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Abstract Background Bevirimat, the prototype Human Immunodeficiency Virus type 1 (HIV-1) maturation inhibitor, is highly potent in cell culture and efficacious in HIV-1 infected patients. In contrast to inhibitors that target the active site of the viral protease, bevirimat specifically inhibits a single cleavage event, the final processing step for the Gag precursor where p25 (CA-SP1) is cleaved to p24 (CA) and SP1. Results In this study, photoaffinity analogs of bevirimat and mass spectrometry were employed to map the binding site of bevirimat to Gag within immature virus-like particles. Bevirimat analogs were found to crosslink to sequences overlapping, or proximal to, the CA-SP1 cleavage site, consistent with previous biochemical data on the effect of bevirimat on Gag processing and with genetic data from resistance mutations, in a region predicted by NMR and mutational studies to have α-helical character. Unexpectedly, a second region of interaction was found within the Major Homology Region (MHR). Extensive prior genetic evidence suggests that the MHR is critical for virus assembly. Conclusions This is the first demonstration of a direct interaction between the maturation inhibitor, bevirimat, and its target, Gag. Information gained from this study sheds light on the mechanisms by which the virus develops resistance to this class of drug and may aid in the design of next-generation maturation inhibitors.

Abstract Background The maturation inhibitor bevirimat (BVM) potently inhibits human immunodeficiency virus type 1 (HIV-1) replication by blocking capsid-spacer peptide 1 (CA-SP1) cleavage. Recent clinical trials demonstrated that a significant proportion of HIV-1-infected patients do not respond to BVM. A patient's failure to respond correlated with baseline polymorphisms at SP1 residues 6-8. Results In this study, we demonstrate that varying levels of BVM resistance are associated with point mutations at these residues. BVM susceptibility was maintained by SP1-Q6A, -Q6H and -T8A mutations. However, an SP1-V7A mutation conferred high-level BVM resistance, and SP1-V7M and T8Δ mutations conferred intermediate levels of BVM resistance. Conclusions Future exploitation of the CA-SP1 cleavage site as an antiretroviral drug target will need to overcome the baseline variability in the SP1 region of Gag.

Abstract Background The HIV-1 Env glycoprotein mediates virus entry by catalyzing direct fusion between the virion membrane and the target cell plasma membrane. Env is composed of two subunits: gp120, which binds to CD4 and the coreceptor, and gp41, which is triggered upon coreceptor binding to promote the membrane fusion reaction. Env on the surface of infected cells is a trimer consisting of three gp120/gp41 homo-dimeric protomers. An emerging question concerns cooperative interactions between the protomers in the trimer, and possible implications for Env function. Results We extended studies on cooperative subunit interactions within the HIV-1 Env trimer, using analysis of functional complementation between coexpressed inactive variants harboring different functional deficiencies. In assays of Env-mediated cell fusion, complementation was observed between variants with a wide range of defects in both the gp120 and gp41 subunits. The former included gp120 subunits mutated in the CD4 binding site or incapable of coreceptor interaction due either to mismatched specificity or V3 loop mutation. Defective gp41 variants included point mutations at different residues within the fusion peptide or heptad repeat regions, as well as constructs with modifications or deletions of the membrane proximal tryptophan-rich region or the transmembrane domain. Complementation required the defective variants to be coexpressed in the same cell. The observed complementation activities were highly dependent on the assay system. The most robust activities were obtained with a vaccinia virus-based expression and reporter gene activation assay for cell fusion. In an alternative system involving Env expression from integrated provirus, complementation was detected in cell fusion assays, but not in virus particle entry assays. Conclusion Our results indicate that Env function does not require every subunit in the trimer to be competent for all essential activities. Through cross-talk between subunits, the functional determinants on one defective protomer can cooperatively interact to trigger the functional determinants on an adjacent protomer(s) harboring a different defect, leading to fusion. Cooperative subunit interaction is a general feature of the Env trimer, based on complementation activities observed for a highly diverse range of functional defects.

Analytical antiretroviral treatment interruption (ATI) is an important feature of HIV research, seeking to achieve sustained viral suppression in the absence of antiretroviral therapy (ART) when the goal is to measure effects of novel therapeutic interventions on time to viral load rebound or altered viral setpoint. Trials with ATIs also intend to determine host, virological, and immunological markers that are predictive of sustained viral control off ART. Although ATI is increasingly incorporated into proof-of-concept trials, no consensus has been reached on strategies to maximise its utility and minimise its risks. In addition, differences in ATI trial designs hinder the ability to compare efficacy and safety of interventions across trials. Therefore, we held a meeting of stakeholders from many interest groups, including scientists, clinicians, ethicists, social scientists, regulators, people living with HIV, and advocacy groups, to discuss the main challenges concerning ATI studies and to formulate recommendations with an emphasis on strategies for risk mitigation and monitoring, ART resumption criteria, and ethical considerations. In this Review, we present the major points of discussion and consensus views achieved with the goal of informing the conduct of ATIs to maximise the knowledge gained and minimise the risk to participants in clinical HIV research., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Over 4 days, more than 270 scientists involved in HIV persistence research convened to share their data and discuss future avenues to control HIV without continuous antiretroviral therapy. This 7(th) International Workshop on HIV Persistence followed the format of the preceding conferences but more time was given for discussing abstracts submitted by the participants and selected by the Steering and Scientific Committees. The topic of the workshop is HIV persistence: consequently, issues of HIV reservoirs and HIV cure are also addressed. In this article we report as closely as possible what was discussed. However, owing to length constraints, not everything is reported here but all the Workshop abstracts can be found online (www.viruseradication.com).

3-O-(3',3'-Dimethylsuccinyl) betulinic acid (DSB), also known as PA-457, bevirimat (BVM), or MPC-4326, is a novel HIV-1 maturation inhibitor. Unlike protease inhibitors, BVM blocks the cleavage of the Gag capsid precursor (CA-SP1) to mature capsid (CA) protein, resulting in the release of immature, noninfectious viral particles. Despite the novel mechanism of action and initial progress made in small-scale clinical trials, further development of bevirimat has encountered unexpected challenges, because patients whose viruses contain genetic polymorphisms in the Gag SP1 (positions 6 to 8) protein do not generally respond well to BVM treatment. To better define the role of amino acid residues in the HIV-1 Gag SP1 protein that are involved in natural polymorphisms to confer resistance to the HIV-1 maturation inhibitor BVM, a series of Gag SP1 chimeras involving BVM-sensitive (subtype B) and BVM-resistant (subtype C) viruses was generated and characterized for sensitivity to BVM. We show that SP1 residue 7 of the Gag protein is a primary determinant of SP1 polymorphism-associated drug resistance to BVM.

Background: The maturation inhibitor bevirimat (BVM) potently inhibits human immunodeficiency virus type 1 (HIV-1) replication by blocking capsid-spacer peptide 1 (CA-SP1) cleavage. Recent clinical trials demonstrated that a significant proportion of HIV-1-infected patients do not respond to BVM. A patient's failure to respond correlated with baseline polymorphisms at SP1 residues 6-8., Results: In this study, we demonstrate that varying levels of BVM resistance are associated with point mutations at these residues. BVM susceptibility was maintained by SP1-Q6A, -Q6H and -T8A mutations. However, an SP1-V7A mutation conferred high-level BVM resistance, and SP1-V7M and T8Delta mutations conferred intermediate levels of BVM resistance., Conclusions: Future exploitation of the CA-SP1 cleavage site as an antiretroviral drug target will need to overcome the baseline variability in the SP1 region of Gag.

Human respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract infection in infants, immunocompromised patients, and the elderly. The RSV fusion (F) protein mediates fusion of the viral envelope with the target cell membrane during virus entry and is a primary target for antiviral drug and vaccine development. The F protein contains two heptad repeat regions, HR1 and HR2. Peptides corresponding to these regions form a six-helix bundle structure that is thought to play a critical role in membrane fusion. However, characterization of six-helix bundle formation in native RSV F protein has been hindered by the fact that a trigger for F protein conformational change has yet to be identified. Here we demonstrate that RSV F protein on the surface of infected cells undergoes a conformational change following exposure to elevated temperature, resulting in the formation of the six-helix bundle structure. We first generated and characterized six-helix bundle-specific antibodies raised against recombinant peptides modeling the RSV F protein six-helix bundle structure. We then used these antibodies as probes to monitor RSV F protein six-helix bundle formation in response to a diverse array of potential triggers of conformational changes. We found that exposure of 'membrane-anchored' RSV F protein to elevated temperature (45-55 degrees C) was sufficient to trigger six-helix bundle formation. Antibody binding to the six-helix bundle conformation was detected by both flow cytometry and cell-surface immunoprecipitation of the RSV F protein. None of the other treatments, including interaction with a number of potential receptors, resulted in significant binding by six-helix bundle-specific antibodies. We conclude that native, untriggered RSV F protein exists in a metastable state that can be converted in vitro to the more stable, fusogenic six-helix bundle conformation by an increase in thermal energy. These findings help to better define the mechanism of RSV F-mediated membrane fusion and have important implications for the identification of therapeutic strategies and vaccines targeting RSV F protein conformational changes.

Development of novel therapeutic targets against HIV-1 is a high research priority owing to the serious clinical consequences associated with acquisition of resistance to current antiretroviral drugs. The HIV-1 structural protein Gag represents a potential new therapeutic target as it plays a central role in virus particle production yet is not targeted by any of the antiretroviral drugs approved at present. The Gag polyprotein precursor multimerizes to form immature particles that bud from the infected cell. Concomitant with virus release, the Gag precursor undergoes proteolytic processing by the viral protease to generate the mature Gag proteins, which include capsid (CA). Once liberated from the Gag polyprotein precursor, CA molecules interact to reassemble into a condensed conical core, which organizes the viral RNA genome and several viral proteins to facilitate virus replication in the next round of infection. Correct Gag proteolytic processing and core assembly are therefore essential for virus infectivity. In this review, we discuss new strategies to inhibit maturation by targeting proteolytic cleavage sites in Gag or CA-CA interactions required for core formation. The identification and development of lead maturation inhibitors are highlighted.

In a continuing study of potent anti-HIV agents, seventeen 28,30-disubstituted betulinic acid (BA, 1) derivatives and seven novel 3,28-disubstituted BA analogues were designed, synthesized, and evaluated for in vitro antiviral activity. Among them, compound 21 showed an improved solubility and equal anti-HIV potency (EC(50) = 0.09 microM) when compared to HIV entry inhibitors 3b (IC9564, (3R,4S)-N'-[N-[3beta-hydroxy-lup-20(29)-en-28-oyl]-8-aminooctanoyl]-4-amino-3-hydroxy-6-methylheptanoic acid) and 4 (A43-D, [[N-[3beta-O-(3',3'-dimethylsuccinyl)-lup-20(29)-en-28-oyl]-7-aminoheptyl]carbamoyl]methane). Using a cyclic secondary amine to form the C-28 amide bond increased the metabolic stability of the derivatives significantly in pooled human liver microsomes. The most potent compounds 47 and 48 displayed potent anti-HIV activity with EC(50) values of 0.007 and 0.006 microM, respectively. These results are slightly better than that of bevirimat (2, 3',3'-dimethylsuccinylbetulinic acid), which is currently in phase IIb clinical trials. Compounds 47 and 48 should serve as attractive promising leads to develop next generation, metabolically stable, 3,28-disubstituted bifunctional HIV-1 inhibitors as clinical trials candidates.

The maturation inhibitor bevirimat [3-O-(3',3'dimethysuccinyl)betulinic acid; BVM; also known as PA-457 or DSB] potently inhibits human immunodeficiency virus type 1 (HIV-1) replication by blocking protease (PR)-mediated cleavage at the junction between capsid (CA) and spacer peptide 1 (SP1) in Gag. We previously isolated a panel of single-amino-acid substitutions that confer resistance to BVM in vitro (C. S. Adamson, S. D. Ablan, I. Boeras, R. Goila-Gaur, F. Soheilian, K. Nagashima, F. Li, K. Salzwedel, M. Sakalian, C. T. Wild, and E. O. Freed, J. Virol. 80:10957-10971, 2006). The BVM resistance mutations cluster at or near the CA-SP1 cleavage site. Because BVM likely will be used clinically in patients harboring viruses resistant to PR inhibitors (PIs), in this study we evaluated the interplay between a PI-resistant (PIR) PR and the BVM resistance mutations in Gag. As expected, the PIR mutations had no effect on inhibition by BVM; however, we observed general processing defects and a slight delay in viral replication in Jurkat T cells associated with the PIR mutations, even in the absence of compound. When combined, most BVM resistance and PIR mutations acted additively to impair viral replication, particularly in the presence of BVM. The BVM-resistant mutant SP1-A1V was an exception, as it supported robust replication in the context of either wild-type (WT) or PIR PR, even at high BVM concentrations. Significantly, the emergence of BVM resistance was delayed in the context of the PIR PR, and the SP1-A1V mutation was acquired most frequently with either WT or PIR PR. These results suggest that resistance to BVM is less likely to emerge in patients who have failed PIs than in patients who are PI naive. We predict that the SP1-A1V substitution is the most likely to emerge in vivo, as this mutant replicates robustly independently of PR mutations or BVM. These findings offer insights into the effect of PIR mutations on the evolution of BVM resistance in PI-experienced patients.

In this study, we evaluated baseline susceptibility to bevirimat (BVM), the first in a new class of antiretroviral agents, maturation inhibitors. We evaluated susceptibility to BVM by complete gag genotypic and phenotypic testing of 20 patient-derived human immunodeficiency virus type 1 isolates and 20 site-directed mutants. We found that reduced BVM susceptibility was associated with naturally occurring polymorphisms at positions 6, 7, and 8 in Gag spacer peptide 1.

Existing antiretroviral treatments for HIV type-1 (HIV-1) disease are limited by problems of resistance and drug-drug interactions. Bevirimat is a novel HIV-1 maturation inhibitor with a mechanism of action that is distinct from other antiretroviral agents. Specific inhibition of the final rate-limiting step in Gag processing by bevirimat prevents release of mature capsid protein from its precursor (CA-SP1), resulting in the production of immature, non-infectious virus particles. Bevirimat inhibits replication of both wild-type and drug-resistant HIV-1 isolates in vitro, achieving similar 50% inhibitory concentration values with both categories. Serial drug passage studies have identified six single amino acid substitutions that independently confer bevirimat resistance. These resistance mutations occur at or near the CA-SP1 cleavage site, which is not a known target for resistance to other antiretroviral drugs. Bevirimat has demonstrated a consistent pharmacokinetic profile in healthy volunteers and HIV-infected patients, with peak plasma concentrations attained approximately 1-3 h after dosing. Plasma concentrations decrease in a log-linear manner with a mean plasma elimination halflife of 58-80 h, supporting once-daily dosing. Animal studies suggest that elimination of bevirimat is primarily by hepatic glucuronidation and hepatobiliary excretion. There is minimal renal elimination, with < 1% of the administered dose appearing in the urine. In responsive patients, bevirimat has demonstrated a robust dosedependent reduction in viral load (> 1.5 log10 copies/ml). Short-term administration (< or = 14 days) of bevirimat is well tolerated, even when used in combination with other antiretroviral agents. Further studies to evaluate the long-term efficacy and tolerability of bevirimat are currently underway.

Bevirimat [3-O-(3',3'-dimethylsuccinyl)betulinic acid] is the first in a new class of anti-human immunodeficiency virus (HIV) drugs that inhibit viral maturation by specifically blocking cleavage of the Gag capsid (CA) precursor, CA-SP1, to mature CA protein, resulting in defective core condensation and release of immature noninfectious virions. Four cohorts of six HIV-infected adults, with CD4 counts of >200 and plasma viral loads of 5,000 to 250,000 transcripts/ml and not currently receiving antiretroviral therapy, were randomized to receive a single oral dose of placebo, 75, 150, or 250 mg of bevirimat. Thirty blood samples for drug concentrations and 20 HIV RNA measures were collected from each subject over a 20-day period. Candidate pharmacokinetic/pharmacodynamic models were fit to individual subjects by maximum likelihood followed by Bayesian estimation; model discrimination was by corrected Akaike's Information Criterion. The bevirimat pharmacokinetics was well described by an oral two-compartment linear model (r(2), 0.98), with a mean (percent coefficient of variation) half-life of 60.3 (13.6) h and apparent oral clearance of bevirimat from the plasma compartment of 0.17 (18) liters/h. HIV RNA was modeled as being produced in infected CD4 cells, with bevirimat inhibiting infection of new CD4 cells thru a Hill-type function (r(2), 0.87). Single oral doses of bevirimat were well tolerated and demonstrated a dose-dependent reduction in viral load. The average maximum reduction from baseline following the 150- and 250-mg doses was greater than 0.45 log(10), with individual patients having reductions of greater than 0.7 log(10). No bevirimat resistance mutations were detected during the course of the study.

The current standard of care for HIV/AIDS in the developed world is HAART therapy, usually a combination of two reverse transcriptase inhibitors and a protease inhibitor. Despite the success of this regimen, there is a continuing need for new drug options to overcome problems with tolerability and the emergence of viral resistance. In this review we discuss the discovery of a potential new class of antiretroviral therapeutics, known as maturation inhibitors, and the development of the first-in-class compound, bevirimat. Bevirimat is distinguished from the currently available antiretrovirals by its unique target and mode of action. While the specific interactions responsible for activity have yet to be fully characterized, it is clear that the target for bevirimat is the Gag polyprotein precursor, the main structural protein responsible for assembly and budding of virion particles. As basic research continues on the precise mechanism of action of bevirimat, clinical development is progressing, with demonstration of both safety and efficacy in early-stage trials. These encouraging results, coupled with the discovery and development of future generations of maturation inhibitors, suggest that maturation inhibitors may be added to the growing set of tools available to control HIV/AIDS.

3-O-(3',3'-dimethylsuccinyl) betulinic acid, also termed PA-457 or DSB, is a novel HIV-1 inhibitor that blocks virus maturation by disrupting cleavage of the capsid precursor, CA-SP1. To better define the molecular target for PA-457, we prepared a panel of mutant viruses with point deletions spanning the CA-SP1 cleavage domain and characterized each of these viruses for PA-457 sensitivity. Our results indicate that amino acid residues in the N-terminal half of SP1 serve as determinants of PA-457 activity, while residues in the C-terminal half of SP1 were not involved in compound activity. These findings support and extend previous observations that PA-457 is a specific inhibitor of CA-SP1 cleavage and identify the CA-SP1 domain as the primary viral determinant for this novel inhibitor of HIV-1 replication.

3-O-(3',3'-dimethylsuccinyl)betulinic acid (PA-457 or bevirimat) potently inhibits human immunodeficiency virus type 1 (HIV-1) maturation by blocking a late step in the Gag processing pathway, specifically the cleavage of SP1 from the C terminus of capsid (CA). To gain insights into the mechanism(s) by which HIV-1 could evolve resistance to PA-457 and to evaluate the likelihood of such resistance arising in PA-457-treated patients, we sought to identify and characterize a broad spectrum of HIV-1 variants capable of conferring resistance to this compound. Numerous independent rounds of selection repeatedly identified six single-amino-acid substitutions that independently confer PA-457 resistance: three at or near the C terminus of CA (CA-H226Y, -L231F, and -L231M) and three at the first and third residues of SP1 (SP1-A1V, -A3T, and -A3V). We determined that mutations CA-H226Y, CA-L231F, CA-L231M, and SP1-A1V do not impose a significant replication defect on HIV-1 in culture. In contrast, mutations SP1-A3V and -A3T severely impaired virus replication and inhibited virion core condensation. The replication defect imposed by SP1-A3V was reversed by a second-site compensatory mutation in CA (CA-G225S). Intriguingly, high concentrations of PA-457 enhanced the maturation of SP1 residue 3 mutants. The different phenotypes associated with mutations that confer PA-457 resistance suggest the existence of multiple mechanisms by which HIV-1 can evolve resistance to this maturation inhibitor. These findings have implications for the ongoing development of PA-457 to treat HIV-1 infection in vivo.

3-O-(3',3'-Dimethysuccinyl) betulinic acid (PA-457) has been shown to potently inhibit human immunodeficiency virus (HIV) replication in culture. In contrast to inhibitors that act upon the viral proteinase, PA-457 appears to block only the final maturational cleavage of p25CA-p2 to p24CA. However, attempts to replicate this effect in vitro using recombinant Gag have failed, leading to the hypothesis that activity is dependent upon the assembly state of Gag. Using a synthesis/assembly system for chimeric HIV type 1 Gag proteins, we have replicated the activity of PA-457 in vitro. The processing of assembled chimeric Gag can be inhibited by the addition of drug with only the final cleavage of p25CA-p2 to p24CA blocked. Consistent with our hypothesis and with previous findings, inhibition appears specific to Gag assembled into an immature capsid-like structure, since synthetic Gag that remains unassembled is properly processed in the presence of the compound. To further analyze the authenticity of the assay, PA-457 was tested in parallel with its inactive parental compound, betulinic acid. Betulinic acid had no effect upon p25 processing in this system. Analysis of a PA-457-resistant mutant, A1V, in this system pointed to more rapid cleavage as a possible mechanism for resistance. However, characterization of additional mutations at the cleavage site and in p2 suggests that resistance does not strictly correlate with the rate of cleavage. With the establishment of an in vitro assay for the detection of PA-457 activity, a more detailed characterization of its mechanism of action will be possible.

While it has been established that peptides modeling the C-helical region of human immunodeficiency virus type 1 gp41 are potent in vivo inhibitors of virus replication, their mechanism of action has yet to be determined. It has been proposed, but never directly demonstrated, that these peptides block virus entry by interacting with gp41 to disrupt the formation or function of a six-helix bundle structure. Using a six-helix bundle-specific monoclonal antibody with isolate-restricted Env reactivity, we provide the first direct evidence that, in receptor-activated viral Env, C-peptide entry inhibitors bind to the gp41 N-helical coiled-coil to form a peptide/protein hybrid structure and, in doing so, disrupt native six-helix bundle formation.

The envelope glycoprotein (Env) of HIV-1 is displayed on the surface of the virion or infected cell as an oligomer of multiple gp120/gp41 complexes. We sought to unravel the relationships between this oligomeric structure and the requirements for sequential interactions with CD4 and coreceptor (CCR5 or CXCR4). We used a quantitative cell fusion assay to examine the effects of coexpressing pairs of Envs, each nonfunctional because of a specific defect in one of the essential properties. We observed efficient fusion activity upon coexpression of two Env variants, one containing a gp41 subunit with a mutated fusion peptide and the other containing a gp120 subunit with a mutated CD4 binding site or a mismatched coreceptor specificity. We also observed fusion upon coexpression of two Env variants with distinct gp120 defects, i.e., a CD4 binding site mutation and the incorrect coreceptor specificity determinants. Coimmunoprecipitation experiments verified the efficient formation of mixed oligomers, suggesting that the observed fusion reflected subunit complementation within the oligomeric complex. These results support a model in which cooperative subunit interactions within the Env oligomer result in concerted conformational changes upon receptor binding, resulting in activation for fusion. The implications of these findings for Env function and virus neutralization are discussed.

We devised an experimental system to examine sequential events by which the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) interacts with CD4 and coreceptor to induce membrane fusion. Recombinant soluble CD4 (sCD4) activated fusion between effector cells expressing Env and target cells expressing coreceptor (CCR5 or CXCR4) but lacking CD4. sCD4-activated fusion was dose dependent, occurred comparably with two- and four-domain proteins, and demonstrated Env-coreceptor specificities parallel to those reported in conventional fusion and infectivity systems. Fusion activation occurred upon sCD4 preincubation and washing of the Env-expressing effector cells but not the coreceptor-bearing target cells, thereby demonstrating that sCD4 exerts its effects by acting on Env. These findings provide direct functional evidence for a sequential two-step model of Env-receptor interactions, whereby gp120 binds first to CD4 and becomes activated for subsequent functional interaction with coreceptor, leading to membrane fusion. We used the sCD4-activated system to explore neutralization by the anti-gp120 human monoclonal antibodies 17b and 48d. These antibodies reportedly bind conserved CD4-induced epitopes involved in coreceptor interactions but neutralize HIV-1 infection only weakly. We found that 17b and 48d had minimal effects in the standard cell fusion system using target cells expressing both CD4 and coreceptor but potently blocked sCD4-activated fusion with target cells expressing coreceptor alone. Both antibodies strongly inhibited sCD4-activated fusion by Envs from genetically diverse HIV-1 isolates. Thus, the sCD4-activated system reveals conserved Env-blocking epitopes that are masked in native Env and hence not readily detected by conventional systems.

We have examined mutations in the ectodomain of the human immunodeficiency virus type 1 transmembrane glycoprotein gp41 within a region immediately adjacent to the membrane-spanning domain for their effect on the outcome of the fusion cascade. Using the recently developed three-color assay (I. Muñoz-Barroso, S. Durell, K. Sakaguchi, E. Appella, and R. Blumenthal, J. Cell Biol. 140:315-323, 1998), we have assessed the ability of the mutant gp41s to transfer lipid and small solutes from susceptible target cells to the gp120-gp41-expressing cells. The results were compared with the syncytium-inducing capabilities of these gp41 mutants. Two mutant proteins were incapable of mediating both dye transfer and syncytium formation. Two mutant proteins mediated dye transfer but were less effective at inducing syncytium formation than was wild-type gp41. The most interesting mutant proteins were those that were not capable of inducing syncytium formation but still mediated dye transfer, indicating that the fusion cascade was blocked beyond the stage of small fusion pore formation. Fusion mediated by the mutant gp41s was inhibited by the peptides DP178 and C34.

Mutations were introduced into the ectodomain of the human immunodeficiency virus type 1 (HIV-1) transmembrane envelope glycoprotein, gp41, within a region immediately adjacent to the membrane-spanning domain. This region, which is predicted to form an alpha-helix, contains highly conserved hydrophobic residues and is unusually rich in tryptophan residues. In addition, this domain overlaps the epitope of a neutralizing monoclonal antibody, 2F5, as well as the sequence corresponding to a peptide, DP-178, shown to potently neutralize virus. Site-directed mutagenesis was used to create deletions, substitutions, and insertions centered around a stretch of 17 hydrophobic and uncharged amino acids (residues 666 to 682 of the HXB2 strain of HIV-1) in order to determine the role of this region in the maturation and function of the envelope glycoprotein. Deletion of the entire stretch of 17 amino acids abrogated the ability of the envelope glycoprotein to mediate both cell-cell fusion and virus entry without affecting the normal maturation, transport, or CD4-binding ability of the protein. This phenotype was also demonstrated by substituting alanine residues for three of the five tryptophan residues within this sequence. Smaller deletions, as well as multiple amino acid substitutions, were also found to inhibit but not block cell-cell fusion. These results demonstrate the crucial role of a tryptophan-rich motif in gp41 during a post-CD4-binding step of glycoprotein-mediated fusion. The basis for the invariant nature of the tryptophans, however, appears to be at the level of glycoprotein incorporation into virions. Even the substitution of phenylalanine for a single tryptophan residue was sufficient to reduce Env incorporation and drop the efficiency of virus entry approximately 10-fold, despite the fact that the same mutation had no significant effect on syncytium formation.

The envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) has been shown to redirect the site of virus assembly in polarized epithelial cells. To test whether localization of the glycoprotein exclusively to the endoplasmic reticulum (ER) could redirect virus assembly to that organelle in nonpolarized cells, an ER -retrieval signal was engineered into an epitope-tagged variant of Env. The epitope tag, attached to the C terminus of Env, did not affect the normal maturation and transport of the glycoprotein or the incorporation of Env into virions. The epitope-tagged Env was also capable of mediating syncytium formation and virus entry with a similar efficiency to that of wild-type Env. When the epitope was modified to contain a consensus K(X)KXX ER retrieval signal, however, the glycoprotein was no longer proteolytically processed into its surface and transmembrane subunits and Env could not be detected at the cell surface by biotinylation. Endoglycosidase H analysis revealed that the modified Env was not transported to the Golgi apparatus. Immunofluorescent staining patterns were also consistent with the exclusion of Env from the Golgi. As expected, cells expressing the modified Env failed to form syncytia with CD4(+) permissive cells. Despite this tight localization of Env to the ER, when the modified Env was expressed in the context of virus, virions continued to be produced efficiently from the plasma membrane of transfected cells. However, these virions contained no detectable glycoprotein and were noninfectious. Electron microscopy revealed virus budding from the plasma membrane of these cells, but no virus was seen assembling at the ER membrane and no assembled virions were found within the cell. These results suggest that the accumulation of Env in an intracellular compartment is not sufficient to redirect the assembly of HIV Gag in nonpolarized cells.