guide individualized treatment choices from initial therapy in the treatment-naive patient to salvage therapy in the highly treatment-experienced individual. The development of effective sequencing strategies will depend upon the continued understanding of drug resistance mutation patterns and their associations with specific HAART combinations. This review summarizes research advances that further the understanding of nucleoside and nucleotide analogue resistance mutations, and their interplay.

The major group of human immunodeficiency viruses (HIV-1) that comprise the current global pandemic have diversified during their worldwide spread and may be divided into at least 10 distinct subtypes or clades, A through J. Subtype B predominates in North America and Europe, subtype E predominates in Southeast Asia, and subtype C predominates in sub-Saharan Africa. Functional distinctions in long terminal repeat (LTR) architecture among HIV subtypes have been identified, thus raising the possibility that regulatory divergence among the subtypes of HIV-1 has occurred. In addition to the transcriptional specificity of the HIV-1 LTR, productive HIV-1 replication is also dependent upon the viral Tat protein. Therefore, we sought to investigate whether interactions between host signaling pathways and the NF-kappaB regions of different HIV-1 subtypes, together with subtype-specific interactions between Tat, TAR, and cellular proteins, modulate the efficiency of HIV-1 clade-specific gene transcription. We demonstrate that the NF-kappaB sites of subtypes B and E both bind NF-kappaB-related complexes. However, the duplicated kappaB sites of the C subtype do not compete for NF-kappaB binding. Also, clade E Tat protein possesses the highest transactivation capacity, regardless of the LTR context. Furthermore, preliminary evidence suggests that the acetylation of subtype-specific Tat proteins may correlate with their transactivation efficiency.

The conversion of the single-stranded RNA genome into double-stranded DNA by virus-coded reverse transcriptase (RT) is an essential step of the retrovirus life cycle. In human immunodeficiency virus type 1 (HIV-1), RT uses the cellular tRNA(Lys)3 to initiate the (-) strand DNA synthesis. Placement of the primer tRNA(Lys)3 involves binding of its 3'-terminal 18 nt to a complementary region of genomic RNA termed PBS. However, the PBS sequence is not the unique determinant of primer usage and additional contacts are important. This placement is believed to be achieved in vivo by the nucleocapsid domain of Gag or by the mature protein NCp. Up to now, structural information essentially arose from heat-annealed primer-template complexes (Isel et al., J Mol Biol, 1995, 247:236-250; Isel et al., EMBO J, 1999, 18:1038-1048). Here, we investigated the formation of the primer-template complex mediated by NCp and compared structural and functional properties of heat- and NCp-annealed complexes. We showed that both heat- and NCp-mediated procedures allow comparable high yields of annealing. Then, we investigated structural features of both kinds of complexes by enzymatic probing, and we compared their relative efficiency in (-) strong stop DNA synthesis. We did not find any significant differences between these complexes, suggesting that information derived from the heat-annealed complex can be transposed to the NCp-mediated complex and most likely to complexes formed in vivo.

Flaviviruses including Zika, Dengue and Hepatitis C virus cause debilitating diseases in humans, and the former are emerging as global health concerns with no antiviral treatments. We investigated Sophora Flavecens, used in Chinese medicine, as a source for antiviral compounds. We isolated Sophoraflavenone G and found that it inhibited Hepatitis C replication, but not Sendai or Vesicular Stomatitis Virus. Pre- and post-infection treatments demonstrated anti-flaviviral activity against Dengue and Zika virus, via viral RNA polymerase inhibition. These data suggest that Sophoraflavenone G represents a promising candidate regarding anti-Flaviviridae research.