The V Protein of Mumps Virus Plays a Critical Role in Pathogenesis

ABSTRACT Constitutive levels of production of STAT-1 were reduced by 10 h postinfection (p.i.) and significantly lost by 24 h p.i. in FL cells acutely infected with mumps virus (MuV). This result was consistent with that observed in previous studies and experiments with cells persistently infected with MuV (FLMT cells). There was a marked decrease in the amount of STAT-1 in cells expressing MuV accessory protein V (MuV-V). Furthermore, single amino acid substitutions in the Cys-rich region of V protein (Vc189a, Vc207a, and Vc214a) showed that each cysteine residue plays an important role in the decrease in STAT-1 production, but substitution of a histidine residue at amino acid position 203 had no effect. These events and the resultant suppression of the alpha interferon (IFN-α) response were confirmed by a luciferase reporter gene assay with five tandem repeats of the IFN-α-stimulated response element as an enhancer element of the firely luciferase gene. STAT-1 production was restored and detectable in FLMT cells treated with a proteosome inhibitor, such as MG132 or lactacystin. In the presence of MG132, ubiquitination of STAT-1 and the interaction of MuV-V with STAT-1 were demonstrated in FLMT cells by immunoprecipitation with anti-STAT-1 antibody. The same results for the interaction and ubiquitination were obtained in experiments with an expression vector for a C-terminal deletion mutant of STAT-1. The truncated STAT-1 molecules were degraded in the presence of MuV-V. Therefore, the C-terminal region (transcriptional activation and Src homology 2 domains) of STAT-1 is not necessary for its degradation caused by MuV-V. Our data suggest that MuV-V promotes ubiquitination and degradation of STAT-1.

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The Carboxyl Segment of the Mumps Virus V Protein Associates with Stat Proteins in Vitro Via a Tryptophan-Rich Motif

Virology ◽

10.1006/viro.2002.1509 ◽

2002 ◽

Vol 300 (1) ◽

pp. 92-99 ◽

Cited By ~ 69

Author(s):

Machiko Nishio ◽

Dominique Garcin ◽

Viviane Simonet ◽

Daniel Kolakofsky

Keyword(s):

Mumps Virus ◽

V Protein ◽

Stat Proteins

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Roles of Phosphorylation of the Nucleocapsid Protein of Mumps Virus in Regulating Viral RNA Transcription and Replication

Journal of Virology ◽

10.1128/jvi.00686-15 ◽

2015 ◽

Vol 89 (14) ◽

pp. 7338-7347 ◽

Cited By ~ 10

Author(s):

James Zengel ◽

Adrian Pickar ◽

Pei Xu ◽

Alita Lin ◽

Biao He

Keyword(s):

Nucleocapsid Protein ◽

Rna Synthesis ◽

Critical Role ◽

Phosphorylation Site ◽

Mumps Virus ◽

Viral Rna ◽

Human Populations ◽

Rna Genome ◽

Major Phosphorylation Site ◽

Transcription And Replication

ABSTRACTMumps virus (MuV) is a paramyxovirus with a negative-sense nonsegmented RNA genome. The viral RNA genome is encapsidated by the nucleocapsid protein (NP) to form the ribonucleoprotein (RNP), which serves as a template for transcription and replication. In this study, we investigated the roles of phosphorylation sites of NP in MuV RNA synthesis. Using radioactive labeling, we first demonstrated that NP was phosphorylated in MuV-infected cells. Using both liquid chromatography-mass spectrometry (LC-MS) andin silicomodeling, we identified nine putative phosphorylated residues within NP. We mutated these nine residues to alanine. Mutation of the serine residue at position 439 to alanine (S439A) was found to reduce the phosphorylation of NP in transfected cells by over 90%. The effects of these mutations on the MuV minigenome system were examined. The S439A mutant was found to have higher activity, four mutants had lower activity, and four mutants had similar activity compared to wild-type NP. MuV containing the S439A mutation had 90% reduced phosphorylation of NP and enhanced viral RNA synthesis and viral protein expression at early time points after infection, indicating that S439 is the major phosphorylation site of NP and its phosphorylation plays an important role in downregulating viral RNA synthesis.IMPORTANCEMumps virus (MuV), a paramyxovirus, is an important human pathogen that is reemerging in human populations. Nucleocapsid protein (NP) of MuV is essential for viral RNA synthesis. We have identified the major phosphorylation site of NP. We have found that phosphorylation of NP plays a critical role in regulating viral RNA synthesis. The work will lead to a better understanding of viral RNA synthesis and possible novel targets for antiviral drug development.

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Mumps Virus Nucleoprotein Enhances Phosphorylation of the Phosphoprotein by Polo-Like Kinase 1

Journal of Virology ◽

10.1128/jvi.02160-15 ◽

2015 ◽

Vol 90 (3) ◽

pp. 1588-1598 ◽

Cited By ~ 3

Author(s):

Adrian Pickar ◽

James Zengel ◽

Pei Xu ◽

Zhuo Li ◽

Biao He

Keyword(s):

Rna Synthesis ◽

Critical Role ◽

Phosphorylation Site ◽

Mumps Virus ◽

Viral Rna ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Rna Complex ◽

Viral Genomic Rna ◽

Negative Sense

ABSTRACTThe viral RNA-dependent RNA polymerases (vRdRps) of nonsegmented, negative-sense viruses (NNSVs) consist of the enzymatic large protein (L) and the phosphoprotein (P). P is heavily phosphorylated, and its phosphorylation plays a critical role in viral RNA synthesis. Since NNSVs do not encode kinases, P is phosphorylated by host kinases. In this study, we investigate the roles that viral proteins play in the phosphorylation of mumps virus (MuV) P. We found that nucleoprotein (NP) enhances the phosphorylation of P. We have identified the serine/threonine kinase Polo-like kinase 1 (PLK1) as a host kinase that phosphorylates P and have found that phosphorylation of P by PLK1 is enhanced by NP. The PLK1 binding site in MuV P was mapped to residues 146 to 148 within the S(pS/T)P motif, and the phosphorylation site was identified as residues S292 and S294.IMPORTANCEIt has previously been shown that P acts as a chaperone for NP, which encapsidates viral genomic RNA to form the NP-RNA complex, the functional template for viral RNA synthesis. Thus, it is assumed that phosphorylation of P may regulate NP's ability to form the NP-RNA complex, thereby regulating viral RNA synthesis. Our work demonstrates that MuV NP affects phosphorylation of P, suggesting that NP can regulate viral RNA synthesis by regulating phosphorylation of P.

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The mumps virus V protein is unstable in virus infected cells

Archives of Virology ◽

10.1007/bf01309756 ◽

1993 ◽

Vol 133 (1-2) ◽

pp. 201-209 ◽

Cited By ~ 2

Author(s):

A. Hu ◽

S. Schwartz ◽

G. Utter ◽

C. �rvell ◽

J. K�vamees ◽

...

Keyword(s):

Mumps Virus ◽

V Protein ◽

Infected Cells

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Association of Mumps Virus V Protein with RACK1 Results in Dissociation of STAT-1 from the Alpha Interferon Receptor Complex

Journal of Virology ◽

10.1128/jvi.76.24.12676-12682.2002 ◽

2002 ◽

Vol 76 (24) ◽

pp. 12676-12682 ◽

Cited By ~ 52

Author(s):

Toru Kubota ◽

Noriko Yokosawa ◽

Shin-ichi Yokota ◽

Nobuhiro Fujii

Keyword(s):

Signal Transduction Pathway ◽

Adaptor Protein ◽

Mumps Virus ◽

Virus Protein ◽

V Protein ◽

Long Form ◽

Infected Cells ◽

Intracellular Target ◽

Two Hybrid ◽

Hybrid Screening

ABSTRACT It has been reported that mumps virus protein V or the C-terminal Cys-rich region of protein V (Vsp) is associated with blocking of the interferon (IFN) signal transduction pathway through a decrease in STAT-1 production. The intracellular target of the V protein was investigated by using a two-hybrid screening system with Vsp as bait. Full-length V protein and Vsp were able to bind to RACK1, and the interaction did not require two WD domains, WD1 and WD2, in RACK1. A significant interaction between V protein and RACK1 was also demonstrated in cells persistently infected with mumps virus (FLMT cells), and the formation of the complex was not affected by treatment with IFN. On the other hand, in uninfected cells, STAT-1 was associated with the long form of the β subunit of the alpha IFN receptor, and this association was mediated by the function of RACK1 as an adaptor protein. Immunoprecipitation and glutathione S-transferase pull-down experiments revealed that the association of RACK1 or mumps virus V protein with the IFN receptor was undetectable in mumps virus-infected cells. Furthermore, RACK1 interacted with mumps virus V protein with a higher affinity than STAT-1 did. Therefore, it is suggested that mumps virus V protein has the ability to interact strongly with RACK1 and consequently to bring about the disruption of the complex formed from STAT-1, RACK1, and the IFN receptor.

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Correction for Yokosawa et al., C-Terminal Region of STAT-1α Is Not Necessary for Its Ubiquitination and Degradation Caused by Mumps Virus V Protein

Journal of Virology ◽

10.1128/jvi.00715-15 ◽

2015 ◽

Vol 89 (17) ◽

pp. 9142-9142

Author(s):

Noriko Yokosawa ◽

Shin-ichi Yokota ◽

Toru Kubota ◽

Nobuhiro Fujii

Keyword(s):

Mumps Virus ◽

Terminal Region ◽

V Protein

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Mumps Virus V Protein Antagonizes Interferon without the Complete Degradation of STAT1

Journal of Virology ◽

10.1128/jvi.79.7.4451-4459.2005 ◽

2005 ◽

Vol 79 (7) ◽

pp. 4451-4459 ◽

Cited By ~ 30

Author(s):

Toru Kubota ◽

Noriko Yokosawa ◽

Shin-ichi Yokota ◽

Nobuhiro Fujii ◽

Masato Tashiro ◽

...

Keyword(s):

Protein Function ◽

Nuclear Translocation ◽

Cysteine Residue ◽

Mumps Virus ◽

Unique Region ◽

V Protein ◽

Complete Degradation ◽

Infected Cells ◽

Induced Signal ◽

Viral Components

ABSTRACT Mumps virus (MuV) has been shown to antagonize the antiviral effects of interferon (IFN) through proteasome-mediated complete degradation of STAT1 by using the viral V protein (T. Kubota et al., Biochem. Biophys. Res. Commun. 283:255-259, 2001). However, we found that MuV could inhibit IFN signaling and the generation of a subsequent antiviral state long before the complete degradation of cellular STAT1 in infected cells. In MuV-infected cells, nuclear translocation and phosphorylation of STAT1 and STAT2 tyrosine residue (Y) at 701 and 689, respectively, by IFN-β were significantly inhibited but the phosphorylation of Jak1 and Tyk2 was not inhibited. The transiently expressed MuV V protein also inhibited IFN-β-induced Y701-STAT1 and Y689-STAT2 phosphorylation, suggesting that the V protein could block IFN-β-induced signal transduction without the aid of other viral components. Finally, a substitution of an alanine residue in place of a cysteine residue in the C-terminal V-unique region known to be required for STAT1 degradation and inhibition of anti-IFN signaling resulted in the loss of V protein function to inhibit the Y701-STAT1 and Y689-STAT2 phosphorylation.

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