The Matrix Protein of Measles Virus Regulates Viral RNA Synthesis and Assembly by Interacting with the Nucleocapsid Protein

ABSTRACT The genome of measles virus (MV) is encapsidated by the nucleocapsid (N) protein and associates with RNA-dependent RNA polymerase to form the ribonucleoprotein complex. The matrix (M) protein is believed to play an important role in MV assembly by linking the ribonucleoprotein complex with envelope glycoproteins. Analyses using a yeast two-hybrid system and coimmunoprecipitation in mammalian cells revealed that the M protein interacts with the N protein and that two leucine residues at the carboxyl terminus of the N protein (L523 and L524) are critical for the interaction. In MV minigenome reporter gene assays, the M protein inhibited viral RNA synthesis only when it was able to interact with the N protein. The N protein colocalized with the M protein at the plasma membrane when the proteins were coexpressed in plasmid-transfected or MV-infected cells. In contrast, the N protein formed small dots in the perinuclear area when it was expressed without the M protein, or it was incapable of interacting with the M protein. Furthermore, a recombinant MV possessing a mutant N protein incapable of interacting with the M protein grew much less efficiently than the parental virus. Since the M protein has an intrinsic ability to associate with the plasma membrane, it may retain the ribonucleoprotein complex at the plasma membrane by binding to the N protein, thereby stopping viral RNA synthesis and promoting viral particle production. Consequently, our results indicate that the M protein regulates MV RNA synthesis and assembly via its interaction with the N protein.

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Transmembrane communication in cells chronically infected with measles virus.

The Journal of Cell Biology ◽

10.1083/jcb.81.2.396 ◽

1979 ◽

Vol 81 (2) ◽

pp. 396-402 ◽

Cited By ~ 42

Author(s):

D L Tyrrell ◽

A Ehrnst

Keyword(s):

Nucleocapsid Protein ◽

Measles Virus ◽

Matrix Protein ◽

Cytochalasin B ◽

Viral Proteins ◽

Surface Proteins ◽

Nucleocapsid Proteins ◽

Viral Antigens ◽

The Matrix ◽

Viral Inclusions

The transmembrane association of the measles virus hemagglutinin and hemolysin surface proteins with intracellular viral antigens was studied. Rabbit antisera monospecific for measles virus matrix and nucleocapsid proteins and a human antiserum containing specificities for both the hemagglutinin and hemolysin proteins were used to study the co-capping of these proteins in human Lu 106 cell-line, chronically infected with measles virus. Capping of the surface-associated envelope components was accompanied by co-capping of the matrix and nucleocapsid proteins, the latter being localized mainly within the inclusions. This demonstrated transmembrane communication between surface-associated envelope components and the intracellular measles virus matrix and nucleocapsid proteins. The results demonstrated the existence of a linkage between viral inclusions and viral proteins associated with cell membranes. In the presence of cytochalasin B (1--2 micrograms/ml), co-capping of the matrix protein was unchanged or slightly enhanced, whereas co-capping of the nucleocapsid protein decreased, indicating that actin filaments may mediate the communication between viral nucleocapsids and the cell membrane.

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Measles virus and canine distemper virus target proteins into a TAP-independent MHC class I-restricted antigen-processing pathway

Journal of General Virology ◽

10.1099/0022-1317-82-2-441 ◽

2001 ◽

Vol 82 (2) ◽

pp. 441-447 ◽

Cited By ~ 25

Author(s):

Claudia Neumeister ◽

Ralph Nanan ◽

Tatjana I. Cornu ◽

Carsten G. K. Lüder ◽

Volker ter Meulen ◽

...

Keyword(s):

Nucleocapsid Protein ◽

Measles Virus ◽

Canine Distemper Virus ◽

Antigen Processing ◽

Matrix Protein ◽

Cytotoxic T Cells ◽

Viral Proteins ◽

Canine Distemper ◽

The Matrix

After infection of CEM174.T2 cells [deficient for the transporter of antigen presentation (TAP)] with measles virus (MV) the nucleocapsid protein is recognized by Ld-restricted cytotoxic T cells in a TAP-independent, chloroquine-sensitive fashion. Presentation via the TAP-independent pathway requires virus replication. During MV infection of the cell the nucleocapsid as well as the matrix protein enter the endolysosomal compartment as indicated by colocalization with the lysosomal-associated membrane protein 1 (LAMP-1). Similarly, the nucleocapsid protein of canine distemper virus (CDV) is recognized in a TAP-independent fashion. In addition, a recombinant MV expressing bacterial β-galactosidase protein is able to introduce the recombinant antigen into the TAP-independent pathway whereas a vaccinia virus expressing β-galactosidase is not. These data and a report about TAP-independent recognition of parainfluenza virus type 1 suggest that members of the Paramyxoviridae family regularly introduce viral proteins into the TAP-independent antigen-processing pathway.

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Measles Virus Circumvents the Host Interferon Response by Different Actions of the C and V Proteins

Journal of Virology ◽

10.1128/jvi.00108-08 ◽

2008 ◽

Vol 82 (17) ◽

pp. 8296-8306 ◽

Cited By ~ 75

Author(s):

Yuichiro Nakatsu ◽

Makoto Takeda ◽

Shinji Ohno ◽

Yuta Shirogane ◽

Masaharu Iwasaki ◽

...

Keyword(s):

Protein Expression ◽

Measles Virus ◽

Rna Synthesis ◽

Virulent Strain ◽

Viral Rna ◽

Interferon Response ◽

Growth Defect ◽

High Morbidity ◽

C Protein ◽

V Protein

ABSTRACT Measles is an acute febrile infectious disease with high morbidity and mortality. The genome of measles virus (MV), the causative agent, encodes two accessory products, V and C proteins, that play important roles in MV virulence. The V but not the C protein of the IC-B strain (a well-characterized virulent strain of MV) has been shown to block the Jak/Stat signaling pathway and counteract the cellular interferon (IFN) response. We have recently shown that a recombinant IC-B strain that lacks C protein expression replicates poorly in certain cell lines, and its growth defect is related to translational inhibition and strong IFN induction. Here, we show that the V protein of the MV IC-B strain also blocks the IFN induction pathway mediated by the melanoma differentiation-associated gene 5 product, thus actively interfering with the host IFN response at two different steps. On the other hand, the C protein per se possesses no activity to block the IFN induction pathway. Our data indicate that the C protein acts as a regulator of viral RNA synthesis, thereby acting indirectly to suppress IFN induction. Since recombinant MVs with C protein defective in modulating viral RNA synthesis or lacking C protein expression strongly stimulate IFN production, in spite of V protein production, both the C and V proteins must be required for MV to fully circumvent the host IFN response.

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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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PPEY Motif within the Rabies Virus (RV) Matrix Protein Is Essential for Efficient Virion Release and RV Pathogenicity

Journal of Virology ◽

10.1128/jvi.00889-08 ◽

2008 ◽

Vol 82 (19) ◽

pp. 9730-9738 ◽

Cited By ~ 56

Author(s):

Christoph Wirblich ◽

Gene S. Tan ◽

Amy Papaneri ◽

Peter J. Godlewski ◽

Jan Marc Orenstein ◽

...

Keyword(s):

Viral Replication ◽

Rabies Virus ◽

Rna Synthesis ◽

Matrix Protein ◽

Virus Production ◽

Wild Type Virus ◽

Wild Type ◽

Conserved Sequence ◽

Virion Release ◽

The Matrix

ABSTRACT Late (L) domains containing the highly conserved sequence PPXY were first described for retroviruses, and later research confirmed their conservation and importance for efficient budding of several negative-stranded RNA viruses. Rabies virus (RV), a member of the Rhabdoviridae family, contains the sequence PPEY (amino acids 35 to 38) within the N terminus of the matrix (M) protein, but the functions of this potential L-domain in the viral life cycle, viral pathogenicity, and immunogenicity have not been established. Here we constructed a series of recombinant RVs containing mutations within the PPEY motif and analyzed their effects on viral replication and RV pathogenicity. Our results indicate that the first proline at position 35 is the most important for viral replication, whereas P36 and Y38 have a lesser but still noticeable impact. The reduction in viral replication was most likely due to inhibition of virion release, because initially no major impact on RV RNA synthesis was observed. In addition, results from electron microscopy demonstrated that the M4A mutant virus (PPEY→SAEA) displayed a more cell-associated phenotype than that of wild-type RV. Furthermore, all mutations within the PPEY motif resulted in reduced spread of the recombinant RVs as indicated by a reduction in focus size. Importantly, recombinant PPEY L-domain mutants were highly attenuated in mice yet still elicited potent antibody responses against RV G protein that were as high as those observed after infection with wild-type virus. Our data indicate that the RV PPEY motif has L-domain activity essential for efficient virus production and pathogenicity but is not essential for immunogenicity and thus can be targeted to increase the safety of rabies vaccine vectors.

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