ABSTRACT
The nef gene of the pathogenic simian immunodeficiency virus (SIV) mac239 clone has been well characterized. Little is known, however, about the function of nef alleles derived from naturally SIVsm-infected sooty mangabeys (Cercocebus atys) and from human immunodeficiency virus type 2 (HIV-2)-infected individuals. Addressing this, we demonstrate that, similarly to the SIVmac239 nef, primary SIVsm and HIV-2 nef alleles down-modulate cell surface expression of human CD4, CD28, CD3, and class I or II major histocompatibility complex (MHC-I or MHC-II, respectively) molecules, up-regulate surface expression of the invariant chain (Ii) associated with immature MHC-II, inhibit early T-cell activation events, and enhance virion infectivity. Both also stimulate viral replication, although HIV-2 nef alleles were less active in this assay than SIVsm nef alleles. Mutational analysis showed that a dileucine-based sorting motif in the C-proximal loop of SIV or HIV-2 Nef is critical for its effects on CD4, CD28, and Ii but dispensable for down-regulation of CD3, MHC-I, and MHC-II. The C terminus of SIV and HIV-2 Nef was exclusively required for down-modulation of MHC-I, further demonstrating that analogous functions are mediated by different domains in Nef proteins derived from different groups of primate lentiviruses. Our results demonstrate that none of the eight Nef functions investigated had been newly acquired after cross-species transmission of SIVsm from naturally infected mangabeys to humans or macaques. Notably, HIV-2 and SIVsm nef alleles efficiently down-modulate CD3 and C28 surface expression and inhibit T-cell activation more efficiently than HIV-1 nef alleles. These differences in Nef function might contribute to the relatively low levels of immune activation observed in HIV-2-infected human individuals.
Subject(s)
Alleles , Genes, nef/physiology , HIV-2/genetics , Simian Immunodeficiency Virus/genetics , Virus Replication , Amino Acid Sequence , Animals , CD28 Antigens/analysis , CD4 Antigens/analysis , Cercocebus atys , HIV-2/pathogenicity , HIV-2/physiology , HeLa Cells , Histocompatibility Antigens Class I/analysis , Humans , Jurkat Cells , Lymphocyte Activation , Molecular Sequence Data , Mutation , Phylogeny , Simian Immunodeficiency Virus/pathogenicity , Simian Immunodeficiency Virus/physiology , T-Lymphocytes/immunologyABSTRACT
CD4(+)- and CD8(+)-T-cell death is a frequent immunological dysfunction associated with the development of human AIDS. We studied a murine model of AIDS, the CD4C/HIV transgenic (Tg) mouse model, to assess the importance of the apoptotic pathway in human immunodeficiency virus type 1 (HIV-1) pathogenesis. In these Tg mice, Nef is the major determinant of the disease and is expressed in immature and mature CD4(+) T cells and in cells of the macrophage/myeloid lineage. We report here a novel AIDS-like phenotype: enhanced death, most likely by apoptosis (as assessed by 7-aminoactinomycin D and annexin V/propidium iodide staining), of Tg thymic and peripheral CD4(+) and CD8(+) T cells. The Tg CD4(+) and CD8(+) T cells were also more susceptible to cell death after activation in vitro in mixed lymph node (LN) cultures. However, activation-induced cell death was not higher in Tg than in non-Tg-purified CD4(+) T cells. In addition, expression of Fas and FasL, assessed by flow cytometry, was increased in CD4(+) and CD8(+) T cells from Tg mice compared to that of non-Tg littermates. Despite the enhanced expression of Fas and FasL on Tg CD4(+) and CD8(+) T cells, Fas (lpr/lpr) and FasL (gld/gld) mutant CD4C/HIV Tg mice developed an AIDS-like disease indistinguishable from lpr/+ and gld/+ CD4C/HIV Tg mice, including loss of CD4(+) T cells. Similarly, CD4C/HIV Tg mice homozygous for mutations of two other genes implicated in cell death (interleukin-1beta-converting enzyme [ICE], tumor necrosis factor receptor 1 [TNFR-1]) developed similar AIDS-like disease as their respective heterozygous controls. Moreover, the double-Tg mice from a cross between the Bcl2/Wehi25 and CD4C/HIV Tg mice showed no major protection against disease. These results represent genetic evidence for the dispensable role of Fas, FasL, ICE, and TNFR-1 on the development of both T-cell loss and organ disease of these Tg mice. They also provide compelling evidence on the lack of protection by Bcl2 against Tg CD4(+)-T-cell death. In view of the high resemblance between numerous phenotypes observed in the CD4C/HIV Tg mice and in human AIDS, our findings are likely to be relevant for the human disease.
Subject(s)
Acquired Immunodeficiency Syndrome/immunology , Apoptosis , Caspase 1/deficiency , Genes, nef/physiology , HIV-1/genetics , Membrane Glycoproteins/metabolism , Proto-Oncogene Proteins/metabolism , T-Lymphocytes/physiology , TNF Receptor-Associated Factor 1/deficiency , fas Receptor/metabolism , Acquired Immunodeficiency Syndrome/genetics , Acquired Immunodeficiency Syndrome/pathology , Animals , Caspase 1/genetics , Disease Models, Animal , Edema/pathology , Fas Ligand Protein , Kidney/pathology , Lymph Nodes/pathology , Membrane Glycoproteins/deficiency , Membrane Glycoproteins/genetics , Mice , Mice, Transgenic , Proto-Oncogene Proteins c-bcl-2 , T-Lymphocytes/immunology , TNF Receptor-Associated Factor 1/genetics , Thymus Gland/pathology , Weight Loss , fas Receptor/geneticsABSTRACT
HIV Nef has a number of important biological effects, including the down-modulation of several immunological important molecules (CD4, major histocompatibility complex [MHC] class I). Down-modulation of CD4 seems to be via clathrin-dependent endocytosis, whereas down-modulation of MHC class I remains unexplained. Several mutant proteins, including mutations in the small GTPase Arf6, have been used to probe membrane traffic pathways. One such mutant has recently been used to propose that Nef acts through Arf6 to activate the endocytosis of MHC class I. Here, we show that MHC class I down-modulation is unaffected by other Arf6 mutants that provide more specific perturbations in the GDP-GTP cycling of Arf6. Inhibition of phosphatidylinositol-3-phosphate kinase, an upstream activator of Arf6, also had no effect on the internalization step, but its activity is required to direct MHC class I to the trans-Golgi network. We conclude that the apparent Arf6 dependency of Nef-mediated MHC class I down-modulation is due to nonspecific perturbations in membrane traffic.
Subject(s)
ADP-Ribosylation Factors/metabolism , Clathrin/metabolism , Genes, MHC Class I/physiology , Genes, nef/physiology , HIV/metabolism , ADP-Ribosylation Factor 6 , Cells, Cultured , Cloning, Molecular , Down-Regulation/physiology , Endocytosis/physiology , Enzyme Activation/physiology , Flow Cytometry , Golgi Apparatus/metabolism , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/metabolism , Humans , Jurkat Cells , Mutation , Phosphatidylinositol 3-Kinases/metabolism , Protein Transport/physiologyABSTRACT
Recently, it has been demonstrated that the human immunodeficiency virus type 1 (HIV-1) Nef from laboratory strains down-modulates cell surface expression of mature major histocompatibility complex class II (MHC-II) molecules, while up-regulating surface expression of the invariant chain (Ii) associated with immature MHC-II (P. Stumptner-Cuvelette, S. Morchoisne, M. Dugast, S. Le Gall, G. Raposo, O. Schwartz, and P. Benaroch, Proc. Natl. Acad. Sci. USA 98:12144-12149, 2001). These Nef functions could contribute to impaired CD4(+)-T-helper-cell responses found in HIV-1-infected patients with progressive disease. However, it is currently unknown whether nef alleles derived from HIV-1-infected individuals or from other primate lentiviruses also modulate MHC-II and Ii. In the present study, we demonstrate that both activities are conserved among primary HIV-1 nef alleles, as well as among HIV-2 and simian immunodeficiency virus (SIV) nef alleles. Down-modulation of mature MHC-II required high levels of Nef expression. In contrast, surface expression of Ii was already strongly increased at low to medium levels of Nef expression. Notably, nef genes derived from two of four HIV-1-infected long-term nonprogressors did not up-regulate Ii, whereas nef alleles derived from 10 individuals with progressive disease were active in this assay. Unlike other in vitro Nef functions, the average activity of Nef in modulating MHC-II and Ii surface expression did not change significantly during the course of infection. Mutational analysis confirmed that MHC-II down- and Ii up-regulation are functionally separable from each other and from other Nef functions and identified acidic residues, located at the base of the flexible C-proximal loop of Nef, that are critical for increased Ii expression. Overall, our results suggest that the ability of Nef to interfere with MHC-II antigen presentation might play a role in AIDS pathogenesis.
Subject(s)
Antigens, Differentiation, B-Lymphocyte/analysis , Genes, nef/physiology , HIV/genetics , Histocompatibility Antigens Class II/analysis , Simian Immunodeficiency Virus/genetics , Acquired Immunodeficiency Syndrome/immunology , Alleles , Amino Acid Sequence , Down-Regulation , Gene Products, nef/chemistry , Gene Products, nef/genetics , Gene Products, nef/physiology , Humans , Jurkat Cells , Molecular Sequence Data , Up-Regulation , nef Gene Products, Human Immunodeficiency VirusABSTRACT
Use of the macaque model of human immunodeficiency virus (HIV) pathogenesis has shown that the accessory genes nef and vpu are important in the pathogenicity of simian immunodeficiency virus (SIV) and simian-human immunodeficiency virus (SHIV). We examined the ability of two nonpathogenic SHIVs, SHIV(PPC) and DeltavpuDeltanefSHIV(PPC), to gain pathogenicity by rapid serial passage in macaques. In this study, each virus was passaged by blood intravenously four times at 4-week intervals in macaques. Animals were monitored for 40 weeks for levels of CD4 T cells and quantitative measures of virus infection. DeltavpuDeltanefSHIV(PPC) maintained a limited phase of productive replication in the four animals, with no loss of CD4(+) T cells, whereas SHIV(PPC) became more pathogenic in later passages, judging by plasma viral load and viral mRNA in lymph nodes, infectious peripheral blood mononuclear cells and CD4(+) T cell loss. The nef, LTR, and env of the SHIV(PPC) viruses underwent numerous mutations, compared to DeltavpuDeltanefSHIV(PPC). This study confirms the seminal role that nef, LTR, and vpu could play in regulation of pathogenesis of HIV infection.
Subject(s)
Acquired Immunodeficiency Syndrome/virology , Genes, nef/physiology , Genes, vpu/physiology , HIV-1 , Reassortant Viruses/genetics , Simian Immunodeficiency Virus , Acquired Immunodeficiency Syndrome/blood , Acquired Immunodeficiency Syndrome/immunology , Amino Acid Sequence , Animals , CD4 Lymphocyte Count , Cell Line , Gene Products, env/genetics , HIV-1/genetics , HIV-1/pathogenicity , Humans , Leukocytes, Mononuclear/virology , Lymph Nodes/virology , Macaca nemestrina , Molecular Sequence Data , Mutation , RNA, Messenger/analysis , RNA, Viral/analysis , RNA, Viral/blood , Reassortant Viruses/isolation & purification , Reassortant Viruses/pathogenicity , Simian Immunodeficiency Virus/genetics , Simian Immunodeficiency Virus/pathogenicity , Viral LoadABSTRACT
Human immunodeficiency virus type 1 (HIV-1) has 4 auxiliary genes, vpr, vpu, nef, and vif, which are dispensable for viral replication in vitro. However, many studies with animal model revealed that these genes play important roles on the viral replication and the development of AIDS in vivo through many complicated mechanisms. Although several key factors involved in the function have been identified, further studies are required for the complete understandings of the action mechanisms. The elucidation of the function of the auxiliary genes on molecular bases leads to the discovery of new therapeutic strategies against HIV and the understanding of basic cellular mechanisms. In this review, we summarize new observations mainly about the interactions between auxiliary genes and host cell functions.
Subject(s)
Genes, nef/physiology , Genes, vif/physiology , Genes, vpr/physiology , Genes, vpu/physiology , HIV-1/genetics , Acquired Immunodeficiency Syndrome/virology , Animals , HIV-1/physiology , Humans , Virus ReplicationABSTRACT
OBJECTIVE: The Nef protein has a major influence on disease pathogenesis in HIV-infected individuals. The objective of the present study was to examine the effects of Nef on T lymphocyte activation and associated signalling events. DESIGN: A recombinant vaccinia expression system was used to express Nef in a human T cell line. Stimulation of these cells with anti-CD28 antibody, and either phorbol 12-myristate 13-acetate (PMA) or anti-CD3, activates signal transduction pathways and results in IL-2 production and IL-2 receptor alpha-chain (CD25) expression. Cellular responses were examined in cells expressing either Nef or an irrelevant control protein. METHODS: Activation of signalling was assessed by immunoblot analysis, or by in-vitro phosphatidylinositol 3-kinase (PI3K) assays. IL-2 production was measured by enzyme-linked immunosorbent assay, and CD25 cell surface expression was examined using flow cytometry. RESULTS: Infection of cells with recombinant vaccinia expressing HIV-nef resulted in a marked increase in the production of IL-2 when cells were activated. The enhanced IL-2 response was accompanied by an increase in the level of PI3K activity. IL-2 production remained sensitive to inhibition with the PI3K competitive inhibitor Ly294002, and to the fungal macrolide, rapamycin. In contrast, CD25 expression was not affected, and there were no measurable changes to nuclear factor kappaB (NFkappaB) activation pathways. CONCLUSION: Enhanced IL-2 production in stimulated T cells expressing HIV-Nef is associated with increased activation of PI3K-dependent signalling pathways. The results support a model in which Nef affects HIV disease progression by distorting T cell responses.
Subject(s)
Gene Products, nef/physiology , HIV-1/genetics , Interleukin-2/biosynthesis , Phosphatidylinositol 3-Kinases/metabolism , T-Lymphocytes/enzymology , T-Lymphocytes/immunology , CD28 Antigens , Enzyme Activation , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Gene Expression Regulation, Viral , Gene Products, nef/genetics , Genes, nef/physiology , Humans , Immunoblotting , Interleukin-2/metabolism , Jurkat Cells , Receptors, Interleukin-2/biosynthesis , Receptors, Interleukin-2/immunology , Signal Transduction , nef Gene Products, Human Immunodeficiency VirusABSTRACT
Transmission of simian immunodeficiency virus SIVmac239Delta(nef) (Delta(nef)) to macaques results in attenuated replication of the virus in most animals and ultimately induces protection against challenge with some pathogenic, wild-type SIV strains. It has been difficult, however, to identify a culture system in which the replication of Delta(nef) is severely reduced relative to that of the wild type. We have utilized a primary culture system consisting of blood-derived dendritic cells (DCs) and autologous T cells. When the DCs were fully differentiated or mature, the DC-CD4(+) T-cell mixtures supported replication of both the parental SIV strain, 239 (the wild type), and its mutant with nef deleted (Delta(nef)), irrespective of virus dose and the cell type introducing the virus to the coculture. In contrast, when immature DCs were exposed to Delta(nef) and cocultured with T cells, virus replication was significantly lower than that of the wild type. Activation of the cultures with a superantigen allowed both Delta(nef) and the wild type to replicate comparably in immature DC-T-cell cultures. Immature DCs, which, it has been hypothesized, capture and transmit SIV in vivo, are deficient in supporting replication of Delta(nef) in vitro and may contribute to the reduced pathogenicity of Delta(nef) in vivo.
Subject(s)
Dendritic Cells/virology , Simian Immunodeficiency Virus/physiology , T-Lymphocytes/virology , Animals , CD4-Positive T-Lymphocytes/virology , Coculture Techniques , Female , Genes, nef/physiology , Macaca mulatta , Male , Simian Immunodeficiency Virus/genetics , Simian Immunodeficiency Virus/pathogenicity , Virus ReplicationABSTRACT
Dementia is a common complication of AIDS which is associated with human immunodeficiency virus type 1 (HIV-1) infection of brain macrophages and microglia. Recent studies have shown that astrocytes are also infected in the brain but HIV-1 replication in these cells is restricted. To determine virus specificity of this restriction we tested the expression of 15 HIV-1 molecular clones in primary human fetal astrocytes by infection and DNA transfection. Infection with cell-free viruses was poorly productive and revealed no clone-specific differences. In contrast, transfected cells produced transiently high levels of HIV-1 p24 core antigen, up to 50 nanograms per ml culture supernatant, and nanogram levels of p24 were detected 3-4 weeks after transfection of some viral clones. The average peak expression of HIV-1 in astrocytes varied as a function of viral clone used by a factor of 15 but the differences and the subsequent virus spread did not correlate with the tropism of the viral clones to T cells or macrophages. Functional vif, vpu, and vpr genes were dispensable for virus replication from transfected DNA, but intact nef provided a detectable enhancement of early viral gene expression and promoted maintenance of HIV-1 infection. We conclude that primary astrocytes present no fundamental barriers to moderate expression of different strains of HIV-1 and that the presence of functional Nef is advantageous to virus infection in these cells.
Subject(s)
Astrocytes/virology , Genes, nef/physiology , HIV-1/genetics , HIV-1/physiology , Cell Culture Techniques , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique, Indirect , HIV Core Protein p24/analysis , HIV-1/isolation & purification , Humans , Sensitivity and Specificity , Time Factors , Transfection , Virus Replication/physiologyABSTRACT
A T-lymphoid cell line termed 221 was derived from a rhesus monkey infected with herpesvirus saimiri. Growth of 221 cells was dependent on the addition of interleukin-2 (IL-2) to the culture medium. In the absence of IL-2, 221 cells arrested in G0-G1 but did not die. Simian immunodeficiency virus (SIV) replicated efficiently in IL-2-stimulated 221 cells whether or not the nef gene was present. In the absence of IL-2, nef-containing SIV replicated 8 to 100 times more efficiently in 221 cells than did the same virus lacking nef. nef-containing virus preferentially stimulated the production of IL-2 from 221 cells. HIV-1 nef and v-ras genes, but not the c-ras gene, were shown to substitute functionally for SIV nef when tested as recombinant viruses in this assay system. These results demonstrate a role for natural nef in causing lymphoid cell activation, and they provide a system for delineating the biochemical mechanisms responsible for this activation.
Subject(s)
Alleles , Genes, nef/physiology , HIV/genetics , Lymphocyte Activation , Simian Immunodeficiency Virus/genetics , Animals , Cell Line , HIV/physiology , Interleukin-2/pharmacology , Macaca mulatta , Simian Immunodeficiency Virus/physiology , Virus ReplicationABSTRACT
Chimeric simian-human immunodeficiency viruses (SHIVs) carrying envelope glycoproteins derived from a T cell-macrophage dual-tropic primary isolate (human immunodeficiency virus type 1 [HIV-1] strain DH12) were constructed. When inoculated into macaque monkeys, SHIV(MD14) carrying simian immunodeficiency virus-derived nef established significantly higher virus loads than did SHIV(MD1), which contains the HIV-1 nef gene. Three patterns of CD4 cell depletion were observed in infected monkeys: exponential and irreversible loss to undetectable levels within 10 weeks of infection; marked reduction during acute infection followed by partial recovery and stabilization (lasting from 10 weeks to > 1 year), with a later decline to undetectable levels in some animals; and a transient loss during acute infection. The induced immunodeficiency was accompanied by CD4 cell counts of < 50 cells/microL and was associated with Pneumocystis carinii pneumonia, cytomegalovirus meningoencephalitis, lymphoid depletion, and thymic atrophy.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , HIV Infections/immunology , HIV Infections/virology , HIV-1/pathogenicity , Simian Immunodeficiency Virus/pathogenicity , Viral Load , Amino Acid Sequence , Animals , Base Sequence , CD4 Lymphocyte Count , CD4-Positive T-Lymphocytes/virology , Cells, Cultured , DNA, Viral/blood , Genes, nef/physiology , HIV-1/genetics , HIV-1/physiology , Humans , Leukocytes, Mononuclear/virology , Macaca , Macrophages/virology , Male , Molecular Sequence Data , Recombinant Fusion Proteins , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Acquired Immunodeficiency Syndrome/virology , Simian Immunodeficiency Virus/genetics , Simian Immunodeficiency Virus/physiology , Virus ReplicationABSTRACT
We have developed an in vitro model to study the influence that human immunodeficiency virus type 1 (HIV-1) may have on the ability of T cells to respond to antigenic challenge. We have examined consequences of HIV-1 gene expression on T-cell activation in antigen-dependent T cells that have stably integrated copies of replication-defective proviral HIV-1. Virus production by HIV-infected, antigen-dependent T cells was induced in response to antigenic stimulation and then decreased as infected cells returned to a state of quiescence. Contrary to the predictions of models proposing that Nef alters signal transduction pathways in T lymphocytes and thereby alters cellular activation, Nef expression in antigen-dependent T-cell clones did not influence their proliferative responses to low or intermediate concentrations of antigen and did not affect other measures of T-cell activation, such as induction of interleukin 2 receptor alpha-chain expression and cytokine production. In addition, we found no evidence for alteration of T-cell responsiveness to antigen by the gag, pol, vif, tat, or rev gene of HIV-1.
Subject(s)
Genes, nef/physiology , HIV-1/genetics , Lymphocyte Activation , T-Lymphocytes/immunology , Antigens/immunology , Cells, Cultured , Cytokines/biosynthesis , Gene Expression , Hemagglutinins, Viral/physiology , Humans , Sensitivity and SpecificityABSTRACT
The pathogenicity of four human immunodeficiency virus type 1 (HIV-1) isolates with nef deleted for SCID mice repopulated with human peripheral blood leukocytes (hu-PBL-SCID mice) was studied. Deletion of nef led to a substantial reduction in CD4-positive T-cell depletion and delayed kinetics of plasma viremia in infected hu-PBL-SCID mice. Deletion of the nef gene impacts both the efficiency of primary infection and the cytopathicity of virus for infected CD4-positive T cells in this animal model of HIV-1 infection.
Subject(s)
Acquired Immunodeficiency Syndrome/immunology , CD4-Positive T-Lymphocytes/physiology , Genes, nef/physiology , HIV-1 , Leukocytes/virology , Animals , Gene Deletion , Humans , Mice , Mice, SCID , RNA, Viral/analysisABSTRACT
Infection by human immunodeficiency virus (HIV)-1 is associated with quantitative and qualitative T cell alterations that severely impair the host's immune defense system. The molecular basis for this immunosuppression remains unclear. Peripheral blood mononuclear cells (PBMC) isolated from patients show markedly decreased interleukin (IL)-2 secretion but unaffected or even increased T helper (Th)2 cytokine production. T cell functional defects were recently reported to correlate more with T cell receptor (TcR) signaling, whereas signals provided by ligation of co-receptors CD27 and CD28 appeared to be preserved. Among the various mechanisms proposed to be involved in HIV-1-induced T cell dysfunction, we and others have reported that the nef gene product exhibited significant immunosuppressive activity. By using an inducible stably integrated nef gene, we demonstrated that Nef specifically down-regulated IL-2 and interferon (IFN)-gama produced upon TcR triggering. Here, using the same experimental system, we extended our initial observations to additional mitogenic signals, and investigated the co-stimulatory function of CD28. Nef down-regulated IL-2, but not IL-4 produced upon induction by combinations of mitogens that mimicked TcR signals together with CD28 mAb or CD28's natural ligand (CD80 and CD86). However, the co-signals provided by CD28 to up-regulate IL-2 induction were unaffected by Nef, since IL-2 produced by nef-transfected cells was proportionally enhanced to the same extent as that of control cells, either upon stimulation by the CD28 mAb or CD80 and CD86. In addition, phosphatidylinositol-3 kinase recruitment induced upon CD28 triggering was also found to be unaltered by nef expression. Together with the observation that similar levels of the Nef protein were detected in nef-transfected cells and upon infection of PBMC, these data suggest a selective immunosuppression induced by nef in human T cells by altering TcR signaling without detectable impact on CD28 co-receptor function. These data agree with the T cell defects observed in PBMC isolated from HIV-infected individuals.
Subject(s)
CD28 Antigens/immunology , Genes, nef/physiology , HIV-1/genetics , Immune Tolerance , Signal Transduction/immunology , T-Lymphocytes/immunology , CD28 Antigens/pharmacology , Enzyme Activation/immunology , Gene Products, nef/metabolism , Humans , Interleukin-2/biosynthesis , Interleukin-4/biosynthesis , Leukemia, T-Cell , Leukocytes, Mononuclear/immunology , Leukocytes, Mononuclear/virology , Ligands , Lymphocyte Activation/genetics , Phosphatidylinositol 3-Kinases , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Protein Binding/immunology , T-Lymphocytes/enzymology , T-Lymphocytes/metabolism , Transfection/immunology , Tumor Cells, Cultured , Up-Regulation/genetics , Up-Regulation/immunology , nef Gene Products, Human Immunodeficiency VirusABSTRACT
The role of the vpx, vpr, and nef genes in the replication of simian immunodeficiency virus (SIV) was investigated using point and deletion mutations in these genes. The effects on replication kinetics of single or combined mutants--vpx, vpr, vpx-vpr, vpx-nef, vpr-nef, and vpx-vpr-nef--in established lymphoid CEMx174 and MT-4 cells were negligible, except that the postinfection appearance of vpx-nef, vpr-nef, and vpx-vpr-nef progeny virus was slightly delayed in MT-4 cells. The vpx, but not the vpr, point mutation reverted to wild-type sequences within 12 days after infection, suggesting that stronger selection pressure for Vpx than for Vpr expression might exist in these established cell lines. In contrast to growth in the lymphoid cell lines, replication of vpx-deleted viruses in macaque peripheral blood mononuclear cells (PBMC) was severely impaired, indicating that Vpx is necessary for efficient replication in PBMC. In contrast, the vpr mutant exhibited different degrees of impairment depending on the donor animal used as a source of PBMC. A virus encoding a Vpx-Vpr fusion protein replicated in PBMC comparably to a vpr deletion mutant virus, whereas a frameshift deletion at the vpx-vpr junction of this mutant eliminated virus replication, suggesting that deletion of the C-terminal half of Vpx was partially compensated by the presence of the large Vpr portion in the fusion protein. Deletion of the nef gene did not affect SIVmac replication in PBMC. The Vpx and Vpr proteins expressed in COS-1 cells were detected in the extracellular medium and did not crossreact with Vpr- and Vpx-specific antisera, in spite of extensive amino acid similarity between these proteins. These studies indicate the importance of Vpx and Vpr in SIVmac infection and suggest that these proteins are antigenically and functionally distinct.
Subject(s)
Genes, nef/physiology , Genes, vpr/physiology , Simian Immunodeficiency Virus/genetics , Viral Regulatory and Accessory Proteins/physiology , Animals , Base Sequence , CD4-Positive T-Lymphocytes/virology , Cell Line , Genes, nef/genetics , Genes, vpr/genetics , Humans , Macaca mulatta , Molecular Sequence Data , Point Mutation , Sequence Deletion , Viral Proteins/analysis , Viral Regulatory and Accessory Proteins/genetics , Virus Replication/physiologyABSTRACT
The gene product of the nef gene of HIV-1 acts both to increase the infectivity of viral particles and to reduce the expression of the CD4 receptor molecule on the cell surface. These two functions of Nef may be related, in that downregulation of CD4 may promote the production of HIV that has greater infectivity.
Subject(s)
Genes, nef/physiology , HIV Infections/virology , HIV-1/genetics , HIV-1/pathogenicity , Animals , CD4 Antigens/metabolism , Down-Regulation , HIV-1/physiology , HeLa Cells , Humans , Lymphocyte Activation , Macaca mulatta , Virus ReplicationABSTRACT
Simian immunodeficiency virus (SIVmac) infectious for rhesus monkeys was altered by site-directed mutagenesis of genes influencing in vivo replication and persistence with the long-term goal to develop attenuated lentiviruses with limited replication capacity in vivo. Double mutants of SIVmac (termed delta-int 1 to 3) were generated by introducing frameshift and deletion mutations into the nef gene and into the pol gene region coding for the integrase protein. Delta-int/delta-nef viruses formed after transfection of CD(4+)-lymphocyte cell lines were unable to establish sustained replication. In contrast, both wild-type SIVmac and mutant SIVmac delta-nef (coding for a truncated NEF protein and a wild-type INT protein) replicated continuously and at a comparable rate. However, a transient and self-limiting infection of the C8166 T-cell line was observed subsequent to transfection of double mutant proviruses into HeLa-tat-III cells. Viruses attenuated by int/nef-double mutation were able to enter the T-cells, initiate synthesis of viral DNA as shown by PCR amplification of closed circular episomes, and express viral antigens in infected cells as demonstrated by immunocytochemical staining. Integration of the int mutant viruses into the chromosome was completely inhibited. Episomal viral DNA was detectable in the infected cells for up to 2 weeks, after which it disappeared. Thus, SIVmac attenuated by int and nef mutation established a transient infection of permissive cells resulting in the expression of viral antigen from episomal viral DNA over a limited period of time.
Subject(s)
Genes, Viral/physiology , Genes, nef/physiology , Mutagenesis, Site-Directed/genetics , Simian Immunodeficiency Virus/genetics , Virus Integration/physiology , Virus Replication/physiology , Antigens, Viral/biosynthesis , Base Sequence , DNA, Viral/biosynthesis , DNA, Viral/genetics , Genes, Viral/genetics , Genes, nef/genetics , HeLa Cells , Humans , Molecular Sequence Data , Mutagenesis, Site-Directed/physiology , Simian Immunodeficiency Virus/physiology , Virus Integration/genetics , Virus Replication/geneticsABSTRACT
Human immunodeficiency virus type 1 (HIV-1) can lead to a profound CD4+ T-cell deficiency. To examine the functional role of HIV-1 Nef protein on the marked loss of CD4+ cells, Nef protein was expressed in and purified from Escherichia coli as a fusion protein with T7 phage gene10 product (Nef-gene10). When peripheral blood mononuclear cells (PBMC) from healthy donors were cultivated in the presence of Nef-gene10 or the gene10 product as well as interleukin-2 (IL-2), it was found that the Nef-gene10, but not the gene10 product, induced a remarkable decline in the CD4/CD8 ratio and in the response to phytohaemagglutinin of PBMC as well as of nylon wool-passed purified T cells. Nef-gene10 inhibited the proliferation of CD4+ cells, but did not kill the cells. This suppression of the IL-2-dependent proliferation of CD4+ cells by Nef-gene10 seemed to be due to enhanced production of several lymphokines, especially of interferon-gamma. Thus, Nef protein might be partly responsible for the selective depletion of CD4+ cells in HIV-1 infection. Furthermore, the Nef-induced decline in the CD4/CD8 ratio was interrupted by anti-Nef antibodies, suggesting the possibility that a vaccine which resulted in the production of such functional Nef antibodies would be useful in the treatment of HIV-1-induced immunodysfunction.
Subject(s)
CD4-Positive T-Lymphocytes/drug effects , Gene Products, nef/physiology , HIV-1 , Animals , Antibodies/pharmacology , Bacteriophage T7/genetics , Base Sequence , CD4-CD8 Ratio/drug effects , CD4-Positive T-Lymphocytes/cytology , CD4-Positive T-Lymphocytes/physiology , Cell Division/drug effects , Cell Division/physiology , Cells, Cultured , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression/genetics , Gene Products, nef/genetics , Gene Products, nef/pharmacology , Genes, nef/genetics , Genes, nef/physiology , HIV-1/genetics , Humans , Interleukin-2/pharmacology , Ligands , Lymphocyte Activation/drug effects , Major Histocompatibility Complex/physiology , Mitogens/pharmacology , Molecular Sequence Data , Phenotype , Rabbits , Receptors, Antigen, T-Cell/metabolism , Viral Fusion Proteins/genetics , Viral Fusion Proteins/pharmacology , Viral Fusion Proteins/physiology , nef Gene Products, Human Immunodeficiency VirusABSTRACT
OBJECTIVE: To study the biochemical properties of the HIV-1 nef gene product. DESIGN: Earlier reports suggested that Nef protein is phosphorylated and has kinase activity. These properties were examined using an in vitro translated product. METHODS: A DNA fragment encoding Nef of HIV-1SF2 was transcribed in vitro under the control of T7 promoter. The generated nef messenger RNA (mRNA) was translated using a rabbit reticulocyte lysate system. Immunoprecipitation was performed with a specific monoclonal antibody to Nef. Kinase activity of the translated product was evaluated using [gamma-32P]GTP and ATP. RESULTS: Translated nef mRNA was found to encode a major protein of 27 kD with two other products, of 25 and 29 kD. Using Nef immune complexes for the kinase reaction, a protein of 46 kD that reacted with the anti-Nef monoclonal antibody was found to be strongly phosphorylated in the presence of Nef. This Nef-associated kinase activity on a p46 cellular substrate was dependent on ATP. CONCLUSIONS: The in vitro-translated nef gene product was found to be associated with phosphorylation of a p46 cellular protein present in rabbit reticulocyte lysate. The Nef protein has been linked to suppression of the replication of certain HIV strains. Identification of cellular targets of Nef activity could help to elucidate the mechanisms for this antiviral effect.