Arangassery Rosemary Bastian

Cell-free HIV-1 virucidal action by modified peptide triazole inhibitors of Env gp120.

Initial entry of HIV-1 into host cells remains a compelling and yet elusive target for developing agents to prevent infection. This step is mediated by a sequence of interactions of a trimeric gp120/gp41 envelope (Env) protein complex with host cells, including initial gp120 encounter with the cellular receptor CD4 and a chemokine co-receptor usually either CCR5 or CXCR4 [1]. A peptide triazole class of entry inhibitor leads has been shown to bind to gp120 with close to nanomolar affinity, to suppress protein ligand interactions of the Env protein at both its CD4 and co-receptor binding sites and to inhibit cell infection by a broad range of virus subtypes [2]. These inhibitors appear to function mechanistically by conformationally entrapping gp120 in an inactivated state, different from either the flexible ground state of gp120 or the highly structured CD4-activated state. This entrapment effectively halts the entry process at the initial binding stages.

Interactions of peptide triazole thiols with Env gp120 induce irreversible breakdown and inactivation of HIV-1 virions

BackgroundWe examined the underlying mechanism of action of the peptide triazole thiol, KR13 that has been shown previously to specifically bind gp120, block cell receptor site interactions and potently inhibit HIV-1 infectivity.ResultsKR13, the sulfhydryl blocked KR13b and its parent non-sulfhydryl peptide triazole, HNG156, induced gp120 shedding but only KR13 induced p24 capsid protein release. The resulting virion post virolysis had an altered morphology, contained no gp120, but retained gp41 that bound to neutralizing gp41 antibodies. Remarkably, HIV-1 p24 release by KR13 was inhibited by enfuvirtide, which blocks formation of the gp41 6-helix bundle during membrane fusion, while no inhibition of p24 release occurred for enfuvirtide-resistant virus. KR13 thus appears to induce structural changes in gp41 normally associated with membrane fusion and cell entry. The HIV-1 p24 release induced by KR13 was observed in several clades of HIV-1 as well as in fully infectious HIV-1 virions.ConclusionsThe antiviral activity of KR13 and its ability to inactivate virions prior to target cell engagement suggest that peptide triazole thiols could be highly effective in inhibiting HIV transmission across mucosal barriers and provide a novel probe to understand biochemical signals within envelope that are involved in membrane fusion.

Mechanism of Multivalent Nanoparticle Encounter with HIV-1 for Potency Enhancement of Peptide Triazole Virus Inactivation

Background: HIV-1 envelope spike protein remains a compelling but elusive target for preventing infection. Results: Gold nanoparticle conjugates of peptide triazole Env inhibitors demonstrated impressive picomolar antiviral potencies. Conclusion: Nanoparticle conjugates enhanced antiviral functions by multivalent attachment to virus Env spikes. Significance: Findings reveal that multispike engagement can exploit the metastability of the virus envelope to irreversibly inactivate HIV-1. Entry of HIV-1 into host cells remains a compelling yet elusive target for developing agents to prevent infection. A peptide triazole (PT) class of entry inhibitor has previously been shown to bind to HIV-1 gp120, suppress interactions of the Env protein at host cell receptor binding sites, inhibit cell infection, and cause envelope spike protein breakdown, including gp120 shedding and, for some variants, virus membrane lysis. We found that gold nanoparticle-conjugated forms of peptide triazoles (AuNP-PT) exhibit substantially more potent antiviral effects against HIV-1 than corresponding peptide triazoles alone. Here, we sought to reveal the mechanism of potency enhancement underlying nanoparticle conjugate function. We found that altering the physical properties of the nanoparticle conjugate, by increasing the AuNP diameter and/or the density of PT conjugated on the AuNP surface, enhanced potency of infection inhibition to impressive picomolar levels. Further, compared with unconjugated PT, AuNP-PT was less susceptible to reduction of antiviral potency when the density of PT-competent Env spikes on the virus was reduced by incorporating a peptide-resistant mutant gp120. We conclude that potency enhancement of virolytic activity and corresponding irreversible HIV-1 inactivation of PTs upon AuNP conjugation derives from multivalent contact between the nanoconjugates and metastable Env spikes on the HIV-1 virus. The findings reveal that multispike engagement can exploit the metastability built into virus the envelope to irreversibly inactivate HIV-1 and provide a conceptual platform to design nanoparticle-based antiviral agents for HIV-1 specifically and putatively for metastable enveloped viruses generally.

Chimeric Cyanovirin-MPER Recombinantly Engineered Proteins Cause Cell-Free Virolysis of HIV-1

ABSTRACT Human immunodeficiency virus (HIV) is the primary etiologic agent responsible for the AIDS pandemic. In this work, we used a chimeric recombinant protein strategy to test the possibility of irreversibly destroying the HIV-1 virion using an agent that simultaneously binds the Env protein and viral membrane. We constructed a fusion of the lectin cyanovirin-N (CVN) and the gp41 membrane-proximal external region (MPER) peptide with a variable-length (Gly4Ser)x linker (where x is 4 or 8) between the C terminus of the former and N terminus of the latter. The His-tagged recombinant proteins, expressed in BL21(DE3)pLysS cells and purified by immobilized metal affinity chromatography followed by gel filtration, were found to display a nanomolar efficacy in blocking BaL-pseudotyped HIV-1 infection of HOS.T4.R5 cells. This antiviral activity was HIV-1 specific, since it did not inhibit cell infection by vesicular stomatitis virus (VSV) or amphotropic-murine leukemia virus. Importantly, the chimeric proteins were found to release intraviral p24 protein from both BaL-pseudotyped HIV-1 and fully infectious BaL HIV-1 in a dose-dependent manner in the absence of host cells. The addition of either MPER or CVN was found to outcompete this virolytic effect, indicating that both components of the chimera are required for virolysis. The finding that engaging the Env protein spike and membrane using a chimeric ligand can destabilize the virus and lead to inactivation opens up a means to investigate virus particle metastability and to evaluate this approach for inactivation at the earliest stages of exposure to virus and before host cell encounter.

Covalent conjugation of a peptide triazole to HIV-1 gp120 enables intramolecular binding site occupancy.

The HIV-1 gp120 glycoprotein is the main viral surface protein responsible for initiation of the entry process and, as such, can be targeted for the development of entry inhibitors. We previously identified a class of broadly active peptide triazole (PT) dual antagonists that inhibit gp120 interactions at both its target receptor and coreceptor binding sites, induce shedding of gp120 from virus particles prior to host–cell encounter, and consequently can prevent viral entry and infection. However, our understanding of the conformational alterations in gp120 by which PT elicits its dual receptor antagonism and virus inactivation functions is limited. Here, we used a recently developed computational model of the PT–gp120 complex as a blueprint to design a covalently conjugated PT–gp120 recombinant protein. Initially, a single-cysteine gp120 mutant, E275CYU-2, was expressed and characterized. This variant retains excellent binding affinity for peptide triazoles, for sCD4 and other CD4 binding site (CD4bs) ligands, and for a CD4-induced (CD4i) ligand that binds the coreceptor recognition site. In parallel, we synthesized a PEGylated and biotinylated peptide triazole variant that retained gp120 binding activity. An N-terminally maleimido variant of this PEGylated PT, denoted AE21, was conjugated to E275C gp120 to produce the AE21–E275C covalent conjugate. Surface plasmon resonance interaction analysis revealed that the PT–gp120 conjugate exhibited suppressed binding of sCD4 and 17b to gp120, signatures of a PT-bound state of envelope protein. Similar to the noncovalent PT–gp120 complex, the covalent conjugate was able to bind the conformationally dependent mAb 2G12. The results argue that the PT–gp120 conjugate is structurally organized, with an intramolecular interaction between the PT and gp120 domains, and that this structured state embodies a conformationally entrapped gp120 with an altered bridging sheet but intact 2G12 epitope. The similarities of the PT–gp120 conjugate to the noncovalent PT–gp120 complex support the orientation of binding of PT to gp120 predicted in the molecular dynamics simulation model of the PT–gp120 noncovalent complex. The conformationally stabilized covalent conjugate can be used to expand the structural definition of the PT-induced “off” state of gp120, for example, by high-resolution structural analysis. Such structures could provide a guide for improving the subsequent structure-based design of inhibitors with the peptide triazole mode of action.

Targeting cell surface HIV-1 Env protein to suppress infectious virus formation

HIV-1 Env protein is essential for host cell entry, and targeting Env remains an important antiretroviral strategy. We previously found that a peptide triazole thiol KR13 and its gold nanoparticle conjugate AuNP-KR13 directly and irreversibly inactivate the virus by targeting the Env protein, leading to virus gp120 shedding, membrane disruption and p24 capsid protein release. Here, we examined the consequences of targeting cell-surface Env with the virus inactivators. We found that both agents led to formation of non-infectious virus from transiently transfected HEK293T cells. The budded non-infectious viruses lacked Env gp120 but contained gp41. Importantly, budded virions also retained the capsid protein p24, in stark contrast to p24 leakage from viruses directly treated by these agents and arguing that the agents led to deformed viruses by transforming the cells at a stage before virus budding. We found that the Env inactivators caused gp120 shedding from the transiently transfected HEK293T cells as well as non-producer CHO-K1-gp160 cells. Additionally, AuNP-KR13 was cytotoxic against the virus-producing HEK293T and CHO-K1-gp160 cells, but not untransfected HEK293T or unmodified CHO-K1 cells. The results obtained reinforce the argument that cell-surface HIV-1 Env is metastable, as on virus particles, and provides a conformationally vulnerable target for virus suppression and infectious cell inactivation.

Disulfide Sensitivity in the Env Protein Underlies Lytic Inactivation of HIV-1 by Peptide Triazole Thiols.

We investigated the mode of action underlying lytic inactivation of HIV-1 virions by peptide triazole thiol (PTT), in particular the relationship between gp120 disulfides and the C-terminal cysteine-SH required for virolysis. Obligate PTT dimer obtained by PTT SH cross-linking and PTTs with serially truncated linkers between pharmacophore isoleucine-ferrocenyltriazole-proline-tryptophan and cysteine-SH were synthesized. PTT variants showed loss of lytic activity but not binding and infection inhibition upon SH blockade. A disproportionate loss of lysis activity vs binding and infection inhibition was observed upon linker truncation. Molecular docking of PTT onto gp120 argued that, with sufficient linker length, the peptide SH could approach and disrupt several alternative gp120 disulfides. Inhibition of lysis by gp120 mAb 2G12, which binds at the base of the V3 loop, as well as disulfide mutational effects, argued that PTT-induced disruption of the gp120 disulfide cluster at the base of the V3 loop is an important step in lytic inactivation of HIV-1. Further, PTT-induced lysis was enhanced after treating virus with reducing agents dithiothreitol and tris (2-carboxyethyl)phosphine. Overall, the results are consistent with the view that the binding of PTT positions the peptide SH group to interfere with conserved disulfides clustered proximal to the CD4 binding site in gp120, leading to disulfide exchange in gp120 and possibly gp41, rearrangement of the Env spike, and ultimately disruption of the viral membrane. The dependence of lysis activity on thiol-disulfide interaction may be related to intrinsic disulfide exchange susceptibility in gp120 that has been reported previously to play a role in HIV-1 cell infection.

Mode of action of the sulfhydryl group in virolytic peptide triazole thiol inhibitors of HIV-1 Env

2 HIV patients were included in this study, who were either on maintenance hemodialysis (n=14) or were renal transplant recipients (n=10). Out of the 24, most were males (85.5%). Their spouses (n=20) were also included in this study. All subjects were interrogated via a questionnaire. All 24 patients were HIV positive. Out of the 20 spouses, 8 were found to be HIV positive (40%). Probable mode of spread of infection was enquired from the patients, 20 patients out of 24 had multiple sexual contacts (83.3%), while blood transfusion and multiple needle pricks due to drug abuse were found in one each (8.4%). 2 patients were not sure of any cause (8.4%). Out of the 20 spouses, 18 were sexually active, out of which 8 were found to be HIV positive and all were of the opinion that they had received the infection due to sexual contact with their spouses who were HIV positive CKD patients. Out of the 18 sexually active couples 12 were using condoms, while 6 spouses who were not using condoms, were found to be HIV positive. Out of the 8 HIV positive spouses, 2 had received the infection in spite of using condoms, thus implying that barrier contraception, though extremely effective, is not a full proof method of protection from the spread of HIV infection. Furthermore 15 patients were chronic alcoholics (62.5%). Most of them thought that social workers are the best way of preventing the disease (n=40, 90.5%), followed by increasing awareness through newspapers and television (n=36, 81.8%). Out of 44 subjects 30 (68.2%) thought that teaching in the school on the subject would be helpful in preventing it, while 28 (63.6%) were of the opinion that the family physicians could prevent the spread of the infection by counseling the families of HIV infected patients. Thus this study concludes that multiple sexual contacts, chronic alcohol abuse, intravenous drug abuse and spouses of patients are at high risk of contracting HIV infection and that social workers may be extremely beneficial in increasing awareness of HIV and restricting its spread.Background: In the setting of progressive immunosuppression, as with a HIV infection, the incidence of cervical dysplasia is increased. Paradoxically, among certain ethnicities and economic levels, HIV-infected women have a lower rate of screening for cervical cancer via Papanicolaou smear (Pap smear) compared to HIV-negative women. Understanding barriers to cervical cancer screening among HIV-infected women is important to develop an intervention to increase adherence to guidelines.H entry is mediated by the interaction of the trimeric envelope glycoprotein (Env) on the virus membrane surface with host cell receptors. However, Env is the only virus-specific protein on the virion surface and is essential for cell receptor interactions and subsequent virus-cell fusion. Therefore, HIV-1 Env is an important target to directly inhibit and thus block the initial steps leading to host cell infection. Our lab has synthesized peptide triazoles, a class of novel entry inhibitors. These peptides contain a substituted triazole derivative formed from a synthetically introduced azido-proline amino acid and bind to gp120 with close to nanomolar affinity. Site-directed mutagenesis and molecular dynamics simulation have shown that peptide triazole binding overlaps the CD4 binding pocket. Peptide triazoles cause cell-independent gp120 shedding, and variants containing C-terminal cysteines cause cell-free virolysis as evidenced by internal p24 capsid release. We are investigating the mode of action by which the sulfhydryl group causes irreversible inactivation. We hypothesize that the thiol interferes with conserved disulfides clustered proximal to the CD4 binding site in gp120 through “disulfide exchange”, which could deform the Env protein spike, and subsequently the viral membrane, leading to p24 release. The process of disulfide exchange has been found to be necessary for HIV viral infection.T study offers a conceptual model which certainly contributes to the literature of discrimination toward people living with HIV (PLHIV) and can provide the theoretical basis for future experimental research as much as intervention actions in order to achieve the aim of Zero discrimination. The health care practitioners’ (HCP) attitudinal factors toward PLHIV as much as the institutional factors should be undertaken in HIV discrimination researches. While the most of researches have relatively done in the field of training and wide-awakening of HCP about HIV transmission in Iran and Asian countries, the process of forming the discriminatory intention among the HCP have not been studied yet. In order to explain the factors affecting the HCPs’ discriminatory intention toward PLHIV, not only the author developed and hypothesized a model using the theory of planned behavior’s (TPB) framework but also proposed a new method of attitude measurement aims to shed a new light on predicting HIV discrimination in the health settings. The hypothesis is including: 1. The belief in the just world (BJW) among the HCPs’ affects their perception of stigma as much as their prejudice and also both of them affect the discriminatory attitude toward PLHIV 2. The perception of stigma affects the HCPs’ socio/cultural risk perception 3. The socio/cultural risk perception affects the HCPs’ discriminatory attitude 4. The transmission risk perception affects the HCPs’ discriminatory attitude 5. The discriminatory attitude affects the HCPs’ discriminatory intention toward PLHIV 6. Fuzzy method compared with the existing conventional measurement methods can measure more precisely the HCPs’ discriminatory attitudeT study determined whether socio-demographic characteristics and social support are associated with quality of life in individuals diagnosed with HIV/AIDS in Ghana. This study was guided by concepts from the intersection domains of social capital, social network, and social support theories. A convenience sample of 300 HIV/AIDS support group members were obtained via cross-sectional design survey. Data were collected from 300 participants selected because they attend support groups meetings, are a convenient sample, and also have experience in participating in research studies. The Medical Outcome Studies (MOS) HIV Health Survey and the MOS Social Support Survey (MOS-SSS) and demographic questionnaire instruments were used to assess quality of life, social support, and demographic information respectively. Multiple regression analysis was performed to determine if socio-demographic factors and social support contribute to quality of life. There was a positive association between overall social support and overall quality of life (r=0.51). The combination of socio-demographic factors and social support related to quality of life. Implications of the findings for practice, research, and policy in Ghana were discussed.T precise molecular mechanisms on how HIV-1 co-infection accelerates HCV-mediated liver disease progression are currently unknown. Our data showed that infectious HIV-1 virus particles failed to enter into human hepatocytic cell lines, and discernible virus replication was not observed, even when the hepatocytes transfected with HIV-1 proviral DNA were co-cultured with Jurkat T cells, suggesting that liver deterioration in the co-infected patients is not due to the replication of HIV-1 in the hepatocytes. Instead, HIV-1 Nef protein can be transferred from Nef-expressing or HIV-1-infected cells to hepatocytes through conduits, not exosomes, and the transferred Nef in the target hepatocytes altered lipid droplet formation, up-regulated subgenomic replicon expression of HCV, augmented reactive oxygen species (ROS) production, and enhanced ethanol-mediated up-regulation of HCV replication. Taken together, these data indicate that HIV-1 Nef plays an integral role in expedition of liver pathogenesis in the HIV-1/HCV co-infected hosts.S the beginning of the 21st century, we are facing the convergence of several epidemics. These include tobacco smoking, tuberculosis (TB) and HIV infection. These epidemics interact by way of increasing disease susceptibility and worsening outcomes. To control these interacting epidemics, it is crucial to better understand each infection and how it influences the others. The association between tobacco smoke and TB was suggested many years ago. Evidence of the impact of tobacco smoking on TB infection has been confounded by its almost universal association with poverty, overcrowding and alcohol usage. Similar pathological mechanisms induced by malnutrition, alcohol abuse and smoking may indeed all predispose an individual to TB. Although both tobacco smoking and HIV infection may be associated through their common associations with poverty and high-risk behavior, tobacco smoking appears to be an independent and important risk factor for contracting HIV. Smoking further raises the extremely high risk of contracting TB in HIV+ individuals. Individuals with HIV/AIDS are at risk for many oral health problems, particularly those who are smokers. People living with HIV/AIDS (PLWHA) are more likely to experience chronic illnesses such as cardiovascular disease and diabetes that are linked to poor oral health status. This presentation will describe the impact of HIV, TB and tobacco use on the oral health of PLWHA; it will discuss the importance of multidisciplinary health teams in the oral care for PLWHA; and it will suggest concrete steps that clinicians can take to promote positive behavioral health changes in PLWHA.