Staphylococcal exotoxin superantigens induce human immunodeficiency virus type 1 expression in naturally infected CD4+ T cells.

A high proportion of Staphylococcus aureus strains of human origin produce one or more exotoxins. In vivo, these toxins may give rise to a variety of clinical syndromes. In vitro, staphylococcal exotoxins have been shown to bind both to human leukocyte antigen (HLA) class II molecules on antigen-presenting cells and to the T-cell receptors on large fractions of T cells. The result of this interaction may be proliferation of the T cells, T-cell anergy, or apoptosis, depending on several factors, including the state of the responding cells and the presence of accessory molecules. Using naturally infected peripheral blood mononuclear cells depleted of CD8+ T cells, we have shown that staphylococcal exotoxins are powerful inducers of human immunodeficiency virus type 1 expression and that they induce expression at low concentrations and with greater efficiency than other T-cell mitogens. Human immunodeficiency virus type 1 was produced entirely by CD4+ T cells in this model; monocytes were expendable both as a source of virus and as a source of HLA class II molecules as long as other cells expressing HLA class II molecules were present. The results suggest that infection by S. aureus may be a cofactor in the immunopathogenesis of AIDS.     

CD4-Negative cells bind human immunodeficiency virus type 1 and efficiently transfer virus to T cells

The ability of human immunodeficiency virus strain MN (HIV(MN)), a T-cell line-adapted strain of HIV, and X4 and R5 primary isolates to bind to various cell types was investigated. In general, HIV(MN) bound to cells at higher levels than did the primary isolates. Virus bound to both CD4-positive (CD4(+)) and CD4-negative (CD4(-)) cells, including neutrophils, Raji cells, tonsil mononuclear cells, erythrocytes, platelets, and peripheral blood mononuclear cells (PBMC), although virus bound at significantly higher levels to PBMC. However, there was no difference in the amount of HIV that bound to CD4-enriched or CD4-depleted PBMC. Virus bound to CD4(-) cells was up to 17 times more infectious for T cells in cocultures than was the same amount of cell-free virus. Virus bound to nucleated cells was significantly more infectious than virus bound to erythrocytes or platelets. The enhanced infection of T cells by virus bound to CD4(-) cells was not due to stimulatory signals provided by CD4(-) cells or infection of CD4(-) cells. However, anti-CD18 antibody substantially reduced the enhanced virus replication in T cells, suggesting that virus that bound to the surface of CD4(-) cells is efficiently passed to CD4(+) T cells during cell-cell adhesion. These studies show that HIV binds at relatively high levels to CD4(-) cells and, once bound, is highly infectious for T cells. This suggests that virus binding to the surface of CD4(-) cells is an important route for infection of T cells in vivo.     

Productive and lytic infection of human CD4+ type 1 helper T cells with macrophage-tropic human immunodeficiency virus type 1.

It is generally recognized that macrophage-tropic human immunodeficiency virus type 1 (HIV-1) is the predominant population during the acute and asymptomatic phases of HIV-1 infection. Here, we compared the proliferation and syncytium-inducing activities of different HIV-1 strains in primary CD4+ T cells expressing various helper T (Th)-type cytokine profiles. The macrophage-tropic HIV-1 strains HIV-1JR-CSF, HIV-1NFN-SX, and HIV-1SF162 could proliferate vigorously and generate syncytia in primary CD4+ T cells irrespective of their Th subtype, in contrast to the T-cell-line-tropic HIV-1 strains HIV-1NL4-3 and HIV-1IIIB, which favored non-type 1 Th conditions. These results indicate that macrophage-tropic HIV-1 may be more invasive and virulent, since it kills more CD4+ Th1 cells than T-cell-line-tropic HIV-1 during the early stages of HIV-1 infection, when the Th1 immune response is dominant.     

CD4-independent infection of human neural cells by human immunodeficiency virus type 1.

A number of studies have indicated that central nervous system-derived cells can be infected with human immunodeficiency virus type 1 (HIV-1). To determine whether CD4, the receptor for HIV-1 in lymphoid cells, was responsible for infection of neural cells, we characterized infectable human central nervous system tumor lines and primary fetal neural cells and did not detect either CD4 protein or mRNA. We then attempted to block infection with anti-CD4 antibodies known to block infection of lymphoid cells; we noted no effect on any of these cultured cells. The results indicate that CD4 is not the receptor for HIV-1 infection of the glioblastoma line U373-MG, medulloblastoma line MED 217, or primary human fetal neural cells.     

The Vif and Gag proteins of human immunodeficiency virus type 1 colocalize in infected human T cells.

The Vif protein of human immunodeficiency virus type 1 (HIV-1) and other lentiviruses is required for efficient replication in primary cells and certain immortalized cell lines in vitro and, in all likelihood, for the establishment of pathogenic infections in vivo. Current hypotheses concerning Vif's mechanism of action posit that it operates in virus-expressing cells during virion assembly, budding, or maturation such that released virions are modified in a manner that enables them to undergo productive infection in subsequent viral challenges. To gain further insight into the mechanism of action of lentivirus Vif proteins, we have performed a variety of in situ localization and biochemical fractionation studies using cells in which Vif is essential for efficient replication. Double-label immunofluorescence analyses of cells productively infected with HIV-1 or feline immunodeficiency virus revealed dramatic patterns of colocalization between Vif and the virally encoded Gag proteins. Subcellular fractionations of human T cells expressing HIV-1 Vif performed in the absence of any detergent demonstrated that greater than 90% of Vif is associated with cellular membranes. Additional purification using a continuous density gradient indicated that the majority of the membrane-bound Vif copurifies with the plasma membrane. Taken together, these observations suggest that lentivirus Vif and Gag proteins colocalize at the plasma membrane as virion assembly and budding take place. As a result, Vif is able to exert its modulatory effect(s) on these late steps of the virus life cycle.     

Inhibition of human immunodeficiency virus replication in acutely infected CD4+ cells by CD8+ cells involves a noncytotoxic mechanism.

The mechanism by which CD8+ T cells from human immunodeficiency virus (HIV)-infected individuals suppress HIV replication in acutely infected CD4+ T cells was investigated. Cytotoxicity was not involved, as the antiviral activity of the CD8+ cells did not correlate with the ability to lyse HIV-infected or uninfected CD4+ T cells. In addition, the frequency of HIV-infected CD4+ cells increased during coculture with CD8+ T cells even in the absence of detectable levels of virus replication. Moreover, separation of the CD4+ and CD8+ cells by a 0.4-micron-pore-size filter delayed HIV replication, indicating a role, at least in part, for a soluble factor. However, cell contact was required for optimal antiviral activity. These results extend further the observation on the mechanism of antiviral HIV activity by CD8+ cells from infected individuals. They support the conclusion that CD8+ cells can play a major role in preventing development of disease in HIV-infected individuals.     

Unintegrated human immunodeficiency virus type 1 DNA in chronically infected cell lines is not correlated with surface CD4 expression.

Using a polymerase chain reaction-based assay on total cell lysates, we have detected unintegrated human immunodeficiency virus type 1 (HIV-1) DNA in chronically infected T-lymphocytic (ACH-2, J1) and promyelocytic (OM-10.1) cell lines. Treatment with 3'-azido-3'-deoxythymidine (AZT) or soluble CD4 inhibited accumulation of unintegrated viral DNA about 10-fold within 72 h; removal of AZT permitted recovery to pretreatment levels within 72 h. Our results indicate that unintegrated HIV-1 DNA is unstable in these cell lines and originates from a continuous process of reinfection. OM-10.1 cells had relatively high levels of surface CD4 by flow cytometry and high levels of unintegrated viral DNA by polymerase chain reaction. ACH-2 cells had very low levels of both surface CD4 and unintegrated viral DNA. However, J1 cells, with surface CD4 below the level of detection of flow cytometry had a high level of unintegrated viral DNA similar to that of OM-10.1 cells. This implies that the number of CD4 receptors is not rate limiting for reinfection.     

Inhibition of human immunodeficiency virus type 1 replication in primary CD4(+) T lymphocytes, monocytes, and dendritic cells by cytotoxic T lymphocytes

We demonstrate that human immunodeficiency virus type 1 (HIV-1)-specific CD8(+) cytotoxic T lymphocytes (CTL) suppress HIV-1 replication in primary lymphocytes, monocytes, and dendritic cells individually. Viral inhibition is significantly diminished in lymphocyte-dendritic cell clusters, suggesting that these clusters in vivo could be sites where viral replication is more difficult to control by CTL.     

Inhibition of human immunodeficiency virus type 1 gp120 presentation to CD4 T cells by antibodies specific for the CD4 binding domain of gp120

Human immunodeficiency virus (HIV)-specific CD4 T-cell responses, particularly to the envelope glycoproteins of the virus, are weak or absent in most HIV-infected patients. Although these poor responses can be attributed simply to the destruction of the specific CD4 T cells by the virus, other factors also appear to contribute to the suppression of these virus-specific responses. We previously showed that human monoclonal antibodies (MAbs) specific for the CD4 binding domain of gp120 (gp120(CD4BD)), when complexed with gp120, inhibited the proliferative responses of gp120-specific CD4 T-cells. MAbs to other gp120 epitopes did not exhibit this activity. The present study investigated the inhibitory mechanisms of the anti-gp120(CD4BD) MAbs. The anti-gp120(CD4BD) MAbs complexed with gp120 suppressed gamma interferon production as well as proliferation of gp120-specific CD4 T cells. Notably, the T-cell responses to gp120 were inhibited only when the MAbs were added to antigen-presenting cells (APCs) during antigen pulse; the addition of the MAbs after pulsing caused no inhibition. However, the anti-gp120(CD4BD) MAbs by themselves, or as MAb/gp120 complexes, did not affect the presentation of gp120-derived peptides by the APCs to T cells. These MAb/gp120 complexes also did not inhibit the ability of APCs to process and present unrelated antigens. To test whether the suppressive effect of anti-gp120(CD4BD) antibodies is caused by the antibodies' ability to block gp120-CD4 interaction, APCs were treated during antigen pulse with anti-CD4 MAbs. These treated APCs remained capable of presenting gp120 to the T cells. These results suggest that anti-gp120(CD4BD) Abs inhibit gp120 presentation by altering the uptake and/or processing of gp120 by the APCs but their inhibitory activity is not due to blocking of gp120 attachment to CD4 on the surface of APCs.     

Compartmentalization of human immunodeficiency virus type 1 between blood monocytes and CD4+ T cells during infection

Distinct sequences of human immunodeficiency virus type 1 (HIV-1) have been found between different tissue compartments or subcompartments within a given tissue. Whether such compartmentalization of HIV-1 occurs between different cell populations is still unknown. Here we address this issue by comparing HIV-1 sequences in the second constant region through the fifth hypervariable region (C2 to V5) of the surface envelope glycoprotein (Env) between viruses in purified blood CD14(+) monocytes and CD4(+) T cells obtained longitudinally from five infected patients over a time period ranging from 117 to 3,409 days postseroconversion. Viral populations in both cell types at early infection time points appeared relatively homogeneous. However, later in infections, all five patients showed heterogeneous populations in both CD14(+) monocytes and CD4(+) T cells. Three of the five patients had CD14(+) monocyte populations with significantly more genetic diversity than the CD4(+) T-cell population, while the other two patients had more genetic diversity in CD4(+) T cells. The cellular compartmentalization of HIV-1 between CD14(+) monocytes and CD4(+) T cells was not seen early during infections but was evident at the later time points for all five patients, indicating an association of viral compartmentalization with the time course of HIV-1 infection. The majority of HIV-1 V3 sequences indicated a macrophage-tropic phenotype, while a V3 sequence-predicted T-cell tropic virus was found in the CD4(+) T cells and CD14(+) monocytes of two patients. These findings suggest that HIV-1 in CD14(+) monocytes could disseminate and evolve independently from that in CD4(+) T cells over the course of HIV-1 infection, which may have implications on the development of new therapeutic strategies.     

Role of vif in replication of human immunodeficiency virus type 1 in CD4+ T lymphocytes.

The viral infectivity factor gene vif of human immunodeficiency virus type 1 has been shown to affect the infectivity but not the production of virus particles. In this study, the effect of vif in the context of the HXB2 virus on virus replication in several CD4+ T-cell lines was investigated. vif was found to be required for replication in the CD4+ T-cell lines CEM and H9 as well as in peripheral blood T lymphocytes. vif was not required for replication in the SupT1, C8166, and Jurkat T-cell lines. The infectivity of vif-defective viruses depended on the cell type in which the virus was produced. In CEM cells, vif was required for production of virus capable of initiating infection in all cell lines studied. vif-defective virus produced by SupT1, C8166, and Jurkat cells and the monkey cell line COS-1 could initiate infection in multiple cell lines, including CEM and H9. These results suggest that vif can compensate for cellular factors required for production of infectious virus particles that are present in some cell lines such as SupT1, C8166, and Jurkat but are absent in others such as CEM and H9 as well as peripheral blood T lymphocytes. The effect of vif was not altered by deletion of the carboxyl terminus of gp41, a proposed target for vif (B. Guy, M. Geist, K. Dott, D. Spehner, M.-P. Kieny, and J.-P. Lecocq, J. Virol. 65:1325-1331, 1991). These studies demonstrate that vif enhances viral infectivity during virus production and also suggest that vif is likely to be important for natural infections.     

Apoptosis induced in CD4+ cells expressing gp160 of human immunodeficiency virus type 1.

In a previous study (Y. Koga, M. Sasaki, H. Yoshida, H. Wigzell, G. Kimura, and K. Nomoto, J. Immunol. 144:94-102, 1990), we demonstrated that the expression of gp160, a precursor form of envelope glycoprotein of human immunodeficiency virus type 1, in CD4+ cells causes the downregulation of surface CD4 and single-cell killing by forming intracellular gp160-CD4 complex. In the present study we investigated the events that lead to cell death in CD4+ cells expressing gp160. We found that apoptosis is induced in cells undergoing single-cell death. Moreover, even the cell clone, which expresses so little gp160 that it does not exhibit any apparent cytopathic effects, such as the inhibition of cell growth, was found to be highly susceptible to the apoptosis induction by the anti-Fas monoclonal antibody.     

Virus-specific interleukin-17-producing CD4+ T cells are detectable in early human immunodeficiency virus type 1 infection

T(H)-17 cells have been shown to play a role in bacterial defense, acute inflammation, and autoimmunity. We examined the role of interleukin 17 (IL-17) production in human immunodeficiency virus type 1 (HIV-1) infection. Both HIV-1- and cytomegalovirus (CMV)-specific IL-17-producing CD4(+) T cells were detectable in early HIV-1 infection but were reduced to nondetectable levels in chronic and nonprogressive HIV-1 infection. IL-17-producing CMV-specific cells were not detected in blood from HIV-1-uninfected normal volunteers. Virus-specific T(H)-17 cells could coexpress other cytokines and could express CCR4 or CXCR3. Although the etiology of these cells has yet to be established, we propose that microbial translocation may induce them.     

Vpr cytopathicity independent of G2/M cell cycle arrest in human immunodeficiency virus type 1-infected CD4+ T cells

The mechanism of CD4(+) T-cell depletion in human immunodeficiency virus type 1 (HIV-1)-infected individuals remains unknown, although mounting evidence suggests that direct viral cytopathicity contributes to this loss. The HIV-1 Vpr accessory protein causes cell death and arrests cells in the G(2)/M phase; however, the molecular mechanism underlying these properties is not clear. Mutation of hydrophobic residues on the surface of its third alpha-helix disrupted Vpr toxicity, G(2)/M arrest induction, nuclear localization, and self-association, implicating this region in multiple Vpr functions. Cytopathicity by virion-delivered mutant Vpr protein correlated with G(2)/M arrest induction but not nuclear localization or self-association. However, infection with whole virus encoding these Vpr mutants did not abrogate HIV-1-induced cell killing. Rather, mutant Vpr proteins that are impaired for G(2)/M block still prevented infected cell proliferation, and this property correlated with the death of infected cells. Chemical agents that inhibit infected cells from entering G(2)/M also did not reduce HIV-1 cytopathicity. Combined, these data implicate Vpr in HIV-1 killing through a mechanism involving inhibiting cell division but not necessarily in G(2)/M. Thus, the hydrophobic region of the third alpha-helix of Vpr is crucial for mediating G(2)/M arrest, nuclear localization, and self-association but dispensable for HIV-1 cytopathicity due to residual cell proliferation blockade mediated by a separate region of the protein.