A trans-receptor mechanism for infection of CD4-negative cells by human immunodeficiency virus type 1.

Chemokine receptors, particularly CCR5 and CXCR4, act as essential coreceptors in concert with CD4 for cellular entry by human immunodeficiency virus type 1 (HIV-1; reviewed in [1]). But infection of CD4(-) cells has also been encountered in various tissues in vivo, including astrocytes, neurons and microvascular endothelial cells of the brain [2] [3] [4] [5] [6], epithelial cells [5] [7], CD4(-) lymphocytes and thymocytes [8] [9], and cardiomyocytes [10]. Here, we present evidence for the infection of CD4(-) cell lines bearing coreceptors by well-known HIV-1 strains when co-cultured with CD4(+) cells. This process requires contact between the coreceptor-bearing and CD4(+) cells and supports the full viral replication cycle within the coreceptor-bearing target cell. Furthermore, CD4 provided in trans facilitates infection of primary human cells, such as brain-derived astrocytes. Although the pathobiological significance of infection of CD4(-) cells in vivo remains to be elucidated, this trans-receptor mechanism may facilitate generation of hidden reservoirs of latent virus that confound antiviral therapies and that contribute to specific AIDS-associated clinical syndromes.     

Chemically induced infection of CD4-negative HeLa cells with HIV-1

Infection with human immunodeficiency virus type-1 (HIV-1) requires the presence of a CD4 molecule and chemokine receptors such as CXCR4 or CCR5 on the surface of target cells. However, it is still not clear how the virus enters the cells. Although CD4 was initially identified as the primary receptor for HIV-1, the expression of CD4 or one of the chemokine receptors alone is not sufficient to render susceptibility to infection with the virus. To ascertain whether or not adsorption of the virus needs charge-to-charge interaction between viral envelope and host cell membrane protein(s) and if binding alone promotes penetration of the virus into the cells, we have developed a chemically induced infection system targeting a CD4-negative and CXCR4-positive HeLa cell clone (N7 HeLa) which is usually not susceptible to infection with the LAI strain of HIV-1. Use of a poly-L-lysine (PLL)-coated culture plate to enhance the attachment of the virus to the cells made N7 HeLa cells infectable with HIV-1 at very low efficiency. PLL alone cannot fully substitute for the function of the CD4 molecule. However, trypsin-treated viruses, which have largely lost infectivity to CD4-positive MT-4 cells that are highly susceptible to HIV-1 infection, enhanced infectivity against N7 HeLa cells when the PLL-coated plate was used. These results provide evidence that infection with HIV-1 requires both high binding affinity between viruses and cells, and then needs a modification of the viral envelope such as cleavage of gp120/160 to enhance the infection, probably resulting in exposure of the hydrophobic fusion domain of gp41. HIV-1 infection of N7 HeLa cells was also enhanced by treatment with low pH, 12-O-tetradecanoylphorbol-13-acetate (TPA) and some factor(s) from the MT-4 cell culture supernatant. Not only tight viral adsorption with cleavage of the viral envelope but also some activated status of the cells may be required for sufficient HIV-1 infection in this artificial condition.     

High level of coreceptor-independent HIV transfer induced by contacts between primary CD4 T cells

Cell-to-cell virus transmission is one of the most efficient mechanisms of human immunodeficiency virus (HIV) spread, requires CD4 and coreceptor expression in target cells, and may also lead to syncytium formation and cell death. Here, we show that in addition to this classical coreceptor-mediated transmission, the contact between HIV-producing cells and primary CD4 T cells lacking the appropriate coreceptor induced the uptake of HIV particles by target cells in the absence of membrane fusion or productive HIV replication. HIV uptake by CD4 T cells required cellular contacts mediated by the binding of gp120 to CD4 and intact actin cytoskeleton. HIV antigens taken up by CD4 T cells were rapidly endocytosed to trypsin-resistant compartments inducing a partial disappearance of CD4 molecules from the cell surface. Once the cellular contact was stopped, captured HIV were released as infectious particles. Electron microscopy revealed that HIV particles attached to the surface of target cells and accumulated in large (0.5-1.0 microm) intracellular vesicles containing 1-14 virions, without any evidence for massive clathrin-mediated HIV endocytosis. The capture of HIV particles into trypsin-resistant compartments required the availability of the gp120 binding site of CD4 but was independent of the intracytoplasmic tail of CD4. In conclusion, we describe a novel mechanism of HIV transmission, activated by the contact of infected and uninfected primary CD4 T cells, by which HIV could exploit CD4 T cells lacking the appropriate coreceptor as an itinerant virus reservoir.     

Dynamics of CD4+ T cells in HIV-1 infection

Mathematical modeling is becoming established in the immunologist's toolbox as a method to gain insight into the dynamics of the immune response and its components. No more so than in the case of the study of human immunodeficiency virus (HIV) infection, where earlier work on the viral dynamics brought significant advances in our understanding of HIV replication and evolution. Here, I review different areas of the study of the dynamics of CD4+ T cells in the setting of HIV, where modeling played important and diverse roles in helping us understand CD4+ T-cell homeostasis and the effect of HIV infection. As the experimental techniques become more accurate and quantitative, modeling should play a more important part in both experimental design and data analysis.     

CD4-derived synthetic peptide blocks the binding of HIV-1 GP120 to CD4-bearing cells and prevents HIV-1 infection

The T cell surface glycoprotein CD4 plays an important role in mediating cellular immunity and serves as the receptor for human immunodeficiency virus. In order to identify primary sequences within the CD4 molecule that may be involved in the binding of the HIV-I envelope, we synthesized various peptides corresponding to the V1, V2, V3, and V4 domains of CD4. We tested the ability of these peptides to block the binding of purified HIV-I gp120 to CD4+ human lymphoblastic leukemia cells (CEM) using fluorescence-activated cell sorting. One of these peptides, corresponding to CD4 amino acids (74-95), when preincubated with gp120, blocked its subsequent binding to CEM cells by 80%. A truncated form of this peptide (81-95), was found to be as efficient as the longer peptide (74-95) in inhibiting the binding of gp120 to CEM cells. The same peptide did not block the binding of OKT4A or Leu3A anti-CD4 monoclonal antibodies, which were previously shown to block HIV-I binding to CD4. The peptides were also tested for their ability to block HIV-I infection of a T cell line in vitro. Only CD4 peptide (74-95) and the shorter fragment (81-95) succeeded in protecting T cells against infection with different HIV-I strains. All the other peptides examined had no effect on gp120 binding to CEM cells and did not block syncytia formation. Goat polyclonal antibodies against the CD4 peptide (74-95) gave modest interference of gp120 binding to CEM cells. These data suggest that the CD4 region (74-95) participates in the CD4-mediated binding and/or internalization of HIV-I virion.     

Regions of the CD4 molecule not involved in virus binding or syncytia formation are required for HIV-1 infection of lymphocytes.

Cell surface-expressed CD4 binds to the envelope glycoprotein of HIV-1 and mediates syncytia formation through interacting with membrane expressed HIV-1 gp120. Further possible roles of the CD4 molecule in the process of cell infection by HIV-1 remain poorly understood. In our study we describe two mAb that recognize the V3/V4 domain of the CD4 molecule. Although these mAb do not inhibit gp120-CD4 binding or HIV-1-induced syncytia formation, they inhibit HIV-1 infection of human PBL. These findings suggest that discrete, definable domains of the CD4 molecule may be involved in interactions after HIV-1 envelope binding that lead to virus entry into the cell.     

Apoptosis of syncytia induced by the HIV-1-envelope glycoprotein complex: influence of cell shape and size

Cells stably transfected with a lymphotropic HIV-1 Env gene form syncytia when cocultured with CD4(+)CXCR4(+) cells. Heterokaryons then spontaneously undergo apoptosis, while manifesting signs of mitochondrial membrane pemeabilization as well as nuclear chromatin condensation. Modulation of cellular geometry was achieved by growing syncytia on self-assembled monolayers of terminally substituted alkanethiolates designed to control the adhesive properties of the substrates. Spreading of syncytia, induced by culturing them on small circular adhesive islets (diameter 5 microm), placed at a distance that cells can bridge (10 microm), inhibited spontaneous and staurosporin-induced signs of apoptosis, both at the mitochondrial and at the nuclear levels, and allowed for the generation of larger syncytia. Transient cell spreading conferred a memory of apoptosis inhibition which was conserved upon adoption of a conventional cell shape. Limiting syncytium size by culturing them on square-shaped planar adhesive islands of defined size (400 to 2500 microm(2)), separated by nonadhesive regions, enhanced the rate of apoptotic cell death, as indicated by an accelerated permeabilization of the outer mitochondrial membrane, loss of the mitochondrial inner transmembrane potential, and an increased frequency of nuclear apoptosis. In conclusion, external constraints on syncytial size and shape strongly modulate their propensity to undergo apoptosis.     

Penicillium marneffei-stimulated dendritic cells enhance HIV-1 trans-infection and promote viral infection by activating primary CD4+ T cells

Penicillium marneffei (P. marneffei) is considered an indicator pathogen of AIDS, and the endemicity and clinical features of P. marneffei have been described. While, how the co-infection of P. marneffei exacerbate deterioration of the immune response remains poorly understood. Here we isolated P. marneffei from the cutaneous lesions of AIDS patients and analyzed its effects on HIV-1-dendritic cells (DCs) interaction. We demonstrated that the monocyte-derived dendritic cells (MDDCs) could be activated by both thermally dimorphic forms of P. marneffei for significantly promoting HIV-1 trans-infection of CD4(+) T cells, while these activated MDDCs were refractory to HIV-1 infection. Mechanistically, P. marneffei-activated MDDCs endocytosed large amounts of HIV-1 and sequestrated the internalized viruses into tetrapasnin CD81(+) compartments potentially for proteolysis escaping. The activated MDDCs increased expression of intercellular adhesion molecule 1 and facilitated the formation of DC-T-cell conjunctions, where much more viruses were recruited. Moreover, we found that P. marneffei-stimulated MDDCs efficiently activated resting CD4(+) T cells and induced more susceptible targets for viral infection. Our findings demonstrate that DC function and its interaction with HIV-1 have been modulated by opportunistic pathogens such as P. marneffei for viral dissemination and infection amplification, highlighting the importance of understanding DC-HIV-1 interaction for viral immunopathogenesis elucidation.     

Effects of microtubule modulators on HIV-1 infection of transformed and resting CD4 T cells

Previous studies have observed fluorescently labeled HIV particles tracking along microtubule networks for nuclear localization. To provide direct evidence for the involvement of microtubules in early steps of HIV infection of human CD4 T cells, we used multiple microtubule modulators such as paclitaxel (originally called taxol; 1 μM), vinblastine (1 and 10 μM), colchicine (10 and 100 μM), and nocodazole (10 and 100 μM) to disturb microtubule networks in transformed and resting CD4 T cells. Although these drugs disrupted microtubule integrity, almost no inhibition of HIV-1 infection was observed. Our results do not appear to support an essential role for microtubules in the initiation of HIV infection of CD4 T cells.     

The leukocyte adhesion glycoprotein CD18 participates in HIV-1-induced syncytia formation in monocytoid and T cells

mAb 60.3 and IB4 to CD18, the common beta-subunit of the human leukocytic cell adhesion molecule family, efficiently inhibit syncytium formation induced by the interaction of HIV type 1 (HIV-1)-infected monocytoid cells and CD4+ T cells. The antibodies also interfere with cellfree HIV-1 infection of U-937 clone 16 cells. Virus-induced aggregation of these cells and the subsequent syncytia formation leading to massive cell death are efficiently blocked, and the number of infected cells remains at a very low level, 2 to 5%, for the entire culture period. However, anti-CD18 mAb do not inhibit binding of the viral envelope glycoprotein gp120 to the cell surface receptor CD4. The results indicate participation of CD18, or of the protein complex CD11a-c/CD18, in addition to CD4, in the infection and cytopathic effect of HIV-1. They also suggest that intercellular adhesion contributes to virus transmission from cell to cell and may be an important mechanism for virus spreading.     

CCL2 increases X4-tropic HIV-1 entry into resting CD4+ T cells

During human immunodeficiency virus type 1 (HIV-1) infection, there is a strong positive correlation between CCL2 levels and HIV viral load. To determine whether CCL2 alters HIV-1 infection of resting CD4(+) T cells, we infected purified resting CD4(+) T cells after incubation with CCL2. We show that CCL2 up-regulates CXCR4 on resting CD4(+) T cells in a CCR2-dependent mechanism, and that this augmentation of CXCR4 expression by CCL2 increases the ability of these cells to be chemoattracted to CXCR4 using gp120 and renders them more permissive to X4-tropic HIV-1 infection. Thus, CCL2 has the capacity to render a large population of lymphocytes more susceptible to HIV-1 late in the course of infection.     

HIV-1-specific mucosal CD8+ lymphocyte responses in the cervix of HIV-1-resistant prostitutes in Nairobi

Understanding how individuals with a high degree of HIV exposure avoid persistent infection is paramount to HIV vaccine design. Evidence suggests that mucosal immunity, particularly virus-specific CTL, could be critically important in protection against sexually acquired HIV infection. Therefore, we have looked for the presence of HIV-specific CD8+ T cells in cervical mononuclear cells from a subgroup of highly HIV-exposed but persistently seronegative female sex workers in Nairobi. An enzyme-linked immunospot assay was used to measure IFN-gamma release in response to known class I HLA-restricted CTL epitope peptides using effector cells from the blood and cervix of HIV-1-resistant and -infected sex workers and from lower-risk uninfected controls. Eleven of 16 resistant sex workers had HIV-specific CD8+ T cells in the cervix, and a similar number had detectable responses in blood. Where both blood and cervical responses were detected in the same individual, the specificity of the responses was similar. Neither cervical nor blood responses were detected in lower-risk control donors. HIV-specific CD8+ T cell frequencies in the cervix of HIV-resistant sex workers were slightly higher than in blood, while in HIV-infected donor cervical response frequencies were markedly lower than blood, so that there was relative enrichment of cervical responses in HIV-resistant compared with HIV-infected donors. HIV-specific CD8+ T cell responses in the absence of detectable HIV infection in the genital mucosa of HIV-1-resistant sex workers may be playing an important part in protective immunity against heterosexual HIV-1 transmission.     

A monoclonal antibody to CD4 domain 2 blocks soluble CD4-induced conformational changes in the envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) and HIV-1 infection of CD4+ cells.

The murine monoclonal antibody (MAb) 5A8, which is reactive with domain 2 of CD4, blocks human immunodeficiency virus type 1 (HIV-1) infection and syncytium formation of CD4+ cells (L. C. Burkly, D. Olson, R. Shapiro, G. Winkler, J. J. Rosa, D. W. Thomas, C. Williams, and P. Chisholm, J. Immunol., in press). Here we show that, in contrast to the CD4 domain 1 MAb 6H10, 5A8 and its Fab fragment do not block soluble CD4 (sCD4) binding to virions, whereas they do inhibit sCD4-induced exposure of cryptic epitopes on gp41 and dissociation of gp120 from virions. Two other MAbs, OKT4 and L120, which are reactive with domains 3 and 4 of CD4, have little or no effect on HIV-1 infection, syncytium formation, or sCD4-induced conformational changes in the envelope glycoproteins. The mechanisms of action of 5A8 and 6H10 can be further distinguished in syncytium inhibition assays: 6H10 blocks competitively, while 5A8 does not. We opine that 5A8 blocks HIV-1 infection and fusion by interfering with conformational changes in gp120/gp41 and/or CD4 that are necessary for virus-cell fusion.     

Type I interferon contributes to CD4+ T cell depletion induced by infection with HIV-1 in the human thymus

Persistent induction of type 1 interferon (IFN) is associated with human immunodeficiency virus type 1 (HIV-1) infection. We report here that the pathogenic HIV strain R3A (HIV-R3A) induced high levels of type 1 IFN, while the nonpathogenic HIV-R3B showed no significant induction in human fetal thymus organ culture (HFTOC). We found that IFN contributed to the depletion of human T cells by HIV-R3A in a fusion-independent fashion. The R3B recombinant with the R3A Env V1V2 domain (R3B/A-V1V2) was able to induce type 1 IFN, which contributed to the increased depletion of T cells. Therefore, type 1 IFN induction plays a significant role in HIV-induced T cell depletion in the human thymus.