Although HIV, the causative agent of AIDS, establishes a lifelong infection that cannot be eradicated even with effective treatment, the host immune system has the ability to contain its replication for many years in which the disease remains asymptomatic. The unexpected encounter, in 1995, between the fields of HIV and chemokines has dramatically advanced our understanding of AIDS pathogenesis, opening new perspectives for the development of effective prophylactic and therapeutic measures against HIV. All known HIV-suppressive chemokines act by recognizing and blocking specific viral coreceptors expressed on the surface of susceptible cells. Thus, RANTES, MIP-1alpha;and MIP-1beta, selectively inhibit HIV isolates that use CCR5 as a coreceptor, while SDF-1 selectively inhibits those using CXCR4. Owing to their inherent antiviral properties, analogues or functional mimics of these chemokines are currently under development as anti-HIV therapeutics or microbicides. 1) Identification of CXCL4 as a novel HIV-suppressive chemokine. Chemokines are produced by various cells that are activated in the course of immunologic and inflammatory responses, including platelets, which are specialized anucleated cells that store in their alpha-granules large amounts of these proteins. We examined the effects of CXCL4, the most abundant protein contained within the platelet alpha-granules, on HIV infection. We found that treatment with either recombinant or native human CXCL4 causes a dose-dependent inhibition of HIV infection in different experimental systems including primary CD4+ T cells and macrophages, as well as neoplastic CD4+ T-cell lines and the MAGI assay. Of note, at variance with other HIV-suppressive chemokines, CXCL4 is active on a broad spectrum of HIV isolates, with similar potency against viruses that use CCR5 and those that use CXCR4 as coreceptors. Analysis of a panel of primary HIV isolates documented different sensitivities to CXCL4, with some isolates displaying a lack of sensitivity to CXCL4 at the concentrations used in our studies (half-maximal inhibitory concentration IC50 range: <25 to >600 nM). However, the sensitivity to CXCL4-mediated inhibition was not correlated with the coreceptor usage phenotype, the genetic subtype or the in vitro adaptation history of such isolates. These results identify CXCL4 as a novel HIV-suppressive chemokine with an atypically broad spectrum of antiviral activity, opening new perspectives for therapy and prevention. 2) Identification of XCL1 as a novel HIV-suppressive chemokine. XCL1/lymphotactin is a member of the C-chemokine family that is produced primarily by activated CD8+ T cells and behaves as a metamorphic protein, interconverting between two structurally distinct conformations (classic and alternative). We found that XCL1 inhibits a broad spectrum of HIV-1 isolates, irrespective of their coreceptor-usage phenotype. 3) Studies on the mechanism of antiviral action of CXCL4 and XCL1. Our initial approach toward elucidating the mechanism of anti-HIV action of CXCL4 and XCL1 was to identify which step in the viral replication cycle is inhibited by these chemokines. Kinetic experiments using the MAGI assay demonstrated that CXCL4 blocks an early step in the viral replication cycle. Thus, we used an HIV-1 entry assay to demonstrate that CXCL4 is a potent inhibitor not only of viral entry but also of virion attachment to the surface of susceptible cells. Of note, inhibition of entry of different HIV-1 isolates correlated with their sensitivity to CXCL4-mediated inhibition in infection assays. Similar results were obtained with XCL1, which potently inhibited both attachment and entry of different HIV-1 isolates. Using a virion-capture assay, we found that both CXCL4 and XCL1 immobilized on the surface of immunomagnetic beads efficiently and specifically bind to intact HIV-1 virions irrespective of their coreceptor-usage phenotype. Further tests demonstrated that CXCL4 and XCL1 are both able to co-immunoprecipitate the gp120 envelope glycoprotein, thus documenting a direct interaction between CXCL4 and native gp120. By virion-capture competition using a panel of gp120-specific mAbs, we were able to map the CXCL4-binding site on gp120 to a region that is in close proximity to, but not overlapping with the CD4-binding domain. In contrast, extensive tests with multiple anti-gp120 antibodies failed to provide information about the putative binding site of XCL1. Altogether, these results demonstrated that CXCL4 inhibits the HIV attachment and entry steps through a novel mechanism that involves direct binding to the viral envelope rather than to the cellular coreceptors. 4) Conformation-specificity of the antiviral activity of XCL1. Experiments with stabilized variants of XCL1 produced in Dr. Volkman's laboratory in Wisconsin demonstrated that HIV-1 inhibition requires access to the alternative, all-βconformation of XCL1, which interacts with proteoglycans but does not bind/activate the specific XCR1 receptor, while the classic conformation is inactive. 5) Correlation between CXCL4 levels and clinical/immunological parameters of disease progression in HIV-infected subjects. CXCL4 is primarily produced by megakaryocytes and platelets, and is promptly released by platelets upon activation. Although its primary function is to promote blood coagulation, CXCL4 has multiple, seemingly unrelated, activities including blockade of angiogenesis, activation of immune cells and, as we discovered, inhibition of HIV. Of note, a variety of platelet abnormalities have been described in patients with HIV infection, whose severity correlates with the progression of the immunodeficiency. As a first step toward elucidating the clinical relevance of CXCL4 as an endogenous HIV-suppressive factor, we measured the serum levels of CXCL4 in a cohort of HIV-infected subjects (n = 279) selected to represent different clinical stages of HIV infection. Linear regression analysis showed that serum CXCL4 concentrations were positively correlated with peripheral blood CD4+ T-cell counts (p = 0.0008), CD8+ T-cell counts (p <0.0001) and platelet counts (p <0.0001), and negatively correlated with levels of HIV plasma viremia (p = 0.0156) and C-reactive protein (p = 0.0001). In multivariate regression analysis, the ranks of CXCL4 were found to be independently associated with the CD4+ T-cell ranks (p = 0.0016). These data are compatible with a potential in vivo protective effect of CXCL4. However, it has to be emphasized that the serum levels of CXCL4 do not represent actual circulating levels of the chemokine, but rather the chemokine reservoir harbored by circulating platelets, indicating that subjects with less advanced HIV disease are endowed with a larger storage pool of CXCL4. Although we cannot exclude that the true plasmatic levels of CXCL4 may also be elevated in the early stages of HIV-1 infection, the current technology does not permit to obtain reliable measurements without the interference of contaminating platelets. Additional studies will be important to elucidate the role of CXCL4 in the natural history of HIV disease.
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