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。     

In vitro phosphorylation and purification of a nonstructural protein of bluetongue virus with affinity for single-stranded RNA.

A phosphorylated, nonstructural protein of bluetongue virus, NS2, is synthesized throughout the replication cycle in comparatively large amounts. The protein was detected in both the soluble and particulate fraction of the cytoplasm of infected cells. The particulate NS2 could be solubilized in 0.5 M NaCl. It was found that NS2 in the particulate fraction and immunoprecipitates of NS2 from the soluble protein fraction could be phosphorylated in vitro. It is not known whether the kinase involved is of cellular or viral origin, but after purification of NS2 by affinity chromatography on poly(U)-Sepharose it could still by phosphorylated in vitro without the addition of exogenous protein kinase. The affinity of NS2 for nucleic acid was also investigated. The protein was found to bind to single-stranded RNA. In the presence of purified bluetongue virus mRNA, NS2 formed a complex with an estimated S value of about 22S.     

The Late-Domain-Containing Protein p6 Is the Predominant Phosphoprotein of Human Immunodeficiency Virus Type 1 Particles

The Gag-derived protein p6 of human immunodeficiency virus type 1 (HIV-1) plays a crucial role in the release of virions from the membranes of infected cells. It is presumed that p6 and functionally related proteins from other viruses act as adapters, recruiting cellular factors to the budding site. This interaction is mediated by so-called late domains within the viral proteins. Previous studies had suggested that virus release from the plasma membrane shares elements with the cellular endocytosis machinery. Since protein phosphorylation is known to be a regulatory mechanism in these processes, we have investigated the phosphorylation of HIV-1 structural proteins. Here we show that p6 is the major phosphoprotein of HIV-1 particles. After metabolic labeling of infected cells with [ortho-32P]phosphate, we found that phosphorylated p6 from infected cells and from virus particles consisted of several forms, suggesting differential phosphorylation at multiple sites. Apparently, phosphorylation occurred shortly before or after the release of p6 from Gag and involved only a minor fraction of the total virion-associated p6 molecules. Phosphoamino acid analysis indicated phosphorylation at Ser and Thr, as well as a trace of Tyr phosphorylation, supporting the conclusion that multiple phosphorylation events do occur. In vitro experiments using purified virus revealed that endogenous or exogenously added p6 was efficiently phosphorylated by virion-associated cellular kinase(s). Inhibition experiments suggested that a cyclin-dependent kinase or a related kinase, most likely ERK2, was involved in p6 phosphorylation by virion-associated enzymes.     

Hepatitis C virus NS5A protein is phosphorylated by casein kinase II

Hepatitis C virus (HCV) has a positive-strand RNA genome that encodes a polyprotein, which is post-translationally processed by cellular and viral proteinases into three structural and six non-structural (NS) proteins. The NS5A protein, expressed in mammalian cells, exists as two phosphorylated forms of 56 kDa and 58 kDa. In this study, we provide evidence for a stable association between NS5A and a protein kinase from rat-1 cells by affinity to immobilized glutathione-S-transferase (GST)-NS5A fusion protein. This protein kinase was associated through the N-terminus of NS5A and was not regulated by cell cycle. The GST-NS5A was also phosphorylated in vitro by the purified casein kinase II (CKII), a member of the CMCG kinase family. Since CKII and the NS5A-associated protein kinase have the same molecular size and property by In-gel kinase assay and an inhibitor treatment test, we conclude that HCV NS5A protein is phosphorylated by CKII.     

Translational control of protein synthesis after infection by vesicular stomatitis virus.

Four hours after infection of BHK cells by vesicular stomatitis virus (VSV), the rate of total protein synthesis was about 65% that of uninfected cells and synthesis of the 12 to 15 predominant cellular polypeptides was reduced to a level about 25% that of control cells. As determined by in vitro translation of isolated RNA and both one- and two-dimensional gel analyses of the products, all predominant cellular mRNA's remained intact and translatable after infection. The total amount of translatable mRNA per cell increased about threefold after infection; this additional mRNA directed synthesis of the five VSV structural proteins. To determine the subcellular localization of cellular and viral mRNA before and after infection, RNA from various sizes of polysomes and nonpolysomal ribonucleoproteins (RNPs) was isolated from infected and noninfected cells and translated in vitro. Over 80% of most predominant species of cellular mRNA was bound to polysomes in control cells, and over 60% was bound in infected cells. Only 2 of the 12 predominant species of translatable cellular mRNA's were localized to the RNP fraction, both in infected and in uninfected cells. The average size of polysomes translating individual cellular mRNA's was reduced about two- to threefold after infection. For example, in uninfected cells, actin (molecular weight 42,000) mRNA was found predominantly on polysomes with 12 ribosomes; after infection it was found on polysomes with five ribosomes, the same size of polysomes that were translating VSV N (molecular weight 52,000) and M (molecular weight 35,000) mRNA. We conclude that the inhibition of cellular protein synthesis after VSV infection is due, in large measure, to competition for ribosomes by a large excess of viral mRNA. The efficiency of initiation of translation on cellular and viral mRNA's is about the same in infected cells; cellular ribosomes are simply distributed among more mRNA's than are present in growing cells. About 20 to 30% of each of the predominant cellular and viral mRNA's were present in RNP particles in infected cells and were presumably inactive in protein synthesis. There was no preferential sequestration of cellular or viral mRNA's in RNPs after infection.     

The NS5A/NS5 Proteins of Viruses from Three Genera of the Family Flaviviridae Are Phosphorylated by Associated Serine/Threonine Kinases

Phosphorylation of the expressed NS5A protein of hepatitis C virus (HCV), a member of the Hepacivirus genus of the family Flaviviridae, has been demonstrated in mammalian cells and in a cell-free assay by an associated kinase activity. In this report, phosphorylation is also shown for the NS5A and NS5 proteins, respectively, of bovine viral diarrhea virus (BVDV) and yellow fever virus (YF), members of the other two established genera in this family. Phosphorylation of BVDV NS5A and YF NS5 was observed in infected cells, transient expression experiments, and a cell-free assay similar to the one developed for HCV NS5A. Phosphoamino acid analyses indicated that all three proteins were phosphorylated by serine/threonine kinases. Similarities in the properties of BVDV NS5A, YF NS5, and HCV NS5A phosphorylation in vitro further suggested that closely related kinases or the same kinase may phosphorylate these viral proteins. Conservation of this trait among three quite distantly related viruses representing three separate genera suggests that phosphorylation of the NS5A/NS5 proteins or their association with cellular kinases may play an important role in the flavivirus life cycle.     

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.     

Identification of SRPK1 and SRPK2 as the major cellular protein kinases phosphorylating hepatitis B virus core protein

Phosphorylation of hepatitis B virus (HBV) core protein has recently been shown to be a prerequisite for pregenomic RNA encapsidation into viral capsids, but the host cell kinases mediating this essential step of the HBV replication cycle have not been identified. We detected two kinases of 95 and 115 kDa in HuH-7 total cell lysates which interacted specifically with the HBV core protein and phosphorylated its arginine-rich C-terminal domain. The 95-kDa kinase was purified and characterized as SR protein-specific kinase 1 (SRPK1) by mass spectrometry. Based on this finding, the 115-kDa kinase could be identified as the related kinase SRPK2 by immunoblot analysis. In vitro, both SRPKs phosphorylated HBV core protein on the same serine residues which are found to be phosphorylated in vivo. Moreover, the major cellular HBV core kinase activity detected in the total cell lysate showed biochemical properties identical to those of SRPK1 and SRPK2, as examined by measuring binding to a panel of chromatography media. We also clearly demonstrate that neither the cyclin-dependent kinases Cdc2 and Cdk2 nor protein kinase C, previously implicated in HBV core protein phosphorylation, can account for the HBV core protein kinase activity. We conclude that both SRPK1 and SRPK2 are most likely the cellular protein kinases mediating HBV core protein phosphorylation during viral infection and therefore represent important host cell targets for therapeutic intervention in HBV infection.     

In Vitro Protein-Synthesizing Activity of Vesicular Stomatitis Virus-Infected Cell Extracts

Crude cytoplasmic extracts from vesicular stomatitis virus (VSV)-infected HeLa cells incorporate radioactive amino acids into hot trichloroacetic acid-precipitable material linearly for 10 to 20 min. The material synthesized in vitro corresponds in molecular weight to four of the five VSV structural proteins. However, synthesis of the viral glycoprotein (G) is significantly reduced, whereas the relative amounts of viral structural proteins L and NS synthesized are increased compared with the ratio of the proteins found in the virion. Fractionation of a VSV-infected crude cytoplasmic extract into a cytoplasmic pellet (20,000 x g for 30 min) and a cytoplasmic supernatant results in a significant reduction in protein synthesizing activity of both fractions, although both contain polysomes. The products synthesized by a cytoplasmic supernatant-directed system included all the VSV structural proteins except the glycoprotein, whereas in an in vitro system directed by the cytoplasmic pellet there is a marked reduction in synthesis of the nucleoprotein (N) and also a small relative increase in synthesis of the glycoprotein. Addition of uninfected, preincubated HeLa or L-cell S10 or a HeLa ribosomal fraction to the VSV-infected cytoplasmic pellet results in a 30- to 60-fold stimulation of (35)S-methionine incorporation. However, these uninfected extracts do not stimulate (35)S-methionine incorporation by the infected crude cytoplasmic extract or the cytoplasmic supernatant. The products synthesized by the stimulated cytoplasmic pellet now include sizeable amounts of the glycoprotein in addition to the other VSV structural proteins.     

Effect of Vesicular Stomatitis Virus Infection on the Histocompatibility Antigen of L Cells

When mouse L cells are infected for 22 hr with vesicular stomatitis virus (VSV), a ribonucleic acid-containing enveloped virus, greater than 70% of the major histocompatibility antigen (H-2), is no longer detectable by the method of inhibition of immune cytolysis. Infected cells prelabeled with (14)C-glucosamine also show a correspondingly greater loss of trichloroacetic acid-insoluble radioactivity than uninfected cells. The loss of H-2 antigenic activity is not due to the viral inhibition of host cell protein synthesis since cells cultured for 18 hr in the presence of cycloheximide have the same amount of H-2 activity as untreated controls. Also, cells infected with encephalomyocarditis virus, a picornavirus, show no loss of H-2 activity at a time when host cell protein synthesis is completely inhibited. VSV structural proteins associated in vitro with uninfected L-cell plasma membranes do not render H-2 sites inaccessible to the assay. Although antibodies may not combine with all the H-2 antigenic sites on the plasma membrane, anti-H-2 serum reacted with L cells before infection does not prevent a normal infection with VSV. H-2 activity can be detected in virus samples purified from the medium of infected L cells; this virus purified after being mixed with L-cell homogenates shows greater H-2 activity than virus purified after being mixed with HeLa cell homogenates. However, VSV made in HeLa cells shows no H-2 activity when mixed with L-cell homogenates.     

Protein Kinase and Phosphoproteins of Vesicular Stomatitis Virus

Protein kinases of similar but not identical activity were found associated with vesicular stomatitis (VS) virions grown in mouse L cells, primary chicken embryo (CE) cells, and BHK-21 cells, as well as being present in VS virions grown in HeLa and Aedes albopictus cells. The virion kinase preferentially phosphorylated the nucleocapsid NS protein in vitro and to a lesser extent the envelope M protein. Other virion proteins were phosphorylated in vitro only after drastic detergent treatment. Partial evidence that the virion kinase is of cellular origin was obtained by finding reduced enzyme activity in virions released from cells pretreated with actinomycin D and cycloheximide. Selective detergent and detergent-salt fractionation of VS virions revealed that the kinase activity was present in the envelope but not the spikes. The virion kinase activity in a Triton-salt-solubilized envelope fraction could be separated from M and G proteins and partially purified by phosphocellulose column chromatography. Virions released from L, CE, and BHK-21 cells infected in the presence of [(32)P]orthophosphate were labeled almost exclusively in the NS protein. Both soluble and nucleocapsid-associated NS phosphoprotein were present in cytoplasmic extracts of VS viral-infected L cells. The origin and function of the NS phosphoprotein remain to be elucidated.     

Regulation of minute virus of mice NS1 replicative functions by atypical PKClambda in vivo

Minute virus of mice NS1 protein is a multifunctional phosphoprotein endowed with a variety of enzymatic and regulatory activities necessary for progeny virus particle production. To regulate all of its different functions in the course of a viral infection, NS1 has been proposed to be modulated by posttranslational modifications, in particular, phosphorylation. Indeed, it was shown that the NS1 phosphorylation pattern is altered during the infectious cycle and that the biochemical profile of the protein is dependent on the phosphorylation state of the polypeptide. Moreover, in vitro approaches have identified members of the protein kinase C (PKC) family, in particular, atypical PKC, as regulators of viral DNA replication through the phosphorylation of NS1 residues T435 and S473, thereby activating the protein for DNA unwinding activities. In order to substantiate these findings in vivo, we produced NS1 in the presence of a dominant-negative PKClambda mutant and characterized the purified protein in vitro. The NS1 protein produced under these conditions was found to be only partially phosphorylated and as a consequence to be deficient for viral DNA replication. However, it could be rescued for this viral function by treatment with recombinant activated PKClambda. Our data clearly demonstrate that NS1 is a target for PKClambda phosphorylation in vivo and that this modification is essential for the helicase activity of the viral polypeptide. In addition, the phosphorylation of NS1 at residues T435 and S473 appeared to occur mainly in the nucleus, providing further evidence for the involvement of PKClambda which, unlike PKCzeta, accumulates in the nuclear compartment of infected cells.     

The severe acute respiratory syndrome coronavirus nucleocapsid protein is phosphorylated and localizes in the cytoplasm by 14-3-3-mediated translocation

The severe acute respiratory syndrome coronavirus(SARS-CoV) nucleocapsid (N) protein is one of the four structural proteins of the virus and is predicted to be a 46-kDa phosphoprotein. Our in silico analysis predicted N to be heavily phosphorylated at multiple residues. Experimentally, we have shown in this report that the N protein of the SARS-CoV gets serine-phosphorylated by multiple kinases, in both the cytoplasm and the nucleus. The phosphoprotein is stable and localizes in the cytoplasm and coprecipitates with the membrane fraction. Also, using specific inhibitors of phosphorylation and an in vitro phosphorylation assay, we show that the nucleocapsid protein is a substrate of cyclin-dependent kinase (CDK), glycogen synthase kinase, mitogen-activated protein kinase, and casein kinase II. Further, we show that the phosphorylated protein is translocated to the cytoplasm by binding to 14-3-3 (tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein). 14-3-3 proteins are a family of highly conserved, ubiquitously expressed eukaryotic proteins that function primarily as adapters that modulate interactions between components of various cellular signaling and cell cycle regulatory pathways through phosphorylation-dependent protein-protein interactions. Coincidentally, the N protein was also found to downregulate the expression of the theta isoform of 14-3-3 (14-3-3theta), leading to the accumulation of phosphorylated N protein in the nucleus, in the absence of growth factors. Using short interfering RNA specific to 14-3-3theta we have inhibited its expression to show accumulation of phosphorylated N protein in the nucleus. Thus, the data presented here provide a possible mechanism for phosphorylation-dependent nucleocytoplasmic shuttling of the N protein. This 14-3-3-mediated transport of the phosphorylated N protein and its possible implications in interfering with the cellular machinery are discussed.     

Assembly of viral membranes. I. Association of vesicular stomatitis virus membrane proteins and membranes in a cell-free system.

We report here an in vitro system designed to study the interactions of vesicular stomatitis virus (VSV) proteins with cellular membranes. We have synthesized the VSV nucleocapsid (N) protein, nonstructural (NS) protein, glycoprotein (G protein), and membrane (M) protein in a wheat germ, cell-free, protein-synthesizing system directed by VSV 12 to 18S RNA. When incubated at low salt concentrations with purified cytoplasmic membranes derived from Chinese hamster ovary cells, the VSV M andG proteins bind to membranes, whereas the VSV N and NS proteins do not. The VSV M protein binds to membranes in low or high divalent cation concentrations, whereas binding of significant amounts of G protein requires at least 5 mM magnesium acetate concentrations.     

Identification of the major phosphorylation site of the hepatitis C virus H strain NS5A protein as serine 2321.

The hepatitis C virus (HCV) NS5A protein is phosphorylated by a cellular, serine/threonine kinase. To identify the major site(s) of NS5A phosphorylation, radiolabeled HCV-H NS5A phosphopeptides were purified and subjected to phosphoamino acid analysis and Edman degradation. These data identified the major intracellular phosphorylation site in the HCV-H NS5A protein as Ser(2321), a result verified by two additional, independent methods: (i) substitution of Ala for Ser(2321) and the concomitant disappearance of the major in vivo phosphorylated peptides and corresponding in vitro phosphorylated peptides; and (ii) comigration of the digestion products of a synthetic peptide phosphorylated on Ser(2321) with the major in vivo phosphorylated NS5A peptides. Site-directed mutagenesis of Ser(2321) suggested that phosphorylation of NS5A is dispensable for previously described interactions with NS4A and PKR, a cellular, antiviral kinase that does not appear to catalyze NS5A phosphorylation. The proline-rich nature of the amino acid sequence flanking Ser(2321) (PLPPPRS(2321) PPVPPPR) suggests that a proline-directed kinase is responsible for the majority of HCV NS5A phosphorylation, consistent with previous kinase inhibitor studies.