X4 HIV-1 Research Articles

Restricted Entry of R5 HIV Type 1 Strains into Eosinophilic Cells

A cell culture system previously developed by our laboratory demonstrated that T cell-tropic (CXCR4-using) but not macrophage-tropic (CCR5-using) HIV-1 strains productively infected eosinophilic cells. In the current study, an improved model was used to determine the level of this viral restriction by assessing viral entry and coreceptor usage. The model was improved by using AML14.3D10 cells that were engineered to express CCR3 in addition to the major HIV-1 coreceptors, CD4, CXCR4, and CCR5, thus making them more like primary eosinophils. A polymerase chain reaction (PCR) assay was used to detect viral entry. In the PCR assay, primers specific for early reverse transcription products were used to amplify minus strand viral DNA from HIV-1-infected AML14.3D10-CCR3 eosinophilic cells. Coreceptor blocking experiments, using the CXCR4 antagonist AMD3100, were performed to determine coreceptor usage by the CXCR4-using (X4) strain known to productively infect the cells. Virus production was measured by p24 immunoassay. As expected, viral DNA was detected in AML14.3D10-CCR3 cells infected with X4 HIV-1, and cell viability was decreased during maximal viral production. Conversely, viral DNA was not detected in eosinophilic cells exposed to a CCR5-using (R5) HIV-1 strain that is also capable of using CCR3, indicating that R5 HIV-1 is unable to enter eosinophilic cells despite the presence of the appropriate coreceptors. Infection of AML14.3D10-CCR3 cells by HTLV-IIIB was completely inhibited by AMD3100, indicating that X4 HIV-1 enters the AML14.3D10- CCR3 cell line by using the CXCR4 coreceptor exclusively. Since X4 strains predominate during the late stages of HIV-1 infection in many patients, when eosinophil numbers also tend to increase, the ability of these HIV-1 strains to infect eosinophilic cells has important implications for the involvement of eosinophils in the pathogenesis of AIDS.

Restricted Entry of R5 HIV Type 1 Strains into Eosinophilic Cells

A cell culture system previously developed by our laboratory demonstrated that T cell-tropic (CXCR4-using) but not macrophage-tropic (CCR5-using) HIV-1 strains productively infected eosinophilic cells. In the current study, an improved model was used to determine the level of this viral restriction by assessing viral entry and coreceptor usage. The model was improved by using AML14.3D10 cells that were engineered to express CCR3 in addition to the major HIV-1 coreceptors, CD4, CXCR4, and CCR5, thus making them more like primary eosinophils. A polymerase chain reaction (PCR) assay was used to detect viral entry. In the PCR assay, primers specific for early reverse transcription products were used to amplify minus strand viral DNA from HIV-1-infected AML14.3D10-CCR3 eosinophilic cells. Coreceptor blocking experiments, using the CXCR4 antagonist AMD3100, were performed to determine coreceptor usage by the CXCR4-using (X4) strain known to productively infect the cells. Virus production was measured by p24 immunoassay. As expected, viral DNA was detected in AML14.3D10-CCR3 cells infected with X4 HIV-1, and cell viability was decreased during maximal viral production. Conversely, viral DNA was not detected in eosinophilic cells exposed to a CCR5-using (R5) HIV-1 strain that is also capable of using CCR3, indicating that R5 HIV-1 is unable to enter eosinophilic cells despite the presence of the appropriate coreceptors. Infection of AML14.3D10-CCR3 cells by HTLV-III(B) was completely inhibited by AMD3100, indicating that X4 HIV-1 enters the AML14.3D10-CCR3 cell line by using the CXCR4 coreceptor exclusively. Since X4 strains predominate during the late stages of HIV-1 infection in many patients, when eosinophil numbers also tend to increase, the ability of these HIV-1 strains to infect eosinophilic cells has important implications for the involvement of eosinophils in the pathogenesis of AIDS.

Constitutive association of cell surface CCR5 and CXCR4 in the presence of CD4

Chemokine receptors CCR5 and CXCR4 are the major coreceptors of HIV-1 infection and also play fundamental roles in leukocyte trafficking, metastasis, angiogenesis, and embyogenesis. Here, we show that transfection of CCR5 into CXCR4 and CD4 expressing 3T3 cells enhances the cell surface level of CXCR4. In CCR5 high expressing cells, cell surface level of CXCR4 was incompletely modulated in the presence of the CXCR4 ligand CXCL12/SDF-1alpha. CCR5 was resistant to ligand-dependent modulation with the CCR5 ligand CCL5/RANTES. Confocal laser microscopy revealed that CCR5 was colocalized with CXCR4 on the cell surface. In CD4 expressing CCR5 and CXCR4 double positive NIH 3T3 cells, immunoprecipitation followed by Western blot analysis revealed that CCR5 was associated with CXCR4 and CD4. CXCR4 and CCR5 were not co-immunoprecipitated in cells expressing CCR5 and CXCR4 but without CD4 expression. Compared to NIH 3T3CD4 cells expressing CXCR4, the entry of an HIV-1 X4 isolate (HCF) into NIH 3T3CD4 expressing both CXCR4 and CCR5 was reduced. Our data indicate that chemokine receptors interact with each other, which may modulate chemokine-chemokine receptor interactions and HIV-1 coreceptor functions.

Alterations in mast cell function and survival following in vitro infection with human immunodeficiency viruses-1 through CXCR4

HIV-1 infection leads to a disease that attacks the central regulatory mechanisms of the immune response. As mucosal tissue is one of the primary sites infected with HIV in vivo, we examined the effects of HIV exposure on human mast cells, important components of mucosal defense. Using the human mast cell line, HMC-1, which expresses CXCR4 but not CCR5 on the cell surface, we found that several HIV-1 X4 tropic lab (IIIB, RF) and primary isolates but not R5 (BAL, ADA) isolates productively infected these cells. Furthermore, stem cell factor-dependent mast cells derived from primary fetal liver or cord blood cultures were also productively infected with both X4 and R5 HIV-1 strains. Infection was blocked at the level of viral entry using monoclonal antibodies to CXCR4 and CD4. Treatment of HMC-1 with TNF-α and TGF-β stimulated cell surface expression of CCR5 and up-regulated expression of both CCR5 and CXCR4 on primary mast cells, leading to increased susceptibility to both X4 and R5 viral isolates. HIV-1 infection also resulted in histamine release from these mast cells, most due in part to HIV-mediated cell death. These results demonstrate that X4 viruses can use CD4 and the CXCR4 receptor to infect mast cells, suggesting that mast cell–T cell interactions may contribute to HIV mediated immune dysfunction in the mucosa.

Mycobacterium tuberculosis-induced CXCR4 and chemokine expression leads to preferential X4 HIV-1 replication in human macrophages.

Opportunistic infections such as pulmonary tuberculosis (TB) increase local HIV-1 replication and mutation. As AIDS progresses, alteration of the HIV-1 gp120 V3 sequence is associated with a shift in viral coreceptor use from CCR5 (CD195) to CXCR4 (CD184). To better understand the effect of HIV/TB coinfection, we screened transcripts from bronchoalveolar lavage cells with high density cDNA arrays and found that CXCR4 mRNA is increased in patients with TB. Surprisingly, CXCR4 was predominately expressed on alveolar macrophages (AM). Mycobacterium tuberculosis infection of macrophages in vitro increased CXCR4 surface expression, whereas amelioration of disease reduced CXCR4 expression in vivo. Bronchoalveolar lavage fluid from TB patients had elevated levels of CCL4 (macrophage inflammatory protein-1beta), CCL5 (RANTES), and CX3CL1 (fractalkine), but not CXCL12 (stromal-derived factor-1alpha). We found that M. tuberculosis infection of macrophages in vitro increased viral entry and RT of CXCR4-using [corrected] HIV-1, but not of CCR5-using [corrected] HIV-1. Lastly, HIV-1 derived from the lung contains CD14, suggesting that they were produced in AM. Our results demonstrate that TB produces a permissive environment for replication of CXCR4-using virus by increasing CXCR4 expression in AM and for suppression of CCR5-using HIV-1 by increasing CC chemokine expression. These changes explain in part why TB accelerates the course of AIDS. CXCR4 inhibitors are a rational therapeutic approach in HIV/TB coinfection.

HIV co-receptors as targets for antiviral therapy.

The chemokine receptors CXCR4 and CCR5 are used as the main co-receptors by the T-cell-tropic (CXCR4-dependent, X4) and macrophage-tropic (CCR5-dependent, R5) HIV-1 strains, respectively, for entering their target cells. The natural ligands for CXCR4, the CXC-chemokine SDF-1 and CCR5, the CC-chemokines RANTES, MIP-1alpha and MIP-1beta are described to inhibit viral entry. In this review we focus on chemokine receptor/HIV co-receptor inhibitors. Modified chemokines such as Met-RANTES and AOP-RANTES showed antiviral activity against R5 viruses. Several low-molecular weight CCR5 antagonists have been described (such as TAK-779 and SCH-C) with potent antiviral activity. The latter compound is also orally available and is able to decrease R5 viral load levels in HIV-infected subjects. Several peptidic compounds, such as T22 (an 18-mer), T134 (a 14-mer), ALX40-4C (a 9-mer) and CGP 64222 (also a 9-mer) have anti-HIV activity and have been identified as CXCR4 antagonists. Also, the HIV-1 Tat protein has been described as a "natural" CXCR4 antagonist with anti-HIV-1 activity. The most potent and specific CXCR4 antagonists are the bicyclam derivatives, which also potently inhibit X4 HIV replication. AMD3100 has proved to be a highly specific CXCR4 antagonist, which consistently blocks X4 viral replication in any target cell-type evaluated so far. AMD3100 was selected as the clinical drug candidate, which, after initial phase I (safety) studies, had proceeded to phase II (efficacy) trials. The compound dose-dependently inhibited X4 viruses after 10 days of continuous infusion of the drug. Recently, the orally bioavailable CXCR4 antagonist, AMD070, is presented as a candidate HIV drug. We believe that chemokine receptor antagonists will become important new antiviral drugs to combat AIDS. Both (CXCR4 and CCR5) chemokine receptor inhibitors will be needed in combination to inhibit viral replication and even in combinations of antiviral drugs that also target other aspects of the HIV replication cycle, such as reverse transcriptase and protease, to obtain optimum therapeutic effects.

The N-terminal of the V3 loop in HIV type 1 gp120 is responsible for its conformation-dependent interaction with cell surface molecules.

The V3 loop of HIV-1 gp120 plays an important role in the interaction of the viral envelope with cellular coreceptors and/or with other cell surface molecules. To clarify this interaction we used a panel of monoclonal antibodies (MAbs) against V3 loop and synthetic looped V3 peptides V3-BH10, V3-ADA, and V3-89.6, derived from the V3 regions of the BH10 clone of IIIB (X4-tropic), ADA (R5-tropic), and 89.6 (R5X4-tropic), respectively. A linear mutant peptide, V3-BH10/CA, was also synthesized as a control. Biotinylated V3-BH10, -BH10/CA, and-ADA were also made. The binding abilities of the biotinylated and nonbiotinylated peptides to various types of cells were investigated by using flow cytometry. Subsequently, the principal region of the V3 loop involved in cell surface binding was analyzed by using MAbs against the tip (447-52D and 694-98D), N-termini (IIIB-V3-21) or C-termini (IIIB-V3-01) of the V3 loop in flow cytometry and enzyme-linked immunoabsorbent assay. We demonstrate that looped V3 peptides of both X4 and R5X4 HIV (V3-BH10 and V3-89.6) can bind to various types of cells irrespective of their CD4 and/or coreceptor expression in a conformation-dependent manner. In contrast, the V3 loop of R5 HIV (V3-ADA) can scarcely bind to the cells. Using MAbs whose epitopes cover the entire V3 loop we found that MAb IIIB-V3-21 can react with platebound but not cell-bound peptides, and the MAb blocked biotin-V3-BH10 binding suggesting that the N-terminal of the V3 loop interacts directly with cell surface molecule(s).

A syndecan-4/CXCR4 complex expressed on human primary lymphocytes and macrophages and HeLa cell line binds the CXC chemokine stromal cell-derived factor-1 (SDF-1).

The stromal cell-derived factor-1 (SDF-1) is a CXC chemokine, which plays critical roles in migration, proliferation, and differentiation of leukocytes. SDF-1 is the only known ligand of CXCR4, the coreceptor of X4 HIV strains. We show that SDF-1 binds to high- and low-affinity sites on HeLa cells. Coimmunoprecipitation studies demonstrate that glycanated and oligomerized syndecan-4 but neither syndecan-1, syndecan-2, betaglycan, nor CD44 forms complexes with SDF-1 and CXCR4 on these cells as well as on primary lymphocytes or macrophages. Moreover, biotinylated SDF-1 directly binds in a glycosaminoglycans (GAGs)-dependent manner to electroblotted syndecan-4, and colocalization of SDF-1 with syndecan-4 was visualized by confocal microscopy. Glycosaminidases pretreatment of the HeLa cells or the macrophages decreases the binding of syndecan-4 to the complex formed by it and SDF-1. In addition, this treatment also decreases the binding of the chemokine to CXCR4 on the primary macrophages but not on the HeLa cells. Therefore GAGs-dependent binding of SDF-1 to the cells facilitates SDF-1 binding to CXCR4 on primary macrophages but not on HeLa cell line. Finally, an SDF-1-independent heteromeric complex between syndecan-4 and CXCR4 was visualized on HeLa cells by confocal microscopy as well as by electron microscopy. Moreover, syndecan-4 from lymphocytes, monocyte derived-macrophages, and HeLa cells coimmunoprecipitated with CXCR4. This syndecan-4/CXCR4 complex is likely a functional unit involved in SDF-1 binding. The role of these interactions in the pathophysiology of SDF-1 deserves further study.

Induction of Intercellular Adhesion Molecule-1 on Human Brain Endothelial Cells by HIV-1 gp120: Role of CD4 and Chemokine Coreceptors

Central nervous system dysfunction is commonly observed in children with HIV-1 infection, but the mechanisms whereby HIV-1 causes encephalopathy are not completely understood. We have previously shown that human brain microvascular endothelial cells (HBMEC) from children are responsive to gp120 derived from X4 HIV-1 by increasing expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule-1. However, the mechanisms involved in gp120-mediated up-regulation of cell adhesion molecule expression is unclear. In the present study, we found that gp120 derived from both X4 and R5 HIV-1 induced increased expression of ICAM-1 on HBMEC, but the degree of this up-regulation differed among the various HBMEC isolates. The up-regulation of ICAM-1 was inhibited by anti-CD4 antibodies as well as by specific antibodies directed against chemokine receptors and small-molecule coreceptor inhibitors. Anti-CD4 antibodies inhibited the increase in ICAM-1 expression mediated by gp120 derived from X4 and R5 HIV-1, whereas antibodies against chemokine receptors displayed a differential inhibition depending on the source of gp120. Both X4 and R5 gp120-induced ICAM-1 expression was sensitive to pertussis toxin and involved the nuclear factor-kB pathway. These findings indicate a direct involvement of CD4 and a differential involvement of chemokine receptors in the activation of pediatric HBMEC by X4 and R5 gp120. The activation of brain endothelium of children by HIV-1 protein gp120 by way of CD4 and chemokine receptors may have implications for the pathogenesis of HIV-1 encephalopathy in the pediatric population.

The antiviral activity of the CXCR4 antagonist AMD3100 is independent of the cytokine-induced CXCR4/HIV coreceptor expression level.

The chemokine receptor CXCR4 is the main coreceptor used by T-tropic X4 HIV-1 strains to infect its target T cells. It has been proven that the CXCR4 expression level in T cells is strongly up-regulated by interleukin (IL)-4, a Th2-type cytokine that is secreted preferentially in HIV-infected patients in a later stage of disease. This results in an enhancement of HIV-1 replication in CD4+ T-lymphocytes. We have now evaluated the potency of the CXCR4 antagonist AMD3100 in phytohemagglutinin (PHA)/IL-2- versus PHA/IL-4-activated T cells in order to determine whether the compound has comparable CXCR4-antagonistic and anti-HIV-1 effects under these different cytokine treatments. We analyzed the CXCR4 expression level and the dose-dependent inhibition of CXCR4 expression by AMD3100, by monitoring the binding of an anti-CXCR4 monoclonal antibody (clone 12G5). We also determined stromal cell-derived factor (SDF)-1-induced intracellular calcium signaling and HIV-1 replication in these cells in the absence and presence of AMD3100. The CXCR4 expression level in PHA/IL-4-stimulated cells was much higher than in PHA/IL-2-stimulated cells. However, the potency of the bicyclam AMD3100 to block anti-CXCR4 mAb binding, SDF-1-induced intracellular calcium signaling, and HIV-1 replication of the X4 NL4.3 strain and three primary isolates remained unchanged. Our data indicate that CXCR4 antagonists such as AMD3100 act independently of the HIV-1 coreceptor expression level. These compounds should therefore be useful in suppressing HIV-1 infection in all stages of the disease.

Cell-Free HIV Type 1 Infection is Restricted in the Human Trophoblast Choriocarcinoma BeWo Cell Line, Even with Expression of CD4, CXCR4 and CCR5

The restriction of cell-free HIV-1 infection has been demonstrated in placental trophoblast choriocarcinoma BeWo cells. We tried to determine the level of the viral replication cycle at which this restriction occurs. BeWo cells produce infectious viruses after transfection with HIV-1 plasmids, independently of viral tropism. CCR5 and CXCR4, but not the CD4 molecule, were detected at the cell surface. We therefore derived CD4-expressing clones from transfected BeWo cells. Cell-free virus infection of these clones resulted in neither virus production nor viral sequence integration, indicating that the restriction occurs before integration of the virus. If we used luciferase reporter viruses pseudotyped with HIV-1 Env R5 and X4 for infection, no luciferase activity was detected, even in the BeWo-CD4+ clone, in contrast to what was observed in VSV-G pseudotyped virus infection. Our results show that infection of trophoblast-derived cells with cell-free virus is at least restricted at the level of entry. Thus, BeWo is an interesting human placental cell line that is resistant to HIV-1, even if CD4, CXCR4, and CCR5 are expressed.

Productive HIV infection of resting CD4+ T cells: role of lymphoid tissue microenvironment and effect of immunomodulating agents.

The ability of resting CD4+ T cells to support HIV replication is relevant to understanding how the reservoir of HIV-1-infected resting CD4+ T cells is generated, maintained and, hopefully, how it might be reduced or eliminated. We have utilized a tonsillar histoculture system to demonstrate that HIV, particularly X4 strains, can productively infect phenotypically resting CD4+ T cells in vitro and that this event is largely dependent on the lymphoid tissue microenvironment. Highly purified CD4+ tonsillar T cells that lack expression of both cell surface and nuclear antigens characteristic of classic T cell activation produce X4 HIV-1 mRNA, p24, and infectious virus while maintaining a resting phenotype when cultured in a tonsillar tissue microenvironment; in contrast, comparable purified resting CD4+ tonsillar T cells that have been exposed to X4 HIV do not support HIV replication when cultured in the absence of a lymphoid tissue microenvironment. HIV production from phenotypically resting CD4+ T cells is dramatically inhibited by anti-proinflammatory cytokine agents or immunosuppressive cytokines, but is only modestly suppressed by an inhibitor of the cell cycle. The ability of resting CD4+ T cells to support HIV replication in the microenvironment of the lymphoid tissue has implications in the pathogenesis of HIV disease and may provide an additional avenue for therapeutic intervention.

The theta-defensin, retrocyclin, inhibits HIV-1 entry.

Retrocyclin is a circular antimicrobial 18-residue peptide encoded in the human genome by a theta-defensin pseudogene. In the human genome, the gene for retrocyclin is inactivated by an in-frame stop codon in its signal sequence but its mature coding sequence is intact. The peptide corresponding to the processed human retrocyclin, generated by solid phase peptide synthesis, inhibited replication of R5 and X4 strains of HIV-1 in human cells. Luciferase reporter virus and Vpr-BLaM entry assays were used to demonstrate that retrocyclin specifically blocked R5 and X4 HIV-1 replication at entry. Surface plasmon resonance demonstrated that retrocyclin bound to soluble CD4 and gp120, but gp120 cell-binding assays revealed that retrocyclin did not fully inhibit the binding of soluble CD4 to gp120. A fluorescent retrocyclin congener localized in cell-surface patches either alone or colocalized with CD4, CXCR4, and CCR5. In the aggregate, these results suggest that retrocyclin blocks an entry step in HIV-1 replication. Retrocyclin represents a new class of small molecule HIV-1 entry inhibitors.

Assessment of oral transmission using cell-free human immunodeficiency virus-1 in mice reconstituted with human peripheral blood leucocyte.

Oral-genital contact is one of the risk factors for the transmission of human immunodeficiency virus (HIV) in adults. In recent reports, oral exposure to simian immunodeficiency virus (SIV) was found to have important implications for the achievement of mucosal transmission of HIV in a rhesus monkey animal model. In the present study, we aimed first to establish a small animal model which did not develop tonsils suitable for HIV oral mucosa transmission, using non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice and NOD/SCID B2m(null) mice grafted with human peripheral blood leucocytes (hu-PBL) and stimulated with interleukin (IL)-4, and second to investigate whether oral exposure to cell-free R5 and X4 HIV-1 could lead to oral transmission of HIV through intact or traumatized mucosal tissues in humanized mice. Both low and high concentrations of cell-free R5 and X4 viruses failed to cause oral transmission with or without trauma in hu-PBL-NOD/SCID and NOD/SCID Beta2m(null) mice, which presented a number of CD4+ cells in gingival tissues and oral cavities with or without tissue injury. The present results show that IL-4-administrated NOD/SCID B2m(null) mice are useful as a small-humanized model for the study of HIV oral infection, which may help to define the window of opportunity for oral transmission by the HIV virus in animal model experiments.

Positive regulation of CXCR4 expression and signaling by interleukin-7 in CD4+ mature thymocytes correlates with their capacity to favor human immunodeficiency X4 virus replication.

The emergence of X4 human immunodeficiency virus type 1 (HIV-1) variants in infected individuals is associated with poor prognosis. One of the possible causes of this emergence might be the selection of X4 variants in some specific tissue compartment. We demonstrate that the thymic microenvironment favors the replication of X4 variants by positively modulating the expression and signaling of CXCR4 in mature CD4(+) CD8(-) CD3(+) thymocytes. Here, we show that the interaction of thymic epithelial cells (TEC) with these thymocytes in culture induces an upregulation of CXCR4 expression. The cytokine secreted by TEC, interleukin-7 (IL-7), increases cell surface expression of CXCR4 and efficiently overcomes the downregulation induced by SDF-1 alpha, also produced by TEC. IL-7 also potentiates CXCR4 signaling, leading to actin polymerization, a process necessary for virus entry. In contrast, in intermediate CD4(+) CD8(-) CD3(-) thymocytes, the other subpopulation known to allow virus replication, TEC or IL-7 has little or no effect on CXCR4 expression and signaling. CCR5 is expressed at similarly low levels in the two thymocyte subpopulations, and neither its expression nor its signaling was modified by the cytokines tested. This positive regulation of CXCR4 by IL-7 in mature CD4(+) thymocytes correlates with their high capacity to favor X4 virus replication compared with intermediate thymocytes or peripheral blood mononuclear cells. Indeed, we observed an enrichment of X4 viruses after replication in thymocytes initially infected with a mixture of X4 (NL4-3) and R5 (NLAD8) HIV strains and after the emergence of X4 variants from an R5 primary isolate during culture in mature thymocytes.

Association between an interleukin-4 promoter polymorphism and the acquisition of CXCR4 using HIV-1 variants.

A polymorphism at position -589 in the interleukin 4 (IL-4) promoter region was recently described as being associated with the presence of syncytium-inducing CXCR4 using (X4) HIV-1 variants. To study the IL-4 promoter polymorphism -589T in relation to HIV-1 disease progression and acquisition of X4 HIV-1 variants. Retrospective longitudinal study among 342 HIV-1-infected homosexual men who participated in the Amsterdam Cohort study. Polymerase chain reaction was used in combination with restriction analysis to identify IL-4 promoter genotypes. Carriers of the -589T allele (either -589 C/T heterozygotes or -589 T/T homozygotes), showed comparable progression to AIDS [relative hazard (RH), 0.94; P = 0.71], and survival (RH IL-4 -589 C/T or T/T, 0.94; P = 0.69) as carriers of the -589 C/C genotype (the reference group). In contrast to a previous study, we found that the -589T polymorphism was associated with a delayed acquisition of X4 HIV-1 variants (RH, 0.56; P = 0.02 for IL-4 -589 C/T or T/T) and a reduced number of CCR5 expressing memory CD4 T cells. In the Amsterdam Cohort of homosexual men with HIV infection, the IL-4 -589T promoter polymorphism was associated with a delayed acquisition of X4 variants but did not affect overall disease progression.

μ-opioid modulation of HIV-1 coreceptor expressionand HIV-1 replication

A substantial proportion of HIV-1-infected individuals are intravenous drug users (IVDUs) who abuse opiates. Opioids induce a number of immunomodulatory effects that may directly influence HIV-1 disease progression. In the present report, we have investigated the effect of opioids on the expression of the major HIV-1 coreceptors CXCR4 and CCR5. For these studies we have focused on opiates which are ligands for the μ-opioid receptor. Our results show that DAMGO, a selective μ-opioid agonist, increases CXCR4 and CCR5 expression in both CD3 + lymphoblasts and CD14 + monocytes three- to fivefold. Furthermore, DAMGO-induced elevation of HIV-1 coreceptor expression translates into enhanced replication of both X4 and R5 viral strains of HIV-1. We have confirmed the role of the μ-opioid receptor based on the ability of a μ-opioid receptor-selective antagonist to block the effects of DAMGO. We have also found that morphine enhances CXCR4 and CCR5 expression and subsequently increases both X4 and R5 HIV-1 infection. We suggest that the capacity of μ-opioids to increase HIV-1 coreceptor expression and replication may promote viral binding, trafficking of HIV-1-infected cells, and enhanced disease progression.

Reconstitution of thymic function in HIV-1 patients treated with highly active antiretroviral therapy

The extent to which highly active antiretroviral therapy can restore thymic function in HIV-1 infected patients is not known. We simulate treatment using a temporal model of thymopoiesis during HIV-1 infection, tracking thymic function by the number of recent thymic emigrants (RTE) exported to the periphery per day. Our results suggest that suppressing viral load in peripheral blood and improving inherent thymic function are necessary for the reconstitution of RTE levels in adult thymic infection with either R5 or X4 HIV-1 strains. This is also the case in pediatric thymic infection with R5 strains. However, recovery of RTE levels during pediatric infection with X4 strains also depends on high drug efficacy within the thymus. We further predict that protease inhibitors have high levels of efficacy directly suppressing viral replication within the thymus, while reverse transcriptase inhibitors have low efficacy.

Reduced Fitness of HIV-1 Resistant to Cxcr4 Antagonists

HIV-1 strains with a syncytium-inducing phenotype that use CXCR4 (X4 strains) have been associated with faster disease progression and AIDS. Antiviral agents designed to block CXCR4 may prevent the emergence of X4 HIV strains but resistant strains that maintain the X4 phenotype can be raised by sequential passage in cell cultures. We have demonstrated that a laboratory adapted strain (NL4-3) and a cloned clinical isolate (CI-1) of HIV-1 cultured in the presence of the CXCR4 antagonist, AMD3100, became resistant to the compound without a change in co-receptor use. These strains became resistant through divergence with respect to the wild-type virus. Conversely, a clinical isolate made resistant to AMD3100 switched co-receptor use from X4 to R5 through a change in diversity from the original virus population. When dual infection competition/heteroduplex tracking assays were performed, all AMD3100-resistant strains, regardless of co-receptor use showed a significantly diminished fitness compared with the wild-type virus. Single virus infections, at a similar multiplicity of infection, also indicated that the wild-type strains possess better replicative ability than their corresponding resistant strains. Thus, viral resistance development to a CXCR4 antagonist such as AMD3100 is associated with reduced viral fitness.

Suppression of chemokine receptor expression by RNA interference allows for inhibition of HIV-1 replication.

Duplexes of 21 base pair RNA, known as short-interfering RNA (siRNA), have been shown to inhibit gene expression by a sequence-specific RNA degradation mechanism termed RNA interference (RNAi). The objective of our study was to evaluate the effect of chemokine receptor gene suppression by RNAi on the entry and replication of HIV-1. A flow cytometry and microscopy evaluation of HIV co-receptor expression of cells transfected with siRNA. An evaluation of the effect of siRNA on HIV entry and replication by intracellular p24 antigen detection, and virus production by infected cells, respectively. siRNA that target CXCR4 and CCR5 could effectively impede cell surface protein expression and their consequent function as HIV co-receptors. The inhibitory effect of RNAi directed to CXCR4 was detected 48 h after transfection of CXCR4+ U87-CD4+ cells. The expression of CXCR4 and CCR5 was blocked in 63 and 48% of positive cells by the corresponding siRNA. However, siRNA directed to CXCR4 or CCR5 did not have an effect on CD4 cells or green fluorescence protein expression. siRNA directed to CXCR4 did not suppress CCR5 expression or vice versa. The suppression of HIV-1 co-receptor expression effectively blocked the acute infection of CXCR4+ or CCR5+ U87-CD4+ cells by X4 (NL4-3) or R5 (BaL) HIV-1 strains. Inhibition of virus replication occurred regardless of the multiplicity of infection employed. Our results demonstrate that RNAi may be used to block HIV entry and replication through the blockade of cellular gene expression. Gene silencing by siRNA may become a valid alternative for HIV intervention.