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1.
Evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts 总被引:11,自引:0,他引:11
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A number of recent studies have demonstrated the significance of detergent-insoluble, glycolipid-enriched membrane domains or lipid rafts, especially in regard to activation and signaling in T lymphocytes. These domains can be viewed as floating rafts composed of sphingolipids and cholesterol which sequester glycosylphosphatidylinositol (GPI)-linked proteins, such as Thy-1 and CD59. CD45, a 200-kDa transmembrane phosphatase protein, is excluded from these domains. We have found that human immunodeficiency virus type 1 (HIV-1) particles produced by infected T-cell lines acquire the GPI-linked proteins Thy-1 and CD59, as well as the ganglioside GM1, which is known to partition preferentially into lipid rafts. In contrast, despite its high expression on the cell surface, CD45 was poorly incorporated into virus particles. Confocal fluorescence microscopy revealed that HIV-1 proteins colocalized with Thy-1, CD59, GM1, and a lipid raft-specific fluorescent lipid, DiIC(16)(3), in uropods of infected Jurkat cells. CD45 did not colocalize with HIV-1 proteins and was excluded from uropods. Dot immunoassay of Triton X-100-extracted membrane fractions revealed that HIV-1 p17 matrix protein and gp41 were present in the detergent-resistant fractions and that [(3)H]myristic acid-labeled HIV Gag showed a nine-to-one enrichment in lipid rafts. We propose a model for the budding of HIV virions through lipid rafts whereby host cell cholesterol, sphingolipids, and GPI-linked proteins within these domains are incorporated into the viral envelope, perhaps as a result of preferential sorting of HIV Gag to lipid rafts. 相似文献
2.
Effects of deletions in the cytoplasmic domain on biological functions of human immunodeficiency virus type 1 envelope glycoproteins. 总被引:40,自引:33,他引:7
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The role of the cytoplasmic domain of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins in virus replication was investigated. Deletion of residues 840 to 856 at the carboxyl terminus of gp41 reduced the efficiency of virus entry during an early step in the virus life cycle between CD4 binding and formation of the DNA provirus without affecting envelope glycoprotein synthesis, processing, or syncytium-forming ability. Deletion of residues amino terminal to residue 846 was associated with decreased stability of envelope glycoproteins made in COS-1 cells, but this phenotype was cell type dependent. The cytoplasmic domain of gp41 was not required for the incorporation of the HIV-1 envelope glycoproteins into virions. These results suggest that the carboxyl terminus of the gp41 cytoplasmic domain plays a role in HIV-1 entry other than receptor binding or membrane fusion. The cytoplasmic domain of gp41 also affects the stability of the envelope glycoprotein in some cell types. 相似文献
3.
Human immunodeficiency virus type 2 glycoprotein enhancement of particle budding: role of the cytoplasmic domain. 总被引:2,自引:2,他引:0
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Previous studies have shown that the glycoprotein cytoplasmic domains of human immunodeficiency virus type 2 (HIV-2) or simian immunodeficiency virus of macaques modulate biological activities of the viral glycoprotein complex, including syncytium formation, exterior glycoprotein conformation, and glycoprotein incorporation into budding virus particles. We have now utilized a recombinant expression system to study interactions of full-length or truncated HIV-2 glycoproteins with coexpressed HIV-2 Gag proteins which self-assemble and bud as virus-like particles. Interestingly, budding of HIV-2 virus-like particles from cells was enhanced 5- to 24-fold when Gag was coexpressed with the full-length HIV-2 glycoprotein, compared with Gag expressed either alone or with a truncated HIV-2 glycoprotein. The results obtained in this model system indicate that an additional effect of the lengthy cytoplasmic domain of the glycoprotein of HIV-2 is enhancement of particle budding. We speculate that the cytoplasmic domain of the viral glycoprotein of HIV-2 enhances budding by (i) potentiation of Gag structure or function or (ii) membrane modulation. 相似文献
4.
Human immunodeficiency virus type 1 and 2 envelope glycoproteins oligomerize through conserved sequences. 总被引:1,自引:1,他引:0
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Hetero-oligomerization between human immunodeficiency virus type 2 (HIV-2) envelope glycoprotein (Env) truncation mutants and epitope-tagged gp160 is dependent on the presence of gp41 transmembrane protein (TM) amino acids 552 to 589, a putative amphipathic alpha-helical sequence. HIV-2 Env truncation mutants containing this sequence were also able to form cross-type hetero-oligomers with HIV-1 Env. HIV-2/HIV-1 hetero-oligomerization was, however, more sensitive to disruption by mutagenesis or increased temperature. The conservation of the Env oligomerization function of the HIV-1 and HIV-2 alpha-helical sequences suggests that retroviral TM alpha-helical motifs may have a universal role in oligomerization. 相似文献
5.
Human immunodeficiency virus type 1 vectors with alphavirus envelope glycoproteins produced from stable packaging cells
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Strang BL Takeuchi Y Relander T Richter J Bailey R Sanders DA Collins MK Ikeda Y 《Journal of virology》2005,79(3):1765-1771
Alphavirus glycoproteins have broad host ranges. Human immunodeficiency virus type 1 (HIV-1) vectors pseudotyped with their glycoproteins could extend the range of tissues that can be transduced in both humans and animal models. Here, we established stable producer cell lines for HIV vectors pseudotyped with alphavirus Ross River virus (RRV) and Semliki Forest virus (SFV) glycoproteins E2E1. RRV E2E1-stable clones could routinely produce high-titer pseudotyped vectors for at least 5 months. SFV E2E1-stable clones, however, produced relatively low titers. We examined the properties of RRV E2E1-pseudotyped vectors [HIV-1(RRV)] and compared them with amphotropic murine leukemia virus Env- and vesicular stomatitis virus glycoprotein G-pseudotyped vectors. HIV-1(RRV) displayed a number of characteristics which would be advantageous in ex vivo and in vivo experiments, including resistance to inactivation by heat-labile components in fresh human sera and thermostability at 37 degrees C. Upon single-step concentration by ultracentrifugation of HIV-1(RRV), we could achieve vector stocks with titers up to 6 x 10(7) IU/ml. HIV-1(RRV) efficiently transduced cells from several different species, including murine primary dendritic cells, but failed to transduce human and murine T cells as well as human hematopoietic stem cells (HSC). These results indicate that HIV-1(RRV) could be used in a number of applications including animal model experiments and suggest that expression of RRV cellular receptors is limited or absent in certain cell types such as T cells and human HSC. 相似文献
6.
Rescue of human immunodeficiency virus type 1 matrix protein mutants by envelope glycoproteins with short cytoplasmic domains. 总被引:22,自引:17,他引:5
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The matrix (MA) protein of human immunodeficiency virus type 1 (HIV-1) forms the outer protein shell directly underneath the lipid envelope of the virion. The MA protein has a key role in different aspects of virus assembly, including the incorporation of the HIV-1 Env protein complex, which contains a transmembrane glycoprotein with an unusually long cytoplasmic tail. In this study, we compared the abilities of HIV-1 MA mutants to incorporate Env protein complexes with long and short cytoplasmic tails. While the mutant particles failed to incorporate the authentic HIV-1 Env protein complex, they retained the ability to efficiently and functionally incorporate the amphotropic murine leukemia virus Env protein complex, which has a short cytoplasmic tail. Moreover, incorporation of the autologous Env protein complex could be restored by a second-site mutation that resulted in the truncation of the cytoplasmic tail of the HIV-1 transmembrane glycoprotein. Remarkably, the second-site mutation also restored the ability of MA mutants to replicate in MT-4 cells. These results imply that the long cytoplasmic tail of the transmembrane glycoprotein is responsible for the exclusion of the HIV-1 Env protein complex from MA mutant particles. 相似文献
7.
Efficient incorporation of HLA class II onto human immunodeficiency virus type 1 requires envelope glycoprotein packaging
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HLA class II DR is one of the most abundant cell surface proteins incorporated onto human immunodeficiency virus type 1 (HIV-1) during budding. The mechanism for HLA class II protein incorporation is not known and may involve a viral protein. To determine whether Env affects HLA class II protein incorporation, HIV-1 virions, either with or without Env on their surface, were produced from HLA class II-expressing cells and analyzed by whole-virus immunoprecipitation with antisera against HLA class II proteins. HLA class II proteins were detected on virions only when wild-type Env was incorporated, while similar experiments showed that HLA class I proteins were incorporated independent of Env packaging. Therefore, the packaging of HIV-1 Env protein is required for the efficient incorporation of HLA class II but not class I proteins into the virion. Analysis of two Env mutants revealed that the presence of a 43-amino-acid sequence between amino acids 708 and 750 in the gp41(TM) cytoplasmic tail was required for efficient incorporation of HLA class II proteins. These data show that HIV-1 actively incorporates HLA class II proteins in a process that, either directly or indirectly, requires Env. 相似文献
8.
Human immunodeficiency virus type 1-derived lentivirus vectors pseudotyped with envelope glycoproteins derived from Ross River virus and Semliki Forest virus 总被引:3,自引:0,他引:3
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Ross River virus (RRV) and Semliki Forest virus (SFV) are two alphaviruses that have a high degree of amino acid homology, as well as a very broad host range. We show here that envelope glycoproteins derived from both viruses can pseudotype human immunodeficiency virus type 1 (HIV-1)-derived lentivirus vectors. Both RRV and SFV glycoproteins considerably expand the host range of the lentivirus vector, and vectors can be efficiently concentrated by ultracentrifugation. A systematic analysis comparing the alphaviral glycoproteins to the vesicular stomatitis virus glycoprotein (VSV-G) revealed that lentivirus vectors incorporate RRV glycoproteins with an efficiency comparable to that of VSV-G. Both pseudotypes have comparable physical titers, but infectious titers with the RRV pseudotype are lower than with VSV-G. Incorporation of SFV glycoproteins into lentivirus vector is less efficient, leading to decreased physical and infectious titers. The transduction rates with VSV-G-, RRV-, and SFV-pseudotyped lentivirus vectors into adherent cell lines can be significantly increased by using a combination of Polybrene and plates coated with CH-296 recombinant fibronectin fragments. Together, our data suggest that RRV and SFV glycoproteins might be suitable as alternatives to VSV-G for pseudotyping lentivirus vectors. 相似文献
9.
Truncation of the cytoplasmic domain of the simian immunodeficiency virus envelope glycoprotein increases env incorporation into particles and fusogenicity and infectivity. 总被引:2,自引:24,他引:2
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Growth of macaque simian immunodeficiency virus (SIVmac) in certain cloned human T-cell lines, such as HUT.78, selects for isolates containing a premature stop codon within the cytoplasmic domain of the transmembrane envelope glycoprotein. In contrast, propagation of virus in macaques or in their cultured T cells favors replication of virus containing the full-length envelope glycoprotein. To elucidate the causes of this phenomenon, we used a human immunodeficiency virus pseudotyping system to assess the effects on infectivity of the cytoplasmic domains of envelope glycoproteins obtained from SIVmac1A11 and SIVmac239. These envelopes contain truncated and full-length cytoplasmic domains, respectively. By analyzing human immunodeficiency virus particles containing selectable genes pseudotyped with each glycoprotein or with chimeric derivatives, we found that truncation of the cytoplasmic domain resulted in a significant advantage in viral entry into HUT.78 T cells and CD4+ U87.MG glial cells. Truncation of the cytoplasmic domain significantly enhanced both envelope density on particles and envelope-mediated cell-to-cell fusion. It is likely that one or both of these effects contribute to the observed differences in infectivity and to the selection of virions with short cytoplasmic tails in human T cells. 相似文献
10.
Role of the cytoplasmic domain of the Newcastle disease virus fusion protein in association with lipid rafts
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To explore the association of the Newcastle disease virus (NDV) fusion (F) protein with cholesterol-rich membrane domains, its localization in detergent-resistant membranes (DRMs) in transfected cells was characterized. After solubilization of cells expressing the F protein with 1% Triton X-100 at 4 degrees C, ca. 40% of total, cell-associated F protein fractionated with classical DRMs with densities of 1.07 to l.14 as defined by flotation into sucrose density gradients. Association of the F protein with this cell fraction was unaffected by the cleavage of F(0) to F(1) and F(2) or by coexpression of the NDV attachment protein, the hemagglutinin-neuraminidase protein (HN). Furthermore, elimination by mutation, of potential palmitate addition sites in and near the F-protein transmembrane domain had no effect on F-protein association with DRMs. Rather, specific deletions of the cytoplasmic domain of the F protein eliminated association with classical DRMs. Comparisons of deletions that affected fusion activity of the protein and deletions that affected DRM association suggested that there is no direct link between the cell-cell fusion activity of the F protein and DRM association. Furthermore, depletion of cholesterol from cells expressing F and HN protein, while eliminating DRM association, had no effect on the ability of these cells to fuse with avian red blood cells. These results suggest that specific localization of the F protein in cholesterol-rich membrane domains is not required for cell-to-cell fusion. Paramyxovirus F-protein cytoplasmic domains have been implicated in virus assembly. The results presented here raise the possibility that the cytoplasmic domain is important in virus assembly at least in part because it directs the protein to cholesterol-rich membrane domains. 相似文献
11.
Expression and characterization of glycophospholipid-anchored human immunodeficiency virus type 1 envelope glycoproteins. 总被引:13,自引:10,他引:3
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Four chimeric human immunodeficiency virus type 1 (HIV-1) env genes were constructed which encoded the extracellular domain of either the wild-type or a cleavage-defective HIV-1 envelope glycoprotein (gp160) fused at one of two different positions in env to a C-terminal glycosyl-phosphatidylinositol (GPI) attachment signal from the mouse Thy-1.1 glycoprotein. All four of the constructs encoded glycoproteins that were efficiently expressed when Rev was supplied in trans, and the two cleavable forms were processed normally to gp120 and a chimeric "gp41." The chimeric glycoproteins, in contrast to the wild-type glycoprotein, could be cleaved from the surface of transfected cells by treatment with phosphatidylinositol-specific phospholipase C, indicating that they were anchored in the plasma membrane by a GPI moiety. These GPI-anchored glycoproteins were transported intracellularly at a rate only slightly lower than that of the full-length HIV-1 glycoprotein and were present on the cell surface in equivalent amounts. Nevertheless, all four glycoproteins were defective in mediating both cell-cell and virus-cell fusion as determined by syncytium formation in COS-1-HeLa-T4 cell mixtures and trans complementation of an env-defective HIV-1 genome. 相似文献
12.
Human immunodeficiency virus type 1 Nef activates p21-activated kinase via recruitment into lipid rafts 总被引:4,自引:0,他引:4
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The Nef protein of human immunodeficiency virus type 1 is an important factor in AIDS pathogenesis. In addition to downregulating CD4 and major histocompatibility complex class I molecules from the cell surface, as well as increasing virion infectivity, Nef triggers activation of the T-cell receptor (TCR) cascade to facilitate virus spread. Signaling pathways that are induced by Nef have been identified; however, it is unclear how and in which subcellular compartment Nef triggers signaling. Nef recruits a multiprotein complex to activate the cellular Pak kinase that mediates downstream effector functions. Since a subpopulation of Nef is present in detergent-insoluble microdomains (lipid rafts) from where physiological TCR signaling is initiated, we tested whether lipid rafts are instrumental for Nef-mediated Pak activation. In flotation analysis, Nef-associated Pak activity exclusively fractionated with lipid rafts. Activation of Pak in the presence of Nef coincided with lipid raft recruitment of the kinase, which was otherwise excluded from detergent-insoluble microdomains. Experimental solubilization of lipid rafts interfered with the association of Pak activity with Nef. To analyze the importance of the raft localization for Nef function more rigorously, we generated a palmitoylated Nef (PalmNef). PalmNef was highly enriched in lipid rafts and associated with significantly higher levels of Pak activity than Nef. Notably, activation of Pak by its physiological activators, Cdc42 and Rac, also occurred in lipid rafts and required raft integrity. Together, these data suggest that Nef induces signal transduction via the recruitment of a signaling machinery including Pak into lipid rafts, thereby mimicking a physiological cellular mechanism to initiate the TCR cascade. 相似文献
13.
Effect of the cytoplasmic domain of the simian immunodeficiency virus envelope protein on incorporation of heterologous envelope proteins and sensitivity to neutralization
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In addition to the viral envelope (Env) proteins, host cell-derived proteins have been reported to be present in human immunodeficiency virus and simian immunodeficiency virus (SIV) envelopes, and it has been postulated that they may play a role in infection. We investigated whether the incorporation of host cell proteins is affected by the structure and level of incorporation of viral Env proteins. To compare the cellular components incorporated into SIV particles and how this is influenced by the structure of the cytoplasmic domain, we compared SIV virions with full-length and truncated Env proteins. The levels of HLA-I and HLA-II molecules were found to be significantly (15- to 25-fold) higher in virions with full-length Env than in those with a truncated Env. Virions with a truncated Env were also found to be less susceptible to neutralization by specific antibodies against HLA-I or HLA-II proteins. We also compared the level of incorporation into SIV virions of a coexpressed heterologous viral glycoprotein, the influenza virus hemagglutinin (HA) protein. We found that SIV infection of cells expressing influenza virus HA resulted in the production of phenotypically mixed SIV virions containing influenza virus HA as well as SIV envelope proteins. The HA proteins were more effectively incorporated into virions with full-length Env than in virions with truncated Env. The phenotypically mixed particles with full-length Env, containing higher levels of HA, were sensitive to neutralization with anti-HA antibody, whereas virions with truncated Env proteins and containing lower levels of HA were more resistant to neutralization by anti-HA antibody. In contrast, SIV virions with truncated Env proteins were found to be highly sensitive to neutralization by antisera to SIV, whereas virions with full-length Env proteins were relatively resistant to neutralization. These results indicate that the cytoplasmic domain of SIV Env affects the incorporation of cellular as well as heterologous viral membrane proteins into the SIV envelope and may be an important determinant of the sensitivity of the virus to neutralizing antibodies. 相似文献
14.
Human immunodeficiency virus type 1 particles pseudotyped with envelope proteins that fuse at low pH no longer require Nef for optimal infectivity
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We have investigated the effects of Nef on infectivity in the context of various viral envelope proteins. These experiments were performed with a minimal vector system where Nef is the only accessory protein present. Our results support the hypothesis that the route of entry influences the ability of Nef to enhance human immunodeficiency virus (HIV) infectivity. We show that HIV particles pseudotyped with Ebola virus glycoprotein or vesicular stomatitis virus glycoprotein (VSV-G), which fuse at low pH, do not require Nef for optimal infectivity. In contrast, Nef significantly enhances the infectivity of virus particles that contain envelope proteins that fuse at neutral pH (CCR5-dependent HIV Env, CXCR4-dependent HIV Env, or amphotropic murine leukemia virus Env). In addition, our results demonstrate that virus particles containing mixed CXCR4-dependent HIV and VSV-G envelope proteins show a conditional requirement for Nef for optimal infectivity, depending on which protein is allowed to facilitate entry. 相似文献
15.
Gag regulates association of human immunodeficiency virus type 1 envelope with detergent-resistant membranes
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Assembly of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein on budding virus particles is important for efficient infection of target cells. In infected cells, lipid rafts have been proposed to form platforms for virus assembly and budding. Gag precursors partly associate with detergent-resistant membranes (DRMs) that are believed to represent lipid rafts. The cytoplasmic domain of the envelope gp41 usually carries palmitate groups that were also reported to confer DRM association. Gag precursors confer budding and carry envelope glycoproteins onto virions via specific Gag-envelope interactions. Thus, specific mutations in both the matrix domain of the Gag precursor and gp41 cytoplasmic domain abrogate envelope incorporation onto virions. Here, we show that HIV-1 envelope association with DRMs is directly influenced by its interaction with Gag. Thus, in the absence of Gag, envelope fails to associate with DRMs. A mutation in the p17 matrix (L30E) domain in Gag (Gag L30E) that abrogates envelope incorporation onto virions also eliminated envelope association with DRMs in 293T cells and in the T-cell line, MOLT 4. These observations are consistent with a requirement for an Env-Gag interaction for raft association and subsequent assembly onto virions. In addition to this observation, we found that mutations in the gp41 cytoplasmic domain that abrogated envelope incorporation onto virions and impaired infectivity of cell-free virus also eliminated envelope association with DRMs. On the basis of these observations, we propose that Gag-envelope interaction is essential for efficient envelope association with DRMs, which in turn is essential for envelope budding and assembly onto virus particles. 相似文献
16.
Human immunodeficiency virus type 1 Vpu protein induces degradation of chimeric envelope glycoproteins bearing the cytoplasmic and anchor domains of CD4: role of the cytoplasmic domain in Vpu-induced degradation in the endoplasmic reticulum. 总被引:2,自引:13,他引:2
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The human immunodeficiency virus type 1 (HIV-1) Vpu protein is a transmembrane phosphoprotein which induces rapid degradation of CD4 in the endoplasmic reticulum (ER). To identify sequences in CD4 for Vpu-induced degradation, we generated four chimeric envelope glycoproteins having the ectodomain of HIV-1 gp160, the anchor domain of CD4, and 38, 25, 24, and 18 amino acids (aa) of the CD4 cytoplasmic domain. Using the vaccinia virus-T7 RNA polymerase expression system, we analyzed the expression of chimeric proteins in the presence and absence of Vpu. In singly transfected cells, the chimeric envelope glycoproteins having 38, 24, and 18 aa of the CD4 cytoplasmic domain were endoproteolytically cleaved and biologically active in the fusion of HeLa CD4+ cells. However, one of the chimeras having 25 aa of the CD4 cytoplasmic tail was retained in the ER using the transmembrane ER retention signal and was defective in membrane fusion. Furthermore, biochemical analyses of the coexpressing cells revealed that the Vpu protein induced degradation of the envelope glycoproteins having 38, 25, and 24 aa of the CD4 cytoplasmic tail and degradation occurred in the ER. Consequently, the fusion-competent glycoproteins did not induce the formation of syncytia in HeLa CD4+ cells expressing Vpu. However, the HIV-1 gp160 and chimeric envelope glycoprotein having the membrane-proximal 18 aa of the CD4 cytoplasmic tail were stable and fusion competent in cells expressing Vpu. In addition, we examined the stability of CD4 molecules in the presence of Vpu. Coexpression analyses revealed that the Vpu protein induced degradation of CD4 whereas mutant CD4 having the membrane-proximal 18 aa of the cytoplasmic domain was relatively stable in the presence of Vpu. Taken together, these studies have elucidated that the Vpu protein requires sequences or sequence determinants in the cytoplasmic domain of CD4 to induce degradation of the glycoproteins in the cell. 相似文献
17.
Independent segregation of human immunodeficiency virus type 1 Gag protein complexes and lipid rafts
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Formation of human immunodeficiency virus type 1 (HIV-1) particles takes place at the plasma membrane of cells and is directed by the Pr55Gag polyprotein. A functional assembly domain (the M domain) within the N-terminal portion of Pr55Gag mediates the interaction of Gag with cellular membranes. However, the determinants that provide specificity for assembly on the plasma membrane, as opposed to intracellular membranes, have not been identified. Recently, it was reported that Pr55Gag interacts with lipid raft microdomains of the plasma membrane. We sought to identify the domains within Pr55Gag that contribute to lipid raft association of Gag. Here we demonstrate that the I domain is required for interaction with detergent-resistant membrane fractions (DRMs). Mutation of key I-domain residues or loss of myristylation abrogated the association of Gag with DRMs. Thus, the I domain and the M domain combine to mediate Gag-lipid raft interactions as defined by these biochemical criteria. However, Gag protein complexes defined by flotation studies were much denser than classical lipid rafts, failed to incorporate classical lipid raft marker proteins, and were not disrupted by cholesterol extraction. Large sheets of Gag protein were identified in DRM fractions upon examination by electron microscopy. These results indicate that HIV-1 Pr55Gag forms detergent-resistant complexes at the cellular periphery that are distinct from lipid raft microdomains. 相似文献
18.
Electrostatic interactions drive membrane association of the human immunodeficiency virus type 1 Gag MA domain
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The assembly of most retroviruses occurs at the plasma membrane. Membrane association is directed by MA, the N-terminal domain of the Gag structural protein. For human immunodeficiency virus type 1 (HIV-1), this association is mediated in part by a myristate fatty acid modification. Conflicting evidence has been presented on the relative importance of myristoylation, of ionic interactions between protein and membrane, and of Gag multimerization in membrane association in vivo. We addressed these questions biochemically by determining the affinity of purified myristoylated HIV-1 MA for liposomes of defined composition, both for monomeric and for dimeric forms of the protein. Myristoylation increases the barely detectable intrinsic affinity of the apo-protein for liposomes by only 10-fold, and the resulting affinity is still weak, similar to that of the naturally nonmyristoylated MA of Rous sarcoma virus. Membrane binding of HIV-1 MA is absolutely dependent on the presence of negatively charged lipid and is abrogated at high ionic strength. Forced dimerization of MA increases its membrane affinity by several orders of magnitude. When green fluorescent protein fusions of monomeric or dimeric MA are expressed in cells, the dimeric but not the monomeric protein becomes strongly membrane associated. Computational modeling supports these results and suggests a molecular mechanism for the modest effect of myristoylation on binding, wherein the membrane provides a hydrophobic environment for the myristate that is energetically similar to that provided by the protein. Overall, the results imply that the driving force for membrane association stems largely from ionic interactions between multimerized Gag and negatively charged phospholipids. 相似文献
19.
Human immunodeficiency virus type 1 Gag contains a dileucine-like motif that regulates association with multivesicular bodies
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Multivesicular bodies (MVBs) are cholesterol-enriched organelles formed by the endocytic pathway. The topology of vesicle formation in MVBs is identical to that of retroviral budding from the plasma membrane, and budding of human immunodeficiency virus type 1 (HIV-1) into MVBs in macrophages has recently been visualized. The Gag proteins from HIV-1, as well as many other retroviruses, contain short motifs that mediate interactions with MVBs and other endocytic components, suggesting that Gag proteins directly interface with the endocytic pathway. Here, we show that HIV-1 Gag contains an internalization signal that promotes endocytosis of a chimeric transmembrane fusion protein. Mutation of this motif within Gag strongly inhibits virus-like particle production. Moreover, wild-type Gag, but not the internalization-defective mutation, can be induced to accumulate within CD63-positive MVBs by treatment of cells with U18666A, a drug that redistributes cholesterol from the plasma membrane to MVBs. We propose that HIV-1 Gag contains a signal that promotes interaction with the cellular endocytic machinery and that the site of particle production is regulated by the subcellular distribution of cholesterol. 相似文献
20.
Human immunodeficiency virus type 1 assembly and lipid rafts: Pr55(gag) associates with membrane domains that are largely resistant to Brij98 but sensitive to Triton X-100
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The assembly and budding of human immunodeficiency virus type 1 (HIV-1) at the plasma membrane are directed by the viral core protein Pr55(gag). We have analyzed whether Pr55(gag) has intrinsic affinity for sphingolipid- and cholesterol-enriched raft microdomains at the plasma membrane. Pr55(gag) has previously been reported to associate with Triton X-100-resistant rafts, since both intracellular membranes and virus-like Pr55(gag) particles (VLPs) yield buoyant Pr55(gag) complexes upon Triton X-100 extraction at cold temperatures, a phenotype that is usually considered to indicate association of a protein with rafts. However, we show here that the buoyant density of Triton X-100-treated Pr55(gag) complexes cannot be taken as a proof for raft association of Pr55(gag), since lipid analyses of Triton X-100-treated VLPs demonstrated that the detergent readily solubilizes the bulk of membrane lipids from Pr55(gag). However, Pr55(gag) might nevertheless be a raft-associated protein, since confocal fluorescence microscopy indicated that coalescence of GM1-positive rafts at the cell surface led to copatching of membrane-bound Pr55(gag). Furthermore, extraction of intracellular membranes or VLPs with Brij98 yielded buoyant Pr55(gag) complexes of low density. Lipid analyses of Brij98-treated VLPs suggested that a large fraction of the envelope cholesterol and phospholipids was resistant to Brij98. Collectively, these results suggest that Pr55(gag) localizes to membrane microdomains that are largely resistant to Brij98 but sensitive to Triton X-100, and these membrane domains provide the platform for assembly and budding of Pr55(gag) VLPs. 相似文献