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.     

Nef-mediated downregulation of CD4 enhances human immunodeficiency virus type 1 replication in primary T lymphocytes

The accessory protein Nef plays a crucial role in primate lentivirus pathogenesis. Nef enhances human immunodeficiency virus type 1 (HIV-1) infectivity in culture and stimulates viral replication in primary T cells. In this study, we investigated the relationship between HIV-1 replication efficiency in CD4(+) T cells purified from human blood and two various known activities of Nef, CD4 downregulation and single-cycle infectivity enhancement. Using a battery of reporter viruses containing point mutations in nef, we observed a strong genetic correlation between CD4 downregulation by Nef during acute HIV-1 infection of activated T cells and HIV-1 replication efficiency in T cells. In contrast, HIV-1 replication ability was not significantly correlated with the ability of Nef to enhance single-cycle virion infectivity, as determined by using viruses produced in cells lacking CD4. These results demonstrate that CD4 downregulation by Nef plays a crucial role in HIV-1 replication in activated T cells and underscore the potential for the development of therapies targeting this conserved activity of Nef.     

Human effector memory CD4+ T cells directly recognize allogeneic endothelial cells in vitro and in vivo

The frequency of circulating alloreactive human memory T cells correlates with allograft rejection. Memory T cells may be divided into effector memory (T(EM)) and central memory (T(CM)) cell subsets, but their specific roles in allograft rejection are unknown. We report that CD4+ T(EM) (CD45RO+ CCR7- CD62L-) can be adoptively transferred readily into C.B-17 SCID/bg mice and mediate the destruction of human endothelial cells (EC) in vascularized human skin grafts allogeneic to the T cell donor. In contrast, CD4+ T(CM) (CD45RO+ CCR7+ CD62L+) are inefficiently transferred and do not mediate EC injury. In vitro, CD4+ T(EM) secrete more IFN-gamma within 48 h in response to allogeneic ECs than do T(CM). In contrast, T(EM) and T(CM) secrete comparable amounts of IFN-gamma in response to allogeneic monocytes (Mo). In the same cultures, both T(EM) and T(CM) produce IL-2 and proliferate in response to IFN-gamma-treated allogeneic human EC or Mo, but T(CM) respond more vigorously in both assays. Blockade of LFA-3 strongly inhibits both IL-2 and IFN-gamma secretion by CD4+ T(EM) cultured with allogeneic EC but only minimally inhibits responses to allogeneic Mo. Blockade of CD80 and CD86 strongly inhibits IL-2 but not IFN-gamma production by in response to allogeneic EC or Mo. Transduction of EC to express B7-2 enhances allogeneic T(EM) production of IL-2 but not IFN-gamma. We conclude that human CD4+ T(EM) directly recognize and respond to allogeneic EC in vitro by secreting IFN-gamma and that this response depends on CD2 but not CD28. Consistent with EC activation of effector functions, human CD4+ T(EM) can mediate allogeneic EC injury 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-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.     

Human immunodeficiency virus type 1 Vpr induces DNA replication stress in vitro and in vivo

The human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) causes cell cycle arrest in G2. Vpr-expressing cells display the hallmarks of certain forms of DNA damage, specifically activation of the ataxia telangiectasia mutated and Rad3-related kinase, ATR. However, evidence that Vpr function is relevant in vivo or in the context of viral infection is still lacking. In the present study, we demonstrate that HIV-1 infection of primary, human CD4+ lymphocytes causes G2 arrest in a Vpr-dependent manner and that this response requires ATR, as shown by RNA interference. The event leading to ATR activation in CD4+ lymphocytes is the accumulation of replication protein A in nuclear foci, an indication that Vpr likely induces stalling of replication forks. Primary macrophages are refractory to ATR activation by Vpr, a finding that is consistent with the lack of detectable ATR, Rad17, and Chk1 protein expression in these nondividing cells. These observations begin to explain the remarkable resilience of macrophages to HIV-1-induced cytopathicity. To study the in vivo consequences of Vpr function, we isolated CD4+ lymphocytes from HIV-1-infected individuals and interrogated the cell cycle status of anti-p24Gag-immunoreactive cells. We report that infected cells in vivo display an aberrant cell cycle profile whereby a majority of cells have a 4N DNA content, consistent with the onset of G2 arrest.     

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.     

Human CD4+ CD25+ regulatory T cells control T-cell responses to human immunodeficiency virus and cytomegalovirus antigens

Regulatory T (T(R)) cells maintain tolerance to self-antigens and control immune responses to alloantigens after organ transplantation. Here, we show that CD4(+) CD25(+) human T(R) cells suppress virus-specific T-cell responses. Depletion of T(R) cells from peripheral blood mononuclear cells enhances T-cell responses to cytomegalovirus and human immunodeficiency virus antigens. We propose that chronic viral infections lead to induction of suppressive T(R) cells that inhibit the antiviral immune response.     

Human immunodeficiency virus type 1 bound to B cells: relationship to virus replicating in CD4+ T cells and circulating in plasma

Human immunodeficiency virus type 1 (HIV-1) virions bind to B cells in the peripheral blood and lymph nodes through interactions between CD21 on B cells and complement-complexed virions. B-cell-bound virions have been shown to be highly infectious, suggesting a unique mode of HIV-1 dissemination by B cells circulating between peripheral blood and lymphoid tissues. In order to investigate the relationship between B-cell-bound HIV-1 and viruses found in CD4+ T cells and in plasma, we examined the genetic relationships of HIV-1 found in the blood and lymph nodes of chronically infected patients with heteroduplex mobility and tracking assays and DNA sequence analysis. In samples from 13 of 15 patients examined, HIV-1 variants in peripheral blood-derived B cells were closely related to virus in CD4+ T cells and more divergent from virus in plasma. In samples from five chronically viremic patients for whom analyses were extended to include lymph node-derived HIV-1 isolates, B-cell-associated HIV-1 and CD4+-T-cell-associated HIV-1 in the lymph nodes were equivalent in their divergence from virus in peripheral blood-derived B cells and generally more distantly related to virus in peripheral blood-derived CD4+ T cells. These results indicates virologic cross talk between B cells and CD4+ T cells within the microenvironment of lymphoid tissues and, to a lesser extent, between cells in lymph nodes and the peripheral blood. These findings also indicate that most of the virus in plasma originates from cells other than CD4(+) T cells in the peripheral blood and lymph nodes.     

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.     

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.     

Human seminal plasma abrogates the capture and transmission of human immunodeficiency virus type 1 to CD4+ T cells mediated by DC-SIGN

Dendritic cell-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is expressed by dendritic cells (DCs) at mucosal surfaces and appears to play an important role in the dissemination of human immunodeficiency virus type 1 (HIV-1) infection. DC-SIGN binds HIV-1 gp120 and efficiently transmits the virus to T CD4⁺ cells, which become the center of viral replication. Semen represents the main vector for HIV-1 dissemination worldwide. In the present study we show that human seminal plasma (SP), even when used at very high dilutions (1:10⁴ to 1:10⁵), markedly inhibits the capture and transmission of HIV-1 to T CD4⁺ cells mediated by both DCs and B-THP-1-DC-SIGN cells. In contrast, SP does not inhibit the capture of HIV-1 by DC-SIGN-negative target cells, such as the T-cell line SupT-1, monocytes, and activated peripheral blood mononuclear cells. The SP inhibitor has a high molecular mass (>100 kDa) and directly interacts with DC-SIGN-positive target cells but not with HIV-1. Moreover, the inhibitor binds to concanavalin A, suggesting that it contains high-mannose N-linked carbohydrates. Of note, using biotin-labeled SP we found that the binding of SP components to DCs was abrogated by mannan, while their interaction with B-THP-1 cells was almost completely dependent on the expression of DC-SIGN. Since epithelium integrity is often compromised after vaginal or anal intercourse, as well as in the presence of ulcerative-sexually transmitted diseases, our results support the notion that components of the SP might be able to access to the subepithelium, inhibiting the recognition of HIV-1 gp120 by DC-SIGN-positive DCs.