Presence of host ICAM-1 in human immunodeficiency virus type 1 virions increases productive infection of CD4+ T lymphocytes by favoring cytosolic delivery of viral material

Although there is now convincing evidence that the infectivity of human immunodeficiency virus type 1 (HIV-1) is increased by incorporation of host intercellular adhesion molecule 1 (ICAM-1) in budding virions, the exact mechanism(s) through which ICAM-1 can so significantly affect HIV-1 biology remains obscure. To address this question, we focused our attention on the most proximal events in the virus life cycle. We made comparative analyses to estimate attachment and internalization of isogenic HIV-1 particles either lacking or bearing host-derived ICAM-1. Using attachment-and-entry assays and confocal fluorescence microscopy, we found that virus binding and uptake were both markedly enhanced by insertion of ICAM-1 within the virus envelope when PM1 lymphoid cells and primary human cells (i.e., peripheral blood lymphocytes and purified CD4(+) T cells) were used as targets. Moreover, ICAM-1-bearing virions entered cells with faster uptake kinetics than viruses devoid of ICAM-1. Experiments conducted with fully competent viruses further confirmed the positive effect of virion-anchored host ICAM-1 on HIV-1 replication. Interestingly, subcellular-fractionation assays revealed that ICAM-1 incorporation modifies the HIV-1 entry route by increasing the level of viral material released in the cytosol, a process of internalization known to be mediated mainly by pH-independent membrane fusion and to result in productive infection. A virion-based fusion assay confirmed that the acquisition of ICAM-1 increases the efficiency of productive HIV-1 entry in primary CD4(+) T lymphocytes. These observations provide new insights into how interactions other than those with gp120 and CD4-coreceptor complex can modulate the process of productive HIV-1 infection in CD4(+) T lymphocytes, a cell target highly relevant to HIV-1 pathogenesis.     

Involvement of Fc gamma RI (CD64) in the mechanism of HIV-1 inhibition by polyclonal IgG purified from infected patients in cultured monocyte-derived macrophages

The aim of this study was to investigate the mechanism of HIV-1 neutralization using monocyte-derived macrophages (MDM) in comparison to PBMC as target cells. For this purpose, we analyzed neutralizing activities of different human polyclonal IgG samples purified from sera of HIV-1-infected individuals using a single cycle infection assay. We found an increase of the neutralizing titer when macrophages vs PBMC were used as target cells. Moreover, polyclonal IgG from HIV-1-infected patients that are not able to neutralize virus when PBMC are used as target cells strongly inhibit MDM infection. Similar results were obtained with neutralizing mAbs. To explore the participation of FcgammaRs in HIV-1 inhibition, F(ab')(2) and Fab of these Igs were produced. Results indicated that both F(ab')(2) and Fab are less effective to inhibit virus replication in MDM. Moreover, competition experiments with Fc fragments of IgG from healthy donors or with purified monoclonal anti-human FcgammaRs Ab strengthen the participation of the FcgammaRs, and in particular of FcgammaRI (CD64) in HIV-1 inhibition on MDM. Mechanisms by which HIV-specific IgG inhibit virus replication in cultured macrophages are proposed and the benefit of inducing such Abs by vaccination is discussed.     

In vitro analysis of complement-dependent HIV-1 cell infection using a model system

Previous studies based on the use of human serum as a source of C have provided evidence for the C-dependent enhancement of cell infection by HIV-1. The present study was undertaken to distinguish C from other serum factors and to identify the proteins and the mechanisms involved in C-dependent cell infection by HIV-1. The classical C activation pathway was reconstituted from the proteins C1q, C1r, C1s, C4, C2, C3, factor H, and factor I; each were purified to homogeneity. A mixture of these proteins at physiological concentrations was shown to reproduce the ability of normal human serum to enhance the infection of MT2 cells by HIV-1 at low doses of virus. This enhancing effect was abolished when heat-inactivated serum and C2- or C3-depleted serum were used, and was restored upon addition of the corresponding purified proteins. A mixture of two synthetic peptides corresponding to positions 10-15 and 90-97 of human C receptor type 2 (CD21) as well as soluble CD4 both inhibited the C-dependent infection process. These data provide unambiguous evidence that HIV-1 triggers a direct activation of the classical C pathway in vitro and thereby facilitates the infection of MT2 cells at low doses of virus. These findings are consistent with a mechanism involving increased interaction between the virus opsonized by C3b-derived fragment(s) and the CD21 cell receptors and subsequent virus entry through CD4 receptors.     

Human immunodeficiency virus type 1 replication in dendritic cell-T-cell cocultures is increased upon incorporation of host LFA-1 due to higher levels of virus production in immature dendritic cells

Dendritic cells (DCs) act as a portal for invasion by human immunodeficiency virus type-1 (HIV-1). Here, we investigated whether virion-incorporated host cell membrane proteins can affect virus replication in DC-T-cell cocultures. Using isogenic viruses either devoid of or bearing host-derived leukocyte function-associated antigen 1 (LFA-1), we showed that HIV-1 production is augmented when LFA-1-bearing virions are used compared to that for viral entities lacking this adhesion molecule. This phenomenon was observed in immature monocyte-derived DCs (IM-MDDCs) only and not in DCs displaying a mature phenotype. The increase is not due to higher virus production in responder CD4(+) T cells but rather is linked with a more important productive infection of IM-MDDCs. We provided evidence that virus-associated host LFA-1 molecules do not affect a late event in the HIV-1 life cycle but rather exert an effect on an early step in virus replication. We demonstrated that the enhancement of productive infection of IM-MDDCs that is conferred by virus-anchored host LFA-1 involves the protein kinase A (PKA) and PKC signal transduction pathways. The biological significance of this phenomenon was established by performing experiments with virus stocks produced in primary human cells and anti-LFA-1 antibodies. Together, our results indicate that the association between some virus-bound host proteins and their natural cognate ligands can modulate de novo HIV-1 production by IM-MDDCs. Therefore, the additional interactions between virus-bound host cell membrane constituents and counter receptors on the surfaces of DCs can influence HIV-1 replication in IM-MDDC-T-cell cocultures.     

Host-derived ICAM-1 glycoproteins incorporated on human immunodeficiency virus type 1 are biologically active and enhance viral infectivity.

Human immunodeficiency virus type 1 (HIV-1) acquires several host cell membrane proteins when it buds from infected cells. To study the effect of virally incorporated host-derived ICAM-1 glycoproteins on the biology of HIV-1, we have developed a transient expression system that has enabled us to produce virus particles differing only in the absence or the presence of virion-bound ICAM-1. By using a single-round infection assay based on an ICAM-1-negative target T-cell line stably transfected with an HIV-1 long terminal repeat driven luciferase gene construct, we have been able to demonstrate that the acquisition of host-derived ICAM-1 by HIV-1 has functional significance, since it leads to a pronounced increase in viral infectivity (4.6- to 9.8-fold) in an ICAM-1/LFA-1-dependent fashion, as shown by blocking with anti-ICAM-1 and -LFA-1 antibodies. The same potentiating effect on viral infectivity was also observed with monocytoid cells. Studies of the kinetics of infection revealed that the positive effect mediated by virally embedded host cell membrane ICAM-1 is due to an increase in the efficiency of early steps in the viral life cycle. These results provide new insights into how incorporation of host proteins can modulate the biological properties of HIV-1. Our findings have direct clinical relevance, considering that ICAM-1 is expressed on the surface of virus-infected cells and, more importantly, that host-derived ICAM-1 has been shown to be acquired by clinical HIV-1 isolates grown on primary mononuclear cells. These data justify a more complete analysis of the other putative role(s) that virally incorporated ICAM-1 may play in the life cycle of HIV-1, for example, at the level of neutralization sensitivity.     

Active cAMP-dependent protein kinase incorporated within highly purified HIV-1 particles is required for viral infectivity and interacts with viral capsid protein

Host cell components, including protein kinases such as ERK-2/mitogen-activated protein kinase, incorporated within human immunodeficiency virus type 1 (HIV-1) virions play a pivotal role in the ability of HIV to infect and replicate in permissive cells. The present work provides evidence that the catalytic subunit of cAMP-dependent protein kinase (C-PKA) is packaged within HIV-1 virions as demonstrated using purified subtilisin-digested viral particles. Virus-associated C-PKA was shown to be enzymatically active and able to phosphorylate synthetic substrate in vitro. Suppression of virion-associated C-PKA activity by specific synthetic inhibitor had no apparent effect on viral precursor maturation and virus assembly. However, virus-associated C-PKA activity was demonstrated to regulate HIV-1 infectivity as assessed by single round infection assays performed by using viruses produced from cells expressing an inactive form of C-PKA. In addition, virus-associated C-PKA was found to co-precipitate with and to phosphorylate the CAp24gag protein. Altogether our results indicate that virus-associated C-PKA regulates HIV-1 infectivity, possibly by catalyzing phosphorylation of the viral CAp24gag protein.     

Nef enhances human immunodeficiency virus type 1 infectivity resulting from intervirion fusion: evidence supporting a role for Nef at the virion envelope

The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef stimulates viral infectivity by facilitating an early event in the HIV-1 life cycle. Although no structural or biochemical defects in Nef-defective HIV-1 particles have been demonstrated, the Nef protein is incorporated into HIV-1 particles. To localize the function of Nef within the virus particle, we developed a novel technology involving fusion of enveloped donor HIV-1 particles bearing core defects with envelope-defective target virions bearing HIV-1 receptors. Although neither virus alone was capable of infecting CD4(+) target cells, the incubation of donor and target virions prior to addition to target cells resulted in infection. This effect, termed "virion transcomplementation," required a functional Env protein on the donor virus and CD4 and an appropriate coreceptor on target virions. To provide evidence for intervirion fusion as the mechanism of complementation, experiments were performed using dual-enveloped HIV-1 particles bearing both HIV-1 and ecotropic murine leukemia virus (E-MLV) Env proteins as donor virions. Infection of CD4-negative target cells bearing E-MLV receptors was prevented by HIV-1 entry inhibitors when added before, but not after, incubation of donor and target virions prior to the addition to cells. When we used Nef(+) and Nef(-) donor and target virions, Nef enhanced infection when present in donor virions. In contrast, no effect of Nef was detected when present in the target virus. These results reveal a potential mechanism for enhancing HIV-1 diversity in vivo through the rescue of defective viral genomes and provide a novel genetic system for the functional analysis of virion-associated proteins in HIV-1 infection.     

Use of a combined ex vivo/in vivo population approach for screening of human genes involved in the human immunodeficiency virus type 1 life cycle for variants influencing disease progression

Humans differ substantially with respect to susceptibility to human immunodeficiency virus type 1 (HIV-1). We evaluated variants of nine host genes participating in the viral life cycle for their role in modulating HIV-1 infection. Alleles were assessed ex vivo for their impact on viral replication in purified CD4 T cells from healthy blood donors (n = 128). Thereafter, candidate alleles were assessed in vivo in a cohort of HIV-1-infected individuals (n = 851) not receiving potent antiretroviral therapy. As a benchmark test, we tested 12 previously reported host genetic variants influencing HIV-1 infection as well as single nucleotide polymorphisms in the nine candidate genes. This led to the proposition of three alleles of PML, TSG101, and PPIA as potentially associated with differences in progression of HIV-1 disease. In a model considering the combined effects of new and previously reported gene variants, we estimated that their effect might be responsible for lengthening or shortening by up to 2.8 years the period from 500 CD4 T cells/mul to <200 CD4 T cells/mul.     

Interactions between human immunodeficiency virus type 1 and human cytomegalovirus in human term syncytiotrophoblast cells coinfected with both viruses.

Human cytomegalovirus (HCMV) and human immunodeficiency virus type 1 (HIV-1) may interact in the pathogenesis of AIDS. The placental syncytiotrophoblast layer serves as the first line of defense of the fetus against viruses. We analyzed the patterns of replication of HIV-1 and HCMV in singly an dually infected human term syncytiotrophoblast cells cultured in vitro. Syncytiotrophoblast cells exhibited restricted permissiveness for HIV-1, while HCMV replication was restricted at the level of immediate-early and early gene products in the singly infected cells. We found that the syncytiotrophoblasts as an overlapping cell population could be coinfected with HIV-1 and HCMV. HIV-1 replication was markedly upregulated by previous or simultaneous infection of the cells with HCMV, whereas prior HIV-1 infection of the cells converted HCMV infection from a nonpermissive to a permissive one. No simultaneous enhancement of HCMV and HIV-1 expression was observed in the dually infected cell cultures. Major immediate-early proteins of HCMV were necessary for enhancement of HIV-1 replication, and interleukin-6 production induced by HCMV and further increased by replicating HIV-1 synergized with these proteins to produce this effect. Permissive replication cycle of HCMV was induced by the HIV-1 tat gene product. We were unable to detect HIV-1 (HCMV) or HCMV (HIV-1) pseudotypes in supernatant fluids from dually infected cell cultures. Our results suggest that interactions between HIV-1 and HCMV in coinfected syncytiotrophoblast cells may contribute to the transplacental transmission of both viruses.     

Enhancement of Human Immunodeficiency Virus Type 1 (HIV-1) Infection via Increased Membrane Fluidity by a Cationic Polymer

Cationic polymers are known to have potent activity against bacteria, but their effects on viral activity have been little studied. We investigated the effect of one such polymer, polyethyleneimine (PEI), on HIV-1 infection. Although virus-cell binding was significantly inhibited by PEI, HIV-1 infection in human T-cell lines such as MT-4 and MOLT-4 was accelerated conversely when the drug treatment was carried out, after the virus had attached to the cells or PEI was simultaneously added to the virus and cell culture system. This paradoxical effect of PEI on HIV-1 infection was examined using HIV-1 chronically infected cells (MOLT-4/HIV-1). Dissociation of the glycoprotein gp120 (as revealed by exposure of transmembrane protein gp41) from MOLT-4/HIV-1 cells and the resultant fusion of these cells was shown to be induced by the addition of PEI. Accordingly, it was suggested that the binding inhibition of HIV-1 to CD4-positive cells by PEI was due to the shedding of gp120 from HIV-1 particles, and this PEI rather promoted membrane fusion between the virus and cells leading to the enhancement of HIV-1 infection. Similarly, dissociation of gp120 from MOLT-4/HIV-1 was also induced by sCD4. The effect of these reagents on changes in membrane fluidity was evaluated by polarization (p) measurements, and it was observed that the acceleration of membrane fluidity occurred only in the PEI system. Therefore, it is likely that PEI accelerates HIV-1 infection by facilitating virus entry into the host cells through an increase in membrane fluidity.     

Neutralizing Monoclonal Antibodies Block Human Immunodeficiency Virus Type 1 Infection of Dendritic Cells and Transmission to T Cells

Prevention of the initial infection of mucosal dendritic cells (DC) and interruption of the subsequent transmission of HIV-1 from DC to T cells are likely to be important attributes of an effective human immunodeficiency virus type 1 (HIV-1) vaccine. While anti-HIV-1 neutralizing antibodies have been difficult to elicit by immunization, there are several human monoclonal antibodies (MAbs) that effectively neutralize virus infection of activated T cells. We investigated the ability of three well-characterized neutralizing MAbs (IgG1b12, 2F5, and 2G12) to block HIV-1 infection of human DC. DC were generated from CD14⁺ blood cells or obtained from cadaveric human skin. The MAbs prevented viral entry into purified DC and the ensuing productive infection in DC/T-cell cultures. When DC were first pulsed with HIV-1, MAbs blocked the subsequent transmission to unstimulated CD3⁺ T cells. Thus, neutralizing antibodies can block HIV-1 infection of DC and the cell-to-cell transmission of virus from infected DC to T cells. These data suggest that neutralizing antibodies could interrupt the initial events associated with mucosal transmission and regional spread of HIV-1.     

Novel activities of cyclophilin A and cyclosporin A during HIV-1 infection of primary lymphocytes and macrophages

Studies conducted in cell lines indicate that cyclophilin A (CypA) is a component of HIV type 1 (HIV-1) virions, and that when CypA incorporation into virions is inhibited by treatment of infected cells with the immunosuppressive agent cyclosporin A (CsA), HIV-1 infection also is inhibited. Because HIV-1 particles assemble along a different pathway and incorporate different host proteins in macrophages than in other cell types, we investigated CypA and CsA activities in HIV-1-infected primary human macrophages, compared with primary human lymphocytes. We tested virus protein production, virion composition and infectivity, and progress through the virus life cycle under perturbation by drug treatment or mutagenesis in infected cells from multiple donors. Our findings from both primary cell types are different from that previously reported in transformed cells and show that the amount of CypA incorporated into virions is variable and that CsA inhibits HIV-1 infection at both early and late phases of virus replication, the stage affected is determined by the sequence of HIV-1 Gag. Because the cell type infected determines the identity of host proteins active in HIV-1 replication and can influence the activity of some viral inhibitors, infection of transformed cells may not recapitulate infection of the native targets of HIV-1.