In vitro exposure to highly cytopathic HIV-1 X4 strains increases expression of mucosa-associated integrins on CD4(+) T cells

To determine whether infection with HIV-1 strains of different tropisms would influence expression of the mucosa-associated integrins alpha 4 beta 7 and alpha E beta 7 or the lymph node homing receptor L-selectin on peripheral T lymphocytes, cells were infected with the CXCR4-tropic (X4)/syncytium-inducing (SI) HIV-1(IIIB) strain or with X4/SI or CCR5-tropic (R5)/non-SI (NSI) primary human isolates. Flow cytometric analyses of CD4(+) T cells from cultures infected with HIV-1(IIIB) and one X4/SI primary HIV-1 isolate revealed a significant increase in surface expression of alpha 4 beta 7 and alpha E beta 7 12 days after infection. L-selectin expression was not significantly affected on CD4(+) T cells. However, infection with another X4/SI and two R5/NSI primary HIV-1 isolates did not significantly alter homing receptor expression on CD4(+) T cells. Since a higher degree of CD4 cytopathicity occurred in those cultures having increased integrin expression, these data suggest that significantly altered mucosal homing receptor expression on CD4(+) T cells may result as a "bystander" effect after infection with some cytopathic isolates of HIV-1.     

Selective HIV-1-induced downmodulation of CD4 and coreceptors

Summary.  CD4 and members of the chemokine receptor family are required for infection of host cells, in vitro and in vivo, by the human immunodeficiency virus type-1. Although it is established that HIV-1 gp120 interacts with CD4 and the coreceptors CCR5 or CXCR4 at the plasma membrane during HIV entry, longer-term interactions taking place between these molecules and HIV Env are less well understood. We have measured the cell surface expression of CD4, CCR5 and CXCR4 on a CD4⁺/CXCR4⁺CCR5⁺ T cell line following infection by cell line-adapted X4 and primary X4, X4R5 and R5 viruses. We report a selective downmodulation of CD4 by X4 and R5X4 viruses, but not by R5 viruses. None of the viruses tested significantly reduced CXCR4 expression at any time after infection. CCR5 protein and mRNA expression was eliminated by chronic infection with R5 viruses. These results indicate that chronic HIV-1 infection has distinct effects on CD4 and coreceptor membrane expression that depends on viral origin and coreceptor usage. Accepted October 25, 1999     

Downregulation of CCR5 on activated CD4 T cells in HIV-infected Indians

BACKGROUND: HIV infection in India is unique as it occurs predominantly by CCR5-utilizing isolates that exhibit no co-receptor switch. OBJECTIVES: To study HIV-1 co-receptor dynamics on T cells and monocytes following viral infection. STUDY DESIGN: HIV co-receptor expression was evaluated by flow cytometry on various cell subsets in HIV-infected Indians and in vitro in human peripheral blood mononuclear cells infected with CCR5- or CXCR4-utilizing HIV-1. Transfection of the T cell line CEM-CCR5 (which expresses CD4, CCR5 and CXCR4) with HIV-1 Nef or Vpu expression vectors, or treatment with recombinant soluble gp120 from CCR5- and CXCR4-tropic HIV-1, was carried out to determine their effects on co-receptor expression. RESULTS: Indian HIV patients had fewer CD4(+)CCR5(+) T cells and CCR5-expressing activated CD4(+) T cells, but higher CXCR4-expressing activated CD4(+) T cells compared with controls. Expression of CCR5 was not different on monocytes in HIV patients as compared to controls. The CCR5 downregulation on T cells was HIV infection specific and was governed by the co-receptor-utilization phenotype of the virus. The Nef and soluble gp120 proteins induced CCR5 downregulation, the latter in a co-receptor-utilization phenotype specific manner. CONCLUSIONS: The HIV-1 co-receptor dynamics in Indian patients is distinct from western patients and depends upon the virus surface protein. We propose this to be a viral survival strategy.     

Asymmetric HIV-1 co-receptor use and replication in CD4(+) T lymphocytes

Susceptibility to infection by the human immunodeficiency virus type-1 (HIV-1), both in vitro and in vivo, requires the interaction between its envelope (Env) glycoprotein gp120 Env and the primary receptor (R), CD4, and Co-R, either CCR5 or CXCR4, members of the chemokine receptor family. CCR5-dependent (R5) viruses are responsible for both inter-individual transmission and for sustaining the viral pandemics, while CXCR4-using viruses, usually dualtropic R5X4, emerge in ca. 50% of individuals only in the late, immunologically suppressed stage of disease. The hypothesis that such a major biological asymmetry is explained exclusively by the availability of cells expressing CCR5 or CXCR4 is challenged by several evidences. In this regard, binding of the HIV-1 gp120 Env to the entry R complex, i.e. CD4 and a chemokine R, leads to two major events: virion-cell membrane fusion and a cascade of cell signaling. While the fusion/entry process has been well defined, the role of R/Co-R signaling in the HIV-1 life cycle has been less characterized. Indeed, depending on the cellular model studied, the capacity of HIV-1 to trigger a flow of events favoring either its own latency or replication remains a debated issue. In this article, we will review the major findings related to the role of HIV R/Co-R signaling in the steps following viral entry and leading to viral spreading in CD4(+) T lymphocytes.     

Restricted replication of primary HIV-1 isolates using both CCR5 and CXCR4 in Th2 but not in Th1 CD4(+) T cells

We have previously reported that CCR5-dependent human immunodeficiency virus type-1 (HIV-1; R5), but not CXCR4-restricted (X4) virus, efficiently replicates in T helper cell type 1 (Th1), Th2, or Th0 polyclonal T cells obtained from human umbilical cord blood (CB lines). The X4 virus restriction was env-dependent but did not occur at the level of viral entry. Here, we describe that in contrast to these monotropic HIVs, primary HIV-1 isolates capable of using CCR5 or CXCR4 indifferently for entry (i.e., R5X4 viruses) efficiently replicated in Th2 but not in Th1 CB lines. Although Th1 cells secreted significantly higher amounts of the three CCR5-binding chemokines in comparison with Th2 cells, this restriction was not explained by a defective infection of Th1 cells. Interferon-gamma (IFN-gamma) down-regulated CCR5 in Th1 cells and inhibited, whereas interleukin-4 (IL-4) up-regulated CXCR4 and enhanced the spreading of R5 and R5X4 viruses in polarized CB lines. However, both cytokines did not rescue the replication of X4 and dualtropic viruses in both types of CB lines or in Th1 cells, respectively, whereas addition of anti-IL-4- or anti-IFN-gamma-neutralizing antibodies did not activate virus expression. These findings together suggest the existence of post-entry restriction pathways influenced by gp120 Env/chemokine coreceptor interaction that may significantly contribute to the superior capacity of R5 and R5X4 HIV-1 strains to spread in vivo in comparison to X4 monotropic viruses.     

Raft localization of CXCR4 is primarily required for X4-tropic human immunodeficiency virus type 1 infection

Human immunodeficiency virus type 1 (HIV-1) infection is initiated by successive interactions of viral envelope glycoprotein gp120 with two cellular surface proteins, CD4 and chemokine receptor. The two most common chemokine receptors that allow HIV-1 entry are the CCR5 and CXCR4. The CD4 and CCR5 are mainly localized to the particular plasma membrane microdomains, termed raft, which is rich in glycolipids and cholesterol. However, the CXCR4 is localized only partially to the raft region. Although the raft domain is suggested to participate in HIV-1 infection, its role in entry of CXCR4-tropic (X4-tropic) virus is still unclear. Here, we used a combination of CD4-independent infection system and cholesterol-depletion-inducing reagent, methyl-β-cyclodextrin (MβCD), to address the requirement of raft domain in the X4-tropic virus infection. Treatment of CD4-negative, CXCR4-positive human cells with MβCD inhibited CD4-independent infection of the X4-tropic strains. This inhibitory effect of the cholesterol depletion was observed even when the CXCR4 was over-expressed on the target cells. Soluble CD4-induced infection was also inhibited by MβCD. The MβCD had no effect on the levels of cell surface expression of CXCR4. In contrast to these infections, MβCD treatment did not inhibit CD4-dependent HIV-1 infection in the wild type CD4-expressing cells. This study and previous reports showing that CD4 mutants localized to non-raft domains function as HIV-1 receptor indicate that CXCR4 clustering in the raft microdomains, rather than CD4, is the key step for the HIV-1 entry.     

Partial inactivation of CCR5- and CXCR4- tropic HIV-1 by human urine

Human urine has been poorly investigated with regard to infection with human immunodeficiency virus (HIV). Here, we have studied the anti-infective functional properties of human urine against HIV. The effect of fresh urine pools on CCR5- and CXCR4-tropic HIV-1 was evaluated by using four in vitro mucosal models: reduction of infectivity of urine-treated HIV-1 particles, HIV-1 attachment to immature monocyte-derived dendritic cells (iMDDC), transfer of HIV-1 particles from iMDDC to autologous CD4 T cells, and HIV-1 transcytosis through epithelial cells. Human urine partially disrupted both CCR5- and CXCR4-tropic HIV-1 particles, moderately decreased the adsorption of HIV-1 on dendritic cells, and partially decreased the transfer of HIV-1 particles from dendritic cells to autologous T cells. These findings demonstrate partial inactivation of HIV infectivity and suggest that voiding urine after coitus could play a potential role in reducing the risk of HIV infection by both mechanically flushing out and neutralizing the infectivity of HIV-1 particles present in the genital tract.     

Selective infection of CD4 effector memory T lymphocytes leads to preferential depletion of memory T lymphocytes in R5 HIV-1-infected humanized NOD/SCID/IL-2Rγ mice

To investigate the events leading to the depletion of CD4⁺ T lymphocytes during long-term infection of human immunodeficiency virus type 1 (HIV-1), we infected human CD34⁺ cells-transplanted NOD/SCID/IL-2Rγ^null mice with CXCR4-tropic and CCR5-tropic HIV-1. CXCR4-tropic HIV-1-infected mice were quickly depleted of CD4⁺ thymocytes and both CD45RA⁺ naïve and CD45RA⁻ memory CD4⁺ T lymphocytes, while CCR5-tropic HIV-1-infected mice were preferentially depleted of CD45RA⁻ memory CD4⁺ T lymphocytes. Staining of HIV-1 p24 antigen revealed that CCR5-tropic HIV-1 preferentially infected effector memory T lymphocytes (T_EM) rather than central memory T lymphocytes. In addition, the majority of p24⁺ cells in CCR5-tropic HIV-1-infected mice were activated and in cycling phase. Taken together, our findings indicate that productive infection mainly takes place in the activated T_EM in cycling phase and further suggest that the predominant infection in T_EM would lead to the depletion of memory CD4⁺ T lymphocytes in CCR5-tropic HIV-1-infected mice.     

Identification of HIV-1 epitopes that induce the synthesis of a R5 HIV-1 suppression factor by human CD4+ T cells isolated from HIV-1 immunized hu-PBL SCID mice

We have previously reported that immunization of the severe combined immunodeficiency (SCID) mice reconstituted with human peripheral blood mononuclear cells (PBMC) (hu-PBL-SCID mice) with inactivated human immunodeficiency virus type-1 (HIV-1)-pulsed-autologous dendritic cells (HIV-DC) elicits HIV-1-reactive CD4(+) T cells that produce an as yet to be defined novel soluble factor in vitro with anti-viral properties against CCR5 tropic (R5) HIV-1 infection. These findings led us to perform studies designed to identify the lineage of the cell that synthesizes such a factor in vivo and define the epitopes of HIV-1 protein that have specificity for the induction of such anti-viral factor. Results of our studies show that this property is a function of CD4(+) but not CD8(+) T cells. Human CD4(+) T cells were thus recovered from the HIV-DC-immunized hu-PBL-SCID mice and were re-stimulated in vitro by co-culture for 2 days with autologous adherent PBMC as antigen presenting cells, APC previously pulsed with inactivated HIV in IL-2-containing medium to expand HIV-1-reactive CD4(+) T cells. Aliquots of these re-stimulated CD4(+) T cells were then co-cultured with similar APC's that were previously pulsed with 10 microg/ml of a panel of HIV peptides for an additional 2 days, and their culture supernatants were examined for the production of both the R5 HIV-1 suppression factor and IFN-gamma. The data presented herein show that the HIV-1 primed CD4(+) T cells produced the R5 suppression factor in response to a wide variety of HIV-1 gag, env, pol, nef or vif peptides, depending on the donor of the CD4(+) T cells. Simultaneous production of human interferon (IFN)-gamma was observed in some cases. These results indicate that human CD4(+) T cells in PBMC of HIV-1 naive donors have a wide variety of HIV-1 epitope-specific CD4(+) T cell precursors that are capable of producing the R5 HIV-1 suppression factor upon DC-based vaccination with whole inactivated HIV-1.     

Antigen stimulation induces HIV envelope gp120-specific CD4(+) T cells to secrete CCR5 ligands and suppress HIV infection

CD4(+) T cells are critical for effective immune responses against HIV, but they are also the main cell type targeted by the virus. To investigate the key factors that could protect these cells from infection, we evaluated the capacity of HIV gp120-specific human CD4(+) T cells to produce chemokines that inhibit HIV and determined their contribution in suppressing infection in the cells. Antigen stimulation of the CD4(+) T cells elicited production of high amounts of CCR5 chemokines MIP-1alpha (CCL3), MIP-1beta (CCL4), and RANTES (CCL5). Production of these CCR5 ligands was more readily and reproducibly detected than that of IFN-gamma or IL-2. Importantly, in association with secretion of the CCR5 ligands, antigen stimulation made these CD4(+) T cells more resistant to CCR5-tropic HIV-1. Conversely, in the absence of antigen stimulation, the cells were readily infected by the virus, and after infection, their capacity to produce MIP-1beta and IFN-gamma rapidly declined. Thus, vaccines that trigger HIV-specific CD4(+) T cells to elicit robust and rapid production of anti-viral chemokines would be advantageous. Such responses would protect virus-specific CD4(+) T cells from HIV infection and preserve their critical functions in mounting and maintaining long-lasting immunity against the virus.     

In Vitro Exposure to Highly Cytopathic HIV-1 X4 Strains Increases Expression of Mucosa-Associated Integrins on CD4 T Cells

To determine whether infection with HIV-1 strains of different tropisms would influence expression of the mucosa-associated integrins α4β7 and αEβ7 or the lymph node homing receptor L-selectin on peripheral T lymphocytes, cells were infected with the CXCR4-tropic (X4)/syncytium-inducing (SI) HIV-1_IIIB strain or with X4/SI or CCR5-tropic (R5)/non-SI (NSI) primary human isolates. Flow cytometric analyses of CD4⁺ T cells from cultures infected with HIV-1_IIIB and one X4/SI primary HIV-1 isolate revealed a significant increase in surface expression of α4β7 and αEβ7 12 days after infection. L-selectin expression was not significantly affected on CD4⁺ T cells. However, infection with another X4/SI and two R5/NSI primary HIV-1 isolates did not significantly alter homing receptor expression on CD4⁺ T cells. Since a higher degree of CD4 cytopathicity occurred in those cultures having increased integrin expression, these data suggest that significantly altered mucosal homing receptor expression on CD4⁺ T cells may result as a “bystander” effect after infection with some cytopathic isolates of HIV-1.     

Differential susceptibility of resting CD4(+) T lymphocytes to a T-tropic and a macrophage (M)-tropic human immunodeficiency virus type 1 is associated with their surface expression of CD38 molecules

Recent evidence has accumulated which definitively shows that chemokine receptors CCR5 and CXCR4 play an essential role as coreceptors for human immunodeficiency virus type 1 (HIV-1) infection. Flow cytometric analysis permitted us to detect CD38, a surface marker of early differentiation, as well as activation of T cells, on about half of healthy donor-derived CD4(+) T cells. In this study, we focused on the susceptibility of CD38(+) and CD38(-) subsets of CD4(+) T cells to HIV-1 infection with different coreceptor tropisms. About 20% of peripheral blood mononuclear cell-derived resting CD4(+) T cells were recovered into the CD38(+) subset fraction by panning with a monoclonal antibody to CD38. Most of the cells in this CD38(high) fraction also expressed CD45RA and CD62L at higher intensities compared with those of CD38(low) fraction. CCR5(+) T cells predominated in the CD38(-) subset, although cell surface expression of CD4 and CXCR4 was almost similar between both subsets. This difference was consistent with a significantly higher susceptibility of the CD38(-) subset to a macrophage (M)-tropic HIV-1 strain. In contrast, it was shown that a T-tropic strain of HIV-1 could replicate more efficiently in the CD38(+) subset, although viral adsorption rates were similar between both subsets. Thus, the differential susceptibility of CD4(+) T cells to M(-) and T-tropic HIV-1 was associated with their surface expression of CD38.     

Replicative fitness of CCR5-using and CXCR4-using human immunodeficiency virus type 1 biological clones

CCR5-tropic viruses cause the vast majority of new HIV-1 infections while about half of the individuals infected with HIV-1 manifest a co-receptor switch (CCR5 (R5) to CXCR4 (X4)) prior to accelerated disease progression. The underlying biological mechanisms of X4 outgrowth in AIDS patients are still poorly understood. Although X4 viruses have been associated with increased "virulence" in vivo, in vitro replication and cytopathicity studies of X4 and R5 viruses have led to conflicting conclusions. We studied the replicative fitness of HIV-1 biological clones with different co-receptor tropism, isolated from four AIDS patients. On average, R5 and X4 clones replicated equally well in mitogen-activated T cells. In contrast, X4 variants were transferred more efficiently from dendritic cells to autologous CD4+ T cells. These observations suggest that interaction between X4 viruses, DC and T cells might contribute to the preferential outgrowth of X4 viruses in AIDS patients.     

Biological parameters of HIV-1 infection in primary intestinal lymphocytes and macrophages

Mucosal surfaces are the portal of entry for most HIV-1 infections and play an important role in disease pathogenesis. To characterize the biological parameters of HIV-1 infection in mucosal cells, we used purified lamina propria lymphocytes and macrophages from normal human small intestine to determine the distribution of the HIV-1 receptor and coreceptors on intestinal mononuclear cells and the permissiveness of these cells to HIV-1 infection. Lamina propria lymphocytes expressed CD4, CCR5, and CXCR4. In contrast, lamina propria macrophages expressed CD4 but not CCR5 or CXCR4. Intestinal lymphocytes supported replication by R5 and X4 isolates of HIV-1, but lamina propria macrophages were permissive to neither. RANTES, macrophage inflammatory protein-1alpha (MIP-1alpha), and MIP-1beta inhibited infection of intestinal lymphocytes by BaL, indicating that R5 infection of the intestinal lymphocytes was mediated by CCR5. Thus, resident lamina propria lymphocytes, not macrophages, are the target mononuclear cell for HIV-1 infection in the intestinal mucosa during early HIV-1 infection.     

HIV-1 coreceptor preference is distinct from target cell tropism: a dual-parameter nomenclature to define viral phenotypes

HIV-1 infection of cells is mediated by engagement between viral envelope glycoproteins (Env) and a receptor complex comprising CD4 and one of two chemokine receptors, CCR5 and CXCR4, expressed on the surface of target cells. Most CD4+-transformed T cell lines express only CXCR4, but primary lymphocytes and macrophages, the main cellular targets for infection in vivo, express both coreceptors. Cell- and viral strain-specific utilization of these coreceptor pathways, rather than coreceptor expression per se, regulates lymphocyte and macrophage entry and tropism. Virus use of coreceptor[s] (R5, X4, or R5 and X4) and its target cell tropism (lymphocytes, macrophages, and/or transformed T cell lines) are related but distinct characteristics of Envs. A comprehensive classification schema of HIV-1 Env phenotypes that addresses both tropism and coreceptor use is proposed. Defining Env phenotype based on both parameters is important in the development of entry inhibitors and vaccines, for understanding changes in Env that evolve over time in vivo, and for discerning differences among viral species that underlie aspects of pathogenesis and transmission. Recognizing how tropism is related to, yet differs from, coreceptor selectivity is critical for understanding the mechanisms by which these viral characteristics impact pathogenesis.     

Entry coreceptor use and fusion inhibitor T20 sensitivity of dual-tropic R5X4 HIV-1 in primary macrophage infection

Macrophages are important targets for HIV-1, and R5X4 strains play a central role in pathogenesis, especially in late-stage patients who may receive the fusion inhibitor T20 (enfuvirtide). Sensitivity to T20 varies markedly among HIV-1 strains and is influenced by viral and cellular factors that affect Env/CD4/coreceptor interactions. We addressed the relation between T20 inhibition and the pathway by which R5X4 HIV-1 infects primary macrophages, which express both coreceptors. In U87/CD4/coreceptor cells, T20 sensitivity for entry through CCR5 and CXCR4 was correlated. In macrophages, the proportion of total entry mediated by each coreceptor differed among isolates. Neither pathway was uniformly more or less sensitive to T20, however, nor did the proportion of entry mediated by each coreceptor predict T20 sensitivity. T20 sensitivity for macrophage infection overall correlated modestly with that for entry through CCR5 but not through CXCR4; however, unlike U87 cells, sensitivity of entry through CCR5 and CXCR4 was not correlated. These results suggest that strain-specific factors influence R5X4 T20 sensitivity regardless of the coreceptor used, an absence of systematic differences in efficiency by which R5X4 strains use the 2 coreceptors, and that efficiency and kinetics of interactions with CCR5 are central determinants of macrophage entry even when both pathways are utilized.     

CD4+ NK cells can be productively infected with HIV, leading to downregulation of CD4 expression and changes in function

NK cells mediate the innate immune response, and HIV-infected individuals demonstrate altered NK cell phenotype and function. We find that CD4+ NK cells are susceptible to HIV infection; this could account for the NK cell dysfunction seen in HIV-infected individuals. CD4+ NK cells express CXCR4 and can be infected with X4-tropic viruses and some primary R5-utilizing viral isolates. Treatment with the CXCR4 ligands AMD3100 and SDF-1α partially blocks infection with X4-tropic virus, treatment with anti-CCL Igs upregulates CCR5 surface expression and enables infection with HIV-Bal. HIV infection of NK cells results in CD4 downregulation and the production of infectious virus. HIV-infected CD4+ NK cells mediate NK cell cytotoxicity, however, HIV infection is associated with decreased chemotaxis towards IL-16. Thus, HIV infection of CD4+ NK cells could account for the NK cell dysfunction observed in HIV-infected individuals. Furthermore infected NK cells could serve as a viral reservoir of HIV in vivo.     

Expression of chemokine receptors on CD4+ T cells in peripheral blood from HIV-infected individuals in Uganda.

CXCR4, a coreceptor for T cell (T)-tropic HIV-1, is preferentially expressed on naive T cells, whereas CCR5, a coreceptor for macrophage (M)-tropic HIV-1, is preferentially expressed on previously activated memory T cells and the Th1 subset of CD4+ T cells. CCR4 is preferentially expressed on the Th2 subset of CD4+ T cells. A cross-sectional flow cytometry study was conducted to evaluate the expression of CXCR4, CCR5, and CCR4 on the peripheral blood CD4+ T cells from African HIV-1-infected and uninfected Ugandan adults. The plasma viral load in HIV-1-infected individuals was also examined. Upregulation of CCR4 and CCR5 expression but no decrease in CXCR4 expression on CD4+ T cells were obtained in peripheral blood from African adults with progression of the disease. Plasma HIV-1 viremia significantly and inversely correlated with the peripheral CD4+ T cell count but did not correlate with the degree of CCR4 and CCR5 expression on the peripheral CD4+ T cells in HIV-1-infected individuals. Our present data suggest an increase in percentage of activated memory CD4+ T cells in the advanced stage of HIV-1 infection among African adults. There was no evidence of a Th1 to Th2 shift in terms of chemokine receptor expression profile with advancing disease in the peripheral blood of these subjects.     

HIV-1 gp120 chemokine receptor-mediated signaling in human macrophages

The chemokine receptors CCR5 and CXCR4 serve as the cellular receptors in conjunction with CD4 for HIV-1 entry and infection of target cells. Although the virus has subverted these molecules for its own use, their natural function is to respond to activation and migration signals delivered by extracellular chemokines. A principal research objective of our laboratory is to understand the consequences of virus-chemokine receptor interactions for cellular function, as well as for entry and infection. We hypothesized that CXCR4-using (X4) and CCR5-using (R5) HIV-1 strains might elicit signals through the chemokine receptors that result in aberrant function and/or regulate virus entry or postentry steps of infection. We have focused on primary human macrophages, which express both CXCR4 and CCR5, because macrophages are a principal target for HIV-1 in vivo, in appropriate macrophage activation appears to play a major role in the pathogenesis of certain sequelae of AIDS, such as HIV encephalopathy, and macrophage infection is regulated at several steps subsequent to entry in ways that are linked to envelope-receptor interactions. This review summarizes our recent findings regarding the mechanisms of chemokine-receptor signaling in macrophages, the role of viral envelope glycoproteins in eliciting macrophage signals, and how these activation pathways may participate in macrophage infection and affect cell functions apart from infection.     

Ezrin, Radixin, and Moesin (ERM) proteins function as pleiotropic regulators of human immunodeficiency virus type 1 infection

Ezrin, radixin, and moesin (ERM) proteins supply functional linkage between integral membrane proteins and cytoskeleton in mammalian cells to regulate membrane protein dynamisms and cytoskeleton rearrangement. To assess potential role of the ERM proteins in HIV-1 lifecycle, we examined if suppression of ERM function in human cells expressing HIV-1 infection receptors influences HIV-1 envelope (Env)-mediated HIV-1-vector transduction and cell-cell fusion. Expression of an ezrin dominant negative mutant or knockdown of ezrin, radixin, or moesin with siRNA uniformly decreased transduction titers of HIV-1 vectors having X4-tropic Env. In contrast, transduction titers of R5-tropic Env HIV-1 vectors were decreased only by radixin knockdown: ezrin knockdown had no detectable effects and moesin knockdown rather increased transduction titer. Each of the ERM suppressions had no detectable effects on cell surface expression of CD4, CCR5, and CXCR4 or VSV-Env-mediated HIV-1 vector transductions. Finally, the individual knockdown of ERM mRNAs uniformly decreased efficiency of cell-cell fusion mediated by X4- or R5-tropic Env and HIV-1 infection receptors. These results suggest that (i) the ERM proteins function as positive regulators of infection by X4-tropic HIV-1, (ii) moesin additionally functions as a negative regulator of R5-tropic HIV-1 virus infection at the early step(s) after the membrane fusion, and (iii) receptor protein dynamisms are regulated differently in R5- and X4-tropic HIV-1 infections.