Eap45 in mammalian ESCRT-II binds ubiquitin via a phosphoinositide-interacting GLUE domain

Ubiquitination serves as a key sorting signal in the lysosomal degradation of endocytosed receptors through the ability of ubiquitinated membrane proteins to be recognized and sorted by ubiquitin-binding proteins along the endocytic route. The ESCRT-II complex in yeast contains one such protein, Vps36, which harbors a ubiquitin-binding NZF domain and is required for vacuolar sorting of ubiquitinated membrane proteins. Surprisingly, the presumptive mammalian ortholog Eap45 lacks the ubiquitin-binding module of Vps36, and it is thus not clear whether mammalian ESCRT-II functions to bind ubiquitinated cargo. In this paper, we provide evidence that Eap45 contains a novel ubiquitin-binding domain, GLUE (GRAM-like ubiquitin-binding in Eap45), which binds ubiquitin with similar affinity and specificity as other ubiquitin-binding domains. The GLUE domain shares similarities in its primary and predicted secondary structures to phosphoinositide-binding GRAM and PH domains. Accordingly, we find that Eap45 binds to a subset of 3-phosphoinositides, suggesting that ubiquitin recognition could be coordinated with phosphoinositide binding. Furthermore, we show that Eap45 colocalizes with ubiquitinated proteins on late endosomes. These results are consistent with a role for Eap45 in endosomal sorting of ubiquitinated cargo.     

Human Tat-specific factor 1 binds the HIV-1 genome and selectively transports HIV-1 RNAs

Molecular Biology Reports - Human immunodeficiency virus type 1 (HIV-1) propagation requires many human cofactors. Multiple groups have demonstrated that Tat-specific factor 1 (Tat-SF1) is an HIV-1...     

The clathrin adaptor complex AP-1 binds HIV-1 and MLV Gag and facilitates their budding

Retroviral assembly is driven by Gag, and nascent viral particles escape cells by recruiting the machinery that forms intralumenal vesicles of multivesicular bodies. In this study, we show that the clathrin adaptor complex AP-1 is involved in retroviral release. The absence of AP-1mu obtained by genetic knock-out or by RNA interference reduces budding of murine leukemia virus (MLV) and HIV-1, leading to a delay of viral propagation in cell culture. In contrast, overexpression of AP-1mu enhances release of HIV-1 Gag. We show that the AP-1 complex facilitates retroviral budding through a direct interaction between the matrix and AP-1mu. Less MLV Gag is found associated with late endosomes in cells lacking AP-1, and our results suggest that AP-1 and AP-3 could function on the same pathway that leads to Gag release. In addition, we find that AP-1 interacts with Tsg101 and Nedd4.1, two cellular proteins known to be involved in HIV-1 and MLV budding. We propose that AP-1 promotes Gag release by transporting it to intracellular sites of active budding, and/or by facilitating its interactions with other cellular partners.     

gp340 expressed on human genital epithelia binds HIV-1 envelope protein and facilitates viral transmission

During sexual transmission of HIV in women, the first cells likely to be infected are submucosal CD4(+) T cells and dendritic cells of the lower genital tract. HIV is segregated from these target cells by an epithelial cell layer that can be bypassed even when healthy and intact. To understand how HIV penetrates this barrier, we identified a host protein, gp340, that is expressed on genital epithelium and binds the HIV envelope via a specific protein-protein interaction. This binding allows otherwise subinfectious amounts of HIV to efficiently infect target cells and allows this infection to occur over a longer period of time after binding. Our findings suggest a mechanism of viral entry during heterosexual transmission where HIV is bound to intact genital epithelia, which then promotes the initial events of infection. Understanding this step in the initiation of infection will allow for the development of tools and methods for blocking HIV transmission.     

Cell-dependent replication potentials of HIV-1 gag mutants

An infectious molecular clone of human immunodeficiency virus type 1 (HIV-1), designated pNLaiKH, which is tropic for both lymphocytic and monocytic cells, was constructed. To study the early function of HIV-1 Gag proteins in two types of cells, the mutations known to give host cell-dependent early defects were introduced into pNLaiKH, and the replication potentials and defective replication sites in the cells of the resultant mutants were monitored. All mutants grew in some lymphocytic cells, but not at all in monocytic cells. A nucleocapsid mutant was found to be defective at an early replication phase in all the cell lines to various extent, as expected. In contrast, a matrix mutant and a capsid mutant displayed a replication defect in a producer-cell-dependent manner. These results demonstrated that complex interactions of cell factors and Gag proteins are involved in an early process of HIV-1 replication.     

Assembly and release of HIV-1 precursor Pr55gag virus-like particles from recombinant baculovirus-infected insect cells

The unprocessed Gag precursor from HIV-1, when expressed in recombinant baculovirus-infected insect cells, is targeted to the plasma membrane and assembles in 100-120 nm particles budding from the cell surface. This process mimics HIV immature particle formation and is dependent on myristoylation of the N-terminal glycine, as deletion of the latter results in particle accumulation in the cytoplasm and, interestingly, in the nucleus, pointing to a potential role of this non-fatty-acid-acylated species in the viral life cycle. Inclusion of the pol gene in the construct results in efficient processing of Pr55gag and a pronounced decrease in particle formation. Deletion of the C terminus (p16) of the Gag precursor, including the finger domains, abolishes particle assembly, but membrane targeting and evagination still occur. Heterologous expression in insect cells may prove very useful for the study of the molecular events leading to retroviral particle morphogenesis.     

Solution properties of murine leukemia virus gag protein: differences from HIV-1 gag

Immature retrovirus particles are assembled from the multidomain Gag protein. In these particles, the Gag proteins are arranged radially as elongated rods. We have previously characterized the properties of HIV-1 Gag in solution. In the absence of nucleic acid, HIV-1 Gag displays moderately weak interprotein interactions, existing in monomer-dimer equilibrium. Neutron scattering and hydrodynamic studies suggest that the protein is compact, and biochemical studies indicate that the two ends can approach close in three-dimensional space, implying the need for a significant conformational change during assembly. We now describe the properties of the Gag protein of Moloney murine leukemia virus (MLV), a gammaretrovirus. We found that this protein is very different from HIV-1 Gag: it has much weaker protein-protein interaction and is predominantly monomeric in solution. This has allowed us to study the protein by small-angle X-ray scattering and to build a low-resolution molecular envelope for the protein. We found that MLV Gag is extended in solution, with an axial ratio of ～7, comparable to its dimensions in immature particles. Mutational analysis suggests that runs of prolines in its matrix and p12 domains and the highly charged stretch at the C terminus of its capsid domain all contribute to this extended conformation. These differences between MLV Gag and HIV-1 Gag and their implications for retroviral assembly are discussed.     

Ficolin-2 binds to HIV-1 gp120 and blocks viral infection

Ficolin-2 is a lectin complement pathway activator present in normal human plasma and usually associated with infectious diseases, but little is known about the role of ficolin-2 in human immunodeficiency virus (HIV) infection. Here, we describe our novel findings that serum ficolin-2 concentrations of 103 HIV-1 patients were much higher compared to those of 57 healthy donors. In vitro analysis showed that HIV-1 infection could enhance ficolin-2 expression. We further demonstrated that recombinant ficolin-2 protein could bind with HIV-1 envelope glycoprotein gp120, and subsequently induce complement dependent cytotoxicity. Moreover, ficolin-2 could block the entry of HIV-1 into target cells (TZM-b1 and MT-2 cells) and infection in a ficolin-2 dosedependent manner. To our knowledge, this is the first report about the protective role of ficolin-2 against HIV-1 infection and our study suggests that ficolin-2 is an important human innate immune molecule against HIV.

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Human ESCRT-II complex and its role in human immunodeficiency virus type 1 release

The budding of many enveloped RNA viruses, including human immunodeficiency virus type 1 (HIV-1), requires some of the same cellular machinery as vesicle formation at the multivesicular body (MVB). In Saccharomyces cerevisiae, the ESCRT-II complex performs a central role in MVB protein sorting and vesicle formation, as it is recruited by the upstream ESCRT-I complex and nucleates assembly of the downstream ESCRT-III complex. Here, we report that the three subunits of human ESCRT-II, EAP20, EAP30, and EAP45, have a number of properties in common with their yeast orthologs. Specifically, EAP45 bound ubiquitin via its N-terminal GRAM-like ubiquitin-binding in EAP45 (GLUE) domain, both EAP45 and EAP30 bound the C-terminal domain of TSG101/ESCRT-I, and EAP20 bound the N-terminal half of CHMP6/ESCRT-III. Consistent with its expected role in MVB vesicle formation, (i) human ESCRT-II localized to endosomal membranes in a VPS4-dependent fashion and (ii) depletion of EAP20/ESCRT-II and CHMP6/ESCRT-III inhibited lysosomal targeting and downregulation of the epidermal growth factor receptor, albeit to a lesser extent than depletion of TSG101/ESCRT-I. Nevertheless, HIV-1 release and infectivity were not reduced by efficient small interfering RNA depletion of EAP20/ESCRT-II or CHMP6/ESCRT-III. These observations indicate that there are probably multiple pathways for protein sorting/MVB vesicle formation in human cells and that HIV-1 does not utilize an ESCRT-II-dependent pathway to leave the cell.     

Analysis of common epitopes on gag proteins of HIV-1, HIV-2 and SIV[AGM] using monoclonal antibodies against HIV-1.

Five monoclonal antibodies (MoAbs) to gag proteins of HIV-1 were prepared in mice. Western blot analyses showed that three clones recognized p24 and the other two p17. Among the three MoAbs recognizing p24, all recognized two of three strains of HIV-2. The spectra of reactions to SIV[AGM] of these MoAbs against p24 were different from one to another; K3-24 recognized all four strains of SIV[AGM], L6-24 three of them, and K5-24 none of them. Of the two MoAbs recognizing p17, K7-17 recognized two of the three strains of HIV-2 but not any SIV[AGM] strain, and the other clone, L14-17 recognized none of analogous proteins of HIV-2 nor of SIV[AGM]. These results demonstrate that the gag proteins of HIV-2 and SIV[AGM] share some common epitopes with those of HIV-1 which are heterogenic in some degree among the different isolates.     

1型和2型外壳蛋白构建的AAV载体携带HIV-1 gag诱导免疫反应的比较研究

比较两种血清型的腺病毒伴随病毒(Adeno-associated virus,AAV)载体携带HIV-1gag基因肌肉注射诱导小鼠免疫反应的特点。分别制备携带EGFP和HIV-1gag基因的重组AAV2/1(AAV1)和AAV2载体。小鼠肌肉注射rAAV1-EGFP和rAAV2-EGFP,观察注射局部EGFP的表达。将rAAV1-gag和rAAV2-gag分别以0、3周初免/加强方式肌肉注射免疫BALB/c小鼠以及新西兰白兔。ELISA法检测抗HIV-1 Gag P24蛋白的特异性抗体,细胞内细胞因子染色法检测Gag特异性的CTL反应。结果表明,rAAV1-EGFP在小鼠肌肉的表达强度显著高于rAAV2-EGFP;用Western blot法和间接免疫荧光法检测rAAV1-gag和rAAV2-gag体外转染的293细胞,均可检测到HIV-1 Gag蛋白的表达;在小鼠体内rAAV1-gag和rAAV2-gag组均可检测到特异性P24抗体,抗体滴度rAAV1-gag组显著高于rAAV2-gag组;而无论rAAV1-gag组还是rAAV2-gag组,特异性CTL反应均较低,与阴性对照组相比均无显著性差异;两种载体免疫兔子也都可检测到特异性P24抗体,同样地,rAAV1-gag组显著高于rAAV2-gag组。结论:携带HIV-1gag基因的rAAV1或rAAV2以肌肉注射方式免疫小鼠主要诱导特异Gag的体液免疫反应;且rAAV1可诱导很强的抗HIV-1 Gag特异性抗体,抗体水平显著高于rAAV2。     

表达HIV-1 gag重组鸡痘病毒的构建与筛选

构建并筛选表达HIV-1 gag蛋白的重组鸡痘病毒,并对其进行鉴定。首先设计引物通过PCR技术扩增HIV-1 gag基因,将其连接到pMD18-T载体上,测序正确后将其克隆入本实验室自行构建的鸡痘病毒穿梭载体pTKET中,获得重组质粒pTKET-HIV gag。然后将其与鸡痘病毒FPV282E4株共转染原代鸡胚成纤维细胞(CEF)进行同源重组,以增强型绿色荧光蛋白(EGFP)为筛选标记,通过噬斑筛选获得重组病毒,应用PCR,RT-PCR,Western blot方法对重组病毒进行鉴定和遗传稳定性分析。结果:通过10次噬斑筛选,PCR检测表明目的基因已整合到重组鸡痘病毒基因组中,RT-PCR,Western blot结果表明HIV-1 gag在感染细胞内成功表达且具有抗原性。连续传代20次,PCR,RT-PCR,Western blot均能检测到外源基因的整合、转录和表达,且未能扩增出FPV-TK基因,表明重组病毒遗传稳定性良好,而且病毒已经纯化。结论:成功获得表达HIV-1gag的重组鸡痘病毒,为进一步免疫试验研究奠定基础。     

Sprouty2 Regulates PI(4,5)P2/Ca Signaling and HIV-1 Gag Release

We reported recently that activation of the inositol 1,4,5-triphosphate receptor (IP3R) is required for efficient HIV-1 Gag trafficking and viral particle release. IP3R activation requires phospholipase C (PLC)-catalyzed hydrolysis of PI(4,5)P₂ to IP3 and diacylglycerol. We show that Sprouty2 (Spry2), which binds PI(4,5)P₂ and PLCγ, interfered with PI(4,5)P₂ in a manner similar to that of U73122, an inhibitor of PI(4,5)P₂ hydrolysis, suggesting that Spry2 negatively regulates IP3R by preventing formation of its activating ligand, IP3. Mutation to Asp of R252, a crucial determinant of PI(4,5)P₂ binding in the C-terminal domain of Spry2, prevented the interference, indicating that binding to the phospholipid is required. By contrast, deletion of the PLCγ binding region or mutation of a critical Tyr residue in the region did not prevent the interference but Spry2-PI(4,5)P₂ colocalization was not detected, suggesting that PLC binding is required for their stable association. Like U73122, Spry2 over-expression inhibited wild type Gag release as virus-like particles. Disrupting either binding determinant relieved the inhibition. IP3R-mediated Ca²⁺signaling, in turn, was found to influence Spry2 subcellular distribution and ERK, a Spry2 regulator. Our findings suggest that Spry2 influences IP3R function through control of PI(4,5)P₂ and IP3R influences Spry2 function by controlling its distribution and ERK activation.     

A simian replication-defective adenoviral recombinant vaccine to HIV-1 gag

In animal models, E1-deleted human adenoviral recombinants of the serotype 5 (AdHu5) have shown high efficacy as vaccine carriers for different Ags including those of HIV-1. Humans are infected by common serotypes of human adenovirus such as AdHu5 early in life and a significant percentage has high levels of neutralizing Abs to these serotypes, which will very likely impair the efficacy of recombinant vaccines based on the homologous virus. To circumvent this problem, a novel replication-defective adenoviral vaccine carrier based on an E1-deleted recombinant of the chimpanzee adenovirus 68 (AdC68) was developed. An AdC68 construct expressing a codon-optimized, truncated form of gag of HIV-1 induces CD8(+) T cells to gag in mice which at the height of the immune response encompass nearly 20% of the entire splenic CD8(+) T cell population. The vaccine-induced immune response provides protection to challenge with a vaccinia gag recombinant virus. Induction of transgene-specific CD8(+) T cells and protection against viral challenge elicited by the AdC68 vaccines is not strongly inhibited in animals preimmune to AdHu5 virus. However, the response elicited by the AdHu5 vaccine is greatly attenuated in AdHu5 preimmune animals.     

Identification of an intracellular trafficking and assembly pathway for HIV-1 gag

Retroviral Gag proteins are membrane-bound polyproteins that are necessary and sufficient for virus-like particle (VLP) formation. It is not known how Gag traffics through the cell or how the site of particle production is determined. Here we use two techniques, biarsenical/tetracysteine (TC) labeling and release from a cycloheximide block, to follow the trafficking of newly synthesized HIV-1 Gag. Gag first appears diffusely distributed in the cytosol, accumulates in perinuclear clusters, passes transiently through a multivesicular body (MVB)-like compartment, and then travels to the plasma membrane (PM). Sequential passage of Gag through these temporal intermediates was confirmed by live cell imaging. Induction of a transient rise in cytoplasmic calcium increased the amounts of Gag, Gag assembly intermediates and VLPs in MVBs, and resulted in a dramatic increase in VLP release. These results define an intracellular trafficking pathway for HIV-1 Gag that uses perinuclear compartments and the MVB as trafficking intermediates. We propose that the regulation of Gag association with MVB-like compartments regulates the site of HIV-1 budding and particle formation.     

HIV-1 gag与gp41基因片段的序列特征与亚型研究

本文对华北地区出入境39例HIV-1阳性样本(中国21例,非洲17例,东南亚1例)的gag和env两个基因片段进行了序列特征和亚型对比分析。发现了A、A1、A3、B、C、G亚型和重组亚型03_AB、01_AE、AG、02_AG、07_BC、08_BC、CD和06_CPX共14个亚型,其中重组亚型占57.2%(8/14)。表明HIV-1基因变异较快,亚型分布广泛,重组亚型有增多趋势。此外发现26.7%(8/30)的样本,其gag和env基因区亚型表现不一致。提示在研究HIV-1亚型中应综合gag和env两个基因区的序列特征进行亚型分析。     

Sprouty1 and Sprouty2 provide a control mechanism for the Ras/MAPK signalling pathway

Sprouty (Spry) inhibits signalling by receptor tyrosine kinases; however, the molecular mechanism underlying this function has not been defined. Here we show that after stimulation by growth factors Spry1 and Spry2 translocate to the plasma membrane and become phosphorylated on a conserved tyrosine. Next, they bind to the adaptor protein Grb2 and inhibit the recruitment of the Grb2-Sos complex either to the fibroblast growth factor receptor (FGFR) docking adaptor protein FRS2 or to Shp2. Membrane translocation of Spry is necessary for its phosphorylation, which is essential for its inhibitor activity. A tyrosine-phosphorylated octapeptide derived from mouse Spry2 inhibits Grb2 from binding FRS2, Shp2 or mouse Spry2 in vitro and blocks activation of the extracellular-signal-regulated kinase (ERK) in cells stimulated by growth factor. A non-phosphorylated Spry mutant cannot bind Grb2 and acts as a dominant negative, inducing prolonged activation of ERK in response to FGF and promoting the FGF-induced outgrowth of neurites in PC12 cells. Our findings suggest that Spry functions in a negative feedback mechanism in which its inhibitor activity is controlled rapidly and reversibly by post-translational mechanisms.     

Binding of human immunodeficiency virus type 1 (HIV-1) RNA to recombinant HIV-1 gag polyprotein.

We have expressed the human immunodeficiency virus type 1 (HIV-1) gag polyprotein (Pr55gag) in bacteria under the control of the T7 phage gene 10 promoter. When the gene encoding the viral protease is included in cis, in the -1 reading frame, the expected proteolytic cleavage products MA and CA are produced. Disruption of the protease-coding sequence prevents proteolytic processing, and full-length polyprotein is produced. Pr55gag, separated from bacterial proteins by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and immobilized on nitrocellulose membranes, binds RNA containing sequences from the 5' end of the HIV-1 genome. This binding is tolerant of a wide range of pH and temperature but has distinct salt preferences. Conditions were identified which prevented nonspecific binding of RNA to bacterial proteins but still allowed binding to Pr55gag. Under these conditions, irrelevant RNA probes lacking HIV-1 sequences bound Pr55gag less efficiently. Quantitation of binding to Pr55gag by HIV-1 RNA probes with deletions mutations demonstrated that there are two regions lying within the HIV-1 gag gene which independently promote binding of RNA to Pr55gag.     

Ceramide binds to the CaLB domain of cytosolic phospholipase A2 and facilitates its membrane docking and arachidonic acid release.

Excessive production of eicosanoids is characteristic of many inflammatory diseases. In this study we show that ceramide, which is an early messenger of inflammatory cytokine action, exerts a dual effect on the cytosolic phospholipase A2 (cPLA2), the rate-limiting enzyme in arachidonic acid release and subsequent eicosanoid formation. Stimulation of renal mesangial cells with exogenous short-chain ceramide analogs for 30 and 60 min leads to a concentration-dependent increase in arachidonic acid release that is not blocked by specific inhibitors of mitogen-activated protein kinase pathways. This suggests that these established upstream activators of cPLA2 are not involved in ceramide-induced arachidonic acid release. By use of photoactivatable ceramide analogs, D- and L-[125I]3-trifluoromethyl-3-(m-iodophenyl)diazirine-ceramides (TID-ceramides), we observed a direct interaction of ceramide with cPLA2. This interaction was independent of the absolute configuration as D- and L-TID-ceramide were equally effective in binding to cPLA2. Moreover, recombinant CaLB domain of cPLA2 as well as a mutant deficient in the connecting 'hinge' domain of cPLA2, efficiently bound D- and L-TID-ceramides, whereas the catalytic domain did not interact with TID-ceramides. In vitro binding assays reveal that stearoyl-arachidonyl-phosphatidylcholine (SAPC)-liposomes containing increasing mol% of ceramide lead to an increased association of recombinant cPLA2 to the liposomes. Furthermore, measurement of cPLA2 activity in vitro shows that the presence of SAPC-liposomes resulted in only weak cPLA2 activity. However, the activity dramatically increases by addition of ceramide to the liposomes. Furthermore, liposomes containing SAPC and sphingomyelin resulted in no better substrate than SAPC liposomes, unless bacterial sphingomyelinase was added to generate ceramide, which then causes a marked increase in cPLA2 activity. These results demonstrate that ceramide can interact directly with cPLA2 via the CaLB domain and thereby serves as a membrane-docking device that facilitates cPLA2 action in inflammatory diseases.