ABSTRACT
A host genetic variant (-35C/T) correlates with increased human leukocyte antigen C (HLA-C) expression and improved control of HIV-1. HLA-C-mediated immunity may be particularly protective because HIV-1 is unable to remove HLA-C from the cell surface, whereas it can avoid HLA-A- and HLA-B-mediated immunity by Nef-mediated down-modulation. However, some individuals with the protective -35CC genotype exhibit high viral loads. Here, we investigated whether the ability of HIV-1 to replicate efficiently in the "protective" high-HLA-C-expression host environment correlates with specific functional properties of Nef. We found that high set point viral loads (sVLs) were not associated with the emergence of Nef variants that had acquired the ability to down-modulate HLA-C or were more effective in removing HLA-A and HLA-B from the cell surface. However, in individuals with the protective -35CC genotype we found a significant association between sVLs and the efficiency of Nef-mediated enhancement of virion infectivity and modulation of CD4, CD28, and the major histocompatibility complex class II (MHC-II)-associated invariant chain (Ii), while this was not observed in subjects with the -35TT genotype. Since the latter Nef functions all influence the stimulation of CD4(+) T helper cells by antigen-presenting cells, they may cooperate to affect both the activation status of infected T cells and the generation of an antiviral cytotoxic T-lymphocyte (CTL) response. In comparison, different levels of viremia in individuals with the common -35TT genotype were not associated with differences in Nef function but with differences in HLA-C mRNA expression levels. Thus, while high HLA-C expression may generally facilitate control of HIV-1, Nef may counteract HLA-C-mediated immune control in some individuals indirectly, by manipulating T-cell function and MHC-II antigen presentation.
Subject(s)
HIV-1/immunology , HLA-C Antigens/immunology , nef Gene Products, Human Immunodeficiency Virus/physiology , CD8 Antigens/genetics , CD8 Antigens/immunology , Cell Line , Genotype , HLA-C Antigens/genetics , Histocompatibility Antigens Class I/genetics , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/immunology , Humans , NFATC Transcription Factors/genetics , NFATC Transcription Factors/metabolism , Polymorphism, Genetic , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Viral Load , nef Gene Products, Human Immunodeficiency Virus/genetics , nef Gene Products, Human Immunodeficiency Virus/immunologyABSTRACT
Mammalian cells are equipped with so-called "restriction factors" that suppress virus replication and help to prevent virus transmission from one species to another. This Essay discusses the host restriction factor tetherin, which blocks the release of enveloped viruses like HIV-1, and the factors evolved by primate lentiviruses, such as Vpu and Nef, that antagonize tetherin's action.
Subject(s)
Antigens, CD/metabolism , HIV-1/metabolism , Membrane Glycoproteins/metabolism , Animals , Antigens, CD/genetics , Antigens, CD/immunology , GPI-Linked Proteins , Gene Products, nef/metabolism , Human Immunodeficiency Virus Proteins/metabolism , Humans , Lentivirus/genetics , Lentivirus Infections/drug therapy , Membrane Glycoproteins/genetics , Membrane Glycoproteins/immunology , Viral Regulatory and Accessory Proteins/metabolismSubject(s)
Calcium/metabolism , Cyclophilins/metabolism , Retroviridae/physiology , Cell Line , Humans , Ligands , Polymerase Chain ReactionABSTRACT
BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) Vpu protein degrades CD4 and counteracts a restriction factor termed tetherin (CD317; Bst-2) to enhance virion release. It has been suggested that both functions can be genetically separated by mutation of a serine residue at position 52. However, recent data suggest that the S52 phosphorylation site is also important for the ability of Vpu to counteract tetherin. To clarify this issue, we performed a comprehensive analysis of HIV-1 with a mutated casein kinase-II phosphorylation site in Vpu in various cell lines, primary blood lymphocytes (PBL), monocyte-derived macrophages (MDM) and ex vivo human lymphoid tissue (HLT). RESULTS: We show that mutation of serine 52 to alanine (S52A) entirely disrupts Vpu-mediated degradation of CD4 and strongly impairs its ability to antagonize tetherin. Furthermore, casein-kinase II inhibitors blocked the ability of Vpu to degrade tetherin. Overall, Vpu S52A could only overcome low levels of tetherin, and its activity decreased in a manner dependent on the amount of transiently or endogenously expressed tetherin. As a consequence, the S52A Vpu mutant virus was unable to replicate in macrophages, which express high levels of this restriction factor. In contrast, HIV-1 Vpu S52A caused CD4+ T-cell depletion and spread efficiently in ex vivo human lymphoid tissue and PBL, most likely because these cells express comparably low levels of tetherin. CONCLUSION: Our data explain why the effect of the S52A mutation in Vpu on virus release is cell-type dependent and suggest that a reduced ability of Vpu to counteract tetherin impairs HIV-1 replication in macrophages, but not in tissue CD4+ T cells.
Subject(s)
HIV-1/physiology , Human Immunodeficiency Virus Proteins/physiology , Macrophages/virology , Membrane Glycoproteins/antagonists & inhibitors , T-Lymphocytes/virology , Viral Regulatory and Accessory Proteins/physiology , Virus Release , Virus Replication , Amino Acid Substitution , Antigens, CD , CD4 Antigens/metabolism , Cell Line , Cells, Cultured , GPI-Linked Proteins , Human Immunodeficiency Virus Proteins/genetics , Humans , Mutagenesis, Site-Directed , Mutant Proteins/genetics , Mutant Proteins/physiology , Viral Regulatory and Accessory Proteins/geneticsABSTRACT
Vpu proteins of pandemic HIV-1 M strains degrade the viral receptor CD4 and antagonize human tetherin to promote viral release and replication. We show that Vpus from SIVgsn, SIVmus, and SIVmon infecting Cercopithecus primate species also degrade CD4 and antagonize tetherin. In contrast, SIVcpz, the immediate precursor of HIV-1, whose Vpu shares a common ancestry with SIVgsn/mus/mon Vpu, uses Nef rather than Vpu to counteract chimpanzee tetherin. Human tetherin, however, is resistant to Nef and thus poses a significant barrier to zoonotic transmission of SIVcpz to humans. Remarkably, Vpus from nonpandemic HIV-1 O strains are poor tetherin antagonists, whereas those from the rare group N viruses do not degrade CD4. Thus, only HIV-1 M evolved a fully functional Vpu following the three independent cross-species transmissions that resulted in HIV-1 groups M, N, and O. This may explain why group M viruses are almost entirely responsible for the global HIV/AIDS pandemic.
Subject(s)
Antigens, CD/physiology , CD4 Antigens/genetics , Membrane Glycoproteins/physiology , Acquired Immunodeficiency Syndrome/epidemiology , Acquired Immunodeficiency Syndrome/virology , Amino Acid Sequence , Animals , Cell Line , Cercopithecus , Evolution, Molecular , GPI-Linked Proteins , Gene Expression Regulation , HIV-1/pathogenicity , HIV-1/physiology , Human Immunodeficiency Virus Proteins/genetics , Humans , Molecular Sequence Data , Sequence Alignment , Simian Immunodeficiency Virus/pathogenicity , Simian Immunodeficiency Virus/physiology , Viral Regulatory and Accessory Proteins/genetics , Zoonoses , nef Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
BACKGROUND: Decreased endothelial nitric oxide (NO) synthase (eNOS) activity and NO production are critical contributors to the endothelial dysfunction and vascular complications observed in many diseases, including diabetes mellitus. Extracellular nucleotides activate eNOS and increase NO generation; however, the mechanism of this observation is not fully clarified. METHODS AND RESULTS: To elucidate the signaling pathway(s) leading to nucleotide-mediated eNOS phosphorylation at Ser-1177, human umbilical vein endothelial cells were treated with several nucleotides, including ATP, UTP, and ADP, in the presence or absence of selective inhibitors. These experiments identified P2Y1, P2Y2, and possibly P2Y4 as the purinergic receptors involved in eNOS phosphorylation and demonstrated that this process was adenosine independent. Nucleotide-induced eNOS phosphorylation and activity were inhibited by BAPTA-AM (an intracellular free calcium chelator), rottlerin (a protein kinase Cdelta inhibitor), and protein kinase Cdelta siRNA. In contrast, blockade of AMP-activated protein kinase, calcium/calmodulin-dependent kinase II, calcium/calmodulin-dependent kinase kinase, serine/threonine protein kinase B, protein kinase A, extracellular signal-regulated kinase 1/2, and p38 mitogen-activated protein kinase did not affect nucleotide-mediated eNOS phosphorylation. CONCLUSIONS: The present study indicates that extracellular nucleotide-mediated eNOS phosphorylation is calcium and protein kinase Cdelta dependent. This newly identified signaling pathway opens new therapeutic avenues for the treatment of endothelial dysfunction.
Subject(s)
Calcium/physiology , Endothelial Cells/metabolism , Endothelium, Vascular/cytology , Nitric Oxide Synthase Type III/metabolism , Protein Kinase C-delta/physiology , Cells, Cultured , Endothelial Cells/enzymology , Humans , Nitric Oxide/biosynthesis , Nucleotides/pharmacology , Phosphorylation , Receptors, Purinergic , Signal TransductionABSTRACT
Recent data suggest that Nef-mediated downmodulation of TCR-CD3 may protect SIVsmm-infected sooty mangabeys (SMs) against the loss of CD4+ T cells. However, the mechanisms underlying this protective effect remain unclear. To further assess the role of Nef in nonpathogenic SIV infection, we cloned nef alleles from 11 SIVsmm-infected SMs with high (>500) and 15 animals with low (<500) CD4+ T-cells/microl in bulk into proviral HIV-1 IRES/eGFP constructs and analyzed their effects on the phenotype, activation, and apoptosis of primary T cells. We found that not only efficient Nef-mediated downmodulation of TCR-CD3 but also of MHC-I correlated with preserved CD4+ T cell counts, as well as with high numbers of Ki67+CD4+ and CD8+CD28+ T cells and reduced CD95 expression by CD4+ T cells. Moreover, effective MHC-I downregulation correlated with low proportions of effector and high percentages of naïve and memory CD8+ T cells. We found that T cells infected with viruses expressing Nef alleles from the CD4low SM group expressed significantly higher levels of the CD69, interleukin (IL)-2 and programmed death (PD)-1 receptors than those expressing Nefs from the CD4high group. SIVsmm Nef alleles that were less active in downmodulating TCR-CD3 were also less potent in suppressing the activation of virally infected T cells and subsequent cell death. However, only nef alleles from a single animal with very low CD4+ T cell counts rendered T cells hyper-responsive to activation, similar to those of HIV-1. Our data suggest that Nef may protect the natural hosts of SIV against the loss of CD4+ T cells by at least two mechanisms: (i) downmodulation of TCR-CD3 to prevent activation-induced cell death and to suppress the induction of PD-1 that may impair T cell function and survival, and (ii) downmodulation of MHC-I to reduce CTL lysis of virally infected CD4+ T cells and/or bystander CD8+ T cell activation.
Subject(s)
CD4-Positive T-Lymphocytes/pathology , Gene Expression Regulation, Viral , Simian Acquired Immunodeficiency Syndrome/metabolism , Simian Immunodeficiency Virus/genetics , Viral Regulatory and Accessory Proteins/genetics , Animals , CD3 Complex/metabolism , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Cercocebus atys , Down-Regulation , Histocompatibility Antigens Class I/metabolism , Humans , Jurkat Cells , Simian Acquired Immunodeficiency Syndrome/immunology , Simian Acquired Immunodeficiency Syndrome/pathology , Simian Immunodeficiency Virus/pathogenicity , Viral Regulatory and Accessory Proteins/metabolism , Virus ReplicationABSTRACT
Human immunodeficiency virus type 1 (HIV-1) Nef downregulates HLA-A and -B molecules, but not HLA-C or -E molecules, based on amino acid differences in their cytoplasmic domains to simultaneously evade cytotoxic T lymphocyte (CTL) and natural killer cell surveillance. Rhesus macaques and sooty mangabeys express orthologues of HLA-A, -B, and -E, but not HLA-C, and many of these molecules have unique amino acid differences in their cytoplasmic tails. We found that these differences also resulted in differential downregulation by primary simian immunodeficiency virus (SIV) SIV(smm/mac) and HIV-2 Nef alleles. Thus, selective major histocompatibility complex class I downregulation is a conserved mechanism of immune evasion for pathogenic SIV infection of rhesus macaques and nonpathogenic SIV infection of sooty mangabeys.
Subject(s)
Alleles , Down-Regulation , Genes, Viral , Genes, nef , HIV-2/genetics , Histocompatibility Antigens Class I/physiology , Simian Immunodeficiency Virus/genetics , Animals , Cercocebus atys , Macaca mulattaABSTRACT
It has been demonstrated that the HIV-1 NL4-3 and IIIB Nef alleles downregulate HLA-A and -B but not -C or -E from the cell surface. It remained elusive, however, whether selective modulation of specific HLA molecules is conserved between different groups of human and simian immunodeficiency viruses, respectively. To address this, we analyzed a large panel of primate lentiviral Nef proteins and we found that this property is conserved among nef alleles from the M, N and O groups of HIV-1, as well as those from SIVcpz, the precursor of HIV-1, and a variety of other highly divergent primate lentiviruses. In conclusion, our data indicate that Nef's ability to selectively downregulate HLA-A and -B alleles to prevent CTL lysis and NK killing of virally infected cells is conserved among different primate lentiviral lineages and preceded the zoonotic transmission of SIVcpz from chimpanzees to humans.
Subject(s)
Alleles , Down-Regulation , Gene Products, nef/metabolism , HLA-A Antigens/metabolism , HLA-B Antigens/metabolism , Lentiviruses, Primate/metabolism , Amino Acid Sequence , Animals , CD8 Antigens/metabolism , Cell Line , Gene Products, nef/chemistry , Gene Products, nef/genetics , HIV-1/metabolism , HLA-A Antigens/chemistry , HLA-B Antigens/chemistry , Humans , Jurkat Cells , Killer Cells, Natural/immunology , Lentiviruses, Primate/classification , Molecular Sequence Data , Simian Immunodeficiency Virus/metabolism , T-Lymphocytes, Cytotoxic/immunology , TransfectionABSTRACT
Sexual intercourse is the major route of HIV transmission. To identify endogenous factors that affect the efficiency of sexual viral transmission, we screened a complex peptide/protein library derived from human semen. We show that naturally occurring fragments of the abundant semen marker prostatic acidic phosphatase (PAP) form amyloid fibrils. These fibrils, termed Semen-derived Enhancer of Virus Infection (SEVI), capture HIV virions and promote their attachment to target cells, thereby enhancing the infectious virus titer by several orders of magnitude. Physiological concentrations of SEVI amplified HIV infection of T cells, macrophages, ex vivo human tonsillar tissues, and transgenic rats in vivo, as well as trans-HIV infection of T cells by dendritic or epithelial cells. Amyloidogenic PAP fragments are abundant in seminal fluid and boost semen-mediated enhancement of HIV infection. Thus, they may play an important role in sexual transmission of HIV and could represent new targets for its prevention.
Subject(s)
Amyloid/physiology , HIV Infections/transmission , Peptide Fragments/physiology , Protein Tyrosine Phosphatases/chemistry , Protein Tyrosine Phosphatases/physiology , Semen/metabolism , Sexually Transmitted Diseases, Viral/metabolism , Acid Phosphatase , Amyloid/isolation & purification , Animals , Animals, Genetically Modified , Humans , Peptide Library , Rats , Semen/enzymology , Semen/physiology , Viral LoadABSTRACT
Nef is a multifunctional accessory protein of primate lentiviruses. Recently, it has been shown that the ability of Nef to downmodulate CD4, CD28, and class I major histocompatibility complex is highly conserved between most or all primate lentiviruses, whereas Nef-mediated downregulation of T-cell receptor-CD3 was lost in the lineage that gave rise to human immunodeficiency virus type 1 (HIV-1). Whether or not other Nef activities are preserved between different groups of primate lentiviruses remained to be determined. Here, we show that nef genes from a large variety of HIVs and simian immunodeficiency viruses (SIVs) enhance virion infectivity and stimulate viral replication in human cells and/or in ex vivo infected human lymphoid tissue (HLT). Notably, nef alleles from unpassaged SIVcpz and SIVsmm enhanced viral infectivity, replication, and cytopathicity in cell culture and in ex vivo infected HLT as efficiently as those from HIV-1 and HIV-2, their human counterparts. Furthermore, nef genes from several highly divergent SIVs that have not been found in humans were also highly active in human cells and/or tissues. Thus, most primate lentiviral Nefs enhance virion infectivity and stimulate viral replication. Moreover, our data show that SIVcpz and SIVsmm Nefs do not require adaptive changes to perform these functions in human cells or tissues and support the idea that nef alleles from other primate lentiviruses would also be capable of promoting efficient virus spread in humans.
Subject(s)
HIV-1/pathogenicity , Simian Immunodeficiency Virus/pathogenicity , Viral Regulatory and Accessory Proteins/metabolism , Virion/pathogenicity , Virus Replication/drug effects , nef Gene Products, Human Immunodeficiency Virus/metabolism , Animals , Cell Line , HIV-1/classification , HIV-1/genetics , HeLa Cells , Humans , Organ Culture Techniques , Palatine Tonsil/virology , Simian Immunodeficiency Virus/classification , Simian Immunodeficiency Virus/genetics , Viral Regulatory and Accessory Proteins/genetics , Viral Regulatory and Accessory Proteins/pharmacology , Virus Replication/ethics , nef Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
Interaction of the human immunodeficiency virus type 1 (HIV-1) Nef protein with p21-activated kinase 2 (PAK2) has been proposed to play a role in T-cell activation, viral replication, apoptosis, and progression to AIDS. However, these hypotheses were based on results obtained using Nef mutants impaired in multiple functions. Recently, it was reported that Nef residue F191 is specifically involved in PAK2 binding. However, only a limited number of Nef activities were investigated in these studies. To further evaluate the role of F191 in Nef function and to elucidate the biological relevance of Nef-PAK2 interaction, we performed a comprehensive analysis of HIV-1 Nef mutants carrying F191H and F191R mutations. We found that the F191H mutation reduces and the F191R mutation disrupts the association of Nef with PAK2. Both mutants upregulated the major histocompatibility complex II (MHC-II)-associated invariant chain and downregulated CD4, MHC-I, and CD28, although with reduced efficiency for the latter. Furthermore, the F191H/R changes neither affected the levels of interleukin-2 receptor expression and apoptosis of HIV-1-infected primary T cells nor reduced Nef-mediated induction of NFAT. Unexpectedly, the F191H change markedly reduced and the F191R mutation disrupted the ability of Nef to enhance virion infectivity in P4-CCR5 indicator cells but not in TZM-bl cells or peripheral blood mononuclear cells. Most importantly, all HIV-1 Nef mutants replicated efficiently and caused CD4+ T-cell depletion in ex vivo-infected human lymphoid tissue. Altogether, our data show that the interaction of Nef with PAK2 does not play a major role in T-cell activation, viral replication, and apoptosis.
Subject(s)
Cytopathogenic Effect, Viral/physiology , HIV-1/immunology , HIV-1/pathogenicity , Lymphoid Tissue/virology , Virus Replication/physiology , nef Gene Products, Human Immunodeficiency Virus/metabolism , p21-Activated Kinases/metabolism , Amino Acid Substitution/genetics , Antigens, Differentiation, B-Lymphocyte/biosynthesis , Apoptosis , CD28 Antigens/biosynthesis , CD4 Antigens/biosynthesis , Cell Line , Histocompatibility Antigens Class I/biosynthesis , Histocompatibility Antigens Class II/biosynthesis , Humans , Mutation, Missense , NFATC Transcription Factors/biosynthesis , Protein Binding , Receptors, Interleukin-2/biosynthesis , nef Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
AMP-activated protein kinase (AMPK) plays a key role in the regulation of energy homeostasis and is activated in response to cellular stress, including hypoxia/ischemia and hyperglycemia. The stress events are accompanied by rapid release of extracellular nucleotides from damaged tissues or activated endothelial cells (EC) and platelets. We demonstrate that extracellular nucleotides (ATP, ADP, and UTP, but not UDP) and adenosine independently induce phosphorylation and activation of AMPK in human umbilical vein EC (HUVEC) by the mechanism that is not linked to changes in AMP:ATP ratio. HUVEC express NTPDases, as well as 5'-nucleotidase; hence, nucleotides can be metabolized to adenosine. However, inhibition of 5'-nucleotidase had no effect on ATP/ADP/UTP-induced phospho- rylation of AMPK, indicating that AMPK activation occurred as a direct response to nucleotides. Nucleotide-evoked phosphorylation of AMPK in HUVEC was mediated by P2Y1, P2Y2, and/or P2Y4 receptors, whereas P2Y6, P2Y11, and P2X receptors were not involved. The nucleotide-induced phosphorylation of AMPK was affected by changes in the concentration of intracellular Ca2+ and by Ca2+/calmodulin-dependent kinase kinase (CaMKK), although most likely it was not dependent on LKB1 kinase. Adenosine-induced phosphorylation of AMPK was not mediated by P1 receptors but required adenosine uptake by equilibrative nucleoside transporters followed by its (intracellular) metabolism to AMP. Moreover, adenosine effect was Ca2+ and CaMKK independent, although probably associated with upstream LKB1. We hypothesize that P2 receptors and adenosine transporters could be novel targets for the pharmacological regulation of AMPK activity and its downstream effects on EC function.