SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx

The primate lentivirus auxiliary protein Vpx counteracts an unknown restriction factor that renders human dendritic and myeloid cells largely refractory to HIV-1 infection. Here we identify SAMHD1 as this restriction factor. SAMHD1 is a protein involved in Aicardi-Goutières syndrome, a genetic encephalopathy with symptoms mimicking congenital viral infection, that has been proposed to act as a negative regulator of the interferon response. We show that Vpx induces proteasomal degradation of SAMHD1. Silencing of SAMHD1 in non-permissive cell lines alleviates HIV-1 restriction and is associated with a significant accumulation of viral DNA in infected cells. Concurrently, overexpression of SAMHD1 in sensitive cells inhibits HIV-1 infection. The putative phosphohydrolase activity of SAMHD1 is probably required for HIV-1 restriction. Vpx-mediated relief of restriction is abolished in SAMHD1-negative cells. Finally, silencing of SAMHD1 markedly increases the susceptibility of monocytic-derived dendritic cells to infection. Our results demonstrate that SAMHD1 is an antiretroviral protein expressed in cells of the myeloid lineage that inhibits an early step of the viral life cycle.     

Isolation of a human gene that inhibits HIV-1 infection and is suppressed by the viral Vif protein

Viruses have developed diverse non-immune strategies to counteract host-mediated mechanisms that confer resistance to infection. The Vif (virion infectivity factor) proteins are encoded by primate immunodeficiency viruses, most notably human immunodeficiency virus-1 (HIV-1). These proteins are potent regulators of virus infection and replication and are consequently essential for pathogenic infections in vivo. HIV-1 Vif seems to be required during the late stages of virus production for the suppression of an innate antiviral phenotype that resides in human T lymphocytes. Thus, in the absence of Vif, expression of this phenotype renders progeny virions non-infectious. Here, we describe a unique cellular gene, CEM15, whose transient or stable expression in cells that do not normally express CEM15 recreates this phenotype, but whose antiviral action is overcome by the presence of Vif. Because the Vif:CEM15 regulatory circuit is critical for HIV-1 replication, perturbing the circuit may be a promising target for future HIV/AIDS therapies.     

The inner-nuclear-envelope protein emerin regulates HIV-1 infectivity

Primate lentiviruses such as human immunodeficiency type 1 (HIV-1) have the capacity to infect non-dividing cells such as tissue macrophages. In the process, viral complementary DNA traverses the nuclear envelope to integrate within chromatin. Given the intimate association between chromatin and the nuclear envelope, we examined whether HIV-1 appropriates nuclear envelope components during infection. Here we show that emerin, an integral inner-nuclear-envelope protein, is necessary for HIV-1 infection. Infection of primary macrophages lacking emerin was abortive in that viral cDNA localized to the nucleus but integration into chromatin was inefficient, and conversion of viral cDNA to non-functional episomal cDNA increased. HIV-1 cDNA associated with emerin in vivo, and the interaction of viral cDNA with chromatin was dependent on emerin. Barrier-to-autointegration factor (BAF), the LEM (LAP, emerin, MAN) binding partner of emerin, was required for the association of viral cDNA with emerin and for the ability of emerin to support virus infection. Therefore emerin, which bridges the interface between the inner nuclear envelope and chromatin, may be necessary for chromatin engagement by viral cDNA before integration.     

Identification of a protein encoded by the vpu gene of HIV-1

Human immunodeficiency virus 1 (HIV-1) is the aetiological agent of AIDS. The virus establishes lytic, latent and non-cytopathic productive infection in cells in culture. The complexity of virus-host cell interaction is reflected in the complex organization of the viral genome. In addition to the genes that encode the virion capsid and envelope proteins and the enzymes required for proviral synthesis and integration common to all retroviruses, HIV-1 is known to encode at least four additional proteins that regulate virus replication, the tat, art, sor and 3' orf proteins, as well as a protein of unknown function from the open reading frame called R. Close examination of the nucleic acid sequences of the genomes of multiple HIV isolates raised the possibility that the virus encodes a previously undetected additional protein. Here we report that HIV-1 encodes a ninth protein and that antibodies to this protein are detected in the sera of people infected with HIV-1. This protein distinguishes HIV-1 isolates from the other human and simian immunodeficiency viruses (HIV-2 and SIV) that do not have the capacity to encode a similar protein.     

Cyclophilin A retrotransposition into TRIM5 explains owl monkey resistance to HIV-1

In Old World primates, TRIM5-alpha confers a potent block to human immunodeficiency virus type 1 (HIV-1) infection that acts after virus entry into cells. Cyclophilin A (CypA) binding to viral capsid protects HIV-1 from a similar activity in human cells. Among New World primates, only owl monkeys exhibit post-entry restriction of HIV-1 (ref. 1). Paradoxically, the barrier to HIV-1 in owl monkey cells is released by capsid mutants or drugs that disrupt capsid interaction with CypA. Here we show that knockdown of owl monkey CypA by RNA interference (RNAi) correlates with suppression of anti-HIV-1 activity. However, reintroduction of CypA protein to RNAi-treated cells did not restore antiviral activity. A search for additional RNAi targets unearthed TRIMCyp, an RNAi-responsive messenger RNA encoding a TRIM5-CypA fusion protein. TRIMCyp accounts for post-entry restriction of HIV-1 in owl monkeys and blocks HIV-1 infection when transferred to otherwise infectable human or rat cells. It seems that TRIMCyp arose after the divergence of New and Old World primates when a LINE-1 retrotransposon catalysed the insertion of a CypA complementary DNA into the TRIM5 locus. This is the first vertebrate example of a chimaeric gene generated by this mechanism of exon shuffling.     

Challenges in the development of an HIV-1 vaccine

The development of a safe and effective human immunodeficiency virus (HIV)-1 vaccine is a critically important global health priority. Despite recent advances in our understanding of HIV-1 pathogenesis and immunology, however, major scientific obstacles remain. Prototype HIV-1 vaccine candidates aimed at eliciting humoral and cellular immune responses have so far failed to protect against HIV-1 infection or to reduce viral loads after infection in clinical efficacy studies. A renewed and coordinated commitment to basic discovery research, preclinical studies and clinical trials will therefore be required to overcome the hurdles currently facing the field. Here I review key challenges and future prospects in the quest to develop a prophylactic HIV-1 vaccine.     

HIV-1 adaptation to NK-cell-mediated immune pressure

Natural killer (NK) cells have an important role in the control of viral infections, recognizing virally infected cells through a variety of activating and inhibitory receptors. Epidemiological and functional studies have recently suggested that NK cells can also contribute to the control of HIV-1 infection through recognition of virally infected cells by both activating and inhibitory killer immunoglobulin-like receptors (KIRs). However, it remains unknown whether NK cells can directly mediate antiviral immune pressure in vivo in humans. Here we describe KIR-associated amino-acid polymorphisms in the HIV-1 sequence of chronically infected individuals, on a population level. We show that these KIR-associated HIV-1 sequence polymorphisms can enhance the binding of inhibitory KIRs to HIV-1-infected CD4(+) T cells, and reduce the antiviral activity of KIR-positive NK cells. These data demonstrate that KIR-positive NK cells can place immunological pressure on HIV-1, and that the virus can evade such NK-cell-mediated immune pressure by selecting for sequence polymorphisms, as was previously described for virus-specific T cells and neutralizing antibodies. NK cells might therefore have a previously underappreciated role in contributing to viral evolution.     

Biologically diverse molecular variants within a single HIV-1 isolate

AIDS is a disorder characterized by a slow progressive impairment of immune function and by infection of human immunodeficiency viruses (HIV-1, HIV-2). Our knowledge of how these viruses cause disease in man, or how the related lentiviruses (visna and equine infectious anaemia virus) cause disease in animals, is still fragmentary. In particular, the significance of genetic variation in HIV-1, occurring within populations, within individuals and over periods of time, and the mechanisms of viral persistence remain unclear. To address these issues we prepared a series of proviral clones of HIV-1 originating from a single patient and compared their biological properties. Here we show that hybrid genomes (in which the envelope region of six viral clones were separately substituted into a prototype HIV-1 genome) generated viruses with widely differing capacity to grow in human T cells, cell lines and monocytoid cultures. These data suggest that extensive biological variation exists in vivo within an infected individual and is in part determined at the level of the viral envelope.     

Herpesvirus latency confers symbiotic protection from bacterial infection

All humans become infected with multiple herpesviruses during childhood. After clearance of acute infection, herpesviruses enter a dormant state known as latency. Latency persists for the life of the host and is presumed to be parasitic, as it leaves the individual at risk for subsequent viral reactivation and disease. Here we show that herpesvirus latency also confers a surprising benefit to the host. Mice latently infected with either murine gammaherpesvirus 68 or murine cytomegalovirus, which are genetically highly similar to the human pathogens Epstein-Barr virus and human cytomegalovirus, respectively, are resistant to infection with the bacterial pathogens Listeria monocytogenes and Yersinia pestis. Latency-induced protection is not antigen specific but involves prolonged production of the antiviral cytokine interferon-gamma and systemic activation of macrophages. Latency thereby upregulates the basal activation state of innate immunity against subsequent infections. We speculate that herpesvirus latency may also sculpt the immune response to self and environmental antigens through establishment of a polarized cytokine environment. Thus, whereas the immune evasion capabilities and lifelong persistence of herpesviruses are commonly viewed as solely pathogenic, our data suggest that latency is a symbiotic relationship with immune benefits for the host.     

Macrophage and T cell-line tropisms of HIV-1 are determined by specific regions of the envelope gp120 gene

Strains of human immunodeficiency virus type 1 (HIV-1) display a high degree of biological heterogeneity which may be linked to certain clinical manifestation of AIDS. They vary in their ability to infect different cell types, to replicate rapidly and to high titre in culture, to down-modulate the CD4 receptor, and to cause cytopathic changes in infected cells. Some of these in vitro properties correlate with pathogenicity of the virus in vivo. To map the viral determinants of the cellular host range of HIV-1, recombinant viruses were generated between biologically active molecular clones of HIV-1 isolates showing differences in infection of primary peripheral blood macrophages and established T-cell lines. We report here that a specific region of the envelope gp120 gene representing 159 amino-acid residues of glycoprotein gp120 seems to determine macrophage tropism, whereas an overlapping region representing 321 amino-acid residues determines T cell-line tropism. These studies provide a basis for relating functional domains of the HIV-1 env gene to pathogenic potential.     

Prevention of HIV-1 infection in chimpanzees by gp120 V3 domain-specific monoclonal antibody.

The acquired immunodeficiency syndrome (AIDS) is the late-stage clinical manifestation of long-term persistent infection with the human immunodeficiency virus type 1 (HIV-1). Immune responses directed against the virus and against virus-infected cells during the persistent infection fail to mediate resolution of the infection. As a result, a successful AIDS vaccine must elicit an immune state that will prevent the establishment of the persistent infection following introduction of the virus into the host. The third hypervariable (V3) domain of the HIV-1 gp120 envelope glycoprotein is a disulphide-linked closed loop of about 30 amino acids which binds and elicits anti-HIV-1 type-specific virus-neutralizing antibodies. The in vitro characteristics of anti-V3 domain antibody suggest that this antibody could by itself prevent HIV-1 infection in vivo, an idea supported by chimpanzee challenge studies in which protection against the HIV-1 persistent infection seemed to correlate with the presence of anti-V3 domain antibody. Here we directly demonstrate the protective efficacy of anti-V3 domain antibody in vivo and propose that this antibody is potentially useful as both a pre- and post-exposure prophylactic agent.     

Extensive variation of human immunodeficiency virus type-1 in vivo

Genotypic variation among independent isolates of human immunodeficiency virus type-1 (HIV-1) is well known, but its molecular basis and biological consequences are poorly understood. We examined the genesis of molecular variation in HIV-1 by sequential virus isolations from two chronically infected individuals and analysis of recombinant HIV-1 genomic clones. In three different virus isolates full-length HIV-1 clones were identified and found to consist, respectively, of 17, 9 and 13 distinguishable, but highly-related, viral genotypes. Thirty-five viral clones derived from two HIV-1 isolates obtained from the same individual but 16 months apart showed progressive change, yet were clearly related. Similar changes in the HIV-1 genome did not occur in vitro during virus isolation and amplification. The results indicate that HIV-1 variation in vivo is rapid, that a remarkably large number of related but distinguishable genotypic variants evolve in parallel and coexist during chronic infection, and that 'isolates' of HIV-1, unless molecularly or biologically cloned, generally consist of complex mixtures of genotypically distinguishable viruses.     

The ectodomain of Toll-like receptor 9 is cleaved to generate a functional receptor

Mammalian Toll-like receptors (TLRs) 3, 7, 8 and 9 initiate immune responses to infection by recognizing microbial nucleic acids; however, these responses come at the cost of potential autoimmunity owing to inappropriate recognition of self nucleic acids. The localization of TLR9 and TLR7 to intracellular compartments seems to have a role in facilitating responses to viral nucleic acids while maintaining tolerance to self nucleic acids, yet the cell biology regulating the transport and localization of these receptors remains poorly understood. Here we define the route by which TLR9 and TLR7 exit the endoplasmic reticulum and travel to endolysosomes in mouse macrophages and dendritic cells. The ectodomains of TLR9 and TLR7 are cleaved in the endolysosome, such that no full-length protein is detectable in the compartment where ligand is recognized. Notably, although both the full-length and cleaved forms of TLR9 are capable of binding ligand, only the processed form recruits MyD88 on activation, indicating that this truncated receptor, rather than the full-length form, is functional. Furthermore, conditions that prevent receptor proteolysis, including forced TLR9 surface localization, render the receptor non-functional. We propose that ectodomain cleavage represents a strategy to restrict receptor activation to endolysosomal compartments and prevent TLRs from responding to self nucleic acids.     

Cytoimmunotherapy for persistent virus infection reveals a unique clearance pattern from the central nervous system

The mechanism(s) by which infectious or malignant material is cleared by the host has long been an area of intensive study. We have used the murine model of infection with lymphocytic choriomeningitis virus (LCMV) to look at immune clearance during persistent infection. LCMV was selected because the mouse is its natural host, it easily induces acute or persistent infection in vivo, and the mechanism by which it is cleared in vivo during acute infection is now well understood. Clearance, although associated with several antiviral immune effector mechanisms, is primarily dependent on the activity of virus-specific cytotoxic T lymphocytes (CTL) restricted by H-2 molecules of the mouse major histocompatibility complex (MHC). If these cells fail to generate or are depleted, progression from acute to persistent infection occurs. Here, using molecular probes, we show that viral nucleic acid sequences, viral proteins and infectious materials can be efficiently and effectively cleared by adoptive transfer of antiviral H-2-restricted lymphocytes bearing the Lyt 2+ phenotype. Viral materials are cleared from a wide variety of tissues and organs where they normally lodge during persistent infection. Unexpectedly, the mode by which viral materials are removed from the central nervous system (CNS) differed markedly from the mechanism of clearance occurring at other sites. These observations indicate the possible use of adoptive lymphocyte therapy for treatment of persistent infections and suggest that immune clearance of products from the CNS probably occurs by a process distinct from those in other organs.     

Antibody neutralization and escape by HIV-1

Neutralizing antibodies (Nab) are a principal component of an effective human immune response to many pathogens, yet their role in HIV-1 infection is unclear. To gain a better understanding of this role, we examined plasma from patients with acute HIV infection. Here we report the detection of autologous Nab as early as 52 days after detection of HIV-specific antibodies. The viral inhibitory activity of Nab resulted in complete replacement of neutralization-sensitive virus by successive populations of resistant virus. Escape virus contained mutations in the env gene that were unexpectedly sparse, did not map generally to known neutralization epitopes, and involved primarily changes in N-linked glycosylation. This pattern of escape, and the exceptional density of HIV-1 envelope glycosylation generally, led us to postulate an evolving 'glycan shield' mechanism of neutralization escape whereby selected changes in glycan packing prevent Nab binding but not receptor binding. Direct support for this model was obtained by mutational substitution showing that Nab-selected alterations in glycosylation conferred escape from both autologous antibody and epitope-specific monoclonal antibodies. The evolving glycan shield thus represents a new mechanism contributing to HIV-1 persistence in the face of an evolving antibody repertoire.     

Prevention of HIV-2 and SIVsm infection by passive immunization in cynomolgus monkeys.

Infection of macaques with simian immunodeficiency virus (SIV) and human immunodeficiency virus type 2 (HIV-2) are useful models for studies of immunotherapy and vaccination against HIV as well as for testing of antiviral drugs. Vaccine research showing protective immunity in immunized monkeys has indicated that it will be possible to develop a vaccine for prevention of human HIV infection, although many hurdles remain. The design of an HIV vaccine would be helped if the basis of the protective immunity could be elucidated. Passive immune prophylaxis offers a means to determine the relative role of antibodies in protection against infection. We have studied whether a transfer of antibodies can prevent HIV-2 and SIVsm (SIV of sooty mangabey origin) infection in cynomolgus monkeys. Sera with high antibody titres were collected, heat-treated and injected into naive animals 6 h before challenge with 10-100 monkey-infectious doses of live homologous virus. All control animals treated with normal monkey serum (n = 6) or no serum (n = 39) became infected by the challenge virus, whereas five out of seven animals pretreated with antibody-containing serum at a dose of 9 ml kg-1 resisted infection. Thus passively transferred antibodies can protect against a low-dose lentivirus challenge in a nonhuman primate.     

Lymphocyte activation by HIV-1 envelope glycoprotein

Cell activation by phytohaemagglutinin, phorbol ester and by the supernatant of phytohaemagglutinin-stimulated peripheral blood mononuclear cells induces the expression and cytopathic effects of latent human immunodeficiency virus type-1 (HIV-1) in vitro. The lymphocyte surface protein CD4 has been identified as a receptor for HIV-1 and binds the viral envelope glycoprotein (gp120). In the light of evidence indicating that one natural function of CD4 is as a growth factor receptor, we examined the ability of native gp120 to activate resting CD4-bearing lymphocytes. Our results indicate that gp120 has innate biological activity as a result of a specific interaction with CD4, inducing increases in intracellular levels of inositol trisphosphate and of calcium, and in interleukin-2 receptor expression and cell motility.     

胞嘧啶脱氨基酶APOBEC1研究进展

为全面理解载脂蛋白B mRNA(ApoB mRNA)编辑酶催化多肽-1(APOBEC1)的作用机制,介绍了APOBEC1和ApoB mRNA的蛋白及核酸序列,总结并绘制了APOBEC1与不同的辅助蛋白的结合模型,阐述了APOBEC1催化ApoB mRNA第6 666位的胞嘧啶(C_(6666))脱氨基化分子机制.列举了啮齿动物APOBEC1抑制多种逆转录病毒的研究报道,介绍了兔源APOBEC1结合人类免疫缺陷病毒1(HIV-1)的病毒粒子并编辑病毒基因组的机理.同时介绍了APOBEC1通过编辑胞嘧啶或与AU富集元件(ARE)结合来调控癌症等疾病相关的细胞因子表达.