Ursane and 24-Noroleanane-Type Triterpenoids with Anti-HIV Activity from the Twigs and Leaves of Antirhea chinensis.

Investigations on the twigs and leaves of Antirhea chinensis have led to the discovery of two undescribed pentacyclic triterpenoids (1 and 2) and nine known analogs. Compounds 1 and 2, each assigned as the ursane and 24-noroleanane-type triterpenoids, featuring similar oxidation pattern of 3ß,6ß,19α-trihydroxy-28-carboxyl. Their structures were elucidated via comprehensive analyses of spectroscopic data. Compound 1 displayed potent anti-HIV activity (EC50 =1.24 µM) and high selectivity index (SI >32.3).

Diverse Types of Diterpenoids with an Aromatized C Ring from the Twigs of Podocarpus imbricatus.

An ethanol extract of the powdered twigs of Podocarpus imbricatus afforded 14 new diterpenoids (1-14), which all share an aromatized C ring. These isolates belong to five diterpenoid types that include abietanes (1-3), semperviranes (4-9), totaranes (10-12), a C-17 norabietane (13), and an icetexane (14). Their structures were assigned mainly by analysis of the spectroscopic data, and the absolute configuration of 1 was determined by X-ray crystallography. A biosynthetic pathway for five of the biogenetically related types of diterpenoids was proposed. Compound 7 showed moderate inhibitory activity against Zika virus with an IC50 value of 2.5 µM.

Antimalarial drugs and their metabolites are potent Zika virus inhibitors.

Studies aimed at repurposing existing drugs revealed that some antimalarial compounds possess anti-Zika virus (anti-ZIKV) activity. Here, we further tested 14 additional antimalarial drugs and their metabolites or analogs for anti-ZIKV activity using a phenotypic screening approach. We identified four compounds with varying anti-ZIKV activity, including a metabolite of amodiaquine termed desethylamodiaquine (DAQ) and N-desethylchloroquine (DECQ), a metabolite of chloroquine, which both exhibited low micromolar effective concentrations against three different ZIKV strains. Two other compounds termed dihydroartemisinin (DHA) and quinidine (QD) exhibited only partial inhibition of ZIKV replication. Characterization of the inhibitory mechanisms of DAQ and DECQ showed that both drugs target the entry step as well as postentry events of the viral replication cycle. These hits represent attractive starting points for future optimization of new anti-ZIKV drug candidates derived from antimalarial drugs and their analogs.

The S230R Integrase Substitution Associated With Virus Load Rebound During Dolutegravir Monotherapy Confers Low-Level Resistance to Integrase Strand-Transfer Inhibitors.

Background: Dolutegravir (DTG) is an integrase strand-transfer inhibitor (INSTI) used for treatment of human immunodeficiency virus (HIV)-infected individuals. Owing to its high genetic barrier to resistance, DTG has been clinically investigated as maintenance monotherapy to maintain viral suppression and to reduce complication and healthcare costs. Our study aims to explain the underlying mechanism related to the emergence of a S230R substitution in patients who experienced virologic failure while using DTG monotherapy. Methods: We evaluated the effect of the S230R substitution in regard to integrase enzyme activity, viral infectivity, replicative capacity, and susceptibility to different INSTIs by biochemical and cell-based assays. Results: The S230R substitution conferred a 63% reduction in enzyme efficiency. S230R virus was 1.29-fold less infectious than wild-type virus but could replicate in PM1 cells without significant delay. Resistance levels against DTG, cabotegravir, raltegravir, and elvitegravir in tissue culture were 3.85-, 3.72-, 1.52-, and 1.21-fold, respectively, in virus with the S230R substitution. Conclusions: Our data indicate that the S230R substitution is comparable to the previously reported R263K substitution in some respects. Virologic failure during DTG monotherapy can occur through the development of the S230R or R263K mutation, without the need for high-level DTG resistance.

JAK-STAT Signaling Pathways and Inhibitors Affect Reversion of Envelope-Mutated HIV-1.

HIV can spread by both cell-free and cell-to-cell transmission. Here, we show that many of the amino acid changes in Env that are close to the CD4 binding pocket can affect HIV replication. We generated a number of mutant viruses that were unable to infect T cells as cell-free viruses but were nevertheless able to infect certain T cell lines as cell-associated viruses, which was followed by reversion to the wild type. However, the activation of JAK-STAT signaling pathways caused the inhibition of such cell-to-cell infection as well as the reversion of multiple HIV Env mutants that displayed differences in their abilities to bind to the CD4 receptor. Specifically, two T cell activators, interleukin-2 (IL-2) and phorbol 12-myristate 13-acetate (PMA), both capable of activation of JAK-STAT pathways, were able to inhibit cell-to-cell viral transmission. In contrast, but consistent with the above result, a number of JAK-STAT and mTOR inhibitors actually promoted HIV-1 transmission and reversion. Hence, JAK-STAT signaling pathways may differentially affect the replication of a variety of HIV Env mutants in ways that differ from the role that these pathways play in the replication of wild-type viruses.IMPORTANCE Specific alterations in HIV Env close to the CD4 binding site can differentially change the ability of HIV to mediate infection for cell-free and cell-associated viruses. However, such differences are dependent to some extent on the types of target cells used. JAK-STAT signaling pathways are able to play major roles in these processes. This work sheds new light on factors that can govern HIV infection of target cells.

The antimalarial drug amodiaquine possesses anti-ZIKA virus activities.

Zika virus (ZIKV) outbreak has emerged as a global health threat, particularly in tropical areas, over the past few years. No antiviral therapy or vaccine is available at present. For these reasons, repurposing clinically approved drugs against ZIKV infection may provide rapid and cost-effective global health benefits. Here, we explored this strategy and screened eight FDA-approved drugs for antiviral activity against ZIKV using a cell-based assay. Our results show that the antimalarial drug amodiaquine has anti-ZIKV activity with EC50 at low micromolar concentrations in cell culture. We further characterized amodiaquine antiviral activity against ZIKV and found that it targets early events of the viral replication cycle. Altogether, our results suggest that amodiaquine may be efficacious for the treatment of ZIKV infection.

Monotherapy with either dolutegravir or raltegravir fails to durably suppress HIV viraemia in humanized mice.

Objectives: To compare the effectiveness of HIV integrase inhibitor monotherapy between raltegravir and dolutegravir as an approach to simplify therapy. Methods: We evaluated and compared the efficacy of 20 week monotherapy with dolutegravir or raltegravir in humanized mice (HSC-NSG) infected with HIVBaL. Plasma HIV RNA was measured by quantitative RT-PCR (limit of detection of 150 copies/45 µL of plasma) and drug levels by LC-MS/MS. Escape viruses were genotyped and analysed for replication capacity and drug susceptibility in tissue culture. Results: Drug-untreated control mice maintained constant viraemia throughout the study. Virus isolates from these mice were susceptible to both raltegravir (EC50 of <8 nM) and dolutegravir (EC50 of <1 nM). Mice treated with raltegravir or dolutegravir had plasma drug levels comparable to those in humans. Monotherapy with raltegravir initially suppressed HIV viraemia, but failed to maintain suppression in 4/4 mice. Viruses from raltegravir failing mice developed mutations G140G/S and Q148H/K, and were resistant to both raltegravir (EC50 values of >100 nM) and dolutegravir (EC50 values ranging from 8.8 to 13.3 nM). Monotherapy with dolutegravir suppressed viraemia in 5/5 of mice, but viraemia rebounded in one animal. The virus from this mouse had mutations E138K, G140S, Q148H, N155H and S230R, was highly resistant to both raltegravir (EC50 of >1000 nM) and dolutegravir (EC50 of 550 nM), and replicated to levels similar to those of control viruses in PBMCs. Conclusions: Monotherapy with either raltegravir or dolutegravir does not consistently maintain HIV suppression, suggesting that dual therapy may be required in simplification strategies.

Purification of Zika virus RNA-dependent RNA polymerase and its use to identify small-molecule Zika inhibitors.

Background: The viral RNA-dependent RNA polymerase (RdRp) enzymes of the Flaviviridae family are essential for viral replication and are logically important targets for development of antiviral therapeutic agents. Zika virus (ZIKV) is a rapidly re-emerging human pathogen for which no vaccine or antiviral agent is currently available. Methods: To facilitate development of ZIKV RdRp inhibitors, we have established an RdRp assay using purified recombinant ZIKV NS5 polymerase. Results: We have shown that both the hepatitis C virus (HCV) nucleoside inhibitor sofosbuvir triphosphate and a pyridoxine-derived non-nucleoside small-molecule inhibitor, DMB213, can act against ZIKV RdRp activity at IC 50 s of 7.3 and 5.2 µM, respectively, in RNA synthesis reactions catalysed by recombinant ZIKV NS5 polymerase. Cell-based assays confirmed the anti-ZIKV activity of sofosbuvir and DMB213 with 50% effective concentrations (EC 50 s) of 8.3 and 4.6 µM, respectively. Control studies showed that DMB213 did not inhibit recombinant HIV-1 reverse transcriptase and showed only very weak inhibition of HIV-1 integrase strand-transfer activity. The S604T substitution in motif B of the ZIKV RdRp, which corresponds to the S282T substitution in motif B of HCV RdRp, which confers resistance to nucleotide inhibitors, also conferred resistance to sofosbuvir triphosphate, but not to DMB213. Enzyme assays showed that DMB213 appears to be competitive with natural nucleoside triphosphate (NTP) substrates. Conclusions: Recombinant ZIKV RdRp assays can be useful tools for the screening of both nucleos(t)ide compounds and non-nucleotide metal ion-chelating agents that interfere with ZIKV replication.

Identification of resveratrol analogs as potent anti-dengue agents using a cell-based assay.

Dengue virus (DENV) causes a variety of difficult-to-treat diseases that threaten almost half of the world's population. Currently, no effective vaccine or antiviral therapy is available. We have examined a series of synthetic resveratrol analogs to identify potential anti-DENV agents. Here, we demonstrate that two resveratrol analogs, PNR-4-44 and PNR-5-02, possess potent anti-DENV activity with EC50 values in the low nanomolar range. These two resveratrol analogs were shown to mainly target viral RNA translation and viral replication, but PNR-5-02 is also likely to target cellular factors inside host cells. Although the precise molecular mechanism(s) mediating anti-DENV activities have not been elucidated, further structure-guided design might lead to the development of newer improved resveratrol derivatives that might have therapeutic value. J. Med. Virol. 89:397-407, 2017. © 2016 Wiley Periodicals, Inc.

Identification of a Pyridoxine-Derived Small-Molecule Inhibitor Targeting Dengue Virus RNA-Dependent RNA Polymerase.

The viral RNA-dependent RNA polymerase (RdRp) activity of the dengue virus (DENV) NS5 protein is an attractive target for drug design. Here, we report the identification of a novel class of inhibitor (i.e., an active-site metal ion chelator) that acts against DENV RdRp activity. DENV RdRp utilizes a two-metal-ion mechanism of catalysis; therefore, we constructed a small library of compounds, through mechanism-based drug design, aimed at chelating divalent metal ions in the catalytic site of DENV RdRp. We now describe a pyridoxine-derived small-molecule inhibitor that targets DENV RdRp and show that 5-benzenesulfonylmethyl-3-hydroxy-4-hydroxymethyl-pyridine-2-carboxylic acid hydroxyamide (termed DMB220) inhibited the RdRp activity of DENV serotypes 1 to 4 at low micromolar 50% inhibitory concentrations (IC50s of 5 to 6.7 µM) in an enzymatic assay. The antiviral activity of DMB220 against DENV infection was also verified in a cell-based assay and showed a 50% effective concentration (EC50) of <3 µM. Enzyme assays proved that DMB220 was competitive with nucleotide incorporation. DMB220 did not inhibit the enzymatic activity of recombinant HIV-1 reverse transcriptase and showed only weak inhibition of HIV-1 integrase strand transfer activity, indicating high specificity for DENV RdRp. S600T substitution in the DENV RdRp, which was previously shown to confer resistance to nucleoside analogue inhibitors (NI), conferred 3-fold hypersusceptibility to DMB220, and enzymatic analyses showed that this hypersusceptibility may arise from the decreased binding/incorporation efficiency of the natural NTP substrate without significantly impacting inhibitor binding. Thus, metal ion chelation at the active site of DENV RdRp represents a viable anti-DENV strategy, and DMB220 is the first of a new class of DENV inhibitor.

Differential effects of the G118R, H51Y, and E138K resistance substitutions in different subtypes of HIV integrase.

UNLABELLED: Dolutegravir (DTG) is the latest antiretroviral (ARV) approved for the treatment of human immunodeficiency virus (HIV) infection. The G118R substitution, previously identified with MK-2048 and raltegravir, may represent the initial substitution in a dolutegravir resistance pathway. We have found that subtype C integrase proteins have a low enzymatic cost associated with the G118R substitution, mostly at the strand transfer step of integration, compared to either subtype B or recombinant CRF02_AG proteins. Subtype B and circulating recombinant form AG (CRF02_AG) clonal viruses encoding G118R-bearing integrases were severely restricted in their viral replication capacity, and G118R/E138K-bearing viruses had various levels of resistance to dolutegravir, raltegravir, and elvitegravir. In cell-free experiments, the impacts of the H51Y and E138K substitutions on resistance and enzyme efficiency, when present with G118R, were highly dependent on viral subtype. Sequence alignment and homology modeling showed that the subtype-specific effects of these mutations were likely due to differential amino acid residue networks in the different integrase proteins, caused by polymorphic residues, which significantly affect native protein activity, structure, or function and are important for drug-mediated inhibition of enzyme activity. This preemptive study will aid in the interpretation of resistance patterns in dolutegravir-treated patients. IMPORTANCE: Recognized drug resistance mutations have never been reported for naive patients treated with dolutegravir. Additionally, in integrase inhibitor-experienced patients, only R263K and other previously known integrase resistance substitutions have been reported. Here we suggest that alternate resistance pathways may develop in non-B HIV-1 subtypes and explain how "minor" polymorphisms and substitutions in HIV integrase that are associated with these subtypes can influence resistance against dolutegravir. This work also highlights the importance of phenotyping versus genotyping when a strong inhibitor such as dolutegravir is being used. By characterizing the G118R substitution, this work also preemptively defines parameters for a potentially important pathway in some non-B HIV subtype viruses treated with dolutegravir and will aid in the inhibition of such a virus, if detected. The general inability of strand transfer-related substitutions to diminish 3' processing indicates the importance of the 3' processing step and highlights a therapeutic angle that needs to be better exploited.

Might dolutegravir be part of a functional cure for HIV?

Antiretroviral therapy (ART) has greatly decreased HIV-related morbidity and mortality. However, HIV can establish viral reservoirs that evade both the immune system and ART. Dolutegravir (DTG) is a second-generation integrase strand transfer inhibitor (INSTI) related to the first-generation INSTIs raltegravir (RAL) and elvitegravir (EVG). DTG shows a higher genetic barrier to the development of HIV-1 resistance than RAL and EVG. More interestingly, clinical resistance mutations to DTG in treatment-naïve patients have not been observed to date. This review summarizes recent studies on strategies toward a cure for HIV, explores resistance profiles of DTG, and discusses how DTG might help in finding a functional cure for HIV.

The connection domain mutation N348I in HIV-1 reverse transcriptase enhances resistance to etravirine and rilpivirine but restricts the emergence of the E138K resistance mutation by diminishing viral replication capacity.

Clinical resistance to rilpivirine (RPV), a novel nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI), is associated an E-to-K mutation at position 138 (E138K) in RT together with an M184I/V mutation that confers resistance against emtricitabine (FTC), a nucleoside RT inhibitor (NRTI) that is given together with RPV in therapy. These two mutations can compensate for each other in regard to fitness deficits conferred by each mutation alone, raising the question of why E138K did not arise spontaneously in the clinic following lamivudine (3TC) use, which also selects for the M184I/V mutations. In this context, we have investigated the role of a N348I connection domain mutation that is prevalent in treatment-experienced patients. N348I confers resistance to both the NRTI zidovudine (ZDV) and the NNRTI nevirapine (NVP) and was also found to be associated with M184V and to compensate for deficits associated with the latter mutation. Now, we show that both N348I alone and N348I/M184V can prevent or delay the emergence of E138K under pressure with RPV or a related NNRTI, termed etravirine (ETR). N348I also enhanced levels of resistance conferred by E138K against RPV and ETR by 2.2- and 2.3-fold, respectively. The presence of the N348I or M184V/N348I mutation decreased the replication capacity of E138K virus, and biochemical assays confirmed that N348I, in a background of E138K, impaired RT catalytic efficiency and RNase H activity. These findings help to explain the low viral replication capacity of viruses containing the E138K/N348I mutations and how N348I delayed or prevented the emergence of E138K in patients with M184V-containing viruses.

A resveratrol analog termed 3,3',4,4',5,5'-hexahydroxy-trans-stilbene is a potent HIV-1 inhibitor.

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.

Limonoids with Anti-HIV Activity from Cipadessa cinerascens.

Sixteen new limonoids, named ciparasins A-P (1-16), comprising three structural categories of trijugin-type (1-7), cipadesin-type (8-15), and prieurianin-type (16) compounds, as well as 15 known limonoid analogues (17-31), were isolated from Cipadessa cinerascens. Ciparasins E-G (5-7) were found to possess a rare γ-hydroxylbutenolide moiety at C-17. Ciparasins B (2) and P (16) showed significant anti-HIV activities, with EC50 values of 5.5 ± 0.6 (SI >7.2) and 6.1 ± 0.7 (SI >6.5) µM, respectively.

Logeracemin A, an anti-HIV Daphniphyllum alkaloid dimer with a new carbon skeleton from Daphniphyllum longeracemosum.

Logeracemin A (1), the first Daphniphyllum alkaloid dimer featuring an unprecedented carbon skeleton with a unique conjugated trispiro[4,5] decane backbone, was isolated from Daphniphyllum longeracemosum. Its structure and absolute configuration were established on the basis of spectroscopic data and X-ray crystallography. Logeracemin A showed significant anti-HIV activity with an EC50 of 4.5 ± 0.1 µM and a selectivity index of 6.2. The structure-activity relationship of the tested compounds was briefly discussed.

The M50I polymorphic substitution in association with the R263K mutation in HIV-1 subtype B integrase increases drug resistance but does not restore viral replicative fitness.

BACKGROUND: First-generation integrase strand-transfer inhibitors (INSTIs), such as raltegravir (RAL) and elvitegravir (EVG), have been clinically proven to be effective antiretrovirals for the treatment of HIV-positive patients. However, their relatively low genetic barrier for resistance makes them susceptible to the emergence of drug resistance mutations. In contrast, dolutegravir (DTG) is a newer INSTI that appears to have a high genetic barrier to resistance in vivo. However, the emergence of the resistance mutation R263K followed by the polymorphic substitution M50I has been observed in cell culture. The M50I polymorphism is also observed in 10-25% of INSTI-naïve patients and has been reported in combination with R263K in a patient failing treatment with RAL. RESULTS: Using biochemical cell-free strand-transfer assays and resistance assays in TZM-bl cells, we demonstrate that the M50I polymorphism in combination with R263K increases resistance to DTG in tissue culture and in biochemical assays but does not restore the viral fitness cost associated with the R263K mutation. CONCLUSIONS: Since the combination of the R263K mutation and the M50I polymorphism results in a virus with decreased viral fitness and limited cross-resistance, the R263K resistance pathway may represent an evolutionary dead-end. Although this hypothesis has not yet been proven, it may be more advantageous to treat HIV-positive individuals with DTG in first-line than in second or third-line therapy.

HIV-1 group O integrase displays lower enzymatic efficiency and higher susceptibility to raltegravir than HIV-1 group M subtype B integrase.

HIV-1 group O (HIV-O) is a rare HIV-1 variant characterized by a high number of polymorphisms, especially in the integrase coding region. As HIV-O integrase enzymes have not previously been studied, our aim was to assess the impact of HIV-O integrase polymorphisms on enzyme function and susceptibility to integrase inhibitors. Accordingly, we cloned and purified integrase proteins from each of HIV-1 group O clades A and B, an HIV-O divergent strain, and HIV-1 group M (HIV-M, subtype B), used as a reference. To assess enzymatic function of HIV-O integrase, we carried out strand transfer and 3' processing assays with various concentrations of substrate (DNA target and long terminal repeats [LTR], respectively) and characterized these enzymes for susceptibility to integrase strand transfer inhibitors (INSTIs) in cell-free assays and in tissue culture, in the absence or presence of various concentrations of several INSTIs. The inhibition constant (Ki) and 50% effective concentration (EC50) values were calculated for HIV-O integrases and HIV-O viruses, respectively, and compared with those of HIV-M. The results showed that HIV-O integrase displayed lower activity in strand transfer assays than did HIV-M enzyme, whereas 3' processing activities were similar to those of HIV-M. HIV-O integrases were more susceptible to raltegravir (RAL) in competitive inhibition assays and in tissue culture than were HIV-M enzymes and viruses, respectively. Molecular modeling suggests that two key polymorphic residues that are close to the integrase catalytic site, 74I and 153A, may play a role in these differences.

Addition of E138K to R263K in HIV integrase increases resistance to dolutegravir, but fails to restore activity of the HIV integrase enzyme and viral replication capacity.

BACKGROUND: The results of several clinical trials suggest that the integrase inhibitor dolutegravir may be less prone than other drugs to the emergence of HIV drug resistance mutations in treatment-naive patients. We have shown that the R263K mutation commonly emerged during tissue culture selection studies with dolutegravir and conferred low levels of resistance to this drug while simultaneously diminishing both HIV replication capacity and integrase enzymatic activity. E138K has been identified as a secondary mutation for dolutegravir in selection studies and has also been observed as a secondary mutation in the clinic for the integrase inhibitors raltegravir and elvitegravir. METHODS: We used biochemical cell-free strand-transfer assays and tissue culture assays to characterize the effects of the E138K/R263K combination of mutations on resistance to dolutegravir, integrase enzyme activity and HIV-1 replication capacity. RESULTS: We show here that the addition of the E138K substitution to R263K increased the resistance of HIV-1 to dolutegravir but failed to restore viral replication capacity, integrase strand-transfer activity and integration within cellular DNA. We also show that the addition of E138K to R263K did not increase the resistance to raltegravir or elvitegravir. The addition of the E138K substitution to R263K was also less detrimental to integrase strand-transfer activity and integration than a different secondary mutation at position H51Y that had also been selected in culture. CONCLUSIONS: The E138K substitution failed to restore the defect in viral replication capacity that is associated with R263K, confirming previous selection studies that failed to identify compensatory mutation(s) for the latter primary mutation. This study suggests that the R263K resistance pathway may represent an evolutionary dead end for HIV in treatment-naive individuals who are treated with dolutegravir and will need to be confirmed by the long-term use of dolutegravir in the clinic.

Identification of an env-defective HIV-1 mutant capable of spontaneous reversion to a wild-type phenotype in certain T-cell lines.

BACKGROUND: Attempts to eradicate HIV from cellular reservoirs are vital but depend on a clear understanding of how viral variants are transmitted and survive in the different cell types that constitute such reservoirs. Mutations in the env gene of HIV may be able to exert a differential influence on viral transmission ability in regard to cell-free and cell-associated viral forms. METHODS: The ability of HIV containing an env G367R mutation in cell-free and cell-associated viruses to cause infection and to revert to wild-type was measured using several T cell lines. To determine factors that might potentially influence the reversion of G367R, we studied each of entry inhibitors, inhibitors of cellular endocytosis, and modulators of cell growth and activation. RESULTS: We demonstrate that an HIV-1 variant containing a G367R substitution within the CD4 binding site of gp120 was non-infectious as free virus in culture but was infectious when infected cells were co-cultured with certain T cell lines or when cells were transfected by a relevant proviral plasmid. Differences in viral infectivity by cell-associated G367R viruses were determined by the type of target cell employed, regardless which type of donor cell was used. Reversion was slowed or inhibited by entry inhibitors and by inhibitors of cellular endocytosis. Interleukin 2 was able to block G367R reversion in only one of the T cell lines studied but not in the other, while phorbol 12-myristate 13-acetate (PMA) inhibited G367R reversion in all the T cell lines. CONCLUSIONS: Env-defective HIV may have a different phenotype as cell-free versus cell-associated virus. The persistence of defective forms can potentially lead to the emergence of virulent forms. The heterogeneity of cell types that constitute the HIV reservoir can contribute to viral variability, even among similar types of cells. This is the first demonstration of a mutation in the HIV envelope, i.e. G367R, that can compromise infection by cell-free virus but less severely by cell-associated virus and that does so in a cell type-dependent manner.