scholarly journals Phosphorylation of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein

2002 ◽  
Vol 76 (20) ◽  
pp. 10569-10576 ◽  
Author(s):  
Sarah K. Wootton ◽  
Raymond R. R. Rowland ◽  
Dongwan Yoo
Keyword(s):  
Nucleocapsid Protein ◽  
Time Course ◽  
Rna Virus ◽  
Respiratory Syndrome Virus ◽  
N Protein ◽  
Two Dimensional Electrophoresis ◽  
Cytoplasmic Rna ◽  
Infected Cells ◽  
Phosphoamino Acid Analysis ◽  
Dimensional Electrophoresis

ABSTRACT Porcine reproductive and respiratory syndrome virus (PRRSV) is a cytoplasmic RNA virus with the unique or unusual feature of having a nucleocapsid (N) protein that is specifically transported to the nucleolus of virus-infected cells. In this communication, we show that the N protein is a phosphoprotein. Phosphoamino acid analysis of authentic and recombinant N proteins demonstrated that serine residues were exclusively phosphorylated. The pattern of phosphorylated N protein cellular distribution in comparison with that of [35S]methionine-labeled N protein suggested that phosphorylation does not influence subcellular localization of the protein. Time course studies showed that phosphorylation occurred during, or shortly after, synthesis of the N protein and that the protein remained stably phosphorylated throughout the life cycle of the virus to the extent that phosphorylated N protein was found in the mature virion. Two-dimensional electrophoresis and acid-urea gel electrophoresis showed that one species of the N protein is predominant in virus-infected cells, suggesting that multiple phosphorylated isoforms of N do not exist.

scholarly journals Antigenic Importance of the Carboxy-Terminal Beta-Strand of the Porcine Reproductive and Respiratory Syndrome Virus Nucleocapsid Protein

2001 ◽  
Vol 8 (3) ◽  
pp. 598-603 ◽  
Author(s):  
Sarah Wootton ◽  
Gabriella Koljesar ◽  
Liuzhan Yang ◽  
Kyoung-Jin Yoon ◽  
Dongwan Yoo
Keyword(s):  
Amino Acids ◽  
Secondary Structure ◽  
Nucleocapsid Protein ◽  
Conformational Epitope ◽  
Single Amino Acid ◽  
Respiratory Syndrome Virus ◽  
Beta Strand ◽  
N Protein ◽  
Terminal Domain ◽  
C Terminus

ABSTRACT Five domains of antigenic importance were previously mapped on the nucleocapsid protein (N) of the porcine reproductive and respiratory syndrome virus (PRRSV), and a domain comprised of the 11 C-terminal-most amino acids (residues 112 to 123) was shown to be essential for binding of N-specific conformation-dependent monoclonal antibodies (MAbs). In the present study, the importance of individual residues within this C-terminal domain for antigenicity was investigated using eight different mutant constructs of N expressed in HeLa cells. Single amino acid substitutions were introduced into the C-terminal domain of the N protein, and the significance of individual amino acids for MAb reactivity was determined by immunoprecipitation. None of the MAbs tested recognized the mutant with a leucine-to-proline substitution at residue 114 (L114P), while V112P, R113P, R113D, I115P, and R116P reduced MAb binding significantly. Conversely, substitution of amino acids at positions 118 (T118S) and 121 (P121A) had little effect on MAb binding. Secondary-structure predictions indicate that amino acids 111 to 117 form a beta-strand. In view of the fact that replacement of beta-strand-forming amino acids with proline elicited the greatest effect on MAb binding, it appears that secondary structure in the C terminus of the N protein is an important determinant of conformational epitope formation. While the crystal structure of the PRRSV N protein remains to be determined, results from these studies broaden our understanding of the secondary structures that make up the PRRSV N protein and shed some light on how they may relate to function.

scholarly journals The Nucleocapsid Protein and Nonstructural Protein 10 of Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Enhance CD83 Production via NF-κB and Sp1 Signaling Pathways

2017 ◽  
Vol 91 (18) ◽  
Author(s):  
Xi Chen ◽  
Qiaoya Zhang ◽  
Juan Bai ◽  
Yongxiang Zhao ◽  
Xianwei Wang ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Nucleocapsid Protein ◽  
Promoter Activity ◽  
Nonstructural Protein ◽  
Immunoglobulin Superfamily ◽  
T Cell Proliferation ◽  
Respiratory Syndrome Virus ◽  
Swine Industry ◽  
N Protein ◽  
Soluble Cd83

ABSTRACT Porcine reproductive and respiratory syndrome, caused by porcine reproductive and respiratory syndrome virus (PRRSV), is a panzootic disease that is one of the most economically costly diseases to the swine industry. A key aspect of PRRSV virulence is that the virus suppresses the innate immune response and induces persistent infection, although the underlying mechanisms are not well understood. The dendritic cell (DC) marker CD83 belongs to the immunoglobulin superfamily and is associated with DC activation and immunosuppression of T cell proliferation when expressed as soluble CD83 (sCD83). In this study, we show that PRRSV infection strongly stimulates CD83 expression in porcine monocyte-derived DCs (MoDCs) and that the nucleocapsid (N) protein and nonstructural protein 10 (nsp10) of PRRSV enhance CD83 promoter activity via the NF-κB and Sp1 signaling pathways. R43A and K44A amino acid substitution mutants of the N protein suppress the N protein-mediated increase of CD83 promoter activity. Similarly, P192-5A and G214-3A mutants of nsp10 (with 5 and 3 alanine substitutions beginning at residues P192 and G214, respectively) abolish the nsp10-mediated induction of the CD83 promoter. Using reverse genetics, four mutant viruses (rR43A, rK44A, rP192-5A, and rG214-3A) and four revertants [rR43A(R), rK44A(R), rP192-5A(R), and rG214-3A(R)] were generated. Decreased induction of CD83 in MoDCs was observed after infection by mutants rR43A, rK44A, rP192-5A, and rG214-3A, in contrast to the results obtained using rR43A(R), rK44A(R), rP192-5A(R), and rG214-3A(R). These findings suggest that PRRSV N and nsp10 play important roles in modulating CD83 signaling and shed light on the mechanism by which PRRSV modulates host immunity. IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically costly pathogens affecting the swine industry. It is unclear how PRRSV inhibits the host's immune response and induces persistent infection. The dendritic cell (DC) marker CD83 belongs to the immunoglobulin superfamily and has previously been associated with DC activation and immunosuppression of T cell proliferation and differentiation when expressed as soluble CD83 (sCD83). In this study, we found that PRRSV infection induces sCD83 expression in porcine MoDCs via the NF-κB and Sp1 signaling pathways. The viral nucleocapsid protein, nonstructural protein 1 (nsp1), and nsp10 were shown to enhance CD83 promoter activity. Amino acids R43 and K44 of the N protein, as well as residues 192 to 196 (P192-5) and 214 to 216 (G214-3) of nsp10, play important roles in CD83 promoter activation. These findings provide new insights into the molecular mechanism of immune suppression by PRRSV.

scholarly journals Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates

2020 ◽  
Author(s):  
Adriana Savastano ◽  
Alain Ibáñez de Opakua ◽  
Marija Rankovic ◽  
Markus Zweckstetter
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Nucleocapsid Protein ◽  
Etiologic Agent ◽  
Liquid Phase Separation ◽  
Viral Rna ◽  
Cytoplasmic Rna ◽  
Infected Cells ◽  
Liquid Liquid Phase Separation

SUMMARYThe etiologic agent of the Covid-19 pandemic is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The viral membrane of SARS-CoV-2 surrounds a helical nucleocapsid in which the viral genome is encapsulated by the nucleocapsid protein. The nucleocapsid protein of SARS-CoV-2 is produced at high levels within infected cells, enhances the efficiency of viral RNA transcription and is essential for viral replication. Here we show that RNA induces cooperative liquid-liquid phase separation of the SARS-CoV-2 nucleocapsid protein. In agreement with its ability to phase separate in vitro, we show that the protein associates in cells with stress granules, cytoplasmic RNA/protein granules that form through liquid-liquid phase separation and are modulated by viruses to maximize replication efficiency. Liquid-liquid phase separation generates high-density protein/RNA condensates that recruit the RNA-dependent RNA polymerase complex of SARS-CoV-2 providing a mechanism for efficient transcription of viral RNA. Inhibition of RNA-induced phase separation of the nucleocapsid protein by small molecules or biologics thus can interfere with a key step in the SARS-CoV-2 replication cycle.

scholarly journals Palmitoylation of the envelope membrane proteins GP5 and M of porcine reproductive and respiratory syndrome virus is essential for virus growth

2021 ◽  
Vol 17 (4) ◽  
pp. e1009554
Author(s):  
Minze Zhang ◽  
Xiaoliang Han ◽  
Klaus Osterrieder ◽  
Michael Veit
Keyword(s):  
Membrane Proteins ◽  
Virus Assembly ◽  
Rna Virus ◽  
Respiratory Syndrome Virus ◽  
Virus Growth ◽  
Close Proximity ◽  
Infected Cells ◽  
Positive Strand Rna Virus ◽  
Positive Strand Rna ◽  
Exocytic Pathway

Porcine reproductive and respiratory syndrome virus (PRRSV), an enveloped positive-strand RNA virus in the Arteiviridae family, is a major pathogen affecting pigs worldwide. The membrane (glyco)proteins GP5 and M form a disulfide-linked dimer, which is a major component of virions. GP5/M are required for virus budding, which occurs at membranes of the exocytic pathway. Both GP5 and M feature a short ectodomain, three transmembrane regions, and a long cytoplasmic tail, which contains three and two conserved cysteines, respectively, in close proximity to the transmembrane span. We report here that GP5 and M of PRRSV-1 and -2 strains are palmitoylated at the cysteines, regardless of whether the proteins are expressed individually or in PRRSV-infected cells. To completely prevent S-acylation, all cysteines in GP5 and M have to be exchanged. If individual cysteines in GP5 or M were substituted, palmitoylation was reduced, and some cysteines proved more important for efficient palmitoylation than others. Neither infectious virus nor genome-containing particles could be rescued if all three cysteines present in GP5 or both present in M were replaced in a PRRSV-2 strain, indicating that acylation is essential for virus growth. Viruses lacking one or two acylation sites in M or GP5 could be rescued but grew to significantly lower titers. GP5 and M lacking acylation sites form dimers and GP5 acquires Endo-H resistant carbohydrates in the Golgi apparatus suggesting that trafficking of the membrane proteins to budding sites is not disturbed. Likewise, GP5 lacking two acylation sites is efficiently incorporated into virus particles and these viruses exhibit no reduction in cell entry. We speculate that multiple fatty acids attached to GP5 and M in the endoplasmic reticulum are required for clustering of GP5/M dimers at Golgi membranes and constitute an essential prerequisite for virus assembly.

scholarly journals Identification and phylogenetic analysis of two canine coronavirus strains

2021 ◽  
Vol 1 (1) ◽  
Author(s):  
Junji Gan ◽  
Ye Tang ◽  
Haifeng Lv ◽  
Wenbin Xiong ◽  
Xiaoyan Tian
Keyword(s):  
Phylogenetic Analysis ◽  
Culture Media ◽  
Rna Virus ◽  
Pcr Analysis ◽  
Cell Culture Media ◽  
N Protein ◽  
Cytopathic Effects ◽  
Canine Coronavirus ◽  
Infected Cells ◽  
Reference Strains

AbstractCanine coronavirus (CCoV), a member of the genus Alphacoronavirus, is an enveloped, single-stranded positive-sense RNA virus that responsible for gastroenteritis in dogs. In this study, two CCoV isolates were successfully propagated from 53 CCoV-positive clinical specimens by serial passaging in A-72 cells. These two strains, CCoV JS1706 and CCoV JS1712, caused cytopathic effects in A-72 cells. The sizes of virus plaque formed by them differed in early passages. Electron microscopy revealed a large quantity of typical coronavirus particles with 80–120 nm in diameter in cell culture media and cytoplasm of infected cells, in which they appeared as inclusion bodies. RT-PCR analysis of S gene indicated that these two isolates were belonged to CCoV IIa subtype. Homology of RdRp, S, M and N proteins between the two strains were 100, 99.6, 99.2 and 100.0%, respectively, whereas they were 99.4–100%, 83.1–95.2%, 88.5–99.2% and 91.9–99.7% identity compared to CCoV II reference strains. Phylogenetic analysis of RdRp, S, M and N protein showed that they were closely related to CCoV II strains. These two subtype IIa isolates will be useful for evaluating the pathogenesis and evolution of CCoV and for developing diagnostic reagents and vaccines.

scholarly journals Lipid Droplet Motility Increases Following Viral Immune Stimulation

2021 ◽  
Vol 22 (9) ◽  
pp. 4418
Author(s):  
Ebony A. Monson ◽  
Donna R. Whelan ◽  
Karla J. Helbig
Keyword(s):  
Lipid Droplets ◽  
Time Course ◽  
Rna Virus ◽  
Poly I:C ◽  
Host Responses ◽  
Immune Stimulation ◽  
Health And Disease ◽  
Infected Cells ◽  
Virus Zika

Lipid droplets (LDs) have traditionally been thought of as solely lipid storage compartments for cells; however, in the last decade, they have emerged as critical organelles in health and disease. LDs are highly dynamic within cells, and their movement is critical in organelle–organelle interactions. Their dynamics are known to change during cellular stress or nutrient deprivation; however, their movement during pathogen infections, especially at very early timepoints, is under-researched. This study aimed to track LD dynamics in vitro, in an astrocytic model of infection. Cells were either stimulated with a dsRNA viral mimic, poly I:C, or infected with the RNA virus, Zika virus. Individual LDs within infected cells were analysed to determine displacement and speed, and average LD characteristics for multiple individual cells calculated. Both LD displacement and mean speed were significantly enhanced in stimulated cells over a time course of infection with an increase seen as early as 2 h post-infection. With the emerging role for LDs during innate host responses, understanding their dynamics is critical to elucidate how these organelles influence the outcome of viral infection.

scholarly journals Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Adriana Savastano ◽  
Alain Ibáñez de Opakua ◽  
Marija Rankovic ◽  
Markus Zweckstetter
Keyword(s):  
Phase Separation ◽  
Liquid Phase ◽  
Nucleocapsid Protein ◽  
Etiologic Agent ◽  
Liquid Phase Separation ◽  
Viral Rna ◽  
Cytoplasmic Rna ◽  
Infected Cells ◽  
Liquid Liquid Phase Separation

AbstractThe etiologic agent of the Covid-19 pandemic is the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The viral membrane of SARS-CoV-2 surrounds a helical nucleocapsid in which the viral genome is encapsulated by the nucleocapsid protein. The nucleocapsid protein of SARS-CoV-2 is produced at high levels within infected cells, enhances the efficiency of viral RNA transcription, and is essential for viral replication. Here, we show that RNA induces cooperative liquid–liquid phase separation of the SARS-CoV-2 nucleocapsid protein. In agreement with its ability to phase separate in vitro, we show that the protein associates in cells with stress granules, cytoplasmic RNA/protein granules that form through liquid-liquid phase separation and are modulated by viruses to maximize replication efficiency. Liquid–liquid phase separation generates high-density protein/RNA condensates that recruit the RNA-dependent RNA polymerase complex of SARS-CoV-2 providing a mechanism for efficient transcription of viral RNA. Inhibition of RNA-induced phase separation of the nucleocapsid protein by small molecules or biologics thus can interfere with a key step in the SARS-CoV-2 replication cycle.

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