共查询到20条相似文献,搜索用时 31 毫秒
1.
Vaccinia virus penetration requires cholesterol and results in specific viral envelope proteins associated with lipid rafts
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Vaccinia virus infects a wide variety of mammalian cells from different hosts, but the mechanism of virus entry is not clearly defined. The mature intracellular vaccinia virus contains several envelope proteins mediating virion adsorption to cell surface glycosaminoglycans; however, it is not known how the bound virions initiate virion penetration into cells. For this study, we investigated the importance of plasma membrane lipid rafts in the mature intracellular vaccinia virus infection process by using biochemical and fluorescence imaging techniques. A raft-disrupting drug, methyl-beta-cyclodextrin, inhibited vaccinia virus uncoating without affecting virion attachment, indicating that cholesterol-containing lipid rafts are essential for virion penetration into mammalian cells. To provide direct evidence of a virus and lipid raft association, we isolated detergent-insoluble glycolipid-enriched membranes from cells immediately after virus infection and demonstrated that several viral envelope proteins, A14, A17L, and D8L, were present in the cell membrane lipid raft fractions, whereas the envelope H3L protein was not. Such an association did not occur after virions attached to cells at 4 degrees C and was only observed when virion penetration occurred at 37 degrees C. Immunofluorescence microscopy also revealed that cell surface staining of viral envelope proteins was colocalized with GM1, a lipid raft marker on the plasma membrane, consistent with biochemical analyses. Finally, mutant viruses lacking the H3L, D8L, or A27L protein remained associated with lipid rafts, indicating that the initial attachment of vaccinia virions through glycosaminoglycans is not required for lipid raft formation. 相似文献
2.
Respiratory syncytial virus F envelope protein associates with lipid rafts without a requirement for other virus proteins
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Fleming EH Kolokoltsov AA Davey RA Nichols JE Roberts NJ 《Journal of virology》2006,80(24):12160-12170
Like many enveloped viruses, human respiratory syncytial virus (RSV) assembles at and buds from lipid rafts. Translocation of the envelope proteins to these membrane subdomains is essential for production of infectious virus, but the targeting mechanism is poorly understood and it is not known if other virus proteins are required. Here we demonstrate that F protein of RSV intrinsically targets to lipid rafts without a requirement for any other virus protein, including the SH and G envelope proteins. Recombinant virus deficient in SH and G but retaining F protein expression was used to demonstrate that F protein still localized in rafts in both A549 and HEp-2 cells. Expression of a recombinant F gene by use of plasmid vectors demonstrated that F contains its own targeting domain and localized to rafts in the absence of other virus proteins. The domain responsible for translocation was then mapped. Unlike most other virus envelope proteins, F is unusual since the target signal is not contained within the cytoplasmic domain nor did it involve fatty acid modified residues. Furthermore, exchange of the transmembrane domain with that of the vesicular stomatitis virus G protein, a nonraft protein, did not alter F protein raft localization. Taken together, these data suggest that domains present in the extracellular portion of the protein are responsible for lipid raft targeting of the RSV F protein. 相似文献
3.
Hydrophobic inactivation of influenza viruses confers preservation of viral structure with enhanced immunogenicity
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Raviv Y Blumenthal R Tompkins SM Humberd J Hogan RJ Viard M 《Journal of virology》2008,82(9):4612-4619
The use of inactivated influenza virus for the development of vaccines with broad heterosubtypic protection requires selective inactivation techniques that eliminate viral infectivity while preserving structural integrity. Here we tested if a hydrophobic inactivation approach reported for retroviruses could be applied to the influenza virus. By this approach, the transmembrane domains of viral envelope proteins are selectively targeted by the hydrophobic photoactivatable compound 1,5-iodonaphthyl-azide (INA). This probe partitions into the lipid bilayer of the viral envelope and upon far UV irradiation reacts selectively with membrane-embedded domains of proteins and lipids while the protein domains that localize outside the bilayer remain unaffected. INA treatment of influenza virus blocked infection in a dose-dependent manner without disrupting the virion or affecting neuraminidase activity. Moreover, the virus maintained the full activity in inducing pH-dependent lipid mixing, but pH-dependent redistribution of viral envelope proteins into the target cell membrane was completely blocked. These results indicate that INA selectively blocks fusion of the virus with the target cell membrane at the pore formation and expansion step. Using a murine model of influenza virus infection, INA-inactivated influenza virus induced potent anti-influenza virus serum antibody and T-cell responses, similar to live virus immunization, and protected against heterosubtypic challenge. INA treatment of influenza A virus produced a virus that is noninfectious, intact, and fully maintains the functional activity associated with the ectodomains of its two major envelope proteins, neuraminidase and hemagglutinin. When used as a vaccine given intranasally (i.n.), INA-inactivated influenza virus induced immune responses similar to live virus infection. 相似文献
4.
Inactivation of retroviruses with preservation of structural integrity by targeting the hydrophobic domain of the viral envelope
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
We describe a new approach for the preparation of inactivated retroviruses for vaccine application. The lipid domain of the viral envelope was selectively targeted to inactivate proteins and lipids therein and block fusion of the virus with the target cell membrane. In this way, complete elimination of the infectivity of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) could be achieved with preservation of antigenic determinants on the surface of the viral envelope. Inactivation was accomplished by modification of proteins and lipids in the viral envelope using the hydrophobic photoinduced alkylating probe 1,5 iodonaphthylazide (INA). Treatment of HIV and SIV isolates with INA plus light completely blocked fusion of the viral envelope and abolished infectivity. The inactivated virus remained structurally unchanged, with no detectable loss of viral proteins. Modifications to envelope and nucleocapsid proteins were detected by changes in their elution pattern on reverse-phase high-performance liquid chromatography. These modifications had no effect on primary and secondary structure epitopes as determined by monoclonal antibodies. Likewise, the inactivated HIV reacted as well as the live virus with the conformation-sensitive and broadly neutralizing anti-HIV type 1 monoclonal antibodies 2G12, b12, and 4E10. Targeting the lipid domain of biological membranes with hydrophobic alkylating compounds could be used as a general approach for inactivation of enveloped viruses and other pathogenic microorganisms for vaccine application. 相似文献
5.
S K Srinivas R V Srinivas G M Anantharamaiah J P Segrest R W Compans 《The Journal of biological chemistry》1992,267(10):7121-7127
The human and simian immunodeficiency virus envelope glycoproteins, which mediate virus-induced cell fusion, contain two putative amphipathic helical segments with large helical hydrophobic moments near their carboxyl-terminal ends. In an attempt to elucidate the biological role of these amphipathic helical segments, we have synthesized peptides corresponding to residues 768-788 and 826-854 of HIV-1/WMJ-22 gp160. Circular dichroism studies of the peptides showed that the alpha helicity of the peptides increased with the addition of dimyristoyl phosphatidylcholine (DMPC) indicating that the peptides form lipid-associating amphipathic helixes. The peptides solubilized turbid suspensions of DMPC vesicles, and electron microscopy of peptide-DMPC mixtures revealed the formation of discoidal complexes, suggesting that the peptides bind to and perturb lipid bilayers. The peptides were found to lyse lipid vesicles and caused carboxyfluorescein leakage from dye-entrapped egg phosphatidylcholine liposomes. The peptides also lysed human erythrocytes and were found to be toxic to cell cultures. At subtoxic concentrations, the peptides effectively inhibited the fusion of CD4+ cells infected with recombinant vaccinia virus expressing human immunodeficiency virus (HIV)-1 envelope proteins. Based on these results, and reported studies on the mutational analysis of HIV envelope proteins, we suggest that the amphipathic helical segments near the carboxyl terminus of HIV envelope proteins may play a role in lysis of HIV-infected cells and also may modulate the extent of cell fusion observed during HIV infection of CD4+ cells. 相似文献
6.
Carl R. Alving Zoltan Beck Nicos Karasavva Gary R. Matyas Mangala Rao 《Molecular membrane biology》2013,30(6):453-465
The human immunodeficiency virus type 1 (HIV-1) is an enveloped virus with a lipid bilayer that contains several glycoproteins that are anchored in, or closely associated with, the membrane surface. The envelope proteins have complex interactions with the lipids both on the host cells and on the target cells. The processes of budding from host cells and entry into target cells occur at sites on the plasma membrane, known as lipid rafts, that represent specialized regions that are rich in cholesterol and sphingolipids. Although the envelope glycoproteins are antigenic molecules that potentially might be used for development of broadly neutralizing antibodies in a vaccine to HIV-1, the development of such antibodies that have broad specificities against primary field isolates of virus has been largely thwarted to date by the ability of the envelope proteins to evade the immune system through various mechanisms. In this review, the interactions of HIV-1 with membrane lipids are summarized. Liposomes are commonly used as models for understanding interactions of proteins with membrane lipids; and liposomes have also been used both as carriers for vaccines, and as antigens for induction of antibodies to liposomal lipids. The possibility is proposed that liposomal lipids, or liposome-protein combinations, could be useful as antigens for inducing broadly neutralizing antibodies to HIV-1. 相似文献
7.
HIV-1, lipid rafts, and antibodies to liposomes: implications for anti-viral-neutralizing antibodies
The human immunodeficiency virus type 1 (HIV-1) is an enveloped virus with a lipid bilayer that contains several glycoproteins that are anchored in, or closely associated with, the membrane surface. The envelope proteins have complex interactions with the lipids both on the host cells and on the target cells. The processes of budding from host cells and entry into target cells occur at sites on the plasma membrane, known as lipid rafts, that represent specialized regions that are rich in cholesterol and sphingolipids. Although the envelope glycoproteins are antigenic molecules that potentially might be used for development of broadly neutralizing antibodies in a vaccine to HIV-1, the development of such antibodies that have broad specificities against primary field isolates of virus has been largely thwarted to date by the ability of the envelope proteins to evade the immune system through various mechanisms. In this review, the interactions of HIV-1 with membrane lipids are summarized. Liposomes are commonly used as models for understanding interactions of proteins with membrane lipids; and liposomes have also been used both as carriers for vaccines, and as antigens for induction of antibodies to liposomal lipids. The possibility is proposed that liposomal lipids, or liposome-protein combinations, could be useful as antigens for inducing broadly neutralizing antibodies to HIV-1. 相似文献
8.
Iwan A. T. Schaap Frédéric Eghiaian Amédée des Georges Claudia Veigel 《The Journal of biological chemistry》2012,287(49):41078-41088
The envelope of the influenza virus undergoes extensive structural change during the viral life cycle. However, it is unknown how lipid and protein components of the viral envelope contribute to its mechanical properties. Using atomic force microscopy, here we show that the lipid envelope of spherical influenza virions is ∼10 times softer (∼0.05 nanonewton nm−1) than a viral protein-capsid coat and sustains deformations of one-third of the virion''s diameter. Compared with phosphatidylcholine liposomes, it is twice as stiff, due to membrane-attached protein components. We found that virus indentation resulted in a biphasic force-indentation response. We propose that the first phase, including a stepwise reduction in stiffness at ∼10-nm indentation and ∼100 piconewtons of force, is due to mobilization of membrane proteins by the indenting atomic force microscope tip, consistent with the glycoprotein ectodomains protruding ∼13 nm from the bilayer surface. This phase was obliterated for bromelain-treated virions with the ectodomains removed. Following pH 5 treatment, virions were as soft as pure liposomes, consistent with reinforcing proteins detaching from the lipid bilayer. We propose that the soft, pH-dependent mechanical properties of the envelope are critical for the pH-regulated life cycle and support the persistence of the virus inside and outside the host. 相似文献
9.
Viruses are pathogenic agents that can infect all varieties of organisms, including plants, animals, and humans. These microscopic particles are genetically simple as they encode a limited number of proteins that undertake a wide range of functions. While structurally distinct, viruses often share common characteristics that have evolved to aid in their infectious life cycles. A commonly underappreciated characteristic of many deadly viruses is a lipid envelope that surrounds their protein and genetic contents. Notably, the lipid envelope is formed from the host cell the virus infects. Lipid-enveloped viruses comprise a diverse range of pathogenic viruses, which often lead to high fatality rates and many lack effective therapeutics and/or vaccines. This perspective primarily focuses on the negative-sense RNA viruses from the order Mononegavirales, which obtain their lipid envelope from the host plasma membrane. Specifically, the perspective highlights the common themes of host cell lipid and membrane biology necessary for virus replication, assembly, and budding. 相似文献
10.
Electron paramagnetic resonance observations were made on nitroxide spin-labeled molecules which were bound to the TC-83 vaccine strain of Venezuelan equine encephalomyelitis virus. Paramagnetic resonance parameters derived from the observations and their dependence on sample temperature were similar but not identical to those which have been reported for these labels dissolved in lipid bilayer membranes of mammalian and bacterial origin. The data are consistent with the existence of a bilayer lipid structure in the virion envelope which has a mechanical rigidity substantially greater than that of bilayers in cellular membranes. A model is presented which assumes the location of the lipid bilayer outside the nucleoprotein capsid and inside a spherical layer of envelope proteins. The model is in accord with Harrison's X-ray diffraction results for Sindbis virus. The model is discussed in terms of its implications with respect to the role played by lipid in viral maturation and infectivity. 相似文献
11.
Zhang W Chipman PR Corver J Johnson PR Zhang Y Mukhopadhyay S Baker TS Strauss JH Rossmann MG Kuhn RJ 《Nature structural biology》2003,10(11):907-912
Improved technology for reconstructing cryo-electron microscopy (cryo-EM) images has now made it possible to determine secondary structural features of membrane proteins in enveloped viruses. The structure of mature dengue virus particles was determined to a resolution of 9.5 A by cryo-EM and image reconstruction techniques, establishing the secondary structural disposition of the 180 envelope (E) and 180 membrane (M) proteins in the lipid envelope. The alpha-helical 'stem' regions of the E molecules, as well as part of the N-terminal section of the M proteins, are buried in the outer leaflet of the viral membrane. The 'anchor' regions of E and the M proteins each form antiparallel E-E and M-M transmembrane alpha-helices, leaving their C termini on the exterior of the viral membrane, consistent with the predicted topology of the unprocessed polyprotein. This is one of only a few determinations of the disposition of transmembrane proteins in situ and shows that the nucleocapsid core and envelope proteins do not have a direct interaction in the mature virus. 相似文献
12.
The nucleocapsid of Sindbis virus, a natural non-infectious complex of the viral RNA and protein molecules can be encapsulated in large, unilamellar vesicles and delivered efficiently to cells in an infectious form. It is shown that high infectivity of the vesicle entrapped nucleocapsids is partly due to the viral envelope proteins which enhance entrapment and liposome cell interaction.We believe that the efficiency of liposome mediated gene transfer of eukaryotic cells can be increased significantly by the insertion of fusogenic viral envelope proteins into the lipid bilayer of liposomes. 相似文献
13.
A method for implanting exogenous membrane proteins into recipient hepatoma cells is described. Red cell band 3 and Sendai virus envelope proteins HN and F were extracted from their respective sources and purified by centrifugation to equilibrium through sucrose step gradients in the presence of octyl-beta-D-glucopyranoside. 0.05-0.15 micron vesicles were formed by adding lipid to combined detergent solubilized, isolated membrane proteins and removing detergent by dialysis. The vesicles were hybrid band 3-Sendai envelope vesicles and not a mixture of two distinct vesicle types as judged by (1) the ability of Sendai specific antibody to immunoprecipitate greater than 99% of band 3 from vesicle suspensions and (2) comigration of band 3 and Sendai envelope proteins on isopyknic sucrose density gradients. The hybrid vesicles (virosomes) were not fusogenic but did bind to cultured hepatoma cells in the cold. Subsequent treatment of virosomes absorbed onto cultured cells with polyethylene glycol resulted in a stable association of 2-10% of added band 3 and Sendai envelope proteins with the cells. Efficient transfer of virosome-associated band 3 to the cells was dependent on both lipid and Sendai envelope proteins. Fluid phase marker transfer, immunofluorescence, and protease digestion experiments demonstrate that the majority of the virosomes were implanted into recipient hepatoma membranes and not simply adsorbed onto their surface or immediately endocytosed. The hybrid membrane protein-viral envelope vesicles thus offer an efficient means for insertion of foreign proteins into the membranes of recipient cultured cells. 相似文献
14.
Solubilization and reconstitution of vesicular stomatitis virus envelope using octylglucoside.
下载免费PDF全文
![点击此处可从《Biophysical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
M Paternostre M Viard O Meyer M Ghanam M Ollivon R Blumenthal 《Biophysical journal》1997,72(4):1683-1694
Reconstituted vesicular stomatitis virus envelopes or virosomes are formed by detergent removal from solubilized intact virus. We have monitored the solubilization process of the intact vesicular stomatitis virus by the nonionic surfactant octylglucoside at various initial virus concentrations by employing turbidity measurements. This allowed us to determine the phase boundaries between the membrane and the mixed micelles domains. We have also characterized the lipid and protein content of the solubilized material and of the reconstituted envelope. Both G and M proteins and all of the lipids of the envelope were extracted by octylglucoside and recovered in the reconstituted envelope. Fusion activity of the virosomes tested either on Vero cells or on liposomes showed kinetics and pH dependence similar to those of the intact virus. 相似文献
15.
Cholesterol is known to play an important role in stabilizing particular cellular membrane structures, so-called lipid or membrane rafts. For several viruses, a dependence on cholesterol for virus entry and/or morphogenesis has been shown. Using flow cytometry and fluorescence microscopy, we demonstrate that infection of cells by canine distemper virus (CDV) was not impaired after cellular cholesterol had been depleted by the drug methyl-beta-cyclodextrin. This effect was independent of the multiplicity of infection and the cellular receptor used for infection. However, cholesterol depletion of the viral envelope significantly reduced CDV infectivity. Replenishment by addition of exogenous cholesterol restored infectivity up to 80%. Thus, we conclude that CDV entry is dependent on cholesterol in the viral envelope. Furthermore, reduced syncytium formation was observed when the cells were cholesterol depleted during the course of the infection. This may be related to the observation that CDV envelope proteins H and F partitioned into cellular detergent-resistant membranes. Therefore, a role for lipid rafts during virus assembly and release as well is suggested. 相似文献
16.
Role of the matrix protein in the virion association of the human immunodeficiency virus type 1 envelope glycoprotein. 总被引:40,自引:36,他引:4
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The matrix (MA) protein of human immunodeficiency virus type 1 (HIV-1) forms an inner coat directly underneath the lipid envelope of the virion. The outer surface of the lipid envelope surrounding the capsid is coated by the viral Env glycoproteins. We report here that the HIV-1 capsid-Env glycoprotein association is very sensitive to minor alterations in the MA protein. The results indicate that most of the MA domain of the Gag precursor, except for its carboxy terminus, is essential for this association. Viral particles produced by proviruses with small missense or deletion mutations in the region coding for the amino-terminal 100 amino acids of the MA protein lacked both the surface glycoprotein gp120 and the transmembrane glycoprotein gp41, indicating a defect at the level of Env glycoprotein incorporation. Alterations at the carboxy terminus of the MA domain had no significant effect on the levels of particle-associated Env glycoprotein or on virus replication. The presence of HIV-1 MA protein sequences was sufficient for the stable association of HIV-1 Env glycoprotein with hybrid particles that contain the capsid (CA) and nucleocapsid (NC) proteins of visna virus. The association of HIV-1 Env glycoprotein with the hybrid particles was dependent upon the presence of the HIV-1 MA protein domain, as HIV-1 Env glycoprotein was not efficiently recruited into virus particles when coexpressed with authentic visna virus Gag proteins. 相似文献
17.
A point mutation in the binding subunit of a retroviral envelope protein arrests virus entry at hemifusion
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The transmembrane subunits of viral envelope proteins are thought to perform all of the functions required for membrane fusion during entry of enveloped viruses. However, changes in a conserved SPHQ motif near the N terminus of the receptor binding subunit of a murine leukemia virus (MLV) envelope protein block infection and induction of cell-cell fusion but not receptor binding. Here we report evidence that a histidine-to-arginine change at position 8 (H8R) in the SPHQ motif of Moloney MLV blocks infection by arresting virus-cell fusion at the hemifusion state. In cell-cell fusion assays, H8R envelope protein induced mixing of membrane outer leaflet lipids but did not lead to content mixing, a finding indicative of fusion pore formation. Kinetic studies of virus-cell fusion showed that lipid mixing of H8R virus membranes begins much later than for wild-type virus. The length of the delay in lipid mixing decreased upon addition of two second-site changes that increase H8R virus infection to 100-fold less than the wild-type virus. Finally, chlorpromazine, dibucaine, and trifluoperazine, agents that induce pores in an arrested hemifusion state, rescued infection by H8R virus to within 2.5-fold of the level of wild-type virus infection and cell-cell fusion to half that mediated by wild-type envelope protein. We interpret these results to indicate that fusion progressed to the hemifusion intermediate but fusion pore formation was inhibited. These results establish that membrane fusion of Moloney MLV occurs via a hemifusion intermediate. We also interpret these findings as evidence that histidine 8 is a key switch-point residue between the receptor-induced conformation changes that expose fusion peptide and those that lead to six-helix bundle formation. 相似文献
18.
Using a panel of amphotropic murine leukemia virus packaging cell lines that differed only in their levels of envelope protein (gp70) expression, we examined the relationship between transduction and the number of envelope proteins per virus. We generated virus stocks that contained different levels of virus-associated envelope proteins, purified them from gp70 that was not associated with the viruses, quantified their titers, and measured the efficiency with which they transduced NIH 3T3, TE671, and HeLa cells. As expected, titers increased monotonically with viral envelope protein number. Titers are measured using highly dilute virus, however, and are often not predictive of gene transfer when high doses of virus are used, as is done in gene therapy protocols. Interestingly, when we used high doses of virus, we observed significantly different trends: gene transfer increased, reached a maximum, and then declined sharply as the number of envelope proteins per virus increased. The highest levels of gene transfer occurred when cells were transduced with a moderate dose of virus that contained low levels of envelope protein. Our results indicate that transduction is inhibited when viruses that contain large numbers of envelope proteins are used. This is most likely because each virus, when it binds to a cell, delivers a large payload of envelope proteins that occupy or inactivate multiple virus receptors, reducing or eliminating the susceptibility of the cell to being transduced by additional viruses. The implications of our findings for the design of improved retroviral vectors for human gene therapy are discussed. 相似文献
19.
Fusogenic liposomes that incorporate Sendai virus envelope proteins, so-called Sendai virosomes, have been developed for in vitro and in vivo genetic modification of animal cells. In this study, several different virosomes of varying lipid compositions were formulated and their in vitro gene-transfer efficiencies compared. The virosomes were prepared by quantitative reconstitution of the Sendai envelope, fusion (F) and hemagglutinin-neuraminidase (HN) proteins into liposomal vesicles. Virosomes that contained luciferase reporter genes were tested in 293 transformed human kidney cells. F/HN-virosomes that were prepared with an artificial Sendai viral envelope (ASVE-virosomes) or phosphatidylserine (PS-virosomes) exhibited an 8- or 6-fold higher gene-transfer efficiency than cationic liposomes that were made with 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). F/HNvirosomes that were prepared with phosphatidic acid (PA-virosomes) instead of PS were less efficient in gene transfer than either ASVE- or PS-virosomes. In addition, the gene-transfer capability of ASVE- and PS-virosomes was maximal at a Ca2+ concentration of 510 mM. These results suggest that the incorporated lipid components significantly affect the in vitro gene transfer that is mediated by Sendai F/HN-virosomes. 相似文献
20.
Role of plasma membrane lipid microdomains in respiratory syncytial virus filament formation
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The fusion protein (F) of respiratory syncytial virus (RSV) is the envelope glycoprotein responsible for the characteristic cytopathology of syncytium formation. RSV has been shown to bud from selective areas of the plasma membrane as pleomorphic virions, including both filamentous and round particles. With immunofluorescent microscopy, we demonstrated evidence of RSV filaments incorporating the fusion protein F and colocalizing with a lipid microdomain-specific fluorescent dye, 1,1-dihexadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate. Western blot analysis of Triton X-100 cold-extracted membrane fractions confirmed the presence of RSV proteins within the lipid microdomains. RSV proteins also colocalized with cellular proteins associated with lipid microdomains, caveolin-1, and CD44, as well as with RhoA, a small GTPase. ADP-ribosylation of RhoA by Clostridium botulinum exotoxin inactivated RhoA signaling and resulted in the absence of RSV-induced syncytia despite no significant change in viral titer. We demonstrated an overall decrease in both the number and length of the viral filaments and a shift in the localization of F to nonlipid microdomain regions of the membrane in the presence of C3 toxin. This suggests that the selective incorporation of RSV proteins into lipid microdomains during virus assembly may lead to critical interactions of F with cellular proteins, resulting in microvillus projections necessary for the formation of filamentous virus particles and syncytium formation. Thus, manipulation of membrane lipid microdomains may lead to alterations in the production of viral filaments and RSV pathogenesis and provide a new pharmacologic target for RSV therapy. 相似文献