小麦丛矮病毒NS蛋白的磷酸化

小麦丛矮病毒是在中国发现的一种植物弹状病毒 ,病毒基因组是由一条单链负链RNA组成并编码 5种病毒结构蛋白质 :表面糖蛋白G、膜基质蛋白M、核衣壳蛋白N、大蛋白L和所谓非结构蛋白NS。后来的研究证明 ,在弹状病毒的模式病毒———水泡性口膜炎病毒中 ,NS蛋白也是一种结构蛋白 ,而且在成熟的病毒粒子中以各种磷酸化形式存在 ,并且证明NS的磷酸化和去磷酸化对病毒基因组的转录和复制的调控起重要的作用。用体外磷酸化方法证明 ,结合于小麦丛矮病毒的核衣壳上的NS蛋白可以被磷酸化 ;同时也证明 ,从大肠杆菌中表达的小麦丛矮病毒的NS蛋白 ,只有在病毒核衣壳存在下才可以体外被磷酸化 ;从而证明 ,小麦丛矮病毒或植物弹状病毒的NS蛋白也是一种磷酸化蛋白质 ,在成熟病毒粒子中可能存在磷酸化和非磷酸化两种形式。病毒的L蛋白除以前报道的具有RNA聚合酶活力外 ,也具有蛋白激酶的活力。     

The 1:1 N-NS protein complex of vesicular stomatitis virus is essential for efficient genome replication.

We studied the effect pH had on the N-NS protein complex to determine its role in vesicular stomatitis virus (VSV) genome replication, as we had previously shown that VSV genome replication in vitro requires the interaction of the viral N and NS proteins into a 1:1 complex. A previous report showed that the growth of VSV in L cells was sensitive to the pH of the environment (M. Fiszman, J. B. Leaute, C. Chany, and M. Girard, J. Virol. 13:801-808, 1974). We hypothesized that low pH might disrupt the N-NS protein complex, and so we investigated the molecular events leading to inhibition of viral RNA replication in vitro from extracts that were prepared from VSV-infected cells incubated at pH 6.6. We found that viral genome RNA synthesis in vitro was reduced when infected cells were maintained at pH 6.6. Through immunoprecipitation analysis of the viral soluble protein pool, we found that a complex that usually exists between the N and NS proteins at pH 7.4 was altered in extracts from infected cells maintained at pH 6.6, and this was responsible for the observed effects on viral replication. The effect of low pH on the N-NS protein complex could not be abolished by increasing the concentration of the altered complex, indicating that the effects is more than simply a decrease in the level of the protein complex in the cell. Our data provide additional evidence that the 1:1 N-NS protein complex, and not the N protein alone, serves as the substrate for viral RNA replication in vivo.     

Phosphorylation of Vesicular Stomatitis Virus In Vivo and In Vitro

The structural protein, NS, of purified vesicular stomatitis virus (VSV) is a phosphoprotein. In infected cells phosphorylated NS is found both free in the cytoplasm and as part of the viral ribonucleoprotein (RNP) complex containing both the 42S RNA and the structural proteins L, N, and NS, indicating that phosphorylation occurs as an early event in viral maturation. VSV contains an endogenous protein kinase activity, probably of host region, which catalyzes the in vitro phosphorylation of the viral proteins NS, M, and L, but not of N or G. The phosphorylated sites on NS appear to be different in the in vivo and in vitro reactions, and are differentially sensitive to alkaline phosphatase. After removal of the membrane components of purified VSV with a dextran-polyethylene glycol two-phase separation, the kinase activity remains tightly associated with the viral RNP. However, viral RNP isolated from infected cells shows only a small amount of kinase activity. The protein kinase enzyme appears to be a cellular contaminant of purified VSV because an activity from the uninfected cell extract can phosphorylate in vitro the dissociated viral proteins NS and M. The virion-associated activity may be derived either from the cytoplasm or the plasma membrane of the host cell since both of these cellular components contain protein kinase activity similar to that found in purified VSV.     

The membrane (M1) protein of influenza virus occurs in two forms and is a phosphoprotein.

The membrane (M1) protein of influenza virus was found to be heterogenous and to occur in two forms in the virus particle. The two forms of M1 were found in virus which was produced both early and late after infection and in infected cells. The two forms could be separated on polyacrylamide gels under specific conditions. The two components of M1 contained similar tryptic peptides. However, a small proteolytic difference between the two proteins could not be ruled out. Both M1 proteins were present in phosphorylated form in the virus particle. The phosphorylated M1 components were not readily distinguished from phosphorylated nonstructural protein (NS1) when cytoplasm of infected cells was analyzed on polyacrylamide gels. The two phosphorylated M1 components could, however, be detected in infected cells by immunoprecipitation. One M1 component contained only phosphoserine whereas the second contained phosphoserine and a small amount of phosphothreonine as well. In addition to the phosphorylated nucleoprotein and M1, a third phosphorylated protein was routinely detected in virus particles. It was a surface component of the virus, since it could be removed from whole virus with chymotrypsin and contained phosphate at serine residues. The identity of this component was not known.     

Ribonucleic Acid Species of Intracellular Nucleocapsids and Released Virions of Vesicular Stomatitis Virus

Infection of chicken embryo cells with vesicular stomatitis (VS) virus resulted in variable production of three classes of intracellular viral ribonucleocapsids with sedimentation coefficients of approximately 140S, 110S, and 80S, as well as three corresponding classes of released virions designated B, LT, and T. Intracellular nucleocapsids of each class contained three proteins of which the major N protein was firmly bound, and the minor L and NS1 proteins were readily dissociated with 0.5 m NaCl. The ribonucleic acid (RNA) species extracted from B, LT, and T virions, and from corresponding intracellular nucleocapsids, contained RNA species with approximate molecular weights of 3.2 x 10(6), 2.0 x 10(6), and 10(6), respectively, as determined by polyacrylamide gel electrophoresis. These values are roughly equivalent to sedimentation coefficients of 42S, 28S, and 23S for each of the virion and nucleocapsid RNA species. Cells infected at high multiplicity with undiluted passage VS virus gave rise primarily to virions and nucleocapsids containing 23S RNA, whereas cells productively infected with purified B virions produced predominantly B and LT virions and nucleocapsids. At late stages in the productive cycle of infection, more virions containing 42S RNA were produced, but the intracellular pool of nucleocapsids containing 28S and 23S RNA remained relatively constant. Additional studies by more refined techniques are required to test the hypothesis that nucleocapsids containing 28S and 23S RNA are precursors of the 42S RNA in infectious VS-B virions and that production of defective T and LT virions results from failure of ligation of the RNA precursors.     

Cellular Proteins Associated with the Interior and Exterior of Vesicular Stomatitis Virus Virions

Virus particles (virions) often contain not only virus-encoded but also host-encoded proteins. Some of these host proteins are enclosed within the virion structure, while others, in the case of enveloped viruses, are embedded in the host-derived membrane. While many of these host protein incorporations are likely accidental, some may play a role in virus infectivity, replication and/or immunoreactivity in the next host. Host protein incorporations may be especially important in therapeutic applications where large numbers of virus particles are administered. Vesicular stomatitis virus (VSV) is the prototypic rhabdovirus and a candidate vaccine, gene therapy and oncolytic vector. Using mass spectrometry, we previously examined cell type dependent host protein content of VSV virions using intact (“whole”) virions purified from three cell lines originating from different species. Here we aimed to determine the localization of host proteins within the VSV virions by analyzing: i) whole VSV virions; and ii) whole VSV virions treated with Proteinase K to remove all proteins outside the viral envelope. A total of 257 proteins were identified, with 181 identified in whole virions and 183 identified in Proteinase K treated virions. Most of these proteins have not been previously shown to be associated with VSV. Functional enrichment analysis indicated the most overrepresented categories were proteins associated with vesicles, vesicle-mediated transport and protein localization. Using western blotting, the presence of several host proteins, including some not previously shown in association with VSV (such as Yes1, Prl1 and Ddx3y), was confirmed and their relative quantities in various virion fractions determined. Our study provides a valuable inventory of virion-associated host proteins for further investigation of their roles in the replication cycle, pathogenesis and immunoreactivity of VSV.     

A membrane glycoprotein that accumulates intracellularly: cellular processing of the large glycoprotein of LaCrosse virus

The intracellular transport and certain posttranslational modifications of the large glycoprotein (G1) of LaCrosse virus (LAC) in BHK cells have been studied. G1 from released LAC virus was characterized by complex oligosaccharides (endo H-resistant) and covalently attached fatty acid. Only a small fraction of total cellular G1 was present on the baby hamster kidney cell surface. Cell-surface G1 contained complex oligosaccharides, while total G1 in infected cells contained largely unprocessed (endo H-sensitive) oligosaccharides. In addition, cell G1 contained significantly less fatty acid than virion-associated G1. Pulse-chase experiments showed that the oligosaccharides of G1 were processed to the complex from much more slowly than the oligosaccharides of the vesicular stomatitis virus (VSV) glycoprotein (G). In addition, transit of LAC G1 to the cell surface and into extracellular virions was two to three fold slower than the transit of VSV G. Thus LAC G1 accumulates intracellularly and is only slowly processed by intracellular processing enzymes. Treatment with monensin caused accumulation in the cell of a form of G1 with partial sensitivity toward endo H, suggesting that monensin may act to inhibit the glycosylation process directly.     

Specific incorporation of host cell surface proteins into budding vesicular stomatitis virus particles

The specific incorporation of cell surface proteins into budding Vesicular Stomatitis Virus (VSV) particles was shown by two approaches. In the first, monolayer cultures of Vero or L cells were labeled by lactoperoxidase-catalyzed iodination and the cells were then infected with VSV. Approximately 2% of the cell surface ¹²⁵1 radioactivity was incorporated into particles which co-purify with normal, infectious virions by both velocity and equilibrium gradient centrifugation and which are precipitated by antiserum specific for the VSV glycoprotein. Control experiments establish that these ¹²⁵I-labeled particles are not cell debris or cellular material which aggregate with or adhere to VSV virions. VSV virions contain only a subset of the 10–15 normal ¹²⁵1-labeled cell surface polypeptides resolved by SDS gel electrophoresis; VSV grown in L cells and Vero cells incorporate different host polypeptides. In a second approach, Vero cells were labeled with ³⁵S-methione, then infected with VSV. Two predominant host polypeptides (molecular weights 110,000 and 20,000) were incorporated into VSV virions. These proteins, like VSV G protein, are exposed to the surface of the virion. They co-migrate with the major incorporated ¹²⁵1 host polypeptides. These host proteins are present in approximately 10 and 80 copies, respectively, per virion. Specific incorporation of host polypeptides into VSV virions does not require the presence of viral glycoprotein. This was shown by use of a ts VSV mutant defective in maturation of VSV G protein to the cell surface. Budding from infected cells are noninfectious particles which contain all the viral proteins except for G; these particles contain the same proportion and spectrum of ¹²⁵1-labeled host surface polypeptides as do wild-type virions. These results extend previous conclusions implicating the submembrane viral matrix protein, or the viral nucleocapsid, as being of primary importance in selecting cell surface proteins for incorporation into budding VSV virions.     

Proteins of Vesicular Stomatitis Virus and of Phenotypically Mixed Vesicular Stomatitis Virus-Simian Virus 5 Virions

The identity of the glycoprotein of vesicular stomatitis virus (VSV) as the spike protein has been confirmed by the removal of the spikes with a protease from Streptomyces griseus, leaving bullet-shaped particles bounded by a smooth membrane. This treatment removes the glycoprotein but does not affect the other virion proteins, apparently because they are protected from the enzyme by the lipids in the viral membrane. The proteins of phenotypically mixed, bullet-shaped virions produced by cells mixedly infected with VSV and the parainfluenza virus simian virus 5 (SV5) have been analyzed by polyacrylamide gel electrophoresis. These virions contain all the VSV proteins plus the two SV5 spike proteins, both of which are glycoproteins. The finding of the SV5 spike glycoproteins on virions with the typical morphology of VSV indicates that there is not a stringent requirement that only the VSV glycoprotein can be used to form the bullet-shaped virion. On the other hand, the SV5 nucleocapsid protein and the major non-spike protein of the SV5 envelope were not detected in the phenotypically mixed virions, and this suggests that a specific interaction between the VSV nucleocapsid and regions of the cell membrane which contain the nonglycosylated VSV envelope protein is necessary for assembly of the bullet-shaped virion.     

Vesicular stomatitis virus growth in Drosophila melanogaster cells: G protein deficiency.

In cultured Drosophila melanogaster cells, vesicular stomatitis virus (VSV) established a persistent, noncytopathic infection. No inhibition of host protein synthesis occurred even though all cells were initially infected. No defective interfering particles were detected, which would explain the establishment of the carrier state. In studies of the time course of viral protein synthesis in Drosophila cells, N, NS, and M viral polypeptides were readily detected within 1 h of infection. The yield of G protein and one of its precursors; G1, was very low at any time of the virus cycle; the released viruses always contained four to five times less G than those produced by chicken embryo cells, whatever the VSV strain or serotype used for infection and whatever the Drosophila cell line used as host. Actinomycin D added to the cells before infection enhanced VSV growth up to eight times. G and G1 synthesis increased much more than that of the other viral proteins when the cells were pretreated with the drug; nevertheless, the released viruses exhibited the same deficiency in G protein as the VSV released from untreated cells. Host cell control on both G-protein maturation process and synthesis at traduction level is discussed in relation to G biological properties.     

Activation of vesicular stomatitis virus fusion with cells by pretreatment at low pH

Fusion of vesicular stomatitis virus (VSV) with Vero cells was measured after exposure of the virus to low pH under a variety of experimental conditions. The method of relief of fluorescence self-quenching of the probe octadecylrhodamine was used to monitor fusion. Incubation of the virus at pH 5.5 prior to binding to cells led to significant enhancement of fusion at the plasma membrane, whereas fusion via the endocytic pathway was inhibited. Fusion of pH 5.5-pretreated VSV showed a similar pH threshold for fusion as nontreated virus, and it was blocked by antibody to VSV G protein. Activation of VSV by pretreatment at low pH was only slightly dependent on temperature. In contrast, when VSV was first bound to target cells and subsequently exposed at 4 degrees C to the low pH, activation of the fusion process did not occur. The pH 5.5-mediated activation of VSV could be reversed by returning the pH to neutral in the absence of target membranes. The low pH pretreatment also led to aggregation of virus; large aggregates could be pelleted by low speed centrifugation and only the effects of the supernatant, which consist of single virions and/or microaggregates, were considered. The data were analyzed in the framework of an allosteric model according to which viral spike glycoproteins undergo a pH-dependent conformational transition to an active (fusion-competent) state. Based on that analysis we conclude that the conformational transition to the active state is rate-limiting for fusion and that the viral spike glycoproteins are fusion-competent only in their protonated form.     

Phenotypic Mixing of Envelope Proteins of the Parainfluenza Virus SV5 and Vesicular Stomatitis Virus

Cells mixedly infected with parainfluenza virus SV5 and vesicular stomatitis virus (VSV) yield phenotypically mixed virions, in addition to both parental types. Two types of phenotypically mixed virions have been identified: 0.6 to 1.2% of the VSV plaque formers were neutralized by SV5 antiserum, but not by VSV antiserum, suggesting the presence of a VSV genome in an SV5 envelope; 9 to 45% of the VSV plaque formers were neutralized by both antisera, indicating the presence of both SV5 and VSV antigens in their envelopes. The presence of SV5 antigen in virions with the typical bullet-shaped appearance of VSV was confirmed with ferritin-labeled anti-SV5 antibody. In contrast to standard VSV, phenotypically mixed virions adsorbed to and eluted from chicken erythrocytes, indicating that these virions contained in their envelopes SV5 hemagglutinin, and possibly neuraminidase. Thus, the VSV nucleocapsid can interact with membranes which contain SV5 proteins in the manner which leads to virus maturation, and the production of a high yield of phenotypically mixed virions with the morphology of VSV indicates that this process can function efficiently. No evidence of genetic recombination between the two viruses was found. These results raise the possibility of an evolutionary relatedness between the paramyxoviruses and the rhabdoviruses.     

Vesicular stomatitis virus G protein acquires pH-independent fusion activity during transport in a polarized endometrial cell line

Entry of vesicular stomatitis virus (VSV), the prototype member of the rhabdovirus family, occurs by receptor-mediated endocytosis. Subsequently, during traversal through the endosomal compartments, the VSV G protein acquires a low-pH-induced fusion-competent form, allowing for fusion of the viral membrane with endosomal and lysosomal membranes. This fusion event releases genomic RNA into the cytoplasm of the cell. Here we provide evidence that the VSV G protein acquires a fusion-competent form during exocytosis in a polarized endometrial cell line, HEC-1A. VSV infection of HEC-1A cells results in high viral yields and giant cell formation. Syncytium formation is blocked in a concentration-dependent manner by treatment with the lysosomotropic weak base ammonium chloride, which raises intravesicular pH. Virus release is somewhat delayed by treatment with ammonium chloride, but virus yields gradually reach those of control cells. In addition, inhibition of vacuolar H(+)-ATPases by treatment with bafilomycin A1 also inhibited cell to cell fusion without altering virus yields. Virions released from infected HEC cells were themselves not fusion competent, since viral entry required an active H(+)-ATPase and a low-pH-induced conformational change in the viral G protein. Thus, the conformation change leading to fusion competence during exocytotic transport is reversible and reverts during or after release of the virion from the infected cell.     

Synthesis in vitro of vesicular stomatitis virus proteins in cytoplasmic extracts of L cells

All five vesicular stomatitis virus (VSV) proteins, namely, L, G,N,NS and M are synthesized $\text{in vitro}$ by a post-nuclear extract from cultured L cells infected with VSV. When, however, membrane bound polysomes are removed from the cytoplasmic extract only four virus specific proteins (L,N,NS and M) were synthesized.     

Immunocytochemical localization of vesicular stomatitis virus proteins N and NS with monoclonal antibodies

The purpose of this paper is to describe the immunocytochemical-localization of N and NS nucleocapsid proteins of vesicular stomatitis virus in the cells throughout the infectious cycle. N protein was detected in the cytoplasm at 2 h after infection and formed small cytoplasmic clusters which progressively increased in size and number. At 5-6 h, it formed large cytoplasmic inclusions. NS protein was detected in the cytoplasm a little later than N protein and showed almost the same immunostaining pattern. However, diffuse background staining of NS protein was identified throughout the cytoplasm by double immunostaining methods. At electron microscopic level, N protein was mostly granular and occasionally organized in strands at 2-3 h. At 5-6 h, numerous immunostained reaction products were organized in strands. The reaction products of NS protein were almost the same as those of N protein with the exception that diffuse background staining was observed. Cos cells, transfected with SV40 vector containing N gene obtained by recombinant DNA technique, showed clusters of N protein, but virtually no strand at electron microscopic levels. The rapid-freezing and deep-etching replica method demonstrated that loosely coiled VSV genome coated with N protein was localized on cytoplasmic sides of cell membranes in the infected cells. These results showed that complete virus genome replication was needed for strand formation of N and NS proteins and suggested that they were bound to VSV genomes in the infected cells.     

Vaccinia virus H2 protein is an essential component of a complex involved in virus entry and cell-cell fusion

The vaccinia virus H2R gene (VACWR 100) is conserved in all sequenced members of the poxvirus family and encodes a protein with a predicted transmembrane domain and four invariant cysteines. A recombinant vaccinia virus, in which expression of the H2 protein is stringently regulated, was unable to replicate without inducer. However, under nonpermissive conditions, all stages of virus morphogenesis appeared normal and extracellular virions were detected at the tips of actin tails. Nevertheless, virus did not spread to neighboring cells nor did syncytia form after low-pH treatment. Purified -H2 and +H2 virions from cells infected in the absence or presence of inducer, respectively, were indistinguishable in microscopic appearance and contained the same complement of major proteins, though only +H2 virions were infectious. The -H2 virions bound to cells, but their cores did not penetrate into the cytoplasm. In addition, exogenously added -H2 virions were unable to mediate the formation of syncytia after low-pH treatment. In contrast, virions lacking the A27 (p14) protein, which was previously considered to have an essential role in fusion, penetrated cells and induced extensive syncytia. The properties of H2, however, are very similar to those recently reported for the A28 protein. Moreover, coimmunoprecipitation experiments indicated an interaction between H2 and A28. Therefore, H2 and A28 are the only proteins presently known to be specifically required for vaccinia virus entry and are likely components of a fusion complex.     

Persistence of vesicular stomatitis virus in cloned interleukin-2-dependent natural killer cell lines.

We have investigated virus-lymphocyte interactions by using cloned subpopulations of interleukin-2-dependent effector lymphocytes maintained in vitro. Cloned lines of H-2-restricted hapten- or virus-specific cytotoxic T lymphocytes (CTL) and alloantigen-specific CTL were resistant to productive infection by vesicular stomatitis virus (VSV). In contrast, cloned lines of natural killer (NK) cells were readily and persistently infected by VSV, a virus which is normally highly cytolytic. VSV-infected NK cells continued to proliferate, express viral surface antigen, and produce infectious virus. Furthermore, persistently infected NK cells showed no marked alteration of normal cellular morphology and continued to lyse NK-sensitive target cells albeit at a slightly but significantly reduced level. The persistence of VSV in NK cells did not appear to be caused by the generation of temperature-sensitive viral mutants, defective interfering particles, or interferon. Consequently, studies comparing the intracellular synthesis and maturation of VSV proteins in infected NK and mouse L cells were conducted. In contrast to L cells, in which host cell protein synthesis was essentially totally inhibited by infection, the infection of NK cells caused no marked diminution in the synthesis of host cell proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates of viral proteins from infected cells showed that the maturation rate and size of VSV surface G glycoprotein were comparable in L cells and NK cells. Nucleocapsid (N) protein synthesis also appeared to be unaffected in NK cells. In contrast, the viral proteins NS and M appeared to be selectively degraded in NK cell extracts. Mixing experiments suggested that a protease in NK cells was responsible for the selective breakdown of VSV NS protein. Finally, VSV-infected NK cells were resistant to lysis by virus-specific CTL, suggesting that persistently infected NK cells may harbor virus and avoid cell-mediated immune destruction in an immunocompetent host.