The transcription and replication of the Newcastle disease virus (NDV) strictly rely on the viral ribonucleoprotein (RNP) complex, which is composed of viral NP, P, L and RNA. However, it is not known whether other viral non-RNP proteins participate in this process for viral self-regulation. In this study, we used a minigenome (MG) system to identify the regulatory role of the viral non-RNP proteins V, M, W, F and HN. Among them, V significantly reduced MG-encoded reporter activity compared with the other proteins and inhibited the synthesis of viral mRNA and cRNA. Further, V interacted with NP. A mutation in residue W195 of V diminished V-NP interaction and inhibited inclusion body (IB) formation in NP-P-L-cotransfected cells. Furthermore, a reverse-genetics system for the highly virulent strain F48E9 was established. The mutant rF48E9-VW195R increased viral replication and apparently enhanced IB formation. In vivo experiments demonstrated that rF48E9-VW195R decreased virulence and retarded time of death. Overall, the results indicate that the V-NP interaction of the W195 mutant V decreased, which regulated viral RNA synthesis, IB formation, viral replication and pathogenicity. This study provides insight into the self-regulation of non-RNP proteins in paramyxoviruses.

Paramyxoviruses are reported to block apoptosis for their replication, but the mechanisms remain unclear. Furthermore, regulation of mitochondrial apoptosis by paramyxoviruses has been hardly reported. We investigated whether and how human parainfluenza virus type 2 (hPIV-2) counteracts apoptosis. Infection of recombinant hPIV-2 carrying mutated V protein showed higher caspase 3/7 activity and higher cytochrome c release from mitochondria than wild type hPIV-2 infection. This indicates that V protein controls mitochondrial apoptosis pathway. hPIV-2 V protein interacted with Bad, an apoptotic promoting protein, and this interaction inhibited the binding of Bad to Bcl-XL. V protein also bound to 14-3-3ε, which was essential for inhibition of 14-3-3ε cleavage. Our data collectively suggest that hPIV-2 V protein has two means of preventing mitochondrial apoptosis pathway: the inhibition of Bad-Bcl-XL interaction and the suppression of 14-3-3ε cleavage. This is the first report of the mechanisms behind how paramyxoviruses modulate mitochondrial apoptosis pathways.

Canine parainfluenza virus (CPIV) is a zoonotic virus that is widely distributed and is the main pathogen causing canine infectious respiratory disease (CIRD), also known as \"kennel cough,\" in dogs. The CPIV-V protein is the only nonstructural protein of the virus and plays an important role in multiple stages of the virus life cycle by inhibiting apoptosis, altering the host cell cycle and interfering with the interferon response. In addition, studies have shown that the V protein has potential applications in the field of immunotherapy in oncolytic virus therapy or self-amplifying RNA vaccines. In this review, the biosynthesis, structural characteristics and functions of the CPIV-V protein are reviewed with an emphasis on how it facilitates viral immune escape and its potential applications in the field of immunotherapy. Therefore, this review provides a scientific basis for research into the CPIV-V protein and its potential applications.

In this study, we recombinantly expressed the V protein of the peste des petits ruminants virus (PPRV) and evaluated its diagnostic value for PPRV infection using an indirect ELISA (i-ELISA). The optimal concentration of the coated antigen of V protein was 15 ng/well at a serum dilution of 1:400, and the optimal positive threshold value was 0.233. A cross-reactivity assay showed that the V protein-based i-ELISA was specific to PPRV with consistent reproducibility and showed a specificity of 82.6% and a sensitivity of 100% with a virus neutralization test. Using the recombinant V protein as an antigen in ELISA is useful for seroepidemiological studies of PPRV infections.

Porcine respirovirus 1 (PRV1), first reported in Hong Kong, is currently widely spread in several countries. Our knowledge of the clinical significance and the pathogenicity of this virus is still limited. In this study, we studied the interactions between PRV1 and host innate immune responses. PRV1 exhibited strong inhibitory effects on the production of interferon (IFN), ISG15, and RIG-I induced by SeV infection. Our data generated in vitro suggest that multiple viral proteins can suppress host type I interferon production and signaling, including N, M, and P/C/V/W. The P gene products disrupt both IRF3 and NF-κB dependent type I IFN production and block type I IFN signaling pathway by sequestering STAT1 in the cytoplasm. The V protein disrupts both MDA5 signaling and RIG-I signaling through interaction with TRIM25 and RIG-I, V protein blocks RIG-I polyubiquitination, which is required for RIG-I activation. V protein also binds to MDA5, which may contribute to its inhibitory effect on MDA5 signaling. These findings indicate that PRV1 antagonizes host innate immune responses using various mechanisms, which provides important insights into the pathogenicity of PRV1.

BACKGROUND: Newcastle disease virus (NDV) is an oncolytic virus that can inhibit cancer cell proliferation and kill cancer cells. The NDV nonstructural V protein can regulate viral replication; however, whether the V protein contributes to NDV oncolysis is unclear.
RESULTS: This study revealed that NDV inhibited tumor cell proliferation and that V protein expression promoted the proliferation of HepG2 cells, as determined at the single-cell level. In addition, to identify the regulatory mechanism of the V protein in HepG2 cells, transcriptome sequencing was performed and indicated that the expression/activation of multiple cell proliferation-related genes/signaling pathways were changed in cells overexpressing the V protein. Hence, the MAPK and WNT signaling pathways were selected for verification, and after blocking these two signaling pathways with inhibitors, the V protein promotion of cell proliferation was found to be attenuated.
CONCLUSIONS: The results showed that the V protein regulated the proliferation of cancer cells through multiple signaling pathways, providing valuable references for future studies on the mechanism by which the V protein regulates cancer cell proliferation.

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.

Nipah virus (NiV) is an emerging and deadly zoonotic paramyxovirus that is responsible for periodic epidemics of acute respiratory illness and encephalitis in humans. Previous studies have shown that the NiV V protein antagonizes host antiviral immunity, but the molecular mechanism is incompletely understood. To address this gap, we biochemically characterized NiV V binding to the host pattern recognition receptor MDA5. We find that the C-terminal domain of NiV V (VCTD) is sufficient to bind the MDA5SF2 domain when recombinantly co-expressed in bacteria. Analysis by hydrogen-deuterium exchange mass spectrometry (HDX-MS) studies revealed that NiV VCTD is conformationally dynamic, and binding to MDA5 reduces the dynamics of VCTD. Our results also suggest that the β-sheet region in between the MDA5 Hel1, Hel2, and Hel2i domains exhibits rapid HDX. Upon VCTD binding, these β-sheet and adjacent residues show significant protection. Collectively, our findings suggest that NiV V binding disrupts the helicase fold and dynamics of MDA5 to antagonize host antiviral immunity.

Henipaviruses are BSL-4 zoonotic pathogens responsible in humans for severe encephalitis. Their V protein is a key player in the evasion of the host innate immune response. We previously showed that the Henipavirus V proteins consist of a long intrinsically disordered N-terminal domain (NTD) and a β-enriched C-terminal domain (CTD). These terminals are critical for V binding to DDB1, which is a cellular protein that is a component of the ubiquitin ligase E3 complex, as well as binding to MDA5 and LGP2, which are two host sensors of viral RNA. Here, we serendipitously discovered that the Hendra virus V protein undergoes a liquid-to-hydrogel phase transition and identified the V region responsible for this phenomenon. This region, referred to as PNT3 and encompassing residues 200-310, was further investigated using a combination of biophysical and structural approaches. Congo red binding assays, together with negative-staining transmisison electron microscopy (TEM) studies, show that PNT3 forms amyloid-like fibrils. Fibrillation abilities are dramatically reduced in a rationally designed PNT3 variant in which a stretch of three contiguous tyrosines, falling within an amyloidogenic motif, were replaced by three alanines. Worthy to note, Congo red staining experiments provided hints that these amyloid-like fibrils form not only in vitro but also in cellula after transfection or infection. The present results set the stage for further investigations aimed at assessing the functional role of phase separation and fibrillation by the Henipavirus V proteins.

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.