Search

We presented our continuing stride to optimize the second-generation NBD entry antagonist targeted to the Phe43 cavity of HIV-1 gp120. We have synthesized thirty-eight new and novel analogs of NBD-14136, earlier designed based on a CH 2 OH "positional switch" hypothesis, and derived a comprehensive SAR. The antiviral data confirmed that the linear alcohol towards the "N" (C4) of the thiazole ring yielded more active inhibitors than those towards the "S" (C5) of the thiazole ring. The best inhibitor, NBD-14273 (compound 13), showed both improved antiviral activity and selectivity index (SI) against HIV-1 HXB2 compared to NBD-14136. We also tested NBD-14273 against a large panel of 50 HIV-1 Env-pseudotyped viruses representing clinical isolates of diverse subtypes. The overall mean data indicate that antiviral potency against these isolates improved by ~3-fold, and SI also improved ~3-fold compared to NBD-14136. This new and novel inhibitor is expected to pave the way for further optimization to a more potent and clinically relevant inhibitor against HIV-1., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

During human immunodeficiency virus type 1 (HIV-1) entry into cells, the viral envelope glycoprotein (Env) trimer [(gp120/gp41) 3 ] binds the receptors CD4 and CCR5 and fuses the viral and cell membranes. CD4 binding changes Env from a pretriggered (state-1) conformation to more open downstream conformations. BMS-378806 (here called BMS-806) blocks CD4-induced conformational changes in Env important for entry and is hypothesized to stabilize a state-1-like Env conformation, a key vaccine target. Here, we evaluated the effects of BMS-806 on the conformation of Env on the surface of cells and virus-like particles. BMS-806 strengthened the labile, noncovalent interaction of gp120 with the Env trimer, enhanced or maintained the binding of most broadly neutralizing antibodies, and decreased the binding of poorly neutralizing antibodies. Thus, in the presence of BMS-806, the cleaved Env on the surface of cells and virus-like particles exhibits an antigenic profile consistent with a state-1 conformation. We designed novel BMS-806 analogues that stabilized the Env conformation for several weeks after a single application. These long-acting BMS-806 analogues may facilitate enrichment of the metastable state-1 Env conformation for structural characterization and presentation to the immune system. IMPORTANCE The envelope glycoprotein (Env) spike on the surface of human immunodeficiency virus type 1 (HIV-1) mediates the entry of the virus into host cells and is also the target for antibodies. During virus entry, Env needs to change shape. Env flexibility also contributes to the ability of HIV-1 to evade the host immune response; many shapes of Env raise antibodies that cannot recognize the functional Env and therefore do not block virus infection. We found that an HIV-1 entry inhibitor, BMS-806, stabilizes the functional shape of Env. We developed new variants of BMS-806 that stabilize Env in its natural state for long periods of time. The availability of such long-acting stabilizers of Env shape will allow the natural Env conformation to be characterized and tested for efficacy as a vaccine., (Copyright © 2020 American Society for Microbiology.)

Objectives: Development of HbAHP-25, a peptide that prevents HIV-1 entry into cells by blocking gp120-CD4 interaction, as a topical anti-HIV drug, necessitates that it is first tested for toxic or abrasive effects on genital epithelial cells and also on the vaginal microbiome. The present study was, therefore, undertaken to investigate whether: (1) HbAHP-25 has any adverse effect on growth and membrane integrity of various cell lines, and (2) HbAHP-25 neutralizes gp120 mediated insults on genital epithelial cells., Methods: MTT and trans-epithelial resistance (TER) assays were performed to assess the viability and integrity of epithelial cells. Real-time PCR and Immunofluorescence/Western blotting were used to decipher the expression of tight junction proteins, at the mRNA and protein levels, respectively. A multiplex cytokine assay was performed to quantify the cytokines., Results: HbAHP-25 had no adverse effect on the viability of VK2/E6E7, End1/E6E7, Ect1/E6E7 and HEC-1A cells, and also on growth of lactobacilli. The barrier integrity of HbAHP-25-treated cells remained unaltered. Expression of tight junction proteins, Claudin-1 and ZO-1, at transcript and protein levels, remained unaltered in HbAHP-25-treated HEC-1A cells. Interestingly, HbAHP-25 treatment prevented the breach of barrier integrity caused by gp120. Further, HbAHP-25 did not elicit the expression of inflammatory cytokines. Instead, the in vitro induction of inflammatory cytokines by gp120 was also abrogated in the presence of HbAHP-25., Conclusion: HbAHP-25 is exceedingly safe to genital epithelial cells and attenuates HIV-1 gp120-mediated barrier dysfunction by limiting excessive inflammation. This study provides significant evidences in the favor of HbAHP-25's potential as a topical anti-HIV agent., (© 2017 British HIV Association.)

Unlabelled: Broadly neutralizing antibodies (bNAbs) specific for conserved epitopes on the HIV-1 envelope (Env) are believed to be essential for protection against multiple HIV-1 clades. However, vaccines capable of stimulating the production of bNAbs remain a major challenge. Given that polyreactivity and autoreactivity are considered important characteristics of anti-HIV bNAbs, we designed an HIV vaccine incorporating the molecular adjuvants BAFF (B cell activating factor) and APRIL (a proliferation-inducing ligand) with the potential to facilitate the maturation of polyreactive and autoreactive B cells as well as to enhance the affinity and/or avidity of Env-specific antibodies. We designed recombinant DNA plasmids encoding soluble multitrimers of BAFF and APRIL using surfactant protein D as a scaffold, and we vaccinated mice with these molecular adjuvants using DNA and DNA-protein vaccination strategies. We found that immunization of mice with a DNA vaccine encoding BAFF or APRIL multitrimers, together with interleukin 12 (IL-12) and membrane-bound HIV-1 Env gp140, induced neutralizing antibodies against tier 1 and tier 2 (vaccine strain) viruses. The APRIL-containing vaccine was particularly effective at generating tier 2 neutralizing antibodies following a protein boost. These BAFF and APRIL effects coincided with an enhanced germinal center (GC) reaction, increased anti-gp120 antibody-secreting cells, and increased anti-gp120 functional avidity. Notably, BAFF and APRIL did not cause indiscriminate B cell expansion or an increase in total IgG. We propose that BAFF and APRIL multitrimers are promising molecular adjuvants for vaccines designed to induce bNAbs against HIV-1., Importance: Recent identification of antibodies that neutralize most HIV-1 strains has revived hopes and efforts to create novel vaccines that can effectively stimulate HIV-1 neutralizing antibodies. However, the multiple immune evasion properties of HIV have hampered these efforts. These include the instability of the gp120 trimer, the inaccessibility of the conserved sequences, highly variable protein sequences, and the loss of HIV-1-specific antibody-producing cells during development. We have shown previously that tumor necrosis factor (TNF) superfamily ligands, including BAFF and APRIL, can be multitrimerized using the lung protein SP-D (surfactant protein D), enhancing immune responses. Here we show that DNA or DNA-protein vaccines encoding BAFF or APRIL multitrimers, IL-12p70, and membrane-bound HIV-1 Env gp140 induced tier 1 and tier 2 neutralizing antibodies in a mouse model. BAFF and APRIL enhanced the immune reaction, improved antibody binding, and increased the numbers of anti-HIV-1 antibody-secreting cells. Adaptation of this vaccine design may prove useful in designing preventive HIV-1 vaccines for humans., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Background: BMS-663068 is the phosphonooxymethyl prodrug of BMS-626529, a small-molecule attachment inhibitor that targets the HIV-1 envelope glycoprotein gp120 preventing it from binding to CD4 T cells. In vitro investigations have demonstrated considerable variation in susceptibility of different HIV-1 isolates to BMS-626529. BMS-663068 monotherapy in HIV-1-infected subjects produced a mean maximum change from baseline of -1.64 log10 copies per milliliter, but the response was variable., Methods: In this analysis, baseline and day 8 samples were analyzed for susceptibility to BMS-626529 and the presence of known HIV-1 attachment inhibitor resistance mutations. In addition, predictors of virological response (maximal HIV-1 RNA decline ≥1 log10 copies per milliliter) and resistance selection were investigated., Results: The only factor associated with reduced virological response was low baseline susceptibility to BMS-626529. There was no apparent relationship between virological response and baseline treatment experience, coreceptor tropism, plasma HIV-1 RNA level, or CD4 T-cell count. Examination of all positions with known BMS-626529 resistance mutations based on in vitro selection studies showed that gp120 M426L was the primary substitution most clearly associated with nonresponse to BMS-663068. There was minimal change in susceptibility to BMS-626529 over the course of the study and no clear evidence of emergence of a known HIV-1 attachment inhibitor resistance mutation in the majority of subjects as measured by standard population-based phenotypic and genotypic approaches., Conclusions: Nonresponse to BMS-663068 was associated with low baseline susceptibility to BMS-626529 and the presence of M426L. In this short-term trial, there was minimal evidence of selection for BMS-626529 high-level resistance over 8 days of monotherapy with BMS-663068 by population-based approaches.

Pradimicins (PRM) are a unique class of nonpeptidic carbohydrate-binding agents that inhibit HIV infection by efficiently binding to the HIV-1 envelope gp120 glycans in the obligatory presence of Ca(2+). Surface plasmon resonance (SPR) data revealed that addition of EDTA dose-dependently results in lower binding signals of PRM-A to immobilized gp120. Pradimicin derivatives that lack the free carboxylic acid group on the C-18 position failed to bind gp120 and were devoid of significant antiviral activity. Ca(2+) was much more efficient for PRM-A binding to gp120 than Cd(2+), Ba(2+) or Sr(2+). Instead, calcium could not be replaced by any other mono- (i.e. K(+)), di- (i.e. Cu(2+), Mg(2+), Mn(2+), Fe(2+), Zn(2+)) or trivalent (i.e. Al,(3+), Fe(3+)) cation without complete loss of gp120 binding. However, Zn(2+), Mg(2+) and Mn(2+) added to a Ca(2+)- pradimicin mixture, prevented pradimicin from efficient binding to gp120 glycans. These data suggest that these bivalent cations may bind to pradimicins but lead to pradimicin-cation complexes that are unable to further coordinate with the glycans of gp120. Thus, in order to afford antiviral activity, only a few cations can (i) bind to pradimicin to form a dimeric complex and (ii) subsequently coordinate the pradimicin/cation interaction with gp120 glycans.

Carbohydrate-binding agents (CBAs) are potential HIV microbicidal agents with a high genetic barrier to resistance. We wanted to evaluate whether two mannose-specific CBAs, recognizing multiple and often distinct glycan structures on the HIV envelope gp120, can interact synergistically against HIV-1, HIV-2, and HIV-1 strains that were selected for resistance against particular CBAs [i.e., 2G12 mAb and microvirin (MVN)]. Paired CBA/CBA combinations mainly showed synergistic activity against both wild-type HIV-1 and HIV-2 but also 2G12 mAb- and MVN-resistant HIV-1 strains as based on the median effect principle with combination indices (CIs) ranging between 0.29 and 0.97. Upon combination, an increase in antiviral potency of griffithsin (GRFT) up to ∼12-fold (against HIV-1), ∼8-fold (against HIV-2), and ∼6-fold (against CBA-resistant HIV-1) was observed. In contrast, HHA/GNA combinations showed additive activity against wild-type HIV-1 and HIV-2 strains, but remarkable synergy with HHA and GNA was observed against 2G12 mAb- and MVN-resistant HIV-1 strains (CI, 0.64 and 0.49, respectively). Overall, combinations of GRFT and other CBAs showed synergistic activity against HIV-1, HIV-2, and even against certain CBA-resistant HIV-1 strains. The CBAs tested appear to have distinct binding patterns on the gp120 envelope and therefore do not necessarily compete with each other's glycan binding sites on gp120. As a result, there might be no steric hindrance between two different CBAs in their competition for glycan binding (except for the HHA/GNA combination). These data are encouraging for the use of paired CBA combinations in topical microbicide applications (e.g., creams, gels, or intravaginal rings) to prevent HIV transmission.

Although antiretrovirals are the mainstay of therapy against HIV infection, neurological complications associated with the virus continue to hamper quality of life of the infected individuals. Drugs of abuse in the infected individuals further fuel the epidemic. Epidemiological studies have demonstrated that abuse of cocaine resulted in acceleration of HIV infection and the progression of NeuroAIDS. Cocaine has not only been shown to play a crucial role in promoting virus replication, but also has diverse but often deleterious effects on various cell types of the CNS. In the neuronal system, cocaine exposure results in neuronal toxicity and also potentiates gp120-induced neurotoxicity. In the astroglia and microglia, cocaine exposure leads to up-regulation of pro-inflammatory mediators such as cytokines and chemokines. These in turn, can lead to neuroinflammation and transmission of toxic responses to the neurons. Additionally, cocaine exposure can also lead to leakiness of the blood-brain barrier that manifests as enhanced transmigraiton of leukocytes/monocytes into the CNS. Both in vitro and in vivo studies have provided valuable tools in exploring the role of cocaine in mediating HIV-associated neuropathogenesis. This review summarizes previous studies on the mechanism(s) underlying the interplay of cocaine and HIV as it relates to the CNS.

HIV-1 infection is a global public health problem with more than 34 million people living with HIV infection. Although great strides have been made in treating this epidemic with therapeutic agents, the increase in patient life span has been coincident with an increase in the prevalence of HIV-associated neurocognitive disorders (HAND). HAND is thought to result from the neurotoxic effects of viral proteins that are shed from HIV-infected microglial cells. One of the primary neurotoxins responsible for this effect is the HIV-1 glycoprotein gp120. Exposure of neurons to gp120 has been demonstrated to cause apoptosis in neurons, as well as numerous indirect effects such as an increase in inflammatory cytokines, an increase in oxidative stress, and an increase in permeability of the blood-brain barrier. In many patients, the use of drugs of abuse (DOA) exacerbates the neurotoxic effects of gp120. Cocaine, methamphetamine and morphine are three DOAs that are commonly used by those infected with HIV-1. All three of these DOAs have been demonstrated to increase oxidative stress in the CNS as well as to increase permeability of the blood-brain barrier. Numerous model systems have demonstrated that these DOAs have the capability of exacerbating the neurotoxic effects of gp120. This review will summarize the neurotoxic effects of gp120, the deleterious effects of cocaine, methamphetamine and morphine on the CNS, and the combined effects of gp120 in the context of these drugs.

One aim for an HIV vaccine is to elicit neutralizing antibodies (Nab) that can limit replication of genetically diverse viruses and prevent establishment of a new infection. Thus, identifying the strengths and weaknesses of Nab during the early stages of natural infection could prove useful in achieving this goal. Here we demonstrate that viral escape readily occurred despite the development of high titer autologous Nab in two subjects with acute/early subtype C infection. To provide a detailed portrayal of the escape pathways, Nab resistant variants identified at multiple time points were used to create a series of envelope (Env) glycoprotein chimeras and mutants within the background of a corresponding newly transmitted Env. In one subject, Nab escape was driven predominantly by changes in the region of gp120 that extends from the beginning of the V3 domain to the end of the V5 domain (V3V5). However, Nab escape pathways in this subject oscillated and at times required cooperation between V1V2 and the gp41 ectodomain. In the second subject, escape was driven by changes in V1V2. This V1V2-dependent escape pathway was retained over time, and its utility was reflected in the virus's ability to escape from two distinct monoclonal antibodies (Mabs) derived from this same patient via introduction of a single potential N-linked glycosylation site in V2. Spatial representation of the sequence changes in gp120 suggested that selective pressure acted upon the same regions of Env in these two subjects, even though the Env domains that drove escape were different. Together the findings argue that a single mutational pathway is not sufficient to confer escape in early subtype C HIV-1 infection, and support a model in which multiple strategies, including potential glycan shifts, direct alteration of an epitope sequence, and cooperative Env domain conformational masking, are used to evade neutralization.

We have previously isolated nucleic acid ligands (aptamers) that bind the surface envelope glycoprotein, gp120, of HIV-1, and neutralize infection of diverse sub-types of virus. Our earlier studies have identified the overall structure of one of these aptamers, B40, and have indicated that it binds to gp120 in a manner that competes with that of the HIV-1 coreceptor, CCR5, and select "CD4i" antibodies with epitopes overlapping this region. Here, we sought to map the B40 binding site on gp120 more precisely by analysing its interaction with a panel of alanine substitution mutants of gp120. Furthermore, we tested our hypothesis concerning the structure of the 40 nucleotide functional core of the aptamer by the solid-phase synthesis of truncated and chemically modified derivatives. The results confirm our structural predictions and demonstrate that aptamer B40 neutralizes a diverse range of HIV-1 isolates as a result of binding to relatively conserved residues on gp120 at the heart of the CCR5-binding site. These structural insights may provide the basis for the development of potential anti-viral agents with high specificity and robustness.

Betulinic acid (BA) derivatives can inhibit human immunodeficiency virus type 1 (HIV-1) entry or maturation depending on side chain modifications. While BA derivatives with antimaturation activity have attracted considerable interest, the anti-HIV-1 profile and molecular mechanism of BA derivatives with anti-HIV-1 entry activity (termed BA entry inhibitors) have not been well defined. In this study, we have found that two BA entry inhibitors, IC9564 and A43D, exhibited a broad spectrum of anti-HIV-1 activity. Both compounds inhibited multiple strains of HIV-1 from clades A, B, and C at submicromolar concentrations. Clade C viruses were more sensitive to the compounds than clade A and B viruses. Interestingly, IC9564 at subinhibitory concentrations could alter the antifusion activities of other entry inhibitors. IC9564 was especially potent in increasing the sensitivity of HIV-1 YU2 Env-mediated membrane fusion to the CCR5 inhibitor TAK-779. Results from this study suggest that the V3 loop of gp120 is a critical determinant for the anti-HIV-1 activity of IC9564. IC9564 escape viruses contained mutations near the tip of the V3 loop. Moreover, IC9564 could compete with the binding of V3 monoclonal antibodies 447-52D and 39F. IC9564 also competed with the binding of gp120/CD4 complexes to chemokine receptors. In summary, these results suggest that BA entry inhibitors can potently inhibit a broad spectrum of primary HIV-1 isolates by targeting the V3 loop of gp120.

Conformational changes in HIV-1 envelope glycoproteins, gp120 and gp41, is a dynamic process essential for HIV-1 entry. Here we show that a small molecule HIV-1 entry inhibitor, IC9564, induces a conformational change in gp120. The conformational change in gp120 is evidenced by a significant increase in the binding of a conformational monoclonal antibody 17b. As a result of the conformational effect, IC9564 significantly enhances the neutralizing activity of 17b. Unlike CD4, IC9564 does not trigger conformational changes in gp41. In fact, IC9564 inhibits CD4-induced conformational changes in gp41. Thus, IC9564 exploits the dynamic nature of gp120 by inducing a nonproductive gp120 conformation that is not able to trigger a conformational change in gp41 for membrane fusion.

Based on the published NMR spectroscopy data, three-dimensional structures of the HIV-1 gp120 protein V3 loop were obtained by computer modeling in the viral strains HIV-Haiti and HIV-MN. In both cases, the secondary structure elements and conformations of irregular stretches were determined for the fragment representing the principal antigenic determinant of the virus, as well as determinants of the cellular tropism and syncytium formation. Notwithstanding the high variability of the amino acid sequence of gp120 protein, more than 50% of the V3 loop residues retained their conformations in the different HIV-1 virions. The combined analysis of the findings and the literature data on the biological activity of the individual residues of the HIV-1 V3 loop resulted in identification of its structurally conservative amino acids, which seem to be promising targets for antiviral drug design by protein engineering approaches.

DC-SIGN (dendritic cell-specific ICAM-3 grabbing non-integrin) is a C-type lectin receptor of dendritic cells and is involved in the initial steps of numerous infectious diseases. Surface plasmon resonance has been used to study the affinity of a glycodendritic polymer with 32 mannoses, to DC-SIGN. This glycodendrimer binds to DC-SIGN surfaces in the submicromolar range. This binding depends on a clustered organization of DC-SIGN mimicking its natural organization as microdomain in the dendritic cells plasma membrane. Moreover, this compound inhibits DC-SIGN binding to the HIV glycoprotein gp120 with an IC50 in the micromolar range and therefore can be considered as a potential antiviral drug.

BMS-488043 is a small-molecule human immunodeficiency virus type 1 (HIV-1) CD4 attachment inhibitor with demonstrated clinical efficacy. The compound inhibits soluble CD4 (sCD4) binding to the 11 distinct HIV envelope gp120 proteins surveyed. Binding of BMS-488043 and that of sCD4 to gp120 are mutually exclusive, since increased concentrations of one can completely block the binding of the other without affecting the maximal gp120 binding capacity. Similarly, BMS-488043 inhibited virion envelope trimers from binding to sCD4-immunoglobulin G (IgG), with decreasing inhibition as the sCD4-IgG concentration increased, and BMS-488043 blocked the sCD4-induced exposure of the gp41 groove in virions. In both virion binding assays, BMS-488043 was active only when added prior to sCD4. Collectively, these results indicate that obstruction of gp120-sCD4 interactions is the primary inhibition mechanism of this compound and that compound interaction with envelope must precede CD4 binding. By three independent approaches, BMS-488043 was further shown to induce conformational changes within gp120 in both the CD4 and CCR5 binding regions. These changes likely prevent gp120-CD4 interactions and downstream entry events. However, BMS-488043 could only partially inhibit CD4 binding to an HIV variant containing a specific envelope truncation and altered gp120 conformation, despite effectively inhibiting the pseudotyped virus infection. Taken together, BMS-488043 inhibits viral entry primarily through altering the envelope conformation and preventing CD4 binding, and other downstream entry events could also be inhibited as a result of these induced conformational changes.

Recent data suggest that heparin sulfates may bind to a CD4 induced epitope in the HIV-1 gp120 that constitutes the coreceptor binding site. We have studied the mechanism of action of ADS-J1, a non-peptidic compound selected by docking analysis to interact with gp41 and to interfere with the formation of N-36/C-34 complexes in sandwich ELISA experiments. We show that ADS-J1 blocked the binding of wild-type HIV-1 NL4-3 strain to MT-4 cells but not virus-cell binding of a polyanion-resistant virus. However, ADS-J1 blocked the replication of polyanion-resistant, T-20- and C34-resistant HIV-1, suggesting a second mechanism of action. Development of resistance to ADS-J1 on the polyanion-resistant HIV-1 led to mutations in gp120 coreceptor binding site and not in gp41. Time of addition experiments confirmed that ADS-J1, but not polyanions such as dextran sulfate or AR177, worked at a step that mimics the activity of an HIV coreceptor antagonist but prior to gp41-dependent fusion. We conclude that ADS-J1 may bind to the HIV coreceptor binding site as its mechanism of anti-HIV activity.

We recently described the isolation and structural characterization of 2'-fluoropyrimidine-substituted RNA aptamers that bind to gp120 of R5 strains of human immunodeficiency virus type 1 and thereby potently neutralize the infectivity of phylogenetically diverse R5 strains. Here we investigate the physical basis of their antiviral action. We show that both N-linked oligosaccharides and the variable loops V1/V2 and V3 are not required for binding of one aptamer, B40, to gp120. Using surface plasmon resonance binding analyses, we show that the aptamer binds to the CCR5-binding site on gp120 in a relatively CD4-independent manner, providing a mechanistic explanation for its neutralizing potency.

The development of drug resistance in HIV compromises the long-term efficacy of current therapies. Furthermore, vaccine development faces huge problems, mainly because of the low antigenicity and immunogenicity of the HIV envelope glycoprotein gp120 and the efficient hiding of highly immunogenic epitopes by its glycans. There is evidence that mutant HIV strains containing glycosylation site deletions trigger the production of specific neutralising antibodies to previously hidden gp120 epitopes. I present a hypothesis that development of resistance against drugs that target the glycans on gp120 would result in a marked enhancement of neutralisation of HIV by the immune system--ie, drugs directed against the carbohydrate component of gp120 will select for mutant virus strains that progressively gain deletions in the glycosylation sites of gp120. Previously hidden epitopes would then be uncovered, and the virus will become highly susceptible to markedly increased immunological neutralisation. I believe this novel approach may become an entirely new therapeutic concept that exploits the high mutation rate of HIV and allows drug therapy to act in concert with a triggered immune response to suppress HIV more efficiently. Moreover, this approach could be applied to treat other chronic infections by viruses that contain a glycosylated envelope (eg, hepatitis B and C).

HIV-1 cell entry is mediated by sequential interactions of the envelope protein gp120 with the receptor CD4 and a coreceptor, usually CCR5 or CXCR4, depending on the individual virion. Considerable efforts on exploiting the HIV coreceptors as drug targets have led to the new class of coreceptor antagonists. While these antiretroviral drugs aim at preventing virus/coreceptor interaction by binding to host proteins, neutralizing antibodies directed against the coreceptor-binding sites on gp120 have attracted attention as possible vaccine candidates. However, both approaches are complicated by the multiple protective mechanisms of gp120 which allow for rapid escape from selective pressures exerted by drugs or antibodies. Thus, advances in rational drug and vaccine design rely heavily on improved insights into the relation between genotype and phenotype, the evolution of coreceptor usage, and, ultimately the structural biology of coreceptor usage and inhibition. The third variable (V3) loop of gp120, crucially involved in all these aspects, will be a major focus of this review.

This paper describes selected modification and structure-activity relationship of the small molecule HIV-1 inhibitor, 4-benzoyl-1-[(4-methoxy-1H-pyrrolo[2,3-b]pyridin-3-yl)oxoacetyl]-2-(R)-methylpiperazine (BMS-378806). The results revealed: i) that both the presence and configuration (R vs. S) of the 3-methyl group on the piperazine moiety are important for the antiviral activity, with the 3-(R)-methyl derivatives showing the highest activity; ii) that the electronegativity of the C-4 substituent on the indole or azaindole ring seems to be important for the activity, with a small, electron-donating group such as a fluoro or a methoxy group showing enhanced activity, while a nitro group diminishes the activity; iii) that the N-1 position of the indole ring is not eligible for modification without losing activity; and iv) that bulky groups around the C-4 position of the indole or azaindole ring diminish the activity, probably due to steric hindrance in the binding. We found that a synthetic bivalent compound with two BMS-378806 moieties being tethered by a spacer demonstrated about 5-fold enhanced activity in an nM range against HIV-1 infection than the corresponding monomeric inhibitor. But the polyacrylamide-based polyvalent compounds did not show inhibitory activity at up to 200 nM.

Fuzeon (also known as T-20 or enfuvirtide), one of the C-peptides derived from the HIV-1 envelope glycoprotein transmembrane subunit gp41 C-terminal heptad repeat (CHR) region, is the first member of a new class of anti-HIV drugs known as HIV fusion inhibitors. It has been widely believed that T-20 shares the same mechanism of action with C34, another C-peptide. The C34 is known to compete with the CHR of gp41 to form a stable 6-helix bundle (6-HB) with the gp41 N-terminal heptad repeat (NHR) and prevent the formation of the fusogenic gp41 core between viral gp41 NHR and CHR, thereby inhibiting fusion between viral and target cell membranes. Here we present data to demonstrate that, contrary to this belief, T-20 cannot form stable 6-HB with N-peptides derived from the NHR region, nor can it inhibit the 6-HB formation of the fusogenic core. Instead, it may interact with N-peptides to form unstable or insoluble complexes. Our data suggest that T-20 has a different mechanism of action from C34. The interaction of T-20 with viral NHR region alone may not prevent the formation of the fusion active gp41 core. We also demonstrate that the T-20-mediated anti-HIV activity can be significantly abrogated by peptides derived from the membrane-spanning domain in gp41 and coreceptor binding site in gp120. These new findings imply that T-20 inhibits HIV-1 entry by targeting multiple sites in gp41 and gp120. Further elucidation of the mechanism of action of T-20 will provide new target(s) for development of novel HIV entry inhibitors.

Amino acid sequences of the HIV-1 surface protein gp120 show extensive variations. Some of the peptide or DNA vaccines using the V3-loop of gp120 can trigger antibody production against some isolates of HIV, but these antibodies are not protective against other isolates. Thus, more conserved peptides from other HIV proteins have also been tried as vaccines. However, antibodies specific for these peptides cannot prevent the normal entry of HIV into the host cells, followed by integration of viral genomes into host chromosomes. Therefore, we decided to make a careful analysis of the aligned amino acid sequences of gp120 stored at Los Alamos National Laboratory. Unexpectedly, we found that there are several, not just one or two, segments of seven or more consecutive and nearly invariant amino acid residues present in the C1, C2 and C5 regions of this protein. Furthermore, four of these segments are physically close to each other in the known three-dimensional structure of the core portion of gp120. The remaining segments are not in that crystallized region; however, two of these are covalently linked with a disulfide bond. Therefore, we propose that these segments may be combined to form the basis of possible HIV vaccine candidates. In addition, it is necessary to incorporate the processed peptide from gp120 or other proteins and to understand the time-dependent effects of the highly variable loops of gp120 on triggering antibody production in human patients.

The key role of gp120 in the cellular entry of HIV makes this glycoprotein an attractive target for new drugs. Various polyanions bind to the positively charged V3 loop of gp120. Here, we consider a series of anionic porphyrins bearing two sulfonate groups and two carboxylic chains with various degree of esterification. These molecules carry an overall negative charge between 4 and 2. Upon activation by light, these compounds, known as photosensitizers, produce highly reactive oxygen species able to damage amino acid chains. The interactions of these molecules with the V3 loop and a positively charged area in the C5 region were investigated in the dark by using specific antibodies and ELISA protocols. Competitive inhibition of the anti-V3 antibody was observed with an increased efficiency for the esterified compounds. No evidence for binding to the C5 region was found. In contrast, when gp120 was irradiated with light in presence of the porphyrin prior to the addition of the antibody, strong inhibition of the anti-C5 antibody was observed revealing irreversible photo-damages in this region. No effect on the V3 loop was observed. Irradiations at two wavelengths made it possible to identify porphyrin monomers as the photoactive forms despite the presence of large excess of dimers in the incubation solution. It is suggested that porphyrins bound to the V3 loop could produce photo-damages at some distance, in particular within the C5 region that contains several photosensitive amino acids.

Background: Epigallocatechin gallate (EGCG), the major component of tea polyphenol, has been reported to have various physiologic modulatory activities. Several reports also have shown that catechin has a protective effect against HIV infection, part of which is mediated by inhibiting virions to bind to the target cell surface., Objective: We investigated the effect of EGCG on the expression of CD4 molecules and on its ability to bind gp120, an envelope protein of HIV-1., Methods: Peripheral blood CD4+ T cells were incubated in the presence of EGCG, and the expression of CD4 was evaluated by means of flow cytometry. The effect of EGCG on the antibody binding to CD4 was investigated by using a sandwich ELISA, and the effect on the gp120 binding to CD4 was analyzed by means of flow cytometry., Results: EGCG efficiently inhibited binding of anti-CD4 antibody to its corresponding antigen. This effect was mediated by the direct binding of EGCG to the CD4 molecule, with consequent inhibition of antibody binding, as well as gp120 binding., Conclusion: The present results suggest a potential preventive effect of EGCG on HIV-1 infection by modulating binding to CD4.

The potential targets of marine sulfated polymannuroguluronate (SPMG) involved in inhibition of HIV-1 entry were investigated by surface plasmon resonance and flow cytometry. Results indicated that binding of SPMG either to soluble oligomeric rgp120 or to complexed rgp120-sCD4 mainly resided in V3 loop region. In addition, SPMG was shown to be less accessible for sCD4 when sCD4 had pre-interacted with rgp120, though SPMG per se multivalently bound to sCD4 with relatively low affinity. While the pre-incubation of SPMG with rgp120 caused a partial blockade of rgp120 binding to sCD4, suggesting that SPMG either shared common binding sites on gp120 with sCD4 or masked the docking sites of gp120 for sCD4. Taken together, V3 domain was demonstrated to be the major site mediating interaction of SPMG with complexed rgp120-sCD4. It seems likely that SPMG binds to both rgp120 and sCD4, but has less accessibility for sCD4 when sCD4 has already bound to rgp120. Nevertheless, addition of SPMG either prior to or after the interaction of rgp120 with sCD4 may suppress rgp120 binding to sCD4. The exact pattern of this trimolecular complex formation at the cell membrane-anchored virus level requires further clarification.

It has been previously reported that insulin-like growth factor I (IGF I) decreases in AIDS patients with wasting, a condition that is partially prevented by combined IGF I growth hormone therapy. By generating bifunctional proteins of IGF I and stromal cell-derived factor 1alpha (SDF-1alpha) or alpha1 proteinase inhibitor (API), two proteins known to prevent HIV infection, it may be possible to improve the therapeutic effectiveness of these compounds for the treatment of AIDS-mediated wasting. SDF-1alpha or the M351E-M358L mutant of API were attached at the C-terminal end of IGF I and synthesized by a stable insect cell expression technique. The IGF I-SDF-1alpha chimera reduced the enhancement of thymidine incorporation into bovine fetal erythroid cells observed in the presence of insect cell produced IGF I alone. It also decreased the SDF-1 and IGF I-stimulated hematopoietic cell migration, without losing the capacity to compete with the binding of HIV-1 (IIIB)-surface glycoprotein gp120. The IGF I-API chimera displayed the same mitogenic activity and a similar, but lower chemotactic activity than IGF I in the assays mentioned above. It had a comparable anti-elastase activity to that observed with a previously described IGF II-API fusion protein with the single mutation M351E. The binding of gp120 to a murine hematopoietic cell line was stimulated by human neutrophil elastase (25-100 nM) and inhibited by IGF I-API. In conclusion, the linkage of IGF I with SDF-1 or API can alter some biological functions of the single components of the chimera while keeping their ability to compete with HIV-1-gp120 binding.

There is an urgent need for new drugs that can kill HIV type 1 (HIV-1)-infected cells. HIV-1 glycoprotein Env, which promotes viral membrane fusion through receptor-mediated conformational changes, is an attractive target for such agents because it is expressed on the surface of both virions and infected cells. Unfortunately, conserved binding elements on this protein frequently are buried under a canopy of flexible, glycosylated peptide loops or exposed only transiently during the fusion process. Here, we investigate the exposure of the C-terminal region of the Env ectodomain outside the context of membrane fusion. This binding element is the target of the 5-Helix protein, a designed entry inhibitor that disrupts conformational changes in Env subunit gp41, essential for the fusion process. We show that 5-Helix is capable of interacting with HIV-1 Env in a receptor-independent fashion and that a chimeric 5-Helix/Pseudomonas exotoxin protein recognizes cells expressing Env from a broad spectrum of HIV-1 strains including primary isolates from clades B, D, E, G, and H. This recombinant toxin selectively kills HIV-1-infected cells and blocks spreading infection while still maintaining potent inhibitory activity against membrane fusion. Our results demonstrate that the C-terminal region of the gp41 ectodomain is an accessible target on HIV-1-infected cells for the development of antiviral therapeutics and neutralizing antibodies.

We previously reported that monoclonal antibodies to protein-disulfide isomerase (PDI) and other membrane-impermeant PDI inhibitors prevented HIV-1 infection. PDI is present at the surface of HIV-1 target cells and reduces disulfide bonds in a model peptide attached to the cell membrane. Here we show that soluble PDI cleaves disulfide bonds in recombinant envelope glycoprotein gp120 and that gp120 bound to the surface receptor CD4 undergoes a disulfide reduction that is prevented by PDI inhibitors. Concentrations of inhibitors that prevent this reduction and inhibit the cleavage of surface-bound disulfide conjugate prevent infection at the level of HIV-1 entry. The entry of HIV-1 strains differing in their coreceptor specificities is similarly inhibited, and so is the reduction of gp120 bound to CD4 of coreceptor-negative cells. PDI inhibitors also prevent HIV envelope-mediated cell-cell fusion but have no effect on the entry of HIV-1 pseudo-typed with murine leukemia virus envelope. Importantly, PDI coprecipitates with both soluble and cellular CD4. We propose that a PDI.CD4 association at the cell surface enables PDI to reach CD4-bound virus and to reduce disulfide bonds present in the domain of gp120 that binds to CD4. Conformational changes resulting from the opening of gp120-disulfide loops may drive the processes of virus-cell and cell-cell fusion. The biochemical events described identify new potential targets for anti-HIV agents.

To investigate the protective effects of lithium against HIV-gp120-mediated toxicity in vivo, mice were exposed to lithium and gp120 and levels of the neuronal markers, microtubule-associated protein-2 and NeuN, and the astrocyte marker, glial fibrillary acidic protein, were determined. In addition, SH-SY5Y neuronal cells exposed to gp120 and lithium were assessed for cell viability. Lithium pretreatment protected the hippocampus of mice from gp120-mediated toxicity. Similarly, preexposure of neuronal cultures to lithium significantly reduced gp120-associated neurotoxicity. However, posttreatment with lithium had minimal neuroprotective effects against gp120, both in vivo and in vitro. The protective effects of lithium in vitro were blocked by LY294002, an inhibitor of the phosphatidylinositol 3-kinase/Akt pathway. Taken together, these results demonstrate that lithium might be neuroprotective against gp120-mediated toxicity and suggest that prophylactic treatment with lithium may prevent the onset/progression of HIV-associated cognitive impairments.

As part of a research effort to design and prepare high affinity ligands for the galactosyl ceramide (GalCer) binding site on the HIV cell surface glycoprotein, gp120, several GalCer analogues have been prepared and characterized. The molecular design of analogues permits independent variations of the carbohydrate, the length of a hydrophilic spacer between the ligand and the lipid, and the composition of the hydrophobic lipid chains. Five different galactosyl analogues were synthesized having hydrophilic spacers of tri-, tetra-, and penta-ethylene glycol separating the carbohydrate from the lipid region which has either oleoyl or stearoyl lipid chains. The synthetic design allows for a convergent synthesis of the three components of the glycolipid conjugate. The structural characterization includes the proton and carbon chemical shifts, which were assigned after analysis of 1D and 2D NMR spectra.

Next to a profound T cell immunodeficiency, HIV-1 infection induces activation and dysfunction of B cells, resulting in hypergammaglobulinemia. Whereas T cell immune reconstitution with potent antiretroviral therapy has been extensively documented, limited data are available on B cell immune reconstitution. We studied the effect of potent antiretroviral therapy on antibody titers to the viral proteins gp120 and p24 and on total IgG concentrations. Three retrospectively chosen groups were studied: a successfully treated group, untreated controls, and subjects with virological failure after several months of successful therapy. In the successfully treated group, the median total IgG concentrations normalized, whereas they remained elevated in the untreated group and rebounded after an initial decline in the therapy failure group. The HIV-1-specific antibody titers declined in the successfully treated group and followed the rebound of the HIV RNA levels in the therapy failure group. With potent antiretroviral therapy the hypergammaglobulinemia normalized whereas HIV-1-specific immune responses were weakened. The weakening of antiviral immunity with therapy may be relevant for current attempts to gain immunological control over the virus through structured treatment interruptions or therapeutic vaccinations.

The virucidal protein cyanovirin-N (CV-N) mediates its highly potent anti-human immunodeficiency virus activity, at least in part, through interactions with the viral envelope glycoprotein gp120. Here we dissect in further detail the mechanism of CV-N's glycosylation-dependent binding to gp120. Isothermal titration calorimetry (ITC) binding studies of CV-N with endoglycosidase H-treated gp120 showed that binding was completely abrogated by removal of high-mannose oligosaccharides from the glycoprotein. Additional ITC and circular dichroism spectral studies with CV-N and other glycoproteins as well showed that CV-N discriminately bound only glycoproteins that contain high-mannose oligosaccharides. Binding experiments with RNase B indicated that the single high-mannose oligosaccharide on that enzyme mediated all of its binding with CV-N (K(d) = 0.602 microM). A finer level of oligosaccharide selectivity of CV-N was revealed in affinity chromatography-liquid chromatography-mass spectrometry experiments, which showed that CV-N preferentially bound only oligomannose-8 (Man-8) and oligomannose-9 isoforms of RNase B. Finally, we biophysically characterized the interaction of CV-N with a purified, single oligosaccharide, Man-8. The binding affinity of Man-8 for CV-N is unusually strong (K(d) = 0.488 microM), several hundredfold greater than observed for oligosaccharides and their protein lectins (K(d) = 1 microM--1 mM), further establishing a critical role of high-mannose oligosaccharides in CV-N binding to glycoproteins.

Cyanovirin-N (CV-N), an 11-kDa protein originally isolated from the cyanobacterium Nostoc ellipsosporum, potently inactivates diverse strains of human immunodeficiency virus type 1 (HIV-1), HIV-2, simian immunodeficiency virus, and feline immunodeficiency virus. It has been well established that the HIV surface envelope glycoprotein gp120 is a molecular target of CV-N. We recently reported that CV-N impaired the binding of virion-associated gp120 to cell-associated CD4 and that CV-N preferentially inhibited binding of the glycosylation-dependent neutralizing monoclonal antibody 2G12 to gp120. However, CV-N did not interfere with the interactions of soluble CD4 (sCD4) with either soluble gp120 (sgp120) or virion-associated gp120. In the present study, we have evaluated the effects of CV-N on the binding of sgp120 to cell-associated CD4 to clarify the experimental basis of the previous binding results, and we further address the detailed mechanism of action of CV-N. Here we present evidence that (i) CV-N impairs both CD4-dependent and CD4-independent binding of sgp120 to the target cells, (ii) CV-N blocks the sCD4-induced binding of sgp120 with cell-associated coreceptor CXCR4, and (iii) CV-N dissociates bound sgp120 from target cells. The results illustrate that the measured effects of CV-N on gp120-CD4 binding interactions depend upon the type of CD4 (soluble or cell associated), but not upon the type of gp120 (soluble or virion associated), employed in the experimental protocol. In addition, this study reinforces that CV-N acts uniquely to prevent essential interactions between the envelope glycoprotein and target cell receptors and further supports the potential broad utility of CV-N as a microbicide to prevent the transmission of HIV and AIDS.

Background: Cellulose acetate phthalate (CAP), a pharmaceutical excipient used for enteric film coating of capsules and tablets, was shown to inhibit infection by the human immunodeficiency virus type 1 (HIV-1) and several herpesviruses. CAP formulations inactivated HIV-1, herpesvirus types 1 (HSV-1) and 2 (HSV-2) and the major nonviral sexually transmitted disease (STD) pathogens and were effective in animal models for vaginal infection by HSV-2 and simian immunodeficiency virus., Methods: Enzyme-linked immunoassays and flow cytometry were used to demonstrate CAP binding to HIV-1 and to define the binding site on the virus envelope., Results: 1) CAP binds to HIV-1 virus particles and to the envelope glycoprotein gp120; 2) this leads to blockade of the gp120 V3 loop and other gp120 sites resulting in diminished reactivity with HIV-1 coreceptors CXCR4 and CCR5; 3) CAP binding to HIV-1 virions impairs their infectivity; 4) these findings apply to both HIV-1 IIIB, an X4 virus, and HIV-1 BaL, an R5 virus., Conclusions: These results provide support for consideration of CAP as a topical microbicide of choice for prevention of STDs, including HIV-1 infection.

The betulinic acid derivative IC9564 is a potent anti-human immunodeficiency virus (anti-HIV) compound that can inhibit both HIV primary isolates and laboratory-adapted strains. However, this compound did not affect the replication of simian immunodeficiency virus and respiratory syncytial virus. Results from a syncytium formation assay indicated that IC9564 blocked HIV type 1 (HIV-1) envelope-mediated membrane fusion. Analysis of a chimeric virus derived from exchanging envelope regions between IC9564-sensitive and IC9564-resistant viruses indicated that regions within gp120 and the N-terminal 25 amino acids (fusion domain) of gp41 are key determinants for the drug sensitivity. By developing a drug-resistant mutant from the NL4-3 virus, two mutations were found within the gp120 region and one was found within the gp41 region. The mutations are G237R and R252K in gp120 and R533A in the fusion domain of gp41. The mutations were reintroduced into the NL4-3 envelope and analyzed for their role in IC9564 resistance. Both of the gp120 mutations contributed to the drug sensitivity. On the contrary, the gp41 mutation (R533A) did not appear to affect the IC9564 sensitivity. These results suggest that HIV-1 gp120 plays a key role in the anti-HIV-1 activity of IC9564.

The neurotoxic mechanism of HIV-1 envelope glycoprotein 120 (gp120) involves glutamatergic (NMDA) receptor/Ca2+-dependent excitotoxicity, mediated in part via glia. Pro-inflammatory cytokines also may have roles. We have reported that pre-exposure of brain cultures to 'physiological' ethanol concentrations (20-30 mM) protects against neuronal damage from HIV-1 gp120, but not from the direct receptor agonist, NMDA. Using lactate dehydrogenase assays and propidium iodide staining of rat organotypic hippocampal-entorhinal cortical slice cultures we determined that ethanol's suppression of gp120 neurotoxicity required at least 4 days of pretreatment. The gp120-induced neurotoxicity was accompanied by interleukin-6 elevations that were not affected by the pretreatment. However, gp120 induced substantial, early increases in extracellular glutamate levels that were blocked by ethanol pretreatment, conceivably abrogating excitotoxicity. Consistent with abrogation of excitotoxic pathways, fura-2 imaging showed selective deficits in gp120-dependent intracellular Ca2+ responses in ethanol-pretreated slices. Gp120 is believed to increase glutamate levels by both stimulating release and inhibiting (re)uptake. Results with a labeled glutamate analog, D-[3H]aspartate, revealed that gp120's inhibition of glutamate uptake, rather than its stimulation of release, was abolished after ethanol. Further studies indicated that two converging effects of ethanol pretreatment may underlie the abolishment of gp120-mediated glutamate uptake inhibition: (a) blockade of gp120-induced release (ostensibly from glia) of arachidonic acid, an inhibitor of astroglial glutamate reuptake, and (b) modest proliferation and activation of astroglia upon gp120 stimulation--which are likely to augment glutamate transporters. Thus, as with gp120 itself, glia and glutamate/arachidonic acid regulation appear to be important targets for ethanol. Since moderate ethanol consumption is as common among HIV-infected individuals as in the general population, this newly recognized neuroprotective (and apparently anti-excitotoxic) effect of ethanol withdrawal in vitro could be important, but it requires further study before its significance, if any, is understood.

HIV-1 particles are decorated with a network of densely arranged envelope spikes on their surface. Each spike is formed of a trimer of heterodimers of the gp120 surface and the gp41 transmembrane glycoproteins. These molecules mediate HIV-1 entry into target cells, initiating the HIV-1 replication cycle. They are a target for entry-blocking drugs and for neutralizing Abs that could contribute to vaccine protection. The crystal structure of the core of gp120 has been recently solved. It reveals the structure of the conserved HIV-1 receptor binding sites and some of the mechanisms evolved by HIV-1 to escape Ab responses. The gp120 consists of three faces. One is largely inaccessible on the native trimer, and two faces are exposed but apparently have low immunogenicity, particularly on primary viruses. We have modeled HIV-1 neutralization by a CD4 binding site monoclonal Ab, and we propose that neutralization takes place by inhibition of the interaction between gp120 and the target cell membrane receptors as a result of steric hindrance. Knowledge of gp120 structure and function should assist in the design of new drugs as well as of an effective vaccine. In the latter case, circumventing the low immunogenicity of the HIV-1 envelope spike is a major challenge.

Background: Human immunodeficiency virus (HIV) infection continues to increase at alarming rates in drug abusers, especially in women. Drugs of abuse can cause long-lasting damage to the brain and HIV infection frequently leads to a dementing illness. To determine how these drugs interact with HIV to cause CNS damage, we used an in vitro human neuronal culture characterized for the presence of dopaminergic receptors, transporters and estrogen receptors. We determined the combined effects of dopaminergic drugs, methamphetamine, or cocaine with neurotoxic HIV proteins, gp120 and Tat., Results: Acute exposure to these substances resulted in synergistic neurotoxic responses as measured by changes in mitochondrial membrane potential and neuronal cell death. Neurotoxicity occurred in a sub-population of neurons. Importantly, the presence of 17beta-estradiol prevented these synergistic neurotoxicities and the neuroprotective effects were partly mediated by estrogen receptors., Conclusion: Our observations suggest that methamphetamine and cocaine may affect the course of HIV dementia, and additionally suggest that estrogens modify the HIV-drug interactions.

The antiviral activity of L-chicoric acid against HIV-1 has been attributed previously to the inhibition of HIV-1 integration. This conclusion was based on the inhibition of integrase activity in enzymatic assays and the isolation of a resistant HIV strain with a mutation (G140S) in the integrase gene. Here we show that the primary antiviral target of L-CA and its analogs in cell culture is viral entry. L- and D-chicoric acid (L-CA and D-CA) and their respective tetra-acetyl esters inhibit the replication of HIV-1 (III(B) and NL4.3) and HIV-2 (ROD) in MT-4 cells at a 50% effective concentration (EC(50)) ranging from 1.7 to 70.6 microM. In a time-of-addition experiment, L-CA, D-CA, L-CATA, and D-CATA were found to interfere with an early event in the viral replication cycle. Moreover, L-CA, D-CA, and their analogs did not inhibit the replication of virus strains that were resistant toward polyanionic and polycationic compounds at subtoxic concentrations. Furthermore, HIV-1 strains resistant to L-CA and D-CA were selected in the presence of L-CA and D-CA, respectively. Mutations were found in the V2, V3, and V4 loop region of the envelope glycoprotein gp120 of the L-CA and D-CA-resistant NL4.3 strains that were not present in the wild-type NL4.3 strain. Recombination of the gp120 gene of the L-CA and D-CA resistant strain in a NL4.3 wild-type molecular clone fully rescued the phenotypic resistance toward L-CA and D-CA. No significant mutations were detected in the integrase gene of the drug-resistant virus strains. Although inhibition of HIV integrase activity by L-CA and its derivatives was confirmed in an oligonucleotide-driven assay, integrase carrying the G140S mutation was inhibited to the same extent as the wild-type integrase.

Cyanovirin-N (CV-N) is a cyanobacterial protein with potent neutralizing activity against human immunodeficiency virus (HIV). CV-N has been shown to bind HIV type 1 (HIV-1) gp120 with high affinity; moreover, it blocks the envelope glycoprotein-mediated membrane fusion reaction associated with HIV-1 entry. However, the inhibitory mechanism(s) remains unclear. In this study, we show that CV-N blocked binding of gp120 to cell-associated CD4. Consistent with this, pretreatment of gp120 with CV-N inhibited soluble CD4 (sCD4)-dependent binding of gp120 to cell-associated CCR5. To investigate possible effects of CV-N at post-CD4 binding steps, we used an assay that measures sCD4 activation of the HIV-1 envelope glycoprotein for fusion with CCR5-expressing cells. CV-N displayed equivalently potent inhibitory effects when added before or after sCD4 activation, suggesting that CV-N also has blocking action at the level of gp120 interaction with coreceptor. This effect was shown not to be due to CV-N-induced coreceptor down-modulation after the CD4 binding step. The multiple activities against the HIV-1 envelope glycoprotein prompted us to examine other enveloped viruses. CV-N potently blocked infection by feline immunodeficiency virus, which utilizes the chemokine receptor CXCR4 as an entry receptor but is CD4 independent. CV-N also inhibited fusion and/or infection by human herpesvirus 6 and measles virus but not by vaccinia virus. Thus, CV-N has broad-spectrum antiviral activity, both for multiple steps in the HIV entry mechanism and for diverse enveloped viruses. This broad specificity has implications for potential clinical utility of CV-N.

The HIV-1 coat protein gp 20, a potent neurotoxin that may underlie AIDS dementia, activates glia to cause neurotoxicity via the NMDA receptor and perhaps other routes. We find that pretreating cultures of rat organotypic cortical/hippocampal slices or cerebellar granule cells subchronically with ethanol in physiological concentrations (20-30 mM; 6 days) largely or even completely inhibits neurodegeneration due to gp120. However, NMDA-induced neurotoxicity appears unaffected by moderate ethanol pretreatment, indicating that ethanol's neuroprotection against gp120 is upstream of the NMDA receptor, possibly at a glial activation stage. The results could lead to a better understanding of relationships between ethanol, glia and neurodegeneration, particularly in AIDS.

Some guanine-rich oligonucleotides inhibit HIV infectivity through interaction with the gp120 glycoprotein. Besides, photoinactivation of viruses attracts attention for blood decontamination. The feasibility of targeting a red light-absorbing chlorin-type photosensitizer to gp120 through covalent coupling with 8-mer phosphodiester oligodeoxynucleotides is investigated. Some conjugates inhibit binding of antibodies directed to gp120. Inhibition is significantly increased upon red light activation. The activity of the conjugates correlates with their ability to self-associate, a process strongly favored by the propensity of the hydrophobic chlorin moiety to dimerize. Thus, the photosensitizer moiety both promotes structures with a higher affinity for gp120 and, upon light activation, can induce site-directed damages to the protein.

Fusion of HIV with its host cell requires the interaction of the viral envelope glycoprotein 120 (gp120) with the chemokine receptor CXCR4 [T cell-tropic (T-tropic) or X4 HIV strains] or CCR5 [macrophage-tropic (M-tropic) or R5 HIV strains] followed by a 'spring-loaded' action of the glycoprotein 41 (gp41) that ensures fusion of the viral and cellular lipid membranes and permits the viral nucleocapsid to enter the cell. The overall fusion process can be blocked by a number of compounds. These include siamycin analogues, SPC 3 (a synthetic peptide derived from the V3 domain of gp120), pentafuside (T 20, DP 178) [a synthetic peptide corresponding to amino acid residues 127 to 162 of gp41], the betulinic acid derivative RPR 103611, TAK 779 (a low molecular weight non-peptide CCR5 antagonist) and a number of compounds (T 22, T 134, ALX40-4C, CGP64222 and AMD 3100) that are targeted at the CXCR4 receptor. In particular, the bicyclam AMD 3100 has proved highly potent and selective as a CXCR4 antagonist that blocks the infectivity of X4 HIV strains in the nanomolar concentration range. The proof-of-concept that fusion inhibitors should be able to suppress viral replication in vivo has been demonstrated with pentafuside. Pentafuside and AMD 3100 have now proceeded to phase II clinical trials.

The scrapie prion protein (PrP(Sc)) has been shown to induce apoptosis of rat cortical neurons in vitro. Here we demonstrate that the toxic effect displayed by PrP(Sc) can be blocked by sulfated colominic acid (polymer of N-acetylneuraminic acid). This compound acts neuroprotectively at a concentration of > or = 0.3 microg/ml when preincubated with the neurons or PrP(Sc). Rat cortical cells also undergo apoptosis after incubation with the HIV-1 coat protein gpl20 in vitro. This effect was abolished also by sulfated colominic acid when preincubated with the cells or gpl20. Addition of 0.3 microg/ml of compound resulted in an increase in cell viability by about 1.6-1.9-fold compared to cultures incubated for 18 h with 30 ng/ml of PrP(Sc) or 20 ng/ml of gpl20 alone (containing about 40% viable cells). Sulfated colominic acid does not act as antagonist of NMDA receptor channels at concentrations of up to 3 microg/ml when co-administered with 100 microg/ml of NMDA. It displayed a strong cytoprotective effect on human T lymphoblastoid CEM cells exposed to HIV-1; a 50% protection occurred after preincubation of the cells with 0.43 microg/ml of compound. At the same concentration, the compound caused an inhibition of HIV-1-induced syncytium formation. Sulfated colominic acid may be a promising compound for treatment of dementia caused by PrP(Sc) and HIV-1 infections.

Concentrations of the potent, HIV(human immunodeficiency virus) inactivating protein, cyanovirin-N (CV-N), which completely inhibit HIV-1 infectivity, do not block the binding of soluble CD4-receptor (sCD4) to HIV-1 lysates nor the attachment of intact HIV-1 virions to several target T-cell lines. Furthermore, in contrast to the known disassociative effects of sCD4 on viral envelope glycoproteins, treatment of HIVRF with high concentrations of CV-N results in complete viral inactivation but without apparent shedding of gp120 or other ultrastructural changes. These results are consistent with the view that the virucidal effects of CV-N result from interference with step(s) in the fusion process subsequent to the initial binding of the virus to target cells.

The blocking effect of curdlan sulfate (CRDS) on human immunodeficiency virus (HIV) infection has been thought to be related to inhibition of the binding of HIV-1 envelope glycoprotein (gp120) and CD4 molecules. However, recent reports have indicated that blocking the binding of gp120 to CD4 by CRDS only makes a small contribution to the inhibition of HIV-1 infection. We report here that the effect of CRDS on the production of beta-chemokines and cytokines might be important in the inhibition of HIV-1 infection, in addition to interference with the binding of gp120 to CD4+ cells.