Steps Of HIV-1 Infection Research Articles

Natural Variation in HIV-1 Entry Kinetics Map to Specific Residues and Reveal an Interdependence Between Attachment and Fusion.

The first step of HIV-1 infection is entry, where virus-cell membrane fusion is driven by the HIV-1 envelope glycoprotein through a series of conformational changes. Some of the most broadly active entry inhibitors work by binding conformations that exist only transiently during entry. The lifetimes of these states and the kinetics of entry are important elements of inhibitor activity for which little is known. We demonstrate a remarkable range of kinetics among 257 diverse HIV-1 isolates and find that this phenotype is highly flexible, with multiple single-residue determinants. Examination of the kinetics of two conformational landmarks shed light on novel kinetic features that offer new details about the role of co-receptor engagement and provide a framework to explain entry inhibitor synergy.

Virion-incorporated PSGL-1 and CD43 inhibit both cell-free infection and transinfection of HIV-1 by preventing virus–cell binding

HIV-1 particles incorporate various host transmembrane proteins in addition to viral Env glycoprotein during assembly at the plasma membrane. In polarized T cells, HIV-1 structural protein Gag localizes to the plasma membrane of uropod, a rear-end protrusion. Notably, uropod transmembrane proteins PSGL-1 and CD43 cocluster specifically with Gag assembling at the plasma membrane even in cells that do not form uropods. Recent reports have shown that expression of either PSGL-1 or CD43 in virus-producing cells reduces the infectivity of progeny virions and that HIV-1 infection reduces the cell surface expression of these proteins. However, the mechanisms for both processes remain to be determined. In this study, we found that virion incorporation of PSGL-1 and CD43 closely correlates with diminished virion infectivity. PSGL-1 and CD43 inhibited virus attachment to CD4+ cells irrespective of the presence of Env. These proteins also inhibited virion attachment to CD4- lymphoid organ fibroblastic reticular cells that mediate transinfection of CD4+ T cells. Consistent with the possibility that highly extended extracellular domains of these proteins physically block virus-cell attachment, the inhibitory effect of PSGL-1 required its full-length ectodomain. HIV-1 encoding Gag mutants that are defective in either coclustering with these host proteins or ESCRT-dependent particle release failed to reduce PSGL-1 on surface of infected cells. This study reveals an anti-HIV-1 mechanism that suppresses virus-cell attachment and a previously unappreciated process of HIV-1-mediated down-regulation of host antiviral proteins, both of which likely require virion incorporation of these proteins.

Identifying a nuclear passport for HIV.

Identification of a protein that pulls HIV into the nucleus explains a key step in HIV infection.

Dynamin-2 Stabilizes the HIV-1 Fusion Pore with a Low Oligomeric State

SummaryOne of the key research areas surrounding HIV-1 concerns the regulation of the fusion event that occurs between the virus particle and the host cell during entry. Even if it is universally accepted that the large GTPase dynamin-2 is important during HIV-1 entry, its exact role during the first steps of HIV-1 infection is not well characterized. Here, we have utilized a multidisciplinary approach to study the DNM2 role during fusion of HIV-1 in primary resting CD4 T and TZM-bl cells. We have combined advanced light microscopy and functional cell-based assays to experimentally assess the role of dynamin-2 during these processes. Overall, our data suggest that dynamin-2, as a tetramer, might help to establish hemi-fusion and stabilizes the pore during HIV-1 fusion.

Inhibition of HIV-1 entry by the tricyclic coumarin GUT-70 through the modification of membrane fluidity

Membrane fusion between host cells and HIV-1 is the initial step in HIV-1 infection, and plasma membrane fluidity strongly influences infectivity. In the present study, we demonstrated that GUT-70, a natural product derived from Calophyllum brasiliense, stabilized plasma membrane fluidity, inhibited HIV-1 entry, and down-regulated the expression of CD4, CCR5, and CXCR4. Since GUT-70 also had an inhibitory effect on viral replication through the inhibition of NF-κB, it is expected to be used as a dual functional and viral mutation resistant reagent. Thus, these unique properties of GUT-70 enable the development of novel therapeutic agents against HIV-1 infection.

Identification of capsid mutations that alter the rate of HIV-1 uncoating in infected cells.

After viral fusion with the cell membrane, the conical capsid of HIV-1 disassembles by a process called uncoating. We recently utilized the cyclosporine (CsA) washout assay, in which TRIM-CypA-mediated restriction of viral replication is used to detect the state of the viral capsid, to study the kinetics of uncoating in HIV-1-infected cells. Here we have extended this analysis to examine the effects of p24 capsid protein (p24(CA)) mutations and cellular environment on the kinetics of uncoating in infected cells. We found that p24(CA) mutations can significantly increase (A92E), delay (E45A and N74D), or have no effect (G94D) on the rate of uncoating and that these alterations are not due to changes in reverse transcription. Inhibition of reverse transcription delayed uncoating kinetics to an extent similar to that of the wild-type virus with all the p24(CA) mutant viruses tested. In addition, we observed differences in uncoating in two cell lines, which suggests that the cellular environment can differentially impact the disassembly of wild-type and mutant capsids. Collectively, these experiments suggest that viral and cellular factors are important for the process of uncoating. Finally, these data support the model whereby early steps in reverse transcription facilitate HIV-1 uncoating. The HIV-1 capsid is a cone-shaped structure, composed of the HIV-1-encoded protein p24(CA), which contains the viral RNA and other proteins needed for infection. After the virus enters a target cell, this capsid must disassemble by a process called uncoating. Uncoating is required for HIV-1 infection to progress, but the details of how this process occurs is not known. In this study, we used an in vivo assay to examine the uncoating process in HIV-1-infected cells. We determined that p24(CA) mutations could increase or decrease the rate of uncoating and that this rate varied in different cell lines. We also found that reverse transcription of the viral RNA altered the process of uncoating before the p24(CA) mutations. Collectively, these experiments provide a better understanding of how viral and cellular factors are involved with a poorly understood step in HIV-1 infection.

Production of Recombinant scFv against p24 of Human Immunodeficiency Virus Type 1 by Phage Display Technology

Phage display has a fundamental role in protein isolation and engineering. Isolated proteins produced with this method can be modified for specific binding and affinity. P24 is the most produced protein during human immune deficiency virus (HIV) replication; especially in the early steps of HIV-1 infection, its evaluation may have diagnostic values. To test the HIV-1 infection, p24 antigen assay appears to be a very promising alternative to RNA assays. In this study, we have generated a recombinant mouse single chain antibody fragment against p24 of the HIV-1 with the use of phage display technology. After isolation of antibody variable-region (V) gene of B cells extracted from the spleen of an immunized mouse, a library of single chain Fv fragments (scFv) was constructed. The library was used in a series of bio-panning processes against recombinant p24 protein expressed from Escherichia coli. The isolated scFv antibody specifically recognizes the HIV-1 capsid protein p24. The affinity constant of the isolated scFv antibody (MF85) was found to be 2×10(-9) M. Our studies showed that the MF85 scFV antibody has similar properties as that of monoclonal antibodies produced by the hybridoma technology.

Single Molecule Study of HIV-1 Reverse Transcriptase Polymerization Activity in the Presence of NC

HIV-1 reverse transcriptase (RT) is a multifunctional polymerase, which synthesizes double-stranded proviral DNA from single-stranded viral RNA by catalyzing RNA- and DNA-dependent DNA polymerization and degrading RNA via its RNase H activity. Reverse transcription is an essential step in HIV-1 infection, and HIV-1 RT is the target of many anti-AIDs therapeutic drugs. HIV-1 nucleocapsid (NC) protein is a nucleic acid chaperone, which facilitates DNA duplex melting and re-annealing, and shows rapid protein-nucleic acid interaction kinetics. The effect of NC on the reverse transcription process is not fully understood. To gain insights into the polymerase activity of RT in the presence of NC protein, we use single molecule force spectroscopy to examine DNA polymerization activity of HIV-RT along long single-stranded DNA (ssDNA) templates with and without NC. Our preliminary observations show the polymerization activity of RT is dependent on the force on ssDNA templates; an increase in the force on ssDNA templates reduces the polymerization activity of HIV-RT. The observed exponential dependence of polymerization activity of RT with force on long ssDNA templates is consistent with previous single molecule studies, and NC appears to enhance the polymerization rate. Our studies will test the polymerization activity of RT in the presence of NC, allowing us to determine the biophysical mechanism by which NC enhances this activity.

The interferon-induced MxB protein inhibits an early step of HIV-1 infection

Background Interferon protects cells from virus infection by inducing the expression of genes with antiviral activities. One such antiviral gene is Mx (myxovirus resistance) that was first identified in mice for its protection against influenza virus infection. Humans carry two Mx genes, MxA and MxB. Although MxA has been reported to inhibit a number of RNA and DNA viruses, the antiviral function of MxB has just begun to be revealed.

CCR5 expression reduces T-tropic HIV-1 strain infection by forming CD4/CXCR4/CCR5 oligomers

Event Abstract Back to Event CCR5 expression reduces T-tropic HIV-1 strain infection by forming CD4/CXCR4/CCR5 oligomers Mario Mellado1* and Laura Martínez-Muñoz1 1 Consejo Superior de Investigaciones Científicas, Immunology & Oncology / Centro Nacional de Biotecnología, Spain The HIV-1 envelope glycoprotein gp120 regulates the initial attachment of viral particles to target cells through its association with CD4 and the chemokine receptors CXCR4 or CCR5. Using several resonance energy transfer techniques, we detected that CD4, CXCR4 and CCR5 form homo- and heterodimers as well as CD4/CXCR4/CCR5 hetero-oligomers. We report that the oligomerization between CD4 and these coreceptors reduces T-tropic HIV 1 infection. FRET and BRET experiments showed that CCR5 coexpression in CD4/CXCR4 cells altered CXCR4 homodimer and CD4/CXCR4 heterodimer conformation. As a result, gp120IIIB-induced viral fusion to host cell membranes was reduced in CD4/CXCR4/CCR5 cells, as was infection by the X4 T-tropic HIV-1NL4.3 strain in human CCR5-expressing CD4+ T cells. When coexpressed in these oligomers, CCR5 blocked gp120IIIB binding to CD4/CXCR4 cells and consequently abrogated actin cytoskeleton rearrangement promoted by gp120IIIB, indicating a mechanism that prevents T tropic HIV infection. Receptor oligomerization is a dynamic process controlled by receptor expression levels that influence the first step in HIV-1 infection, and might be a target for new therapeutic approaches for AIDS intervention. Acknowledgements This work was supported in part by grants from the Spanish Ministry of Science and Innovation (SAF 2011-27370), the RETICS Program (RD08/0075/0010; RD12/0009/009; RIER), the Madrid regional government (S2010/BMD-2350; RAPHYME) and the European Union (FP7-integrated project Masterswitch 223404). Keywords: Oligomerization, HIV-1 Infection, CCR5, CXCR4, HIV-1 entry Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Host-pathogen interactions Citation: Mellado M and Martínez-Muñoz L (2013). CCR5 expression reduces T-tropic HIV-1 strain infection by forming CD4/CXCR4/CCR5 oligomers. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00615 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 12 Jun 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Mario Mellado, Consejo Superior de Investigaciones Científicas, Immunology & Oncology / Centro Nacional de Biotecnología, Madrid, Madrid, 28049, Spain, mmellado@cnb.csic.es Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Mario Mellado Laura Martínez-Muñoz Google Mario Mellado Laura Martínez-Muñoz Google Scholar Mario Mellado Laura Martínez-Muñoz PubMed Mario Mellado Laura Martínez-Muñoz Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

HIV-1 induces NALP3-inflammasome expression and interleukin-1β secretion in dendritic cells from healthy individuals but not from HIV-positive patients

NALP3-inflammasome is an innate mechanism, alternative to type-1 interferon, which is able to recognize nucleic acids and viruses in the cytoplasm and to induce pro-inflammatory response. Here, we hypothesized the involvement of inflammasome in the early defense against HIV-1 and in the full maturation of dendritic cells: for this, we evaluated the response of dendritic cells pulsed with HIV-1 in terms of inflammasome activation in healthy donors. Moreover, inflammasome response to HIV was evaluated in HIV-infected individuals. Monocyte-derived dendritic cells isolated from 20 healthy individuals (HC-DC) and 20 HIV-1-infected patients (HIV-DC) were pulsed with alditrithiol-2-inactivated HIV-1. We then analyzed inflammasome genes expression and interleukin-1β (IL-1β) secretion. In HC-DC, HIV-1 induced higher NLRP3/NALP3 mRNA expression compared with other inflammasome genes such as NALP1/NLRP1 or IPAF/NLRC4 (P < 0.001). This augmented expression was accompanied by CASP1-increased and IL1B-increased mRNA levels and by a significant increment of IL-1β secretion (P < 0.05). Otherwise, HIV-1 failed to activate inflammasome and cytokine production in HIV-DC. HIV-DC showed an increased NLRP3/NALP3 basal expression, suggesting a chronic inflammatory profile of patients' immune cells. HIV-1 was able to induce a NALP3-inflammasome response in healthy individuals, indicating that this inflammasome could play a role in the first steps of HIV-1 infection; the consequent inflammatory process may be important for directing host immune response against the virus and/or disease progression. HIV-DC seemed to be chronically activated, but unresponsive against pathogens. Our findings could be of interest considering the ongoing research about dendritic cell manipulation and therapeutic strategies for AIDS involving dendritic cell-based immune-vaccines.

Determinants of Lipid Mixing Membrane Fusion by HIV gp41

A critical step in HIV infection involves membrane fusion between the enveloped virion and the target cell plasma membrane, catalyzed by the viral membrane protein gp41 at or near physiologic pH. Specific global gp41 conformations (i.e. six-helix-bundle and coiled-coil) and specific regions (i.e. fusion peptide) are implicated in steps of fusion catalysis. Our goal is to elucidate the molecular mechanism of gp41 catalyzed membrane fusion. One challenge to biophysical analysis of structure and function in hydrophobic gp41 is solubility. Low pH (∼3.0) is utilized to enhance protein solubility by significantly increasing overall positive charge. Interestingly, the cationic six-helix-bundle region enhances/inhibits lipid mixing of anionic vesicles at low/neutral pH, which correlates with close association (∼5A) between membrane headgroup phosphates and a large/small proportion or population of helical bundles based on solid-state NMR analysis. The apolar trimeric fusion-peptide region contributes to lipid mixing and is predominantly β-sheet (∼75%) with a large fraction of β-strands located close to (∼5-7A)/far from (>∼10A) membrane headgroup phosphates at residues Ala-1 and Ala-14/Leu-7 and Met-19 at low pH. Swapping to neutral pH does not affect fusion-peptide conformation, but does lower the fraction of β-strands close to the membrane surface. Partial shedding of fusion-peptides from membranes, poor protein solubility, and protein lipid mixing inactivity at neutral pH are reversed upon pH swap back to low, indicating irreversible aggregation is not occurring at neutral pH. Our solid-state NMR findings indicate that abrogation of lipid-mixing fusion function by gp41 in six-helix-bundle conformation at the physiologic pH of viral fusion is determined primarily by stearic and electrostatic barriers to close membrane apposition imposed by the six-helix-bundle region, and minimally by membrane location or conformation of the fusion-peptide region.

Characterization of the barrier conferred by type I interferon on the early steps of HIV-1 infection in primary cells

Background Type I interferon (IFN) induces blocks to various steps of viral life cycles. It has been known for a long time that treatment of certain cell types with IFN confers a potent block to both the early and late steps of HIV-1 replication. However, the early block has remained poorly characterized so far. Material and methods We have investigated in detail the effect of type I IFN on HIV-1 infection in cell types relevant to natural infection, including primary CD4+ T cells, monocytederived macrophages, as well as several monocytic and T cell lines. Results We have found that IFN treatment of macrophages, THP-1 cells, and to a lesser extent CD4+ T cells, has a profound effect on the outcome of HIV-1 infection, whereas the effect is much less pronounced in U937 cells and minimal in various T cell lines. We have also shown that IFN exerts potent anti-viral effects against a broad range of retroviruses, including diverse HIV-1 strains (IIIB, BK132, NL4-3, YU-2, Ba-L), HIV-1 derived lentiviral vectors (LVs), SIVmac and FIV LVs, as well as B-MLV. Using a BlaM-Vpr entry assay, we have observed that viral entry is not affected by IFN treatment. More specifically, the block directly correlates with a strong decrease in the accumulation of viral cDNAs. As APOBEC3 proteins are IFN regulated, we have asked whether they have the capacity to edit reverse transcripts generated by incoming wild type HIV-1 viruses in macrophages. Using single genome sequencing, we have shown that viral cDNAs from IFN treated macrophages bear a low but statistically significant amount of G-to-A mutations that are acquired postentry. Based on the sites that are subjected to editing, we predict that APOBEC3 proteins beyond APOBEC3G play a role in this process. Conclusions Taken together, our results indicate that one or several effectors of the type I IFN response are able to block viral DNA accumulation of distant retroviruses in primary macrophages and CD4+T cells. In addition, we have shown that the classic hallmark of APOBEC3mediated editing is observed on viral cDNAs recovered from IFN treated macrophages. Given the low mutational frequency noted in these experiments, it seems unlikely that the editing shown here is an essential effector of the potent IFN induced antiviral effect. Instead it is more likely to provide an additional mechanism to augment other sources of HIV-1 sequence diversification.

Trimeric HIV-1 glycoprotein gp140 immunogens and native HIV-1 envelope glycoproteins display the same closed and open quaternary molecular architectures.

The initial step in HIV-1 infection occurs with the binding of cell surface CD4 to trimeric HIV-1 envelope glycoproteins (Env), a heterodimer of a transmembrane glycoprotein (gp41) and a surface glycoprotein (gp120). The design of soluble versions of trimeric Env that display structural and functional properties similar to those observed on intact viruses is highly desirable from the viewpoint of designing immunogens that could be effective as vaccines against HIV/AIDS. Using cryoelectron tomography combined with subvolume averaging, we have analyzed the structure of SOSIP gp140 trimers, which are cleaved, solubilized versions of the ectodomain of trimeric HIV-1 Env. We show that unliganded gp140 trimers adopt a quaternary arrangement similar to that displayed by native unliganded trimers on the surface of intact HIV-1 virions. When complexed with soluble CD4, Fab 17b, which binds to gp120 at its chemokine coreceptor binding site, or both soluble CD4 and 17b Fab, gp140 trimers display an open conformation in which there is an outward rotation and displacement of each gp120 protomer. We demonstrate that the molecular arrangements of gp120 trimers in the closed and open conformations of the soluble trimer are the same as those observed for the closed and open states, respectively, of trimeric gp120 on intact HIV-1 BaL virions, establishing that soluble gp140 trimers can be designed to mimic the quaternary structural transitions displayed by native trimeric Env.

Differential activity of candidate microbicides against early steps of HIV-1 infection upon complement virus opsonization

BackgroundHIV-1 in genital secretions may be opsonized by several molecules including complement components. Opsonized HIV-1 by complement enhances the infection of various mucosal target cells, such as dendritic cells (DC) and epithelial cells.ResultsWe herein evaluated the effect of HIV-1 complement opsonization on microbicide candidates' activity, by using three in vitro mucosal models: CCR5-tropic HIV-1JR-CSF transcytosis through epithelial cells, HIV-1JR-CSF attachment on immature monocyte-derived dendritic cells (iMDDC), and infectivity of iMDDC by CCR5-tropic HIV-1BaL and CXCR4-tropic HIV-1NDK. A panel of 10 microbicide candidates [T20, CADA, lectines HHA & GNA, PVAS, human lactoferrin, and monoclonal antibodies IgG1B12, 12G5, 2G12 and 2F5], were investigated using cell-free unopsonized or opsonized HIV-1 by complements. Only HHA and PVAS were able to inhibit HIV trancytosis. Upon opsonization, transcytosis was affected only by HHA, HIV-1 adsorption on iMDDC by four molecules (lactoferrin, IgG1B12, IgG2G5, IgG2G12), and replication in iMDDC of HIV-1BaL by five molecules (lactoferrin, CADA, T20, IgG1B12, IgG2F5) and of HIV-1NDK by two molecules (lactoferrin, IgG12G5).ConclusionThese observations demonstrate that HIV-1 opsonization by complements may modulate in vitro the efficiency of candidate microbicides to inhibit HIV-1 infection of mucosal target cells, as well as its crossing through mucosa.

Increased α-Defensins 1-3 Production by Dendritic Cells in HIV-Infected Individuals Is Associated with Slower Disease Progression

BackgroundDefensins are natural endogenous antimicrobial peptides with potent anti-HIV activity and immuno-modulatory effects. We recently demonstrated that immature dendritic cells (DC) produce α-defensins1-3 and that α-defensins1-3 modulate DC generation and maturation. Since DC-HIV interaction plays a critical role during the first steps of HIV infection, we investigated the possible impact of α-defensins1-3 production by DC on disease progression.Methodology/Principal FindingsMonocyte-derived DC (MDDC) were analyzed comparatively in healthy controls (HC) and HIV-infected patients, including untreated “elite” and “viremic” controllers, untreated viremic non-controllers and antiretroviral-treated patients. We found that production of α-defensins1-3 was significantly increased in MDDC from HIV-infected patients versus HC, and this increase was mainly due to that observed in controllers, while in non-controllers the increase was not statistically significant (controllers vs. HC, p<0.005; controllers vs. non-controllers p<0.05). Secreted α-defensins1-3 by immature MDDC positively correlated with CD4 T cell counts in controllers, but not in non-controllers. Moreover, independently of their clinical classification, HIV-infected patients with higher α-defensins1-3 secretion by immature MDDC showed slower disease progression, measured as no decrease in the number of CD4+ T-cells below 350 cell/mm3, lower increase of plasma viral load and no initiation of treatment over time. Plasma alpha-defensins1-3 levels lacked any relationship with immunologic and virologic parameters.Conclusions/SignificanceHigh production of α-defensins1-3 by immature DCs appears as a host protective factor against progression of HIV-1infection, suggesting potential diagnostic, therapeutic and preventive implications. This protective effect may arise from the activity of α-defensins1-3 to damage the virions prior and/or after their internalization by immature DC, and hence favoring a more efficient viral processing and presentation to HIV-specific CD4+ T cells, without or with a minor rate of transmission of infectious HIV-1 virions.

A strong correlation between fusogenicity and membrane insertion depth of the HIV fusion peptide

Fusion between the membrane of HIV and the membrane of a host cell is a crucial step in HIV infection and is catalyzed by the binding of the fusion peptide domain (HFP) of the HIV gp41 protein to the host cell membrane. The HFP by itself induces vesicle fusion and is a useful model system to understand the fusion peptide/host cell membrane interaction. This article reports an experimental correlation between the membrane locations of different HFP constructs and their fusogenicities. The constructs were the HFP monomer with Val-2 to Glu-2 mutation (HFPmn_mut), wild type HFP monomer (HFPmn), and wild type HFP trimer (HFPtr). All constructs have predominant beta sheet structure in membranes with physiologically relevant cholesterol content. HFPmn_mut does not fuse vesicles, HFPmn has moderate fusion rate, and HFPtr has the putative oligomerization state of HIV gp41 and a very rapid fusion rate. The HFP membrane locations were probed with solid-state NMR measurements of distances between labeled carbonyl ((13)CO) nuclei in the HFP backbone and lipid nuclei in the surface or interior regions of the membrane bilayer. HFPmn_mut is located at the membrane surface, HFPmn is inserted into a single membrane leaflet, and HFPtr is the most deeply inserted construct with contact with the center of the membrane. These results show a clear positive correlation between the insertion depths and the fusion activities of the HFP constructs. Other disease-causing enveloped viruses contain fusion peptides and this correlation may be a general structure-function model for these peptides.

Dichotomous effects of the cofilin kinase LIMK1 on the early steps of HIV-1 infection of CD4 T cells

We have previously demonstrated that binding of HIV-1 to the chemokine coreceptor, CXCR4, on resting CD4 T cells activates an actin-depolymerizing factor cofilin to promote the cortical actin dynamic critical for HIV latent infection [1]. The LIM domain kinase 1 (LIMK1) directly phosphorylates cofilin and regulates the actin cytoskeleton. Here, we investigated the role of LIMK1 in HIV-1 infection of resting CD4 T cells and found that HIV-1 infection triggered a rapid, transient activation of LIMK1. We also used siRNA knockdown to inhibit LIMK1 activity and found that knockdown of LIMK1 caused a decrease of F-actin and T cell chemotaxis. LIMK1 also had dichotomous effects on the chemokine coreceptor CXCR4 cycling (Figure ​(Figure1)1) and viral DNA synthesis (Figure ​(Figure2).2). Our findings are consistent with a model suggesting a multi-functional role of the cortical actin in mediating chemokine coreceptor density, HIV-1 DNA synthesis and intracellular migration. Figure 1 Endocytosis of CXCR4 analyzed by Western blot. Figure 2 Real-time PCR analysis HIV-1DNA synthesis in LIMK1 knockdown cells (007-LIMK knockdown cells, NTC-control cells).

Docking and 3D-QSAR studies of BMS-806 analogs as HIV-1 gp120 entry inhibitors

BMS-378806 (BMS-806) is a small molecule that blocks the binding of host-cell CD4 with viral gp120 protein and therefore inhibits the first steps of HIV-1 infection. Recently, 36 analogs compounds of BMS-806 were synthesized and their biological activity evaluated. Based on these compounds, a molecular docking was firstly performed with BMS-806 to the gp120 cavity in order to get a representative ligand conformation for the 3D-QSAR process. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were then conducted for these 36 compounds. CoMFA and CoMSIA models give reliable correlative and predictive abilities but the CoMFA model performance was slightly better than CoMSIA. CoMFA contours were analysed and have been correlated to the gp120 viral protein. The discussion indicates several key fragment positions on the ligands and their implications on the gp120 protein binding. The computational approach used in this paper provides reliable clues for further design of small molecules gp120/CD4 inhibitors based on the BMS-806.

Donor variability in HIV binding to peripheral blood mononuclear cells

BackgroundHIV infection of cells varies greatly between individuals, with multiple steps in the replication cycle potentially contributing to the variability. Although entry and post-entry variability of HIV infection levels in cells has been demonstrated, variability in HIV binding has not been examined. In this study, we examined variability of HIV binding to peripheral blood mononuclear cells (PBMC) from different donors.ResultsHIV binding to PBMC varied up to 3.9-fold between individuals and was independent of CD4. Replication of HIV in donor PBMC required CD4 and paralleled virus binding trends of donor PBMC. To assess the stability of virus binding phenotypes over time, HIV was bound to donors with low- and high-binding phenotypes. The binding phenotypes were maintained when tested weekly over a 4-week period for 3 of 4 donors, while one high-binding donor decreased to lower binding on the 4th week. The low- and high-binding phenotypes were also preserved across different HIV strains. Experiments performed to determine if there was an association between HIV binding levels and specific cell subset levels within PBMC showed no correlation, suggesting that HIV binds to multiple cell subsets.ConclusionThese results show that differences exist in HIV binding to donor PBMC. Our data also show that HIV binding to donor PBMC is CD4-independent and can change over time, suggesting that virus binding variability is due to differences in the expression of changeable cell-surface host factors. Taken together, this study highlights the impact of cell-surface factors in HIV binding to, and infection of, PBMC which likely represents an important step in HIV infection in vivo.