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Sendai virus V protein is a known antagonist of RIG-I-like receptors (RLRs) RIG-I and MDA5, which activate transcription factors IRF3, leading to activation of ISGF3 and NF-κB. These transcription factors are known activators of inducible NO synthase (iNOS) and increase the production of nitric oxide (NO). By inhibiting ISGF3 and NF-κB, the V protein acts as an indirect negative regulator of iNOS and NO. Here we report that the V gene knockout Sendai virus [SeV V(−)] markedly enhanced iNOS expression and subsequent NO production in infected macrophages compared to wild-type SeV. The knockout of RIG-I in cells inhibited SeV V(−)-induced iNOS expression and subsequent NO production. To understand the underlying mechanism of the V protein-mediated negative regulation of iNOS activation, we transfected HEK293T cells with RIG-I and the RIG-I regulatory protein TRIM25. Our results demonstrated that the V protein inhibited iNOS activation via the RIG-I/TRIM25 pathway. Moreover, the V protein inhibited TRIM25-mediated K63-linked ubiquitination of RIG-I, as well as its CARD-dependent interaction with mitochondrial antiviral signaling (MAVS) molecules. These results suggest that the V protein downregulates iNOS activation and inhibits NO production by preventing the RIG-I-MAVS interaction, possibly through its effect on the ubiquitination status of RIG-I. [ABSTRACT FROM AUTHOR]

Newcastle disease virus (NDV) V protein is crucial for viral interferon (IFN) antagonism and virulence, determining its host range restriction. However, little information is available on the B cell epitopes of V protein and the subcellular movement of V protein in the process of NDV infection. In this study, the monoclonal antibody (mAb) clone 3D7 against genotype VII NDV V protein was generated by immunizing mice with a purified recombinant His-tagged carboxyl-terminal domain (CTD) region of V protein. Fine epitope mapping analysis and B-cell epitope prediction indicated that mAb 3D7 recognized a linear epitope 152 RGPAELWK 159 , which is located in the V protein CTD region. Sequence alignment showed that the mAb clone 3D7-recognized epitope is highly conserved among Class II genotype VII NDV strains, but not among other genotypes, suggesting it could serve as a genetic marker to differentiate NDV genotypes. Furthermore, the movement of V protein during NDV replication in infected cells were determined by using this mAb. It was found that V protein localized around the nucleus during virus replication. The establishment of V protein-specific mAb and identification of its epitope extend our understanding of the antigenic characteristics of V protein and provide a basis for the development of epitope-based diagnostic assays. [ABSTRACT FROM AUTHOR]

The genetic variation and molecular evolution of the V gene of the class II Newcastle disease virus (NDV) isolates with genotypes I–XVIII were determined using bioinformatics. Results indicated that low homology existed in different genotype viruses, whereas high homology often for the same genotypes, exception may be existed within genotypes I, V, VI, and XII. Sequence analysis showed that the genetic variation of V protein was consistent with virus genotype, and specific signatures on the V protein for nine genotypes were identified. Phylogenetic analysis demonstrated that the phylogenetic trees were highly consistent between the V and F genes, with slight discrepancies in the sub-genotypes. Evolutionary rate analyses based on V and F genes revealed the evolution rates varied in genotypes. These data indicate that the genetic variation of V protein is genotype-related and will help in elucidating the molecular evolution of NDV. [ABSTRACT FROM AUTHOR]

• Establishment of an efficient rescue system for attenuated canine distemper virus. • Functional l-protein required a glutamate residue at position 13. • Attaching mNeonGreen to the N-protein via a P2A motif impaired viral growth. • A combinatorial mutation approach to delete V-protein expression. To provide insights into the biology of the attenuated canine distemper virus (CDV) Onderstepoort (OP) strain (large plaque forming variant), design next-generation multivalent vaccines, or further investigate its promising potential as an oncolytic vector, we employed contemporary modifications to establish an efficient OP-CDV-based reverse genetics platform. Successful viral rescue was obtained however only upon recovery of a completely conserved charged residue (V13E) residing at the N-terminal region of the large protein (L). Although L-V13 and L-V13E did not display drastic differences in cellular localization and physical interaction with P, efficient polymerase complex (P+ L) activity was recorded only with L-V13E. Interestingly, grafting mNeonGreen to the viral N protein via a P2A ribosomal skipping sequence (OPneon) and its derivative V-protein-knockout variant (OPneon-Vko) exhibited delayed replication kinetics in cultured cells. Collectively, we established an efficient OP-CDV-based reverse genetics system that enables the design of various strategies potentially contributing to veterinary medicine and research. [ABSTRACT FROM AUTHOR]

Abstract: Laboratory-adapted and vaccine strains of measles virus (MV) induce type I interferon (IFN) in infected cells to a far greater extent than wild-type strains. We investigated the mechanisms for this differential type I IFN production in cells infected with representative MV strains. The overexpression of the wild-type V protein suppressed melanoma differentiation-associated gene 5 (MDA5)-induced IFN-β promoter activity, while this was not seen in A549 cells expressing CD150 transfected with the V protein of the vaccine strain. The V proteins of the wild-type also suppressed poly I:C-induced IFN regulatory factor 3 (IRF-3) dimerization. The V proteins of the wild-type and vaccine strain did not affect retinoic acid-inducible gene 1 (RIG-I)- or toll-IL-1R homology domain-containing adaptor molecule 1 (TICAM-1)-induced IFN-β promoter activation. We identified an amino acid substitution of the cysteine residue at position 272 (which is conserved among paramyxoviruses) to an arginine residue in the V protein of the vaccine strain. Only the V protein possessing the 272C residue binds to MDA5. The mutation introduced into the wild-type V protein (C272R) was unable to suppress MDA5-induced IRF-3 nuclear translocation and IFN-β promoter activation as seen in the V proteins of the vaccine strain, whereas the mutation introduced in the vaccine strain V protein (R272C) was able to inhibit MDA5-induced IRF-3 and IFN-β promoter activation. The other 6 residues of the vaccine strain V sequence inconsistent with the authentic sequence of the wild-type V protein barely affected the IRF-3 nuclear translocation. These data suggested that the structural difference of vaccine MV V protein hampers MDA5 blockade and acts as a nidus for the spread/amplification of type I IFN induction. Ultimately, measles vaccine strains have two modes of IFN-β-induction for their attenuation: V protein mutation and production of defective interference (DI) RNA. [ABSTRACT FROM AUTHOR]

Abstract: The paramyxovirus P gene encodes accessory proteins antagonistic to interferon (IFN). Viral proteins responsible for the IFN antagonism, however, are distinct among paramyxoviruses. Here we determine bovine parainfluenza virus type 3 (bPIV3) IFN antagonists that suppress IFN-β production, and investigate the underlying molecular mechanism. Of bPIV3 P gene products, C and V proteins were found to suppress double-stranded RNA-stimulated IFN-β production. The V protein of bPIV3 and Sendai virus in the same genus Respirovirus significantly inhibits double-stranded RNA-stimulated IFN-β production and the IFN-β promoter activation enhanced by overexpression of MDA5 but not RIG-I, and yet does not suppress IFN-β production induced by TRIF, TBK1, and IKKi. The V protein of both viruses specifically binds to MDA5 but not RIG-I. These results suggest that the V protein targets MDA5 for blockage of the IFN-β gene activation signal. On the other hand, both bPIV3 and Sendai virus C proteins modestly inhibited IFN-β production irrespective of a species of the signaling molecules used as an inducer. Interestingly, reporter gene expression driven by various promoters was also suppressed by the C proteins irrespective of the promoter species. These results demonstrate that the target of the respirovirus C protein is undoubtedly different from that of the V protein. [Copyright &y& Elsevier]

The gene encoding the phosphoprotein of the vaccine strain of Peste des petits ruminants (PPR) virus (Nigeria 75/1 vaccine strain) has been cloned and its nucleotide sequence been determined. This gene is 1655 nucleotides long and encodes two overlapping open reading frames (ORFs). Translation from the first AUG would produce a polypeptide of 509 amino acid residues with a predicted molecular mass of 54.9 kDa, the longest of the published morbillivirus P proteins. Translation from the second AUG would produce a protein of 177 amino acid residues with a predicted molecular mass of 20.3 kDa, analogous to the C proteins of other morbilliviruses. Evidence was found for the production of two types of P mRNA transcript, one a faithful transcript of the gene and the other with an extra G residue inserted at position 751. Translation from the first AUG of this second mRNA would produce a protein of 298 amino acids, with a predicted molecular mass 32.3 kDa, analogous to the V protein produced by other morbilliviruses. Sequences of the predicted P, C and V proteins were compared with those of the other morbillivirus sequences available to date. The P protein was found to be the most poorly conserved of the morbillivirus proteins, the amino acid identity ranging from 54% in case of Canine distemper virus (CDV) to 60% in the case of the Dolphin morbillivirus (DMV). [Copyright &y& Elsevier]

In this paper we show that the porcine rubulavirus LPMV phosphoprotein (P) and V protein (V) both interact with the nucleoprotein (NP). There are also indications for an interaction between P and V with L protein. Further analysis of the domains of the P and V which are necessary for interaction with the NP protein demonstrates that the interaction is not mediated from their common part but instead from their unique C-terminal parts, respectively. The common N-terminus of P and V appear to mediate the interaction with L. We also map the regions of NP that are necessary for interaction with P and V, respectively. Both P and V interact with regions of NP, which reside in the N-terminal part but appear not to overlap. [Copyright &y& Elsevier]

• lentogenic V protein is more proficient in enhancing viral replication. • lentogenic V protein inhibits IFN responses more efficiently. • Velogenic V protein triggered a higher apoptosis rate than lentogenic V protein. • V protein is involved in the formation of syncytia. The V protein of Newcastle disease virus (NDV) has been shown to inhibit the secretion of interferon (IFN) during infection, which is responsible for the promotion of NDV pathogenicity. However, the ability of the V protein to suppress host innate immunity is not well understood. In this study, we explored the function of V protein and its relationship with virulence by generating V protein-inserted recombinant (r) NDVs. Using rNDVs as a model, we examined the efficiency of infection, IFN responses, and apoptosis of host cells during infection. We found that viral propagation occurred smoothly when V protein from lentogenic NDV is inserted instead of the V protein from the velogenic strain. The infection of lentogenic V protein-inserted rNDV induced less expression of IFNs and downstream antiviral proteins via efficient degradation of p-STAT1 and MDA5. Moreover, velogenic V protein triggered a higher apoptosis rate during infection thereby restricting the replication of NDV. Conversely, lentogenic V protein inhibits IFN responses efficiently and induces less apoptosis compared to the velogenic strain. Our findings provide a novel understanding of the role of V protein in NDV pathogenicity. [ABSTRACT FROM AUTHOR]

• This is the first report that provides evidence that hnRNP H1 plays a positive role in NDV replication. • hnRNPH1 can interact with NDV V protein and colocalized in the cytoplasm of DF-1 cells. • NDV V protein could regulate hnRNPH1 expression at the protein level instead of the transcription level. • NDV V protein may regulate cell proliferation by controlling the expression of hnRNP H1. The interactions between host cellular proteins and viral proteins are important for successful infection by viruses. Previous studies from our group have identified various host cellular proteins that can interact with the Newcastle disease virus V protein (Chu et al., 2018a), but their function in NDV replication has not been fully determined. The present study reports that heterogenous nuclear ribonucleoprotein H1 (hnRNP H1) can interact with NDV V protein in yeast. The immunofluorescence results showed that hnRNP H1 and V protein could colocalize in the cytoplasm of a chicken embryo fibroblast cell line (DF-1 cells). Co-immunoprecipitation assays further verified the interaction of these two proteins. The effects of overexpression and knockdown of hnRNP H1 on NDV replication were evaluated in DF-1 cells through real time quantitative PCR (RT-qPCR) and plaque assays. The regulation of V protein on hnRNP H1 expression was also examined. The results indicated that overexpression of hnRNP H1 facilitated NDV replication, while knockdown of hnRNP H1 decreased NDV replication. It was also shown that V protein could regulate hnRNP H1 expression at the protein level instead of the transcription level. The effect of V protein and hnRNP H1 on the DF-1 cell cycle was also tested and the results revealed that V protein may regulate cell proliferation by controlling the expression of hnRNP H1. Taken together, these results suggest that NDV V protein could promote viral replication by interacting with hnRNP H1. [ABSTRACT FROM AUTHOR]