Stages Of HIV Life Cycle Research Articles

Drug Re-positioning Studies for Novel HIV-1 Inhibitors Using Binary QSAR Models and Multi-target-driven In Silico Studies.

Current antiretroviral therapies against HIV involve the usage of at least two drugs that target different stages of HIV life cycle. However, potential drug interactions and side effects pose a problem. A promising concept for complex disease treatment is 'one molecule-multiple target' approach to overcome undesired effects of multiple drugs. Additionally, it is beneficial to consider drug re-purposing due to the cost of taking a drug into the market. Taking these into account, here potential anti-HIV compounds are suggested by virtually screening small approved drug molecules and clinical candidates. Initially, binary QSAR models are used to predict the therapeutic activity of around 7900 compounds against HIV and to predict the toxicity of molecules with high therapeutic activities. Selected compounds are considered for molecular docking studies against two targets, HIV-1 protease enzyme, and chemokine co-receptor CCR5. The top docking poses for all 549 molecules are then subjected to short (1 ns) individual molecular dynamics (MD) simulations and they are ranked based on their calculated relative binding free energies. Finally, 25 molecules are selected for long (200 ns) MD simulations, and 5 molecules are suggested as promising multi-target HIV agents. The results of this study may open new avenues for the designing of new dual HIV-1 inhibitor scaffolds.

Short Communication: Elevated Labile Iron Levels in CD4 and CD8 T Cells from HIV-Positive Individuals with Undetectable Viral Load.

Iron is a key factor at various stages of HIV life cycle and determines the progression of HIV infection. Data about cellular labile iron pool (LIP) in the settings of contemporary antiretroviral therapy (cART) are lacking. Yet LIP is directly related to the generation of reactive oxygen species, and may contribute to immune activation, dysfunction, and exhaustion. Using multiparameter flow cytometry, we evaluated LIP in CD4 and CD8 T cells from HIV+ patients with sustained viral suppression (SVS) as a result of continuous long-term cART. Based on the recovery of CD4/CD8 ratio, two patients' subgroups were defined: A (n = 26), CD4/CD8 > 0.9, and B (n = 37), CD4/CD8 < 0.9, with significantly differing CD4 absolute count (AC) (mean 752 vs. 571 cells/μL, p < .05). Although hemoglobin and serum iron had recovered in all patients, CD4 T cell LIP and CD8 T cell LIP were significantly higher than that of controls, both in the subgroup with complete (A) and with incomplete (B) immune recovery [mean CD4 mean fluorescence intensity (ΔMFI) 318.7 and 777.8 vs. 157.6; mean CD8 ΔMFI 359.5 and 628.7 vs. 179.2, analysis of variance p < .05 for both]. CD4 LIP correlated inversely with CD4 AC (R = -0.4, p < .01), and both CD4 LIP and CD8 LIP-with CD4/CD8 ratio (R = -0.4, p < .01). Thus, increased CD4 T cell LIP and CD8 T cell LIP in the settings of SVS and immune recovery are a sensitive marker of residual immune activation and may predict immune exhaustion in long-term cART-treated patients.

Metal cofactor modulated folding and target recognition of HIV-1 NCp7.

The HIV-1 nucleocapsid 7 (NCp7) plays crucial roles in multiple stages of HIV-1 life cycle, and its biological functions rely on the binding of zinc ions. Understanding the molecular mechanism of how the zinc ions modulate the conformational dynamics and functions of the NCp7 is essential for the drug development and HIV-1 treatment. In this work, using a structure-based coarse-grained model, we studied the effects of zinc cofactors on the folding and target RNA(SL3) recognition of the NCp7 by molecular dynamics simulations. After reproducing some key properties of the zinc binding and folding of the NCp7 observed in previous experiments, our simulations revealed several interesting features in the metal ion modulated folding and target recognition. Firstly, we showed that the zinc binding makes the folding transition states of the two zinc fingers less structured, which is in line with the Hammond effect observed typically in mutation, temperature or denaturant induced perturbations to protein structure and stability. Secondly, We showed that there exists mutual interplay between the zinc ion binding and NCp7-target recognition. Binding of zinc ions enhances the affinity between the NCp7 and the target RNA, whereas the formation of the NCp7-RNA complex reshapes the intrinsic energy landscape of the NCp7 and increases the stability and zinc affinity of the two zinc fingers. Thirdly, by characterizing the effects of salt concentrations on the target RNA recognition, we showed that the NCp7 achieves optimal balance between the affinity and binding kinetics near the physiologically relevant salt concentrations. In addition, the effects of zinc binding on the inter-domain conformational flexibility and folding cooperativity of the NCp7 were also discussed.

P15.01 Innate mucosal serpin inhibits late stages of hiv life cycle and reduces cellular proliferation

Background Antiproteases, specifically serpins, are up-regulated within cervicovaginal fluid (CVF), of highly-exposed sero-negative women (HESN), within the Pumwani cohort in Nairobi, Kenya. These proteins are presumed to contribute to the overall susceptibility of a woman to HIV-1. We hypothesise that CVF from HESN women will exhibit stronger HIV neutralisation activity, compared to HIV susceptible women. We believe that overabundant serpins within CVF are capable of neutralising HIV infection and will do so through both direct and indirect anti-HIV mechanisms and are potential novel microbicide candidates. Methodology HIV-1 neutralisation assays were performed with individual CVL samples from HESN and HIV-susceptible women. Real time-PCR was utilised to determine the level of viral DNA as well as viral mRNA following treatment with the serpin of interest. ACH2 cells, determined the effect of the antiprotease on late stages of the viral life cycle and confocal microscopy revealed levels of cellular entry and specific localization of the exogenously added serpin. Lastly, flow cytometry was employed to determine the effect of the protein on cellular proliferation. Results No significant difference was observed in the neutralisation capacity of CVF between HESN and HIV-susceptible women. However, various serpins within the CVF, when isolated and added exogenously to cell cultures, did exhibit significant HIV-neutralising effects. Following numerous mechanistic studies on one such serpin, it was apparent that it not only interferes with proper cellular proliferation but also directly in late stages of the virus lifecycle, (post-transcriptionally) likely during translation or assembly/budding of the virion. Conclusion Cervical vaginal fluid contains a myriad of immunomodulatory proteins that may contribute to a woman’s overall susceptibility to HIV infection. The serpin studied within this project demonstrates both broad (cellular proliferation) and narrow (HIV specific) mechanisms of action, making it a potentially effective microbicide candidate. Disclosure of interest statement Nothing to declare.

Journey describing the discoveries of anti-HIV triterpene acid families targeting HIV-entry/fusion, protease functioning and maturation stages.

HIV infection/AIDS, is a fatal disease multiplying rapidly in virtually every country. Extensive creations are in progress to arrest the replication of the HIV, following the destruction of either particular step involved in the progression of HIV infection. In such endeavors, mechanistically more diverse antiviral therapies were showcased using naturally occurring triterpene acid and their derivatives acting at various stages of HIV life cycle like entry or fusion, function of HIV protease enzyme and finally at maturation. The present article holds an extensive step-by-step summary of anti-HIV breakthroughs of triterpene acid analogues and their derivatives with synthetic and activity aspects, featuring fertile clues for novel anti-HIV drug design, which helps to develop unprecedented opportunities to discover the next-generation anti- HIV armamentarium.

Universal modular system for in vitro screening of potential inhibitors of HIV-1 replication

Here we describe a system based on recombinant lentiviral vectors for the safe screening of potential anti-HIV drugs. The system allows to evaluate the sensitivity of HIVl-1 reverse transcriptase and integrase (wild-type as well as mutant forms of these enzymes detected in drug-resistant virus isolates) towards different drugs and substances, but also to screen inhibitors of other stages of HIV-1 life cycle.

New Antiretroviral Therapies and Potential Drug Interactions in HIVInfected Drug Abusers

Substance abuse remains a major obstacle in treating HIV-infected patients. The high prevalence of drug use is responsible for lack of adherence and severe drug interactions to antiretroviral therapy in HIV positive individuals. Over the past decade, newer therapies for HIV have been developed by targeting different stages of HIV life cycle. Of importance, targeting initial binding with receptor and genome integration by CCR5 antagonists and integrase inhibitors, respectively have been effective in reducing viral loads in clinical trials. However, most of these recently approved or investigational antiretroviral drugs are also metabolized by cytochrome P450 (CYP) enzymes. Thus, substance abuse mediated changes in level of CYP enzymes (induction or inhibition) may result in severe drug interactions, failure of therapy, rapid progression to AIDS, and increased mortality in HIV patients who are treated with regimens containing CCR5 antagonists and integrase inhibitors. In this review, we have discussed the pharmacokinetic data, efficacy, lack of adherence to these therapies in drug abusers, and CYP-mediated potential drug interactions for CCR5 antagonists and integrase inhibitors in HIV-infected drug abusers.

Screening strategies to identify host factors associated with the early and late stages of HIV-1 life cycle

Dear Editor, The HIV-1 infection in humans has now become a pandemic and in the absence of treatment it often leads to acquired immunodeficiency syndrome (AIDS). Although significant progress has been made to reduce the HIV-1 viral load in the blood of infected patients, challenges still exist in...

Neisseria gonorrhoeae Enhances HIV-1 Infection of Primary Resting CD4+ T Cells through TLR2 Activation

Sexually transmitted infections increase the likelihood of HIV-1 transmission. We investigated the effect of Neisseria gonorrheae (gonococcus [GC]) exposure on HIV replication in primary resting CD4(+) T cells, a major HIV target cell during the early stage of sexual transmission of HIV. GC and TLR2 agonists, such as peptidylglycan (PGN), Pam(3)CSK(4), and Pam(3)C-Lip, a GC-derived synthetic lipopeptide, but not TLR4 agonists including LPS or GC lipooligosaccharide enhanced HIV-1 infection of primary resting CD4(+) T cells after viral entry. Pretreatment of CD4(+) cells with PGN also promoted HIV infection. Anti-TLR2 Abs abolished the HIV enhancing effect of GC and Pam(3)C-Lip, indicating that GC-mediated enhancement of HIV infection of resting CD4(+) T cells was through TLR2. IL-2 was required for TLR2-mediated HIV enhancement. PGN and GC induced cell surface expression of T cell activation markers and HIV coreceptors, CCR5 and CXCR4. The maximal postentry HIV enhancing effect was achieved when PGN was added immediately after viral exposure. Kinetic studies and analysis of HIV DNA products indicated that GC exposure and TLR2 activation enhanced HIV infection at the step of nuclear import. We conclude that GC enhanced HIV infection of primary resting CD4(+) T cells through TLR2 activation, which both increased the susceptibility of primary CD4(+) T cells to HIV infection as well as enhanced HIV-infected CD4(+) T cells at the early stage of HIV life cycle after entry. This study provides a molecular mechanism by which nonulcerative sexually transmitted infections mediate enhancement of HIV infection and has implication for HIV prevention and therapeutics.

HIV-1 Gene Therapy at Pre-Integration and Provirus DNA Levels

AIDS is the result of infection by a lentivirus, HIV-1, which primarily infects CD4+ T cells and macrophages. There is presently no vaccine and none will be available in the foreseeable future. Highly active antiretroviral drug therapy has led to a dramatic reduction of viral load in many infected individuals, and has decreased mortality in the developing world. However, besides long-term drug toxicity and eventual emergence of drug-resistant strains, withdrawal from the therapy (even after effective and continuous treatment) results in re-emergence of the virus since cells harbouring the latent viral reservoirs persist. These issues highlight the need for alternative therapies, e.g. gene therapy. This review summarizes various gene therapy strategies that target early stages of HIV-1 life cycle. We will cover strategies that allow interference at the level of the released virion RNA, reverse transcriptase, pre-integration complex, integrase, dsDNA and provirus DNA in gene-modified cells.

Flazinamide, a novel β-carboline compound with anti-HIV actions

A β-carboline compound, flazin isolated from Suillus granulatus has been shown weak anti-HIV-1 activity. Based on the structure of flazin, flazinamide [1-(5′- hydromethyl-2′-furyl)-β-carboline-3-carboxamide] was synthesized and its anti-HIV activities were evaluated in the present study. The cytotoxicity of flazinamide was about 4.1-fold lower than that of flazin. Flazinamide potently reduced syncytium formation induced by HIV-1IIIB with EC50 value of 0.38 μM, the EC50 of flazinamide was about 6.2-fold lower than that of flazin. Flazinamide also inhibited HIV-2ROD and HIV-2CBL-20 infection with EC50 values of 0.57 and 0.89 μM, respectively. Flazinamide reduced p24 antigen expression in HIV-1IIIB acute infected C8166 and in clinical isolated strain HIV-1KM018 infected PBMC, with EC50 values of 1.45 and 0.77 μM, respectively. Flazinamide did not suppress HIV-1 replication in chronically infected H9 cells. Flazinamide blocked the fusion between normal cells and HIV-1 or HIV-2 chronically infected cells. It weakly inhibited activities of recombinant HIV-1 reverse transcriptase, protease or integrase at higher concentrations. In conclusion, the conversion of the carboxyl group in 3 position of flazin markedly enhanced the anti-viral activity (TI value increased from 12.1 to 312.2) and flazinamide might interfere in the early stage of HIV life cycle.

The Host Cell MAP Kinase ERK-2 Regulates Viral Assembly and Release by Phosphorylating the p6gag Protein of HIV-1

The host cell MAP kinase ERK-2 incorporated within human immunodeficiency virus type 1 particles plays a critical role in virus infectivity by phosphorylating viral proteins. Recently, a fraction of the virus incorporated late (L) domain-containing p6(gag) protein, which has an essential function in the release of viral particles from the cell surface, was reported to be phosphorylated by an unknown virus-associated cellular protein kinase (Muller, B., Patschinsky, T., and Krausslich, H. G. (2002) J. Virol. 76, 1015-1024). The present study demonstrates the contribution of the MAP kinase ERK-2 in p6(gag) phosphorylation. According to mutational analysis, a single ERK-2-phosphorylated threonine residue, belonging to a highly conserved phosphorylation MAP kinase consensus site, was identified at position 23 within p6(gag). Substitution by an alanine of the Thr(23) phosphorylable residue within the pNL4.3 molecular clone was found to decrease viral release from various cell types. As observed from electron microscopy experiments, most virions produced from this molecular clone remained incompletely separated from the host cell membrane with an immature morphology and displayed a reduced infectivity in single round infection experiments. Analysis of protein processing by Western blotting experiments revealed an incomplete Pr55(gag) maturation and a reduction in the virion-associated reverse transcriptase proteins was observed that was not related to differences in intracellular viral protein expression. Altogether, these data suggest that phosphorylation of p6(gag) protein by virus-associated ERK-2 is involved in the budding stage of HIV-1 life cycle.