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

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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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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Structural insight into Marburg virus nucleoprotein-RNA complex formation

10.1101/2021.07.13.452116 ◽

2021 ◽

Author(s):

Yoko Fujita-Fujiharu ◽

Yukihiko Sugita ◽

Yuki Takamatsu ◽

Kazuya Houri ◽

Manabu Igarashi ◽

...

Keyword(s):

Rna Synthesis ◽

Ebola Virus ◽

Mutational Analysis ◽

Marburg Virus ◽

Viral Rna ◽

Structural Basis ◽

Close Relative ◽

Rna Complex ◽

Key Residues ◽

Viral Genomic Rna

The nucleoprotein (NP) of Marburg virus (MARV), a close relative of Ebola virus (EBOV), encapsidates the single-stranded, negative-sense viral genomic RNA (vRNA) to form the helical NP-RNA complex. The NP-RNA complex serves as a scaffold for the assembly of the nucleocapsid that is responsible for viral RNA synthesis. Although appropriate interactions among NPs and RNA are required for the formation of nucleocapsid, the structural basis of the helical assembly remains largely elusive. Here, we show the structure of the MARV NP-RNA complex determined using cryo-electron microscopy at a resolution of 3.1 angstrom. The structures of the asymmetric unit, a complex of an NP and six RNA nucleotides, was very similar to that of EBOV, suggesting that both viruses share common mechanisms for the nucleocapsid formation. Structure-based mutational analysis of both MARV and EBOV NPs identified key residues for the viral RNA synthesis as well as the helical assembly. Importantly, most of the residues identified were conserved in both viruses. These findings provide a structural basis for understanding the nucleocapsid formation and contribute to the development of novel antivirals against MARV and EBOV.

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Akt Plays a Critical Role in Replication of Nonsegmented Negative-Stranded RNA Viruses

Journal of Virology ◽

10.1128/jvi.01520-07 ◽

2007 ◽

Vol 82 (1) ◽

pp. 105-114 ◽

Cited By ~ 58

Author(s):

Minghao Sun ◽

Sandra M. Fuentes ◽

Khalid Timani ◽

Dengyun Sun ◽

Chris Murphy ◽

...

Keyword(s):

Rna Synthesis ◽

Rna Viruses ◽

Critical Role ◽

Simian Virus ◽

Viral Rna ◽

Serine Threonine Kinase ◽

V Protein ◽

C Terminus ◽

P Proteins ◽

Interfering Rna

ABSTRACT The order Mononegavirales (comprised of nonsegmented negative-stranded RNA viruses or NNSVs) contains many important pathogens. Parainfluenza virus 5 (PIV5), formerly known as simian virus 5, is a prototypical paramyxovirus and encodes a V protein, which has a cysteine-rich C terminus that is conserved among all paramyxoviruses. The V protein of PIV5, like that of many other paramyxoviruses, plays an important role in regulating viral RNA synthesis. In this work, we show that V interacts with Akt, a serine/threonine kinase, also known as protein kinase B. Both pharmacological inhibitors and small interfering RNA against Akt1 reduced PIV5 replication, indicating that Akt plays a critical role in PIV5 replication. Furthermore, treatment with Akt inhibitors also reduced the replication of several other paramyxoviruses, as well as vesicular stomatitis virus, the prototypical rhabdovirus, indicating that Akt may play a more universal role in NNSV replication. The phosphoproteins (P proteins) of NNSVs are essential cofactors for the viral RNA polymerase complex and require heavy phosphorylation for their activity. Inhibition of Akt activity reduced the level of P phosphorylation, suggesting that Akt is involved in regulating viral RNA synthesis. In addition, Akt1 phosphorylated a recombinant P protein of PIV5 purified from bacteria. The finding that Akt plays a critical role in replication of NNSV will lead to a better understanding of how these viruses replicate, as well as novel strategies to treat infectious diseases caused by NNSVs.

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Ulk1 plays a critical role in the autophagic clearance of mitochondria and ribosomes during reticulocyte maturation

Blood ◽

10.1182/blood-2008-02-137398 ◽

2008 ◽

Vol 112 (4) ◽

pp. 1493-1502 ◽

Cited By ~ 351

Author(s):

Mondira Kundu ◽

Tullia Lindsten ◽

Chia-Ying Yang ◽

Junmin Wu ◽

Fangping Zhao ◽

...

Keyword(s):

Red Blood Cells ◽

Blood Cells ◽

Critical Role ◽

Serine Threonine Kinase ◽

Newborn Mice ◽

Atp Production ◽

Threonine Kinase ◽

Selective Elimination ◽

Reticulocyte Maturation ◽

Erythroid Maturation

Abstract Production of a red blood cell's hemoglobin depends on mitochondrial heme synthesis. However, mature red blood cells are devoid of mitochondria and rely on glycolysis for ATP production. The molecular basis for the selective elimination of mitochondria from mature red blood cells remains controversial. Recent evidence suggests that clearance of both mitochondria and ribosomes, which occurs in reticulocytes following nuclear extrusion, depends on autophagy. Here, we demonstrate that Ulk1, a serine threonine kinase with homology to yeast atg1p, is a critical regulator of mitochondrial and ribosomal clearance during the final stages of erythroid maturation. However, in contrast to the core autophagy genes such as atg5 and atg7, expression of ulk1 is not essential for induction of macroautophagy in response to nutrient deprivation or for survival of newborn mice. Together, these data suggest that the ATG1 homologue, Ulk1, is a component of the selective autophagy machinery that leads to the elimination of organelles in erythroid cells rather that an essential mechanistic component of autophagy.

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An RNA Pseudoknot in the 3′ End of the Arterivirus Genome Has a Critical Role in Regulating Viral RNA Synthesis

Journal of Virology ◽

10.1128/jvi.00747-07 ◽

2007 ◽

Vol 81 (17) ◽

pp. 9426-9436 ◽

Cited By ~ 15

Author(s):

Nancy Beerens ◽

Eric J. Snijder

Keyword(s):

Rna Synthesis ◽

Critical Role ◽

Equine Arteritis Virus ◽

Viral Rna ◽

Replicase Gene ◽

Loop Structure ◽

Minus Strand ◽

Stem Loop ◽

Loop Region ◽

Stem Loop Structure

ABSTRACT In the life cycle of plus-strand RNA viruses, the genome initially serves as the template for both translation of the viral replicase gene and synthesis of minus-strand RNA and is ultimately packaged into progeny virions. These various processes must be properly balanced to ensure efficient viral proliferation. To achieve this, higher-order RNA structures near the termini of a variety of RNA virus genomes are thought to play a key role in regulating the specificity and efficiency of viral RNA synthesis. In this study, we have analyzed the signals for minus-strand RNA synthesis in the prototype of the arterivirus family, equine arteritis virus (EAV). Using site-directed mutagenesis and an EAV reverse genetics system, we have demonstrated that a stem-loop structure near the 3′ terminus of the EAV genome is required for RNA synthesis. We have also obtained evidence for an essential pseudoknot interaction between the loop region of this stem-loop structure and an upstream hairpin residing in the gene encoding the nucleocapsid protein. We propose that the formation of this pseudoknot interaction may constitute a molecular switch that could regulate the specificity or timing of viral RNA synthesis. This hypothesis is supported by the fact that phylogenetic analysis predicted the formation of similar pseudoknot interactions near the 3′ end of all known arterivirus genomes, suggesting that this interaction has been conserved in evolution.

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Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein Interacts with Nsp9 and Cellular DHX9 To Regulate Viral RNA Synthesis

Journal of Virology ◽

10.1128/jvi.03216-15 ◽

2016 ◽

Vol 90 (11) ◽

pp. 5384-5398 ◽

Cited By ~ 24

Author(s):

Long Liu ◽

Jiao Tian ◽

Hao Nan ◽

Mengmeng Tian ◽

Yuan Li ◽

...

Keyword(s):

Rna Synthesis ◽

Nonstructural Protein ◽

Critical Role ◽

Continuous Extension ◽

Rna Helicase ◽

Viral Rna ◽

Respiratory Syndrome Virus ◽

Nascent Transcript ◽

Multifunctional Protein ◽

N Protein

ABSTRACTPorcine reproductive and respiratory syndrome virus (PRRSV) nucleocapsid (N) protein is the main component of the viral capsid to encapsulate viral RNA, and it is also a multifunctional protein involved in the regulation of host cell processes. Nonstructural protein 9 (Nsp9) is the RNA-dependent RNA polymerase that plays a critical role in viral RNA transcription and replication. In this study, we demonstrate that PRRSV N protein is bound to Nsp9 by protein-protein interaction and that the contacting surface on Nsp9 is located in the two predicted α-helixes formed by 48 residues at the C-terminal end of the protein. Mutagenesis analyses identified E646, E608, and E611 on Nsp9 and Q85 on the N protein as the pivotal residues participating in the N-Nsp9 interaction. By overexpressing the N protein binding fragment of Nsp9 in infected Marc-145 cells, the synthesis of viral RNAs, as well as the production of infectious progeny viruses, was dramatically inhibited, suggesting that Nsp9-N protein association is involved in the process of viral RNA production. In addition, we show that PRRSV N interacts with cellular RNA helicase DHX9 and redistributes the protein into the cytoplasm. Knockdown of DHX9 increased the ratio of short subgenomic mRNAs (sgmRNAs); in contrast, DHX9 overexpression benefited the synthesis of longer sgmRNAs and the viral genomic RNA (gRNA). These results imply that DHX9 is recruited by the N protein in PRRSV infection to regulate viral RNA synthesis. We postulate that N and DHX9 may act as antiattenuation factors for the continuous elongation of nascent transcript during negative-strand RNA synthesis.IMPORTANCEIt is unclear whether the N protein of PRRSV is involved in regulation of the viral RNA production process. In this report, we demonstrate that the N protein of the arterivirus PRRSV participates in viral RNA replication and transcription through interacting with Nsp9 and its RdRp and recruiting cellular RNA helicase to promote the production of longer viral sgmRNAs and gRNA. Our data here provide some new insights into the discontinuous to continuous extension of PRRSV RNA synthesis and also offer a new potential anti-PRRSV strategy targeting the N-Nsp9 and/or N-DHX9 interaction.

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Modulation of Cell Wall Structure and Antimicrobial Susceptibility by a Staphylococcus aureus Eukaryote-Like Serine/Threonine Kinase and Phosphatase

Infection and Immunity ◽

10.1128/iai.01499-08 ◽

2009 ◽

Vol 77 (4) ◽

pp. 1406-1416 ◽

Cited By ~ 89

Author(s):

Amanda M. Beltramini ◽

Chitrangada D. Mukhopadhyay ◽

Vijay Pancholi

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Gene Knockout ◽

Critical Role ◽

Cell Wall Structure ◽

Wall Structure ◽

Cellular Functions ◽

Serine Threonine Kinase ◽

Threonine Kinase ◽

Irregular Cell

ABSTRACT It is well established that prokaryotes and eukaryotes alike utilize phosphotransfer to regulate cellular functions. One method by which this occurs is via eukaryote-like serine/threonine kinase (ESTK)- and phosphatase (ESTP)-regulated pathways. The role of these enzymes in Staphylococcus aureus has not yet been examined. This resilient organism is a common cause of hospital-acquired and community-associated infections, infecting immunocompromised and immunocompetent hosts alike. In this study, we have characterized a major functional ESTK (STK) and ESTP (STP) in S. aureus and found them to be critical modulators of cell wall structure and susceptibility to cell wall-acting β-lactam antibiotics. By utilizing gene knockout strategies, we created S. aureus N315 mutants lacking STP and/or STK. The strain lacking both STP and STK displayed notable cell division defects, including multiple and incomplete septa, bulging, and irregular cell size, as observed by transmission electron microscopy. Mutants lacking STP alone displayed thickened cell walls and increased resistance to the peptidoglycan-targeting glycylglycine endopeptidase lysostaphin, compared to the wild type. Additionally, mutant strains lacking STK or both STK and STP displayed increased sensitivity to cell wall-acting cephalosporin and carbapenem antibiotics. Together, these results indicate that S. aureus STK- and STP-mediated reversible phosphorylation reactions play a critical role in proper cell wall architecture, and thus the modulation of antimicrobial resistance, in S. aureus.

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Oligomerization of Mumps Virus Phosphoprotein

Journal of Virology ◽

10.1128/jvi.01719-15 ◽

2015 ◽

Vol 89 (21) ◽

pp. 11002-11010 ◽

Cited By ~ 6

Author(s):

Adrian Pickar ◽

Andrew Elson ◽

Yang Yang ◽

Pei Xu ◽

Ming Luo ◽

...

Keyword(s):

Rna Synthesis ◽

Mutational Analysis ◽

Mumps Virus ◽

Viral Rna ◽

Large Protein ◽

Terminal Domain ◽

P Protein ◽

Minigenome System ◽

Oligomerization Domain

ABSTRACTThe mumps virus (MuV) genome encodes a phosphoprotein (P) that is important for viral RNA synthesis. P forms the viral RNA-dependent RNA polymerase with the large protein (L). P also interacts with the viral nucleoprotein (NP) and self-associates to form a homotetramer. The P protein consists of three domains, the N-terminal domain (PN), the oligomerization domain (PO), and the C-terminal domain (PC). While PNis known to relax the NP-bound RNA genome, the roles of POand PCare not clear. In this study, we investigated the roles of POand PCin viral RNA synthesis using mutational analysis and a minigenome system. We found that PNand PCfunctions can betrans-complemented. However, this complementation requires PO, indicating that POis essential for P function. Using thistrans-complementation system, we found that P forms parallel dimers (PNto PNand PCto PC). Furthermore, we found that residues R231, K238, K253, and K260 in POare critical for P's functions. We identified PCto be the domain that interacts with L. These results provide structure-function insights into the role of MuV P.IMPORTANCEMuV, a paramyxovirus, is an important human pathogen. The P protein of MuV is critical for viral RNA synthesis. In this work, we established a novel minigenome system that allows the domains of P to be complemented intrans. Using this system, we confirmed that MuV P forms parallel dimers. An understanding of viral RNA synthesis will allow the design of better vaccines and the development of antivirals.

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Staufen1 Interacts with Multiple Components of the Ebola Virus Ribonucleoprotein and Enhances Viral RNA Synthesis

mBio ◽

10.1128/mbio.01771-18 ◽

2018 ◽

Vol 9 (5) ◽

Cited By ~ 8

Author(s):

Jingru Fang ◽

Colette Pietzsch ◽

Palaniappan Ramanathan ◽

Rodrigo I. Santos ◽

Philipp A. Ilinykh ◽

...

Keyword(s):

Rna Synthesis ◽

Binding Proteins ◽

Ebola Virus ◽

Rna Binding ◽

Rna Binding Proteins ◽

Critical Role ◽

Host Factors ◽

Viral Rna ◽

Virion Protein ◽

Public Health Importance

ABSTRACTEbola virus (EBOV) genome and mRNAs contain long, structured regions that could hijack host RNA-binding proteins to facilitate infection. We performed RNA affinity chromatography coupled with mass spectrometry to identify host proteins that bind to EBOV RNAs and identified four high-confidence proviral host factors, including Staufen1 (STAU1), which specifically binds both 3′ and 5′ extracistronic regions of the EBOV genome. We confirmed that EBOV infection rate and production of infectious particles were significantly reduced in STAU1-depleted cells. STAU1 was recruited to sites of EBOV RNA synthesis upon infection and enhanced viral RNA synthesis. Furthermore, STAU1 interacts with EBOV nucleoprotein (NP), virion protein 30 (VP30), and VP35; the latter two bridge the viral polymerase to the NP-coated genome, forming the viral ribonucleoprotein (RNP) complex. Our data indicate that STAU1 plays a critical role in EBOV replication by coordinating interactions between the viral genome and RNA synthesis machinery.IMPORTANCEEbola virus (EBOV) is a negative-strand RNA virus with significant public health importance. Currently, no therapeutics are available for Ebola, which imposes an urgent need for a better understanding of EBOV biology. Here we dissected the virus-host interplay between EBOV and host RNA-binding proteins. We identified novel EBOV host factors, including Staufen1, which interacts with multiple viral factors and is required for efficient viral RNA synthesis.

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