Protein Composition of the Structural Components of Vesicular Stomatitis Virus

Digitonin, a sterol glycoside which complexes with cholesterol, stripped off the envelope of vesicular stomatitis (VS) virions and liberated two viral structural proteins, 83% of P6 and 53% of P4. Deoxycholate also disrupted VS virions but released nucleocapsid cores which could be identified by higher buoyant density, ratio of incorporated (3)H-uridine to (14)C-protein, and electron microscopy. The major nucleocapsid protein was P5 but varying amounts of the minor protein aggregate P2 were present, depending on the concentration of urea used for extraction. P2 appeared to be a polymer of P5. Two other minor structural proteins, P1 and P3, could not be located in the virion. From these data, we conclude that the three microscopically identifiable structures of VS virions are each composed primarily of a single major protein, as follows: P6 = envelope protein, P4 = protein of underlying "shell," and P5 = nucleocapsid protein.     

Fusion of Vesicular Stomatitis Virus with the Cytoplasmic Membrane of L Cells

At an early stage in infection, vesicular stomatitis viruses were attached to the surface of L cells by fusion of the viral and cell membranes.     

Effects of Psoralen-Derivatized Oligonucleoside Methylphosphonates on Vesicular Stomatitis Virus (VSV) Protein Synthesis In Vitro and in VSV-Infected Cells

Abstract

Oligodeoxyribonucleoside methylphosphonates (16-mers) targeted to VSV mRNAs were derivatized with 4′-{[N-(aminoethyl)amino]methyl}-4,5′8-trimethylpsoralen. These oligomers specifically inhibit translation of their targeted mRNAs in vitro following UV irradiation of the oligomer-mRNA complexes. Psoralen-derivatized oligonucleoside methylphosphonates are stable in cells and can inhibit VSV protein synthesis in culture following UV-irradiation of VSV-infected cells.

     

Localization of Membrane-Associated Proteins in Vesicular Stomatitis Virus by Use of Hydrophobic Membrane Probes and Cross-Linking Reagents

The location of membrane-associated proteins of vesicular stomatitis virus was investigated by using two monofunctional and three bifunctional probes that differ in the degree to which they partition into membranes and in their specific group reactivity. Two hydrophobic aryl azide probes, [(125)I]5-iodonaphthyl-1-azide and [(3)H]pyrenesulfonylazide, readily partitioned into virion membrane and, when activated to nitrenes by UV irradiation, formed stable covalent adducts to membrane constituents. Both of these monofunctional probes labeled the glyco-protein G and matrix M proteins, but [(125)I]5-iodonaphthyl-1-azide also labeled the nucleocapsid N protein and an unidentified low-molecular-weight component. Protein labeling of intact virions was unaffected by the presence of cytochrome c or glutathione, but disruption of membrane by sodium dodecyl sulfate greatly enhanced the labeling of all viral proteins except G. Labeling of G protein was essentially restricted to the membrane-embedded, thermolysin-resistant tail fragment. Three bifunctional reagents, tartryl diazide, dimethylsuberimidate, and 4,4'-dithiobisphenylazide, were tested for their capacity to cross-link proteins to membrane phospholipids of virions grown in the presence of [(3)H]palmitate. Only G and M proteins of intact virions were labeled with (3)H-phospholipid by these cross-linkers; the reactions were not affected by cytochrome c but were abolished by disruption of virus with sodium dodecyl sulfate. Dimethylsuberimidate, which reacts with free amino groups, cross-linked (3)H-phospholipid to both G and M protein. In contrast, the hydrophilic tartryl diazide cross-linked phospholipid primarily to the M protein, whereas the hydrophobic 4,4'-dithiobisphenylazide cross-linked phospholipid primarily to the intrinsic G protein. These data support the hypothesis that the G protein traverses the virion membrane and that the M protein is membrane associated but does not penetrate very deeply, if at all.     

Contributions of Hydrology to Vesicular Stomatitis Virus Emergence in the Western USA

Ecosystems - Relationships between environmental variables associated with the spread of vector-borne pathogens, such as RNA viruses transmitted to humans and animals, remain poorly understood....     

Membrane fluidity of Toxoplasma gondii: a fluorescence polarization study

Toxoplasma gondii membrane fluidity was investigated by fluorescence polarization. We used 1,6-diphenyl 1,3,5-hexatriene (DPH) as a fluorescent hydrophobic probe. Fluorescence anisotropy (r) and degree of order (s) showed high fluidity properties. Chemical analysis was performed on this parasite. We found a low cholesterol/phospholipid ratio, many unsaturated fatty acids chains, and high phosphatidylcholine and low sphingomyelin amounts. These results were in good agreement with the observed high fluidity. This may be related to the great adaptability of Toxoplasma gondii in infesting a wide variety of host cells.     

Use of fluorescence anisotropy determinations for indicating the physiological status of hybridoma cell cultures

The evolution of lipid compartment fluidity during culture of hybridoma cells was studied by fluorescence polarization measurements. The probe partition between the plasma membrane and intracytoplasmic compartments was determined by a quenching fluorescence method. A progressive decrease of the plasma membrane fluidity was observed during the growth phase with an increase during stationary and degeneration phases of the culture. These data suggest that fluidity parameters could be used to follow the behaviour of hybridoma cell cultures.     

The Lipidomes of Vesicular Stomatitis Virus,Semliki Forest Virus,and the Host Plasma Membrane Analyzed by Quantitative Shotgun Mass Spectrometry

Although enveloped virus assembly in the host cell is a crucial step in the virus life cycle, it remains poorly understood. One issue is how viruses include lipids in their membranes during budding from infected host cells. To analyze this issue, we took advantage of the fact that baby hamster kidney cells can be infected by two different viruses, namely, vesicular stomatitis virus and Semliki Forest virus, from the Rhabdoviridae and Togaviridae families, respectively. We purified the host plasma membrane and the two different viruses after exit from the host cells and analyzed the lipid compositions of the membranes by quantitative shotgun mass spectrometry. We observed that the lipid compositions of these otherwise structurally different viruses are virtually indistinguishable, and only slight differences were detected between the viral lipid composition and that of the plasma membrane. Taken together, the facts that the lipid compositions of the two viruses are so similar and that they strongly resemble the composition of the plasma membrane suggest that these viruses exert little selection in including lipids in their envelopes.Enveloped viruses acquire their lipid envelope from the membranes of host cells (43). In this process, the nucleocapsid or the nucleocapsid-matrix complex of the viruses buds out of the cell and becomes enveloped by a segment of the host membrane. This membrane segment is modified during the budding process, such that virally encoded membrane proteins are included in the viral envelope, while most host proteins are excluded. Since viruses usually do not carry lipid-synthesizing enzymes, the lipids in the viral envelope are derived from the host membrane. The lipid compositions of enveloped viruses have been studied for years (2, 15, 17, 18, 23, 25, 34, 36, 38, 40). One question that remains to be answered is whether the lipids are included passively, and thus the lipid composition of the envelope reflects the lipid composition of the host membrane, or whether lipid sorting occurs, leading to selective inclusion of some lipids and exclusion of others. This issue has been complicated by the fact that the lipid bilayer is no longer considered a homogenous liquid but contains fluctuating nanoscale assemblies of sphingolipids, saturated phospholipids, cholesterol (Chol), and proteins, called lipid rafts (13, 44). Lipid rafts can be induced to coalesce—usually by protein-protein interactions—into larger, dynamic platforms that function in signal transduction, intracellular membrane transport, and other membrane functions (45). It was also proposed that viruses make use of these membrane domains during their exit from cells (29, 32).A major complication in comparing viral envelopes with host cell membranes is the difficulty in obtaining host cell membranes of purity similar to that of the easily purified viruses. Many studies are faulted by the impurity of the cell membranes analyzed. Moreover, the early work in this field employed conventional analytical methods (such as thin-layer chromatography) that provide only semiquantitative estimates of the total abundance of the major lipid classes. Most importantly, lipid species diversity could not be analyzed. Recent developments in mass spectrometry (MS) have enabled comprehensive and quantitative analyses of lipidomes at the level of individual molecular species. The lipidomes of human immunodeficiency virus (HIV), murine leukemia virus (6, 7), and several bacteriophages (20, 21) were recently analyzed by these new methods.This paper focuses on two well-characterized enveloped viruses, Semliki Forest virus (SFV) and vesicular stomatitis virus (VSV). SFV is an RNA virus belonging to the Togaviridae family of the Alphaviridae that acquires its envelope by budding from the host cell plasma membrane (PM) (46). Early studies analyzed the lipid composition of the viral envelope and also that of the host cell PM (39, 40). These studies revealed strong similarity between the envelope of SFV and the host PM, but one important discrepancy was the higher Chol-to-phospholipid ratio in the virus.VSV is an RNA virus belonging to the Rhabdoviridae family and also hijacks its envelope from the host cell PM (35), but the lipid specificity of the budding process remains controversial. The most recent studies claim that VSV buds from localized regions that do not reflect the average composition of the PM (23, 36). It has also been claimed that lipid rafts are involved in VSV envelope assembly during budding (37).We used BHK-21 cells as host cells to purify SFV and VSV. The purposes of this study were (i) to establish a robust, comprehensive, and quantitative method to analyze lipidomes, including the full complement of glycerolipid, glycerophospholipid, and sphingolipid species as well as Chol; (ii) to establish a protocol for purification of PM suitable for MS analysis; and (iii) to analyze and compare the lipidomes of SFV, VSV, and the BHK-21 PM.We found that the lipidomes of SFV and VSV are similar in molecular composition and are closely related to that of the BHK-21 PM. The small differences observed could be explained by the high degrees of curvature generated during the viral budding process.     

栀子提取物ZG对副流感病毒1型感染后宿主细胞膜的影响

为了探讨栀子提取物ZG抗病毒作用的生物学机制,观察了栀子提取物ZG对副流感病毒1型(PIV-1)感染后宿主细胞膜电位、膜Na -K -ATP酶活性和膜流动性的影响。以氯化乙酰胆碱为阳性对照,采用荧光探针Di-BAC4(3)标记Hep-2细胞膜电位,借助流式细胞仪检测膜电位;定磷法,分光光度计检测Na -K -ATP酶活性;荧光探针NBD-C6-HPC标记细胞膜磷脂,以荧光漂白恢复法和激光扫描共聚焦显微镜检测膜流动性。结果显示:PIV-1感染后宿主细胞膜电位下降,处于超极化状态;膜Na -K -ATP酶活性显著增加,膜流动性显著降低。栀子提取物ZG作用后,对宿主细胞膜的超极化状态没有明显影响;对膜Na -K -ATP酶活性没有明显影响;而对膜流动性则有明显的恢复作用。阳性对照药乙酰胆碱能明显改善病毒感染后膜电位的超极化状态。PIV-1感染后膜电位、Na -K -ATP酶活性和膜流动性等细胞膜能态和功能的改变,可能为病毒感染的生物学机制之一;栀子提取物ZG可能是通过改善细胞膜流动性,维持细胞膜的正常功能来发挥抗病毒感染的作用,而与膜电位和膜Na -K -ATP酶活性等能态来源的环节可能无关。     

Structure of the Vesicular Stomatitis Virus L Protein in Complex with Its Phosphoprotein Cofactor

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铝和铝+钙对小麦幼苗根尖质膜、液泡膜微囊ATP酶和膜流动性的影响

研究了铝和铝 钙对小麦幼苗根尖质膜、液泡膜微囊H ATP酶、Ca2 ATP酶、Mg2 ATP酶活性及其动力学参数和膜流动性的影响。在质膜和液泡膜微囊制剂中加入 1.0mmol/L的Al3 (AlCl3)时 ,H ATP酶、Ca2 ATP酶、Mg2 ATP酶活性和酶促反应的Vmax及膜流动性下降 ,而酶促反应的最适pH和Km 均不受影响。提高酶促反应介质的Ca2 (CaCl2 )浓度可以缓解Al3 对膜ATP酶活性和膜流动性的影响。推测Al3 可能通过与膜的结合而抑制膜ATP酶的活性     

Suppression of Vesicular Stomatitis Virus Defective Intefering Particle Generation by a Function(s) Associated with Human Chromosome 16

Human-mouse somatic cell hybrids were made between adenine phosphoribosyltransferase-deficient mouse L cells and a strain of human primary fibroblasts and selected in medium containing alanosine and adenine (J. A. Tischfield and F. H. Ruddle, Proc. Natl. Acad. Sci. U.S.A. 71:45-49, 1974). These hybrids were tested for the generation of defective interfering (DI) particles of vesicular stomatitis virus to determine whether or not a host gene controls the induction of DI particles. None of the seven independently arising hybrid clones tested generated detectable DI particles during 13 successive undiluted passages. In addition, the parental human cells also failed to generate DI particles. In contrast, the parental mouse cells generated a detectable level of DI particles during continuous passage. Thus, failure to generate DI particles appears to act in a dominant fashion in these hybrids. Human chromosome 16 and adenine phosphoribosyltransferase were present, as a direct consequence of the selection system, in all of the hybrid clones that failed to generate DI particles. It was the only human chromosome observed in the cells of every hybrid clone. This was verified by both isozyme and karyotype analyses. After hybrids were back-selected (with 2,6-diaminopurine) for loss of human adenine phosphoribosyltransferase and chromosome 16, they gained the ability to generate DI particles. Replication of DI particles already present in virus stocks, however, was normal in all of the hybrid clones and the parental human cells. This suggests that the induction, but not the replication, of DI particles is affected by the human genome and that a factor on human chromosome 16 seems to selectively suppress the mouse cell's ability to generate DI particles in the hybrids. These results support the idea that the induction of DI particles is controlled in part by host cell function(s), as suggested previously (C. Y. Kang and R. Allen, J. Virol. 25:202-206, 1978).     

水分胁迫对甘蔗叶片线粒体膜流动性的影响及其与膜脂过氧化的关系

用荧光探剂ANS对抗旱性不同的甘蔗品种在水分胁迫下叶片线粒体膜流动性的变化进行的研究表明,水分胁迫降低了线粒体膜的流动性,抗旱性强的甘蔗品种Co 617和F.Y.79-9的下降幅度分别小于抗旱性弱的Co 740和M.T.77-208;水分胁迫下线粒体膜流动性的下降与膜脂过氧化产物丙二醛含量的增加有密切关系。外源自由基处理试验也表明,甘蔗叶片线粒体膜流动性的下降与膜脂过氧化作用有关。     

The Glycoprotein of Vesicular Stomatitis Virus Is the Antigen That Gives Rise to and Reacts with Neutralizing Antibody

The glycoprotein, but no other virion protein, of vesicular stomatitis virus was solubilized by the nonionic detergent Triton X-100 in low ionic strength buffer. The solubilized viral glycoprotein induced the synthesis of antibody that formed a single precipitin line with the glycoprotein and that neutralized the infectivity of the virus. The neutralizing activity of the antibody was efficiently blocked by purified glycoprotein.     

Antiviral activity of antisense oligonucleotides linked to poly(L-lysine):targets on genomic RNA and/or mRNA of Vesicular Stomatitis Virus

Abstract

Synthetic oligonucleotides provide a rational approach to viral genes control. Conjugation of antisense oligodeoxyribonucleotides (directed against several viral sequences) to poly(L-lysine) brings about specific protection against Vesicular Stomatitis Virus at concentrations lower than 1 uM.     

Translation of mRNAs from Vesicular Stomatitis Virus and Vaccinia Virus Is Differentially Blocked in Cells with Depletion of eIF4GI and/or eIF4GII

Cytolytic viruses abrogate host protein synthesis to maximize the translation of their own mRNAs. In this study, we analyzed the eukaryotic initiation factor (eIF) 4G requirement for translation of vesicular stomatitis virus (VSV) and vaccinia virus (VV) mRNAs in HeLa cells using two different strategies: eIF4G depletion by small interfering RNAs or cleavage of eIF4G by expression of poliovirus 2A protease. Depletion of eIF4GI or eIF4GII moderately inhibits cellular protein synthesis, whereas silencing of both factors has only a slightly higher effect. Under these conditions, the extent of VSV protein synthesis is similar to that of nondepleted control cells, whereas VV expression is substantially reduced. Similar results were obtained when eIF4E was depleted. On the other hand, eIF4G cleavage by poliovirus 2A protease strongly inhibits translation of VV protein expression, whereas translation directed by VSV mRNAs is not abrogated, even though VSV mRNAs are capped. Therefore, the requirement for eIF4F activity is different for VV and VSV, suggesting that the molecular mechanism by which their mRNAs initiate their translation is also different. Consistent with these findings, eIF4GI does not colocalize with ribosomes in VSV-infected cells, while eIF2α locates at perinuclear sites coincident with ribosomes.     

Increase in lipid microviscosity of unilamellar vesicles upon the creation of transmembrane potential

Summary Diffusion potential of potassium ions was formed in unilamellar vesicles of phosphatidyl choline. The vesicles, which included potassium sulfate buffered with potassium phosphate, were diluted into an analogous salt solution made of sodium sulfate and sodium phosphate. The diffusion potential was created by the addition of the potassium-ionophore, valinomycin. The change in lipid microviscosity, ensuing the formation of membrane potential, was measured by the conventional method of fluorescence depolarization with 1,6-diphenyl-1,3,5-hexatriene as a probe. Lipid microviscosity was found to increase with membrane potential in a nonlinear manner, irrespective of the potential direction. Two tentative interpretations are proposed for this observation. The first assumes that the membrane potential imposes an energy barrier on the lipid flow which can be treated in terms of Boltzmann-distribution. The other interpretation assumes a decrease in lipid-free volume due to the pressure induced by the electrical potential. Since increase in lipid viscosity can reduce lateral and rotational motions, as well as increase exposure of functional membrane proteins, physiological effects induced by transmembrane potential could be associated with such dynamic changes.