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At least 19 recordsLinked to original sources
PubMed2026

PRMT3 restricts porcine epidemic diarrhea virus replication by disrupting the interaction between VAPA and the viral nucleocapsid protein.

Porcine epidemic diarrhea virus (PEDV) represents a severe threat to the global swine industry. Its infection process involves intricate virus-host interactions and immune evasion mechanisms, but effective therapeutic targets remain elusive. In this study, we identified protein arginine methyltransferase 3 (PRMT3) as a novel regulatory factor that significantly modulates PEDV infection via genome-wide CRISPR/Cas9 knockout library screening. Knockout or inhibition of PRMT3 markedly enhanced PEDV infection in multiple cell lines, including LLC-PK1, IPEC-J2, and primary porcine intestinal epithelial cells. Mechanistic investigations revealed that PRMT3 can restrict PEDV infection by interacting with vesicle-associated membrane protein-associated protein A (VAPA). Further analysis revealed that VAPA facilitates cholesterol transport through binding to oxysterol-binding protein (OSBP) and inhibits the autophagic degradation of the viral nucleocapsid (N) protein, with both processes being critical for promoting PEDV infection in host cells. A detailed analysis revealed that K52 within its major sperm protein (MSP) domain interacts with D404 and D405 in the two phenylalanines in an acidic tract (FFAT)-like motifs of the N protein, and these interactions proved essential for PEDV infection. In summary, this is the first study to identify and validate the PRMT3-VAPA-N protein autophagic degradation axis as a key pathway through which PRMT3 suppresses PEDV infection, with VAPA acting as an essential host factor for PEDV pathogenesis. These findings uncover novel signaling pathways and molecular targets for the development of anti-PEDV therapeutics.

Animals↗
PubMed1975

Specific cleavage of Sendai virus nucleocapsid protein subunits during virus storage.

The alteration of whole Sendai virus and especially of its nucleocapsid polypeptides, during storage of the virus at 4 degree C in the allantoic fluids in which it was cultivated, has cultivated, has been studied by sodium dodecyl sulfate gel electrophoresis. During virus storage the nucleocapsid protein subunits with a molecular weight of 60,000 and the putative inner envelope protein with a molecular weight of 38,000 were mainly affected. Both virus components were partially degraded to smaller components. Examination of nucleocapsids isolated from "stored" virus showed that, in addition to the 60,000-molecular weight polypetide component, a smaller polypeptide component with a molecular weight of 46,000 appeared. The relative proportion of the small component increased with the storage period: a kind of specific conversion of large to small components occurred during storage. Since viruses kept in the absence of allantoic fluids revealed no similar modifications of their polypeptides, we concluded that a cellular component present in the allantoic fluids - very likely of enzymatic nature - is responsible for the observed cleavage of virus polypeptides.

Body Fluids↗
PubMed1978

Small viral RNA segment of bunyaviruses codes for viral nucleocapsid protein.

Tryptic peptide analyses have been undertaken on the nucleocapsid (N) protein of snowshoe hare (SSH) and La Crosse (LAC) bunyaviruses. Similar analyses have been performed on the N proteins of two recombinant viruses which have the large/medium/small RNA genome configurations: SSH/LAC/LAC and SSH/LAC/SSH. The results provide conclusive evidence that the S RNA of bunyaviruses codes for the the viral N protein.

Arboviruses↗
PubMed2025

The R203M and D377Y mutations of the nucleocapsid protein promote SARS-CoV-2 infectivity by impairing RIG-I-mediated antiviral signaling.

The viral protein mutations can modify virus-host interactions during virus evolution, and thus alter the extent of infection or pathogenicity. Studies indicate that nucleocapsid (N) protein of SARS-CoV-2 participates in viral genome assembly, intracellular signal regulation and immune interference. However, its biological function in viral evolution is not well understood. SARS-CoV-2 N protein mutations were analyzed in Delta, Omicron, and original strains. Two mutations with a methionine (M) residue at site 203 and a tyrosine (Y) residue at site 377 of the N protein were found in Delta strain but not in Omicron and original strains, and promoted SARS-CoV-2 infection therein. Those mutations, R203M and D377Y, enhanced the inhibitory impact of N protein on the impairment of RIG-I-mediated antiviral signaling, such as IRF3 phosphorylation and IFN-β activation. The viral RNA-binding activity of N protein was promoted by these mutations, effectively attenuating the recognition and interaction of RIG-I with viral RNA compared to the original or other variants. The R203M/D377Y mutations thus enhanced the suppressive activity of the N protein on RIG-I-mediated interferon induction both in vitro and in vivo, which in turn promoted viral replication. This study helps to understand the variability of SARS-CoV-2 in regulating host immunity.

SARS-CoV-2↗
PubMed1979

Production of monoclonal antibodies against nucleocapsid proteins of herpes simplex virus types 1 and 2.

We prepared mouse hybrid cell lines which produced antibodies against herpes simplex virus type 1 and 2 nucleocapsids. Cell lines 1D4 and 3E1, respectively, secreted immunoglobulin G1 herpes simplex virus type 1 and immunoglobulin G1 herpes simplex virus type 2 antibodies which immunoprecipitated proteins designated p40 and p45 from homologous nucleocapsid preparations but precipitated no proteins from heterologous preparations. In contrast, guinea pig antisera prepared against either herpes simplex virus type 1 or 2 p40 precipitated p40 and p45 from both homologous and heterologous preparations. These findings suggest that p40 and p45 possess similar antigenic determinants and that the monoclonal antibodies that were tested reacted preferentially with the homologous determinants.

Antibodies, Viral↗
PubMed1979

Phosphoproteins of murine hepatitis viruses.

Four strains of the coronavirus murine hepatitis virus were examined for the presence of phosphorylated proteins. The nucleocapsid protein was determined to contain phosphate covalently linked to serine but not to threonine residues. The nucleocapsid protein was the only phosphorylated protein detected in these strains of murine hepatitis virus.

Capsid↗
PubMed1977

Antigenic characterization of flavivirus structural proteins separated by isoelectric focusing.

Isoelectrofocusing of nonionic-detergent-disrupted flaviviruses separated the envelope glycoprotein of 53,000 to 58,000 daltons and the nucleocapsid protein of 14,000 daltons. The envelope protein and nucleocapsid protein were isolated at isoelectric points of pI 7.8 and 10.3, respectively. The antigenic determinants of St. Louis encephalitis, Japanese encephalitis, and dengue virus envelope and nucleocapsid proteins were examined by solid-phase competition radioimmunoassay. By the appropriate selection of antiserum and competing proteins, it was possible to distinguish type-specific, complex-reactive and flavivirus group-reactive antigenic determinants. The envelope glycoproteins of St. Louis encephalitis, Japanese encephalitis, and dengue viruses were found to contain each of these three classes of antigenic determinants. Most of the determinants on the envelope protein were type specific, some were complex reactive, and a small fraction were flavivirus group reactive. The nucleocapsid protein contained only flavivirus group-reactive antigenic determinants.

Cell Line↗
PubMed1978

Determinants of measles virus (hamster neurotropic strain) replication in mouse brain.

In newborn mice the hamster neurotropic strain of measles virus produces a severe meningoencephalitis with readily recoverable virus, while in weanling mice a fatal encephalopathy is produced with scant histopathology and no viral infectivity in brain homogenates. In this study various host factors that may change with maturation and determine the restriction of viral expression were investigated, including immune response, interferon production, host temperature, and the possible role of proteases. None of these factors appeared to be responsible for host restriction of viral expression. Recovery of virus from brains of weanling mice was not significantly enhanced by cocultivation with Vero cells, complementation with temperature-sensitive mutants, or phenotypic mixing with the Edmonston strain of measles virus. These data, combined with our previous observations of production of viral proteins including nucleocapsid proteins without development of recognizable nucleocapsids in neurons of weanling mice, suggest that with maturation the neural cells fail to replicate sufficient amounts of encapsidated viral ribonucleic acid.

Aging↗
PubMed1978

Cross-linking of Newcastle disease virus (NDV) proteins.

The proxomity and spatial relationships of the structural proteins of Newcastle disease virus (NDV) were studied by chemical cross-linking with a series of imidoesters. When the virions were reacted by the cross-linker with a distance 6.1A or longer between the functional groups and analyzed by polyacrylamide gel electrophoresis, remarkable changes were observed in the migration patterns of the viral proteins. The most striking one was the extensive decrease in the intensity of the M protein band, and although not so strikingly, glycoprotein and nucleocapsid protein bands were reduced significantly. Instead, several protein complexes appeared at and near the top of the gels. The protein complexes formed by a reversible cross-linker, dimethyl-3,3'-dithiobispropionimidate (DTBP), were analyzed by two dimensional electrophoresis; the complexes on the first-dimension cylindrical gels were cleaved by reduction with 2-mercaptoethanol and electrophoresed laterally on the second-dimension slab gels. The results indicated that homodimers of glycoprotein, nucleocapsid protein and M protein were generated under the condition of the most gentle cross-linking employed. At the same time, however, trimer and higher homopolymers of M protein were already detectable. Under the more extensive conditions, the bulk of M protein was cross-linked to form a large protein complex with very high molecular weight. Further, small but significant amounts of glycoprotein and nucleocapsid protein were always detected in this complex. These results suggest that M protein may be present in the virion in close enough proximity to interact with each other and may further have some interactions with glycoprotein and nucleocapsid protein. On the basis of these findings possible roles of M protein in virus assembly were discussed.

Dimethyl Adipimidate↗
PubMed1979

Transmembrane communication in cells chronically infected with measles virus.

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.

Antigens, Surface↗
PubMed1978

Influenza virus proteins: identity, synthesis, and modification analyzed by two-dimensional gel electrophoresis.

A modification of the two-dimensional protein electrophoresis system of O'Farrell was used to resolve influenza A virus proteins from each other and from host proteins in infected cells. Viral protein spots corresponding to the hemagglutinin proteins, neuraminidase, nucleocapsid protein, and nonstructural protein, were identified on the two-dimensional electrophoretogram. Use of the two-dimensional separation has allowed us to identify glycoprotein heterogeneity, to demonstrate directly the synthesis of neuraminidase, to analyze some viral proteins early after infection, and to demonstrate that the influenza virus NP and NS proteins are phosphorylated in infected MDCK cells.

Cells, Cultured↗
PubMed2004

A bunyamwera virus minireplicon system in mosquito cells.

Artificial minigenomes are powerful tools for studying the replication and transcription of negative-strand RNA viruses. Bunyamwera virus (BUN; genus Orthobunyavirus, family Bunyaviridae) is an arbovirus that shows fundamental biological differences when replicating in mammalian versus mosquito cells. To study BUN RNA synthesis in mosquito cells, we developed a bacteriophage T7 RNA polymerase-based minireplicon system similar to that described previously for mammalian cells. An Aedes albopictus C6/36-derived mosquito cell line stably expressing T7 RNA polymerase was established. Viral proteins and artificial minigenomes (containing Renilla luciferase as a reporter) were transcribed and expressed in these cells from transfected T7 promoter-containing plasmids. Transcription of the minigenome required two viral proteins, the nucleocapsid protein N and the RNA-dependent RNA polymerase L, a situation similar to that in mammalian cells. However, unlike the situation in mammalian cells, the viral polymerase was not inhibited by the viral nonstructural protein NSs. We also report that promoter strength is different for vertebrate versus invertebrate cells. The development of this system opens the way for a detailed comparison of bunyavirus replication in cells of disparate phylogeny.

Aedes↗
PubMed1979

Replication of standard and defective Ross River virus in BHK cells: patterns of viral RNA and polypeptide synthesis.

Virus-specific macromolecule synthesis has been examined in BHK cells infected with Ross River virus. Unpassaged virus (R-0) and tenth-passage virus (R-10) have been compared. In infected cells R-0 generates i) 45S, 28S, 33S and 26S viral RNAs, ii) virus-specific precursor polypeptides of mol. wt. 127,000, 95,000 and 61,000 and iii) viral envelope proteins (mol. wts. 52,000 and 49,000) and nucleocapsid protein (mol. wt. 32,000). Thus in terms of virus-specific RNA and polypeptide synthesis, the replication of standard RRV is analogous to that of Semliki Forest virus and Sindbis virus. R-10 interferes with the replication of standard Ross River virus and generates large amounts of 19S and 24S defective RNA species; 45S and 26S RNA synthesis was not markedly affected. Defective RNAs are associated with RNAse-sensitive, 50S cytoplasmic particles which contain a variety of (mainly host) proteins but no nucleocapsid protein. No evidence for translation of defective RNAs was obtained. R-10 infection is also characterized by a relatively early shut down of host protein syntehsis and by a reduction in virus-specific polypeptide synthesis and nucleocapsid formation. The data suggest that defective Ross River virus interferes primarily at the translational level.

Animals↗
PubMed1975

Inhibition of viral transcriptase by immunoglobulin directed against the nucleocapsid NS protein of vesicular stomatitis virus.

In search of an anti-transcriptase, antibody was raised in rabbits to partially purified, soluble NS protein present in cytoplasmic extracts of cells infected with the Indiana serotype of vesicular stomatitis (VSInd) virus. This antiserum gave specific reactions of identity by agar immunodiffusion with both cytoplasmic and virion NS protein. NS antiserum also preferentially precipitated NS 3-H-labeled protein from infected cytoplasmic extracts, whereas anti-whole VSInd virion serum also precipitated N 3-H-labeled protein from extracts both of infected cytoplasm and virion nucleocapsids. Transcriptase activity of VSInd cytoplasmic or virion-derived nucleocapsids was effectively inhibited by ribonuclease-free immunoglubulin prepared from homologous NSInd antiserum or from anti-whole vesicular stomatitis virus serum. Transcriptase activity of heterologous New Jersey serotype (VSNJ) nucleocapsids and virions was not appreciably affected by anti-NSInd or by anti-whole VSInd virion gamma globulin. Anti-NS gamma glubulin immediately switched off RNA synthesis by actively transcribing VSInd nucleocapsids, a finding which suggests that NS antibody inhibits RNA chain elongation.

Animals↗
PubMed1977

Formation of recombinants between snowshoe hare and La Crosse bunyaviruses.

Wild-type recombinants were obtained at high frequency from coinfections of BHK cells involving temperature-sensitive, conditional-lethal mutants of snowshoe hare (SSH) and La Crosse (LAC) bunyaviruses. Analyses of two of the recombinants indicated that they have the genome compositions SSH/LAC/SSH and SSH/LAC/LAC for their respective L, M, and S virion RNA species. This evidence, together with that for the genetic stability of the recombinants, indicates that they were derived by segment reassortment of the competent genome pieces of the parental viruses. The SSH/LAC/SSH recombinant appears, from polypeptide analysis, to have the SSH type of nucleocapsid protein (N), whereas the SSH/LAC/LAC recombinant has the LAC nucleocapsid protein, suggesting that the viral S RNA codes for the N protein.

Animals↗
PubMed1979

Cell-free synthesis of measles virus proteins.

Polyadenylated mRNA extracted from cytoplasm of measles virus-infected Vero cells was translated in a cell-free system. Three of the polypeptides obtained corresponded to nucleocapsid protein, phosphoprotein, and membrane protein of measles virions. A fourth polypeptide, present in measles virus-infected cells, could be generated by addition of Vero cytoplasmic extract and was identified as a cleavage product of the nucleocapsid protein.

Capsid↗
PubMed1977

Host induced modifications of Newcastle disease virus virion polypeptides.

The polypeptide composition of Newcastle Disease Virus (NDV) virions grown in two host cell cystems--chorioallantlic membrane (CAM) and BHK-21 cells--was studied. Two strains of virus were compared, one highly virulent, the other completely avirulent. No significant differences in the polypeptide composition of the two strains of virus could be detected. However, differences were found in virions grown in different hosts, the same differences being found in both strains. An additional polypeptide is found in BHK grown virus which is not present in CAM grown virus and this is associated with a decreased relative amount of nucleocapsid protein in BHK grown virus. The possibility of this new polypeptide being a degradation product of the nucleocapsid protein is discussed. BHK grown virions also contain increased amounts of a polypeptide migrating to a position which might be expected of the FO precursor glycoprotein. However, in contrast to the FO polypeptide, this polypeptide does not appear to be glycosylated.

Animals↗
PubMed1977

RNA synthesis of vesicular stomatitis virus. VII. Complete separation of the mRNA's of vesicular stomatitis virus by duplex formation.

Full-length virion RNA and complementary mRNA's of vesicular stomatitis virus can be annealed to each other, digested with RNases, and then separated as five unique duplex RNA molecules on polyacrylamide slab gels. Similar RNA duplexes were detected whether mRNA or virion RNA was the radioactive component and whether the mRNA was synthesized in vitro or in vivo. The sharp banding pattern of these RNA molecules was dependent on treatment with RNase T2, suggesting that removal of poly(A) is necessary. Identification of the coding region contained in each RNA duplex was based on their previous identification as single-stranded mRNA on formamide-containing, polyacrylamide gels. Because the two smallest mRNA'S had not been previously separated, their identification was based on their in vitro transcriptional gene order. In the order of increasing mobilities on the slab gels, the RNA duplexes are identified as the hybrid of the region of the genome RNA hybridized to the complementary mRNA coding for the large protein, the glycoprotein, the nucleocapsid protein, the core-associated NS protein, and the matrix protein (L,G,N,NS, and M). Several lines of evidence support the presence of undegraded complete mRNA, excluding poly(A), in these RNA duplexes. Also, the two smallest mRNA's, separated by duplex formation, were denatured, and their individual oligonucleotide fingerprints were determined. From chemical length determinations, the molecular weights of the mRNA, minus poly(A), are 2.78 X 10(5) and 2.5 X 10(5), respectively, for the mRNA's of the NS and M proteins.

Cell Line↗