Tzanko S. Stantchev

Human immunodeficiency virus type-1 and chemokines: beyond competition for common cellular receptors.

The chemokines and their receptors have been receiving exceptional attention in recent years following the discoveries that some chemokines could specifically block human immunodeficiency virus type 1 (HIV-1) infection and that certain chemokine receptors were the long-sought coreceptors which, along with CD4, are required for the productive entry of HIV-1 and HIV-2 isolates. Several chemokine receptors or orphan chemokine receptor-like molecules can support the entry of various viral strains, but the clinical significance of the CXCR4 and CCR5 coreceptors appear to overshadow a critical role for any of the other coreceptors and all HIV-1 and HIV-2 strains best employ one or both of these coreceptors. Binding of the HIV-1 envelope glycoprotein gp120 subunit to CD4 and/or an appropriate chemokine receptor triggers conformational changes in the envelope glycoprotein oligomer that allow it to facilitate the fusion of the viral and host cell membranes. During these interactions, gp120 appears to be capable of inducing a variety of signaling events, all of which are still not defined in detail. In addition, the more recently observed dichotomous effects, of both inhibition and enhancement, that chemokines and their receptor signaling events elicit on the HIV-1 entry and replication processes has once again highlighted the intricate and complex balance of factors that govern the pathogenic process. Here, we will review and discuss these new observations summarizing the potential significance these processes may have in HIV-1 infection. Understanding the complexities and significance of the signaling processes that the chemokines and viral products induce may substantially enhance our understanding of HIV-1 pathogenesis, and perhaps facilitate the discovery of new ways for the prevention and treatment of HIV-1 disease.

Identification of Hendra Virus G Glycoprotein Residues That Are Critical for Receptor Binding

ABSTRACT Hendra virus (HeV) is an emerging paramyxovirus capable of infecting and causing disease in a variety of mammalian species, including humans. The virus infects its host cells through the coordinated functions of its fusion (F) and attachment (G) glycoproteins, the latter of which is responsible for binding the virus receptors ephrinB2 and ephrinB3. In order to identify the receptor binding site, a panel of G glycoprotein constructs containing mutations was generated using an alanine-scanning mutagenesis strategy. Based on a predicted G structure, charged amino acids residing in regions that could be homologous to those in the measles virus H attachment glycoprotein known to be involved in its protein receptor interaction were targeted. Using a coprecipitation-based assay, seven single-amino-acid substitutions in HeV G were identified as having significantly impaired binding to both the ephrinB2 and ephrinB3 viral receptors: D257A, D260A, G439A, K443A, G449A, K465A, and D468A. The impairment of receptor interaction conferred a concomitant diminution in their abilities to promote membrane fusion when coexpressed with F. The G glycoprotein mutants were also recognized by three or more conformation-dependent monoclonal antibodies of a panel of five, were expressed on the cell surface, and retained their abilities to bind and coprecipitate F. Interestingly, some of these mutant G glycoproteins coprecipitated with F more efficiently than wild-type G. Taken together, these data provide strong biochemical and functional evidence that some of these residues could be part of a conformation-dependent, discontinuous, and overlapping ephrinB2 and -B3 binding domain within the HeV G glycoprotein.

Coreceptor Competition for Association with CD4 May Change the Susceptibility of Human Cells to Infection with T-Tropic and Macrophagetropic Isolates of Human Immunodeficiency Virus Type 1

ABSTRACT The chemokine receptors CCR5 and CXCR4 were found to function in vivo as the principal coreceptors for M-tropic and T-tropic human immunodeficiency virus (HIV) strains, respectively. Since many primary cells express multiple chemokine receptors, it was important to determine if the efficiency of virus-cell fusion is influenced not only by the presence of the appropriate coreceptor (CXCR4 or CCR5) but also by the levels of other coreceptors expressed by the same target cells. We found that in cells with low to medium surface CD4 density, coexpression of CCR5 and CXCR4 resulted in a significant reduction in the fusion with CXCR4 domain (X4) envelope-expressing cells and in their susceptibility to infection with X4 viruses. The inhibition could be reversed either by increasing the density of surface CD4 or by antibodies against the N terminus and second extracellular domains of CCR5. In addition, treatment of macrophages with a combination of anti-CCR5 antibodies or β-chemokines increased their fusion with X4 envelope-expressing cells. Conversely, overexpression of CXCR4 compared with CCR5 inhibited CCR5-dependent HIV-dependent fusion in 3T3.CD4.401 cells. Thus, coreceptor competition for association with CD4 may occur in vivo and is likely to have important implications for the course of HIV type 1 infection, as well as for the outcome of coreceptor-targeted therapies.

Cell adhesion-dependent membrane trafficking of a binding partner for the ebolavirus glycoprotein is a determinant of viral entry.

Ebolavirus is a hemorrhagic fever virus associated with high mortality. Although much has been learned about the viral lifecycle and pathogenesis, many questions remain about virus entry. We recently showed that binding of the receptor binding region (RBR) of the ebolavirus glycoprotein (GP) and infection by GP pseudovirions increase on cell adhesion independently of mRNA or protein synthesis. One model to explain these observations is that, on cell adhesion, an RBR binding partner translocates from an intracellular vesicle to the cell surface. Here, we provide evidence for this model by showing that suspension 293F cells contain an RBR binding site within a membrane-bound compartment associated with the trans-Golgi network and microtubule-organizing center. Consistently, trafficking of the RBR binding partner to the cell surface depends on microtubules, and the RBR binding partner is internalized when adherent cells are placed in suspension. Based on these observations, we reexamined the claim that lymphocytes, which are critical for ebolavirus pathogenesis, are refractory to infection because they lack an RBR binding partner. We found that both cultured and primary human lymphocytes (in suspension) contain an intracellular pool of an RBR binding partner. Moreover, we identified two adherent primate lymphocytic cell lines that bind RBR at their surface and strikingly, support GP-mediated entry and infection. In summary, our results reveal a mode of determining viral entry by a membrane-trafficking event that translocates an RBR binding partner to the cell surface, and they suggest that this process may be operative in cells important for ebolavirus pathogenesis (e.g., lymphocytes and macrophages).

Cell Adhesion Promotes Ebola Virus Envelope Glycoprotein-Mediated Binding and Infection

ABSTRACT Ebola virus infects a wide variety of adherent cell types, while nonadherent cells are found to be refractory. To explore this correlation, we compared the ability of pairs of related adherent and nonadherent cells to bind a recombinant Ebola virus receptor binding domain (EboV RBD) and to be infected with Ebola virus glycoprotein (GP)-pseudotyped particles. Both human 293F and THP-1 cells can be propagated as adherent or nonadherent cultures, and in both cases adherent cells were found to be significantly more susceptible to both EboV RBD binding and GP-pseudotyped virus infection than their nonadherent counterparts. Furthermore, with 293F cells the acquisition of EboV RBD binding paralleled cell spreading and did not require new mRNA or protein synthesis.

Consistent and Significant Inhibition of Human Immunodeficiency Virus Type 1 Envelope-Mediated Membrane Fusion by μ-Chemokines (RANTES) in Primary Human Macrophages

Infection and entry of CD4(+) cells by human immunodeficiency virus type 1 (HIV-1) requires a coreceptor molecule, which, in concert with CD4, interacts with the viral envelope glycoprotein (Env), leading to membrane fusion. The principal coreceptors are the CCR5 and CXCR4 chemokine receptors. The suppressive effect of beta-chemokines, principally RANTES, on certain HIV-1 isolates was established before the discovery of the CCR5 receptor, and there have since been multiple reports confirming this initial observation. However, the inhibitory effect of beta-chemokines on HIV-1 infection of macrophages has been controversial. The current study focused on this issue in detail, with a reductionist approach, using assays that measure the effect of beta-chemokines solely on Env-mediated fusion. It is shown that under a variety of culture and differentiation conditions, RANTES maintains a significant and consistent inhibitory effect on CCR5-dependent Env-mediated fusion, and the role of these findings is discussed in relation to the role of beta-chemokines in HIV pathogenesis.

Expression of Human CD4 and chemokine receptors in cotton rat cells confers permissiveness for productive HIV infection

BackgroundCurrent small animal models for studying HIV-1 infection are very limited, and this continues to be a major obstacle for studying HIV-1 infection and pathogenesis, as well as for the urgent development and evaluation of effective anti-HIV-1 therapies and vaccines. Previously, it was shown that HIV-1 can infect cotton rats as indicated by development of antibodies against all major proteins of the virus, the detection of viral cDNA in spleen and brain of challenged animals, the transmission of infectious virus, albeit with low efficiency, from animal to animal by blood, and an additional increase in the mortality in the infected groups.ResultsUsing in vitro experiments, we now show that cotton rat cell lines engineered to express human receptor complexes for HIV-1 (hCD4 along with hCXCR4 or hCCR5) support virus entry, viral cDNA integration, and the production of infectious virus.ConclusionThese results further suggest that the development of transgenic cotton rats expressing human HIV-1 receptors may prove to be useful small animal model for HIV infection.

Human TOP1 residues implicated in species specificity of HIV-1 infection are required for interaction with BTBD2, and RNAi of BTBD2 in old world monkey and human cells increases permissiveness to HIV-1 infection

BackgroundHost determinants of HIV-1 viral tropism include factors from producer cells that affect the efficiency of productive infection and factors in target cells that block infection after viral entry. TRIM5α restricts HIV-1 infection at an early post-entry step through a mechanism associated with rapid disassembly of the retroviral capsid. Topoisomerase I (TOP1) appears to play a role in HIV-1 viral tropism by incorporating into or otherwise modulating virions affecting the efficiency of a post-entry step, as the expression of human TOP1 in African Green Monkey (AGM) virion-producing cells increased the infectivity of progeny virions by five-fold. This infectivity enhancement required human TOP1 residues 236 and 237 as their replacement with the AGM counterpart residues abolished the infectivity enhancement. Our previous studies showed that TOP1 interacts with BTBD1 and BTBD2, two proteins which co-localize with the TRIM5α splice variant TRIM5δ in cytoplasmic bodies. Because BTBD1 and BTBD2 interact with one HIV-1 viral tropism factor, TOP1, and co-localize with a splice variant of another, we investigated the potential involvement of BTBD1 and BTBD2 in HIV-1 restriction.ResultsWe show that the interaction of BTBD1 and BTBD2 with TOP1 requires hu-TOP1 residues 236 and 237, the same residues required to enhance the infectivity of progeny virions when hu-TOP1 is expressed in AGM producer cells. Additionally, interference with the expression of BTBD2 in AGM and human 293T target cells increased their permissiveness to HIV-1 infection two- to three-fold.ConclusionsThese results do not exclude the possibility that BTBD2 may modestly restrict HIV-1 infection via colocation with TRIM5 variants in cytoplasmic bodies.

Electron-Beam-Lithographed Nanostructures as Reference Materials for Label-Free Scattered-Light Biosensing of Single Filoviruses

Optical biosensors based on scattered-light measurements are being developed for rapid and label-free detection of single virions captured from body fluids. Highly controlled, stable, and non-biohazardous reference materials producing virus-like signals are valuable tools to calibrate, evaluate, and refine the performance of these new optical biosensing methods. To date, spherical polymer nanoparticles have been the only non-biological reference materials employed with scattered-light biosensing techniques. However, pathogens like filoviruses, including the Ebola virus, are far from spherical and their shape strongly affects scattered-light signals. Using electron beam lithography, we fabricated nanostructures resembling individual filamentous virions attached to a biosensing substrate (silicon wafer overlaid with silicon oxide film) and characterized their dimensions with scanning electron and atomic force microscopes. To assess the relevance of these nanostructures, we compared their signals across the visible spectrum to signals recorded from Ebola virus-like particles which exhibit characteristic filamentous morphology. We demonstrate the highly stable nature of our nanostructures and use them to obtain new insights into the relationship between virion dimensions and scattered-light signal.

Chemokine Receptors and HIV/AIDS

There have been tremendous advances made toward our understanding of chemokine receptor biology over the past decade. Much of the research conducted in this area was fueled by discoveries that certain chemokine receptor ligands (chemokines) could specifically block human immunodeficiency virus type 1 (HIV-1) infection and that certain chemokine receptors were the long-sought coreceptors that, together with CD4, were required for the productive entry of HIV-1, HIV-2, and simian immuno-deficiency virus (SIV) isolates. In the current survey, along with the interactions of the HIV-1 envelope glycoprotein with chemokine receptors, we have focused on the relationship certain chemokine receptors have with particular cellular systems such as lipid rafts, chemokine receptor-mediated signaling, and the actin cytoskeleton; all of which appear to play roles in or influence the establishment of HIV-1 infection. We will also discuss the dichotomous effects that chemokines display at both entry and postentry levels of HIV-1 infection and their potential significance for HIV-1 pathogenesis and acquired immunodeficiency syndrome (AIDS).