scholarly journals Serine 105 and 120 are important phosphorylation sites for porcine reproductive and respiratory syndrome virus N protein function

2018 ◽  
Vol 219 ◽  
pp. 128-135 ◽  
Author(s):  
Yao Chen ◽  
Xiulin Xing ◽  
Qi Li ◽  
Songlin Feng ◽  
Xiaoliang Han ◽  
...  
Keyword(s):  
Protein Function ◽  
Respiratory Syndrome Virus ◽  
Phosphorylation Sites ◽  
N Protein

MicroRNA-376b-3p Promotes Porcine Reproductive and Respiratory Syndrome Virus Replication by Targeting Viral Restriction Factor TRIM22

2021 ◽  
Author(s):  
Jing Chen ◽  
Shijie Zhao ◽  
Zhiying Cui ◽  
Wen Li ◽  
Pengli Xu ◽  
...  
Keyword(s):  
Viral Infections ◽  
Antiviral Effect ◽  
Economic Losses ◽  
Respiratory Syndrome Virus ◽  
Restriction Factor ◽  
Transcriptional Level ◽  
Swine Industry ◽  
Human Virus ◽  
N Protein ◽  
Novel Strategy

Porcine reproductive and respiratory syndrome virus is a major economically significant pathogen and has evolved several strategies to evade host's antiviral response and provide favorable conditions for survival. In the present study, we demonstrated that a host microRNA, miR-376b-3p, was upregulated by PRRSV infection through the viral components, nsp4 and nsp11, and miR-376b-3p can directly target tripartite motif-containing 22 (TRIM22) to impair its anti-PRRSV activity, thus facilitating the replication of PRRSV. Meanwhile, we found that TRIM22 induced degradation of the nucleocapsid protein (N) of PRRSV by interacting with N protein to inhibit PRRSV replication, and further study indicated that TRIM22 could enhance the activation of lysosomal pathway by interacting with LC3 to induce lysosomal degradation of N protein. In conclusion, PRRSV increased miR-376b-3p expression and hijacked the host miR-376b-3p to promote PRRSV replication by impairing the antiviral effect of TRIM22. Therefore, our finding outlines a novel strategy of immune evasion exerted by PRRSV, which is helpful for better understanding the pathogenesis of PRRSV. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) causes enormous economic losses each year in the swine industry worldwide. MicroRNAs (miRNAs) play important roles during viral infections via modulating the expression of viral or host genes at post-transcriptional level. TRIM22 has recently been identified as a key restriction factor that inhibited the replication of a number of human virus such as HIV, ECMV, HCV, HBV, IAV, and RSV. Here we showed that host miR-376b-3p could be up-regulated by PRRSV and functioned to impair the anti-PRRSV role of TRIM22 to facilitate PRRSV replication. Meanwhile, we found that TRIM22 inhibited the replication of PRRSV by interacting with viral N protein and accelerating its degradation through the lysosomal pathway. Collectively, the paper described a novel mechanism that PRRSV exploited the host miR-376b-3p to evade antiviral responses and provided a new insight into the study of virus-host interactions.

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

2016 ◽  
Vol 90 (11) ◽  
pp. 5384-5398 ◽  
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.

scholarly journals Respiratory Syncytial Virus M2-1 Protein Requires Phosphorylation for Efficient Function and Binds Viral RNA during Infection

2001 ◽  
Vol 75 (24) ◽  
pp. 12188-12197 ◽  
Author(s):  
Tara L. Cartee ◽  
Gail W. Wertz
Keyword(s):  
Protein Function ◽  
Consensus Sequence ◽  
Casein Kinase ◽  
Rnase A ◽  
Site Directed Mutagenesis ◽  
Binding Motif ◽  
Casein Kinase I ◽  
N Protein ◽  
Syncytial Virus

ABSTRACT The M2-1 protein of respiratory syncytial (RS) virus is a transcriptional processivity and antitermination factor. The M2-1 protein has a Cys3His1 zinc binding motif which is essential for function, is phosphorylated, and has been shown to interact with the RS virus nucleocapsid (N) protein. In the work reported here, we determined the sites at which the M2-1 protein was phosphorylated and investigated the importance of these phosphorylated residues for M2-1 function in transcription. By combining protease digestion, matrix-assisted laser desorption ionization–time of flight mass spectrometry, and site-directed mutagenesis, we identified the phosphorylated residues as serines 58 and 61, not threonine 56 and serine 58 as previously reported. Serines 58 and 61 and the surrounding amino acids are in a consensus sequence for phosphorylation by casein kinase I. Consistent with this, we showed that the unphosphorylated M2-1 protein synthesized in Escherichia coli could be phosphorylated in vitro by casein kinase I. The effect of eliminating phosphorylation by site-specific mutagenesis of serines 58 and 61 on the function of the M2-1 protein in transcription of RS virus subgenomic replicons was assayed. The activities of the M2-1 protein phosphorylation mutants in transcriptional antitermination were tested over a range of concentrations and were found to be substantially inhibited at all concentrations. The data show that phosphorylation is important for the M2-1 protein function in transcription. However, mutation of the M2-1 phosphorylation sites did not interfere with the ability of the M2-1 protein to interact with the N protein in transfected cells. The interaction of the M2-1 and N proteins in cotransfected cells was found to be sensitive to RNase A, indicating that the M2-1–N protein interaction was mediated via RNA. Furthermore, the M2-1 protein was shown to bind monocistronic and polycistronic RS virus mRNAs during infection.

scholarly journals The Major Phosphorylation Sites of the Respiratory Syncytial Virus Phosphoprotein Are Dispensable for Virus Replication In Vitro

2002 ◽  
Vol 76 (21) ◽  
pp. 10776-10784 ◽  
Author(s):  
Bin Lu ◽  
Chien-Hui Ma ◽  
Robert Brazas ◽  
Hong Jin
Keyword(s):  
Respiratory Syncytial Virus ◽  
Virus Replication ◽  
Vero Cells ◽  
Phosphorylation Sites ◽  
N Protein ◽  
P Protein ◽  
Infected Cells ◽  
Syncytial Virus

ABSTRACT The phosphoprotein (P protein) of respiratory syncytial virus (RSV) is a key component of the viral RNA-dependent RNA polymerase complex. The protein is constitutively phosphorylated at the two clusters of serine residues (116, 117, and 119 [116/117/119] and 232 and 237 [232/237]). To examine the role of phosphorylation of the RSV P protein in virus replication, these five serine residues were altered to eliminate their phosphorylation potential, and the mutant proteins were analyzed for their functions with a minigenome assay. The reporter gene expression was reduced by 20% when all five phosphorylation sites were eliminated. Mutants with knockout mutations at two phosphorylation sites (S232A/S237A [PP2]) and at five phosphorylation sites (S116L/S117R/S119L/S232A/S237A [PP5]) were introduced into the infectious RSV A2 strain. Immunoprecipitation of 33Pi-labeled infected cells showed that P protein phosphorylation was reduced by 80% for rA2-PP2 and 95% for rA2-PP5. The interaction between the nucleocapsid (N) protein and P protein was reduced in rA2-PP2- and rA2-PP5-infected cells by 30 and 60%, respectively. Although the two recombinant viruses replicated well in Vero cells, rA2-PP2 and, to a greater extent, rA2-PP5, replicated poorly in HEp-2 cells. Virus budding from the infected HEp-2 cells was affected by dephosphorylation of P protein, because the majority of rA2-PP5 remained cell associated. In addition, rA2-PP5 was also more attenuated than rA2-PP2 in replication in the respiratory tracts of mice and cotton rats. Thus, our data suggest that although the major phosphorylation sites of RSV P protein are dispensable for virus replication in vitro, phosphorylation of P protein is required for efficient virus replication in vitro and in vivo.

scholarly journals Evolution and Engineering of Allosteric Regulation in Protein Kinases

2017 ◽  
Author(s):  
David Pincus ◽  
Jai P. Pandey ◽  
Pau Creixell ◽  
Orna Resnekov ◽  
Kimberly A. Reynolds
Keyword(s):  
Cell Signaling ◽  
Active Site ◽  
Protein Function ◽  
Kinase Activity ◽  
Allosteric Regulation ◽  
Cellular Responses ◽  
Phosphorylation Sites ◽  
Spatially Distributed ◽  
Regulatory Dynamics

ABSTRACTAllosteric regulation – the control of protein function by sites far from the active site – is a common feature of proteins that enables dynamic cellular responses. Reversible modifications such as phosphorylation are well suited to mediate such regulatory dynamics, yet the evolution of new allosteric regulation demands explanation. To understand this, we mutationally scanned the surface of a prototypical kinase to identify readily evolvable phosphorylation sites. The data reveal a set of spatially distributed “hotspots” that coevolve with the active site and preferentially modulate kinase activity. By engineering simple consensus phosphorylation sites at these hotspots we successfully rewired in vivo cell signaling. Beyond synthetic biology, the hotspots are frequently used by the diversity of natural allosteric regulatory mechanisms in the kinase family and exploited in human disease.ONE SENTENCE SUMMARYCell signaling is easily rewired by introducing new phosphoregulation at latent allosteric surface sites.

scholarly journals Phosphorylation modulates liquid-liquid phase separation of the SARS-CoV-2 N protein

2020 ◽  
Author(s):  
Christopher R. Carlson ◽  
Jonathan B. Asfaha ◽  
Chloe M. Ghent ◽  
Conor J. Howard ◽  
Nairi Hartooni ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Protein Function ◽  
Viral Genome ◽  
Rna Binding ◽  
Viral Gene ◽  
Protein Protein Interactions ◽  
N Protein ◽  
Binding Domains ◽  
Genome Transcription ◽  
Liquid Liquid Phase Separation

The nucleocapsid (N) protein of coronaviruses serves two major functions: compaction of the RNA genome in the virion and regulation of viral gene transcription in the infected cell1–3. The N protein contains two globular RNA-binding domains surrounded by regions of intrinsic disorder4. Phosphorylation of the central disordered region is required for normal viral genome transcription5,6, which occurs in a cytoplasmic structure called the replication transcription complex (RTC)7–11. It is not known how phosphorylation controls N protein function. Here we show that the N protein of SARS-CoV-2, together with viral RNA, forms biomolecular condensates12–15. Unmodified N protein forms partially ordered gel-like structures that depend on multivalent RNA-protein and protein-protein interactions. Phosphorylation reduces a subset of these interactions, generating a more liquid-like droplet. We speculate that distinct oligomeric states support the two functions of the N protein: unmodified protein forms a structured oligomer that is suited for nucleocapsid assembly, and phosphorylated protein forms a liquid-like compartment for viral genome processing. Inhibitors of N protein phosphorylation could therefore serve as antiviral therapy.

scholarly journals Arginine Methylation Regulates SARS-CoV-2 Nucleocapsid Protein Function and Viral Replication

2021 ◽  
Author(s):  
Ting Cai ◽  
Zhenbao Yu ◽  
Zhen Wang ◽  
Chen Liang ◽  
Stephane Richard
Keyword(s):  
Protein Function ◽  
Rna Binding ◽  
Binding Capacity ◽  
Viral Proteins ◽  
Arginine Methylation ◽  
Host Protein ◽  
Hek293 Cells ◽  
Type I ◽  
Dependent Manner ◽  
N Protein

Viral proteins are known to be methylated by host protein arginine methyltransferases (PRMTs) playing critical roles during viral infections. Herein, we show that PRMT1 methylates SARS-CoV-2 nucleocapsid (N) protein at residues R95 and R177 within RGG/RG sequences. Arginine methylation of N protein was confirmed by immunoblotting viral proteins extracted from SARS-CoV-2 virions isolated by cell culture. We demonstrate that arginine methylation of N protein is required for its RNA binding capacity, since treatment with a type I PRMT inhibitor (MS023) or substitution of R95K or R177K inhibited interaction with the 5'-UTR of the SARS-CoV-2 genomic RNA. We defined the N interactome in HEK293 cells with or without MS023 treatment and identified PRMT1 and many of its RGG/RG substrates including the known interactor, G3BP1, and other components of stress granules (SG). Methylation of N protein at R95 regulates another function namely its property to suppress the formation of SGs. MS023 treatment or R95K substitution blocked N-mediated suppression of SGs. Also, the co-expression of methylarginine reader TDRD3 quenched N-mediated suppression of SGs in a dose-dependent manner. Finally, pre-treatment of VeroE6 cells with MS023 significantly reduced SARS-CoV-2 replication. With type I PRMT inhibitors being in clinical trials for cancer treatment, inhibiting arginine methylation to target the later stages of the viral life cycle such as viral genome packaging and assembly of virions may be an additional therapeutic application of these drugs.

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