Search

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

Abstract 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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Newcastle disease is a viral infection causing serious economic losses to the global poultry industry. The V protein of Newcastle disease virus (NDV) is a pathogenicity determinant having various functions such as the suppression of apoptosis and replication of the NDV. This study was designed to assess the resistance potential of plant flavonoids against the V protein of Newcastle disease virus. Sequence analysis was performed using EXPASY and ProtParam tools. To build the three-dimensional structure of V protein, a homology-modeling method was used. Plant flavonoids with formerly reported therapeutic benefits were collected from different databases to build a library for virtual screening. Docking analysis was performed using the modeled structure of V protein on MOE software. Interaction analysis was also performed by MOE to explain the results of docking. Sequence analysis and physicochemical properties showed that V protein is negatively charged, acidic in nature, and relatively unstable. The 3D structure of the V protein showed eight β-pleated sheets, three helices, and ten coiled regions. Based on docking score, ten flavonoids were selected as potential inhibitors of V protein. Furthermore, a common configuration was obtained among these ten flavonoids. The interaction analysis also identified the atoms involved in every interaction of flavonoid and V protein. Molecular dynamics (MD) simulation confirmed the stability of two compounds, quercetin-7-O-[α-L-rhamnopyranosyl(1→6)-β-D-galactopyranoside] and luteolin 7-O-neohesperidoside, at 100 ns with V protein. The identified compounds through molecular docking and MD simulation could have potential as NDV-V protein inhibitor after further validation. This study could be useful for the designing of anti-NDV drugs. [ABSTRACT FROM AUTHOR]

Newcastle disease virus (NDV) is a paramyxovirus that is highly pathogenic to poultry causing severe economic loss worldwide. The non-structural V protein is one of the virulence factors of the virus. It antagonises the interferon of the host innate immunity in order to allow successful virus replication in the host cells. However, detailed investigation of recombinant NDV expressing mutated V protein is scarce. In this study, a mesogenic recombinant NDV expressing GFP (rAF-GFP) was used to investigate the relation of V protein mutation on virus pathogenicity. Site-directed mutagenesis was performed using overlapping PCR to introduce four premature stop codons 456G>T, 537G>T, 624C>T and 642G>T in the V gene reading frame. The virus was then rescued and propagated in embryonated chicken eggs. However, instead of the substituted thymine, this nucleotide was mutated into cytosine in three rescued mutants, while 537G>T mutant could not be rescued. As a result, the premature stop codon was substituted with other amino acid and the V protein was expressed in full length. The pathogenicity type of the rAF (456G>T>C), rAF (624C>T>C), and rAF (642G>T>C) mutants remained to be as in mesogenic strains, suggesting that substituted amino acids were functionally interchangeable with the original amino acids present in V protein. It appears that an intact V protein is important for the virus survival. This study explored the possibility of V protein mutation in NDV through exploiting genetic engineering and warrants a further investigation on modifying mutations on a conserved protein in NDV or other paramyxoviruses. [ABSTRACT FROM AUTHOR]

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-X L. 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-X L interaction and the suppression of 14-3-3ε cleavage. This is the first report of the mechanisms behind how paramyxoviruses modulate mitochondrial apoptosis pathways. • Two ways of inhibition of mitochondrial apoptosis pathway by human parainfluenza virus type 2 V protein. • The two ways: prevention of Bad/Bcl-X L interaction and inhibition of 14-3-3ε cleavage. • The first report of mechanisms behind how paramyxoviruses modulate mitochondrial apoptosis-related proteins. [ABSTRACT FROM AUTHOR]

Intracellular iron concentration is tightly controlled for cell viability. It is known to affect the growth of several viruses, but the molecular mechanisms are not well understood. We found that iron chelators inhibit growth of human parainfluenza virus type 2 (hPIV-2). Furthermore, infection with hPIV-2 alters ferritin localization from granules to a homogenous distribution within cytoplasm of iron-stimulated cells. The V protein of hPIV-2 interacts with ferritin heavy chain 1 (FTH1), a ferritin subunit. It also binds to nuclear receptor coactivator 4 (NCOA4), which mediates autophagic degradation of ferritin, so-called ferritinophagy. V protein consequently interferes with interaction between FTH1 and NCOA4. hPIV-2 growth is inhibited in FTH1 knockdown cell line where severe hPIV-2-induced apoptosis is shown. In contrast, NCOA4 knockdown results in the promotion of hPIV-2 growth and limited apoptosis. Our data collectively suggest that hPIV-2 V protein inhibits FTH1-NCOA4 interaction and subsequent ferritinophagy. This iron homeostasis modulation allows infected cells to avoid apoptotic cell death, resulting in effective growth of hPIV-2. [ABSTRACT FROM AUTHOR]

Cavin proteins have important roles in the formation of caveolae in lipid raft microdomains. Pulse-chase experiments of cells infected with human parainfluenza virus type 2 (hPIV-2) showed decreased proteasomal degradation of Cavin3. Overexpression of hPIV-2 V protein alone was sufficient to inhibit Cavin3 degradation. Immunoprecipitation analysis revealed that V protein bound to Cavin3. Trp residues within C-terminal region of V protein, as well as the N-terminal region of Cavin3, are important for V–Cavin3 interaction. Cavin3 knockdown suppressed hPIV-2 growth without affecting its entry, replication, transcription, or translation. Higher amounts of Cavin3 were observed in V protein-overexpressing cells than in control cells in lipid raft microdomains. Our data collectively suggest that hPIV-2 V protein binds to and stabilizes Cavin3, which in turn facilitates assembly and budding of hPIV-2 in lipid raft microdomains.

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]

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). The CTD is 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.

Newcastle disease virus (NDV) causes serious economic losses to the poultry industry. In our previous study, we found that NDV induced a strong innate immune response in the chicken embryo and bursa of Fabricius (BF). However, the underlying mechanisms by which NDV escapes the host innate immunity are not well-understood. The suppressor of cytokine signaling 3 (SOCS3) inhibits the type I interferon-dependent antiviral signaling pathway by utilizing a feedback loop. In this study, we analyzed the transcriptome data of the chicken embryo and BF infected with NDV and found significant upregulation of SOCS3. Next, we demonstrated that NDV infection and nonstructural V protein induced the up-regulation of SOCS3. Furthermore, we showed that overexpression of SOCS3 facilitated viral replication and reduced the expression of phosphorylation STAT1, MX1, and OASL, while inhibition of SOCS3 with siRNAs reduced virus replication and promoted the expression of phosphorylation STAT1, MX1, and OASL. Finally, we demonstrated that the MEK/ERK signaling pathway was involved in the expression of SOCS3 mediated by NDV infection and V protein transfection, and using specific inhibitor U0126 to block this signaling pathway attenuated SOCS3 expression and inhibited NDV replication through promoting the expression of type I interferon, OASL and MX1. Taken together, these data demonstrate that NDV infection and NDV nonstructural V protein activates the expression of SOCS3 at the mRNA and protein level through a mechanism dependent on the MEK/ERK signaling pathway, which benefits virus replication.

Tight junctions enable epithelial cells to form physical barriers that act as an innate immune defense against respiratory infection. However, the involvement of tight junction molecules in paramyxovirus infections, which include various respiratory pathogens, has not been examined in detail. Human parainfluenza virus type 2 (hPIV2) infects airway epithelial cells and causes respiratory illness. In the present study, we found that hPIV2 infection of cultured cells induces expression of claudin-1 (CLDN1), an essential component of tight junctions. This induction seemed to be intrinsically restricted by V, an accessory protein that modulates various host responses, to enable efficient virus propagation. By generating CLDN1 over-expressing and knockout cell lines, we showed that CLDN1 is involved in the restriction of hPIV2 spread via cell-to-cell contact. Taken together, we identified CLDN1 an inhibitory factor for hPIV2 dissemination, and that its V protein acts to counter this. [ABSTRACT FROM AUTHOR]

Human parainfluenza virus type 2 phosphoprotein (P) is an essential component of viral polymerase. The P gene encodes both P and accessory V proteins by a specific gene editing mechanism. Therefore, the N-terminal 164 amino acids of P protein are common to V protein. Interestingly, while P protein is located in the cytoplasm, V protein is found mainly in the nucleus. Using deletion mutants, we show the presence of a nuclear localization signal (NLS) in the P/V common domain, and a nuclear export signal (NES) in the C-terminal P specific region. The NLS region makes a complex with importin α5 or 7. In the presence of leptomycin B, P protein is retained in the nucleus, indicating that it contains a CRM1-dependent NES. We identified the NLS (65PVKPRRKK72) and the NES (225IIELLKGLDL234) using β-galactosidase fusion proteins. Moreover, nucleocytoplasmic shuttling of P protein appears to be important for efficient viral polymerase activity. [ABSTRACT FROM AUTHOR]

Newcastle disease virus (NDV) has been classified by the World Organization for Animal Health (OIE) as a notable disease-causing virus, and this virus has the ability to infect a wide range of birds. V protein is a non-structural protein of NDV. V protein has been reported to inhibit cell apoptosis (Park et al., 2003a) and promote viral replication (Huang et al., 2003), however, the mechanisms of action of V protein have not been elucidated. In the present study, a yeast two-hybrid screen was performed, and V protein was found to interact with the CacyBP/SIP protein. The results of co-immunoprecipitation and immuno-colocalization assays confirmed the interaction between V protein and CacyBP/SIP. The results of quantitative-PCR and viral plaque assays showed that overexpression of CacyBP/SIP inhibited viral replication in DF-1 cells. Overexpression of CacyBP/SIP in DF-1 cells induced caspase3-dependent apoptosis. The effect of knocking down CacyBP/SIP by siRNA was the opposite of that observed upon overexpression. Moreover, it is known that NDV induces cell apoptosis via multiple caspase-dependent pathways. Furthermore, V protein inhibited cell apoptosis and downregulated CacyBP/SIP expression in DF-1 cells. Taken together, the findings of the current study indicate that V protein interacts with CacyBP/SIP, thereby regulating cell apoptosis and viral replication.

Poultry industry facing problems in vaccination of chicken against Newcastle disease virus (NDV) due to the difficulty of differentiating the vaccinated and naturally infected birds. If we could find a key marker that could differentiate, we would bring a great achievement to the industry. NDV genome contains genes for six major structural proteins and two non-structural proteins. The non-structural protein V could be a useful antigen for differentiating chickens infected with NDV from those vaccinated with inactivated whole virus. We have constructed a recombinant plasmid using the C-terminal region of the non-structural V protein. Another recombinant plasmid using the C-terminal polypeptide of structural P protein was developed as a control. We succeeded in expressing the unique C-terminal part of the V protein in E. coli as a fusion protein with GST (GST-V), 40.8 kDa, as well as the C-terminal part of the P protein (GST-P), 55.4 kDa. Purified NDV-V and NDV-P proteins had sizes of 14.8 kDa and 29.4 kDa, respectively. The antigenicity of recombinant V protein was confirmed with Western blotting assay, which makes the using of this protein as a biological marker prospective. [ABSTRACT FROM AUTHOR]

Inflammasomes play a key role in host innate immune responses to viral infection by caspase-1 (Casp-1) activation to facilitate interleukin-1β (IL-1β) secretion, which contributes to the host antiviral defense. The NLRP3 inflammasome consists of the cytoplasmic sensor molecule NLRP3, adaptor protein ASC, and effector protein pro-caspase-1 (pro-Casp-1). NLRP3 and ASC promote pro-Casp-1 cleavage, leading to IL-1β maturation and secretion. However, as a countermeasure, viral pathogens have evolved virulence factors to antagonize inflammasome pathways. Here we report that V gene knockout Sendai virus [SeV V(-)] induced markedly greater amounts of IL-1β than wild-type SeV in infected THP1 macrophages. Deficiency of NLRP3 in cells inhibited SeV V(-)-induced IL-1β secretion, indicating an essential role for NLRP3 in SeV V(-)-induced IL-1β activation. Moreover, SeV V protein inhibited the assembly of NLRP3 inflammasomes, including NLRP3-dependent ASC oligomerization, NLRP3-ASC association, NLRP3 self-oligomerization, and intermolecular interactions between NLRP3 molecules. Furthermore, a high correlation between the NLRP3-binding capacity of V protein and the ability to block inflammasome complex assembly was observed. Therefore, SeV V protein likely inhibits NLRP3 selfoligomerization by interacting with NLRP3 and inhibiting subsequent recruitment of ASC to block NLRP3-dependent ASC oligomerization, in turn blocking full activation of the NLRP3 inflammasome and thus blocking IL-1β secretion. Notably, the inhibitory action of SeV V protein on NLRP3 inflammasome activation is shared by other paramyxovirus V proteins, such as Nipah virus and human parainfluenza virus type 2. We thus reveal a mechanism by which paramyxovirus inhibits inflammatory responses by inhibiting NLRP3 inflammasome complex assembly and IL-1β activation. [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]

Newcastle disease virus (NDV) has been classified by the World Organization for Animal Health (OIE) as a notable disease-causing virus, and this virus has the ability to infect a wide range of birds. V protein is a non-structural protein of NDV. V protein has been reported to inhibit cell apoptosis (Park et al., 2003a) and promote viral replication (Huang et al., 2003), however, the mechanisms of action of V protein have not been elucidated. In the present study, a yeast two-hybrid screen was performed, and V protein was found to interact with the CacyBP/SIP protein. The results of co-immunoprecipitation and immuno-colocalization assays confirmed the interaction between V protein and CacyBP/SIP. The results of quantitative-PCR and viral plaque assays showed that overexpression of CacyBP/SIP inhibited viral replication in DF-1 cells. Overexpression of CacyBP/SIP in DF-1 cells induced caspase3-dependent apoptosis. The effect of knocking down CacyBP/SIP by siRNA was the opposite of that observed upon overexpression. Moreover, it is known that NDV induces cell apoptosis via multiple caspase-dependent pathways. Furthermore, V protein inhibited cell apoptosis and downregulated CacyBP/SIP expression in DF-1 cells. Taken together, the findings of the current study indicate that V protein interacts with CacyBP/SIP, thereby regulating cell apoptosis and viral replication. [ABSTRACT FROM AUTHOR]

Newcastle disease virus (NDV) can infect a wide range of domestic and wild bird species. The non-structural V protein of NDV plays an important role in antagonizing innate host defenses to facilitate viral replication. However, there is a lack of knowledge related to the mechanisms through which the V protein regulates viral replication. The extracellular signal-regulated kinase (ERK) signaling pathway in the host is involved in a variety of functions and is activated by several stimuli, including viral replication. In this study, we show that both the lentogenic strain, La Sota, and the velogenic strain, F48E9, of NDV activate the mitogen-activated protein kinase (MEK)/ERK signaling pathway. The pharmacological inhibition of ERK1/2 phosphorylation using the highly selective inhibitors U0126 and SCH772984 resulted in the reduced levels of NDV RNA in cells and virus titers in the cell supernatant, which established an important role for the MEK/ERK signaling pathway in NDV replication. Moreover, the overexpression of the V protein in HeLa cells increased the phosphorylation of ERK1/2 and induced the transcriptional changes in the genes downstream of the MEK/ERK signaling pathway. Taken together, our results demonstrate that the V protein is involved in the ERK signaling pathway-mediated promotion of NDV replication and thus, can be investigated as a potential antiviral target. [ABSTRACT FROM AUTHOR]

Excessive interferon (IFN) production and signaling can lead to immunological and developmental defects giving rise to autoimmune diseases referred to collectively as “type I interferonopathies.” A subset of these diseases is caused by monogenic mutations affecting proteins involved in nucleic acid sensing, homeostasis, and metabolism. Interferonopathic mutations in the cytosolic antiviral sensor MDA5 render it constitutively hyperactive, resulting in chronic IFN production and IFN-stimulated gene expression. Few therapeutic options are available for patients with interferonopathic diseases, but a large number of IFN evasion and antagonism strategies have evolved in viral pathogens that can counteract IFN production and signaling to enhance virus replication. To test the hypothesis that these natural IFN suppressors could be used to subdue the activity of interferonopathic signaling proteins, hyperactive MDA5 variants were assessed for susceptibility to a family of viral MDA5 inhibitors. In this study, Paramyxovirus V proteins were tested for their ability to counteract constitutively active MDA5 proteins. Results indicate that the V proteins are able to bind to and disrupt the signaling activity of these MDA5 proteins, irrespective of their specific mutations, reducing IFN production and IFN-stimulated gene expression to effectively suppress the hyperactive antiviral response. [ABSTRACT FROM AUTHOR]

Tetherin is an anti-viral factor that restricts viral budding through tethering virions to the cell surface. The human parainfluenza virus type 2 (hPIV-2) V protein decreases cell surface tetherin in HeLa cells, which constitutively express endogenous tetherin. However, the role of the hPIV-2 V protein in tetherin induction remains unclear. Here, we examined whether hPIV-2 infection itself induces tetherin in HEK293 cells that have no basal expression of tetherin. Unlike influenza A virus (IAV) infection, hPIV-2 infection induced neither tetherin mRNA nor protein expression. In contrast, robust tetherin induction was observed by infection of rPIV-2s carrying V mutants, in which either three Trp residues (W178H/W182E/W192A) or Cys residues (C209/211/214A) that are important for decreasing cell surface tetherin are mutated. hPIV-2 infection also inhibited the induction of tetherin expression by IFN-α and IAV infection. Furthermore, hPIV-2 V protein but not P and V suppressed tetherin induction. Our data collectively suggest that the hPIV-2 V protein inhibits tetherin expression induced by several external stimuli. [ABSTRACT FROM AUTHOR]

Tetherin (BST-2/CD317/HM1.24) is an anti-viral factor that restricts the budding of several enveloped viruses. Most of these viruses have evolved to encode tetherin antagonists. Our previous study demonstrated that the growth of human parainfluenza virus type 2 (hPIV-2), a member of the genus Rubulavirus in the family Paramyxoviridae, was inhibited by tetherin, and its V protein decreases the amount of cell surface tetherin by the interaction. In the present study, we investigated whether tetherin inhibits the growth of other rubulaviruses including PIV-5, mumps virus (MuV), simian virus 41, and hPIV-4, and whether their V proteins act as tetherin antagonists. Plaque assay demonstrated that the growth of PIV-5 and MuV was inhibited by tetherin. Flow cytometry and immunoblot analyses revealed that the infection of PIV-5 and MuV caused reduction of cell surface tetherin without affecting total amount of tetherin. Immunoprecipitation analysis showed that all V proteins of rubulaviruses tested bound to tetherin. These results suggest that tetherin antagonism by V proteins is common among the genus Rubulavirus. [ABSTRACT FROM AUTHOR]

Newcastle disease (ND) is one of the most important diseases of poultry worldwide. In the last decades, molecular research has gained a lot of new information about its causative agent, newcastle disease virus (NDV). In poultry industry, certain strains of NDV have been used for preventive vaccination for more than 60 years. NDV has also been applied to cancer patients with beneficial effects for about 50 years, but this is less well known. The molecular basis for these differential effects of NDV in birds and man have been elucidated in the last decades and are explained in this review. The anti-neoplastic and immune-stimulatory properties in non-permissive hosts such as mouse and man have to do with the strong type I interferon responses induced in these foreign species. Additionally, NDV has the potential to break various types of tumor resistances and also to affect liver fibrosis. A main section is devoted to the benefits of clinical application of NDV and NDV-based vaccines to cancer patients. Reverse genetics technology allowed developing NDV into a vector suitable for gene therapy. Examples will be provided in which genetically engineered NDV is being used successfully as vector against new emerging viruses. [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]

Peste des petits ruminants virus (PPRV) causes a fatal disease in small ruminants. V protein of PPRV plays a pivotal role in interfering with host innate immunity by blocking IFNs signaling through interacting with STAT1 and STAT2. In the present study, the results demonstrated that PPRV V protein blocks IFN actions in a dose dependent manner and restrains the translocation of STAT1/2 proteins. We speculate that the translocation inhibition might be caused by the interfering of the downstream of STAT protein. Mutagenesis defines that Cys cluster and Trp motif of PPRV V protein are essential for STAT-mediated IFN signaling. These findings give a new sight for the further studies to understand the delicate mechanism of PPRV to escape the IFN signaling. [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 HPIV2 V protein inhibits type I interferon (IFN) induction and signaling. To manipulate the V protein, whose coding sequence overlaps that of the polymerase-associated phosphoprotein (P), without altering the P protein, we generated an HPIV2 virus in which P and V are expressed from separate genes (rHPIV2-P+V). rHPIV2-P+V replicated like HPIV2-WT in vitro and in non-human primates. HPIV2-P+V was modified by introducing two separate mutations into the V protein to create rHPIV2-L101E/L102E and rHPIV2-Δ122–127. In contrast to HPIV2-WT, both mutant viruses were unable to degrade STAT2, leaving virus-infected cells susceptible to IFN. Neither mutant, nor HPIV2-WT, induced significant amounts of IFN-β in infected cells. Surprisingly, neither rHPIV2-L101E/L102E nor rHPIV2-Δ122–127 was attenuated in two species of non-human primates. This indicates that loss of HPIV2''s ability to inhibit IFN signaling is insufficient to attenuate virus replication in vivo as long as IFN induction is still inhibited. [Copyright &y& Elsevier]

Abstract: In wild-type human parainfluenza virus type 2 (WT HPIV2), one gene (the P/V gene) encodes both the polymerase-associated phosphoprotein (P) and the accessory V protein. We generated a HPIV2 virus (rHPIV2-Vko) in which the P/V gene encodes only the P protein to examine the role of V in replication in vivo and as a potential live attenuated virus vaccine. Preventing expression of V protein severely impaired virus recovery from cDNA and growth in vitro, particularly in IFN-competent cells. rHPIV2-Vko, unlike WT HPIV2, strongly induced IFN-β and permitted IFN signaling, leading to establishment of a robust antiviral state. rHPIV2-Vko infection induced extensive syncytia and cytopathicity that was due to both apoptosis and necrosis. Replication of rHPIV2-Vko was highly restricted in the respiratory tract of African green monkeys and in differentiated primary human airway epithelial (HAE) cultures, suggesting that V protein is essential for efficient replication of HPIV2 in organized epithelial cells and that rHPIV2-Vko is over-attenuated for use as a live attenuated vaccine. [Copyright &y& Elsevier]

Newcastle disease virus (NDV) is not only one of the most economically important pathogen of poultry but also has a potential as anticancer virotherapy. The role of NDV V protein in virus-production kinetics was investigated using DF-1 cell-based production system. The presence of an anti-interferon (IFN)-alpha antibody resulted in enhanced NDV production kinetics in a dose-dependent manner by blocking binding of NDV-induced IFN to its receptor. To prepare DF-1 cell whose cellular IFN signaling is blocked efficiently, stable cell lines expressing either lentogenic or velogenic NDV V protein known as an IFN antagonist were established. The overexpression of NDV V protein enhanced NDV production kinetics and expedited the rate of NDV production, while it had no effect on Japanese encephalitis virus production. NDV V protein functions as an IFN antagonist by inhibiting the increase in type I IFNs by NDV infection. The IFN signals in cells expressing NDV V protein were weakened by decreased activation or expression of the dsRNA-activated enzymes. These IFN antagonist activities enhance rapid virus replication and spread in the early phase of viral infection and will be useful in improving the production of viral vaccine strains. [ABSTRACT FROM AUTHOR]

Abstract: Measles virus (MV) causes various responses including the induction of immune responses, transient immunosuppression and establishment of long-lasting immunity. To obtain a comprehensive view of the effects of MV infection on target cells, DNA microarray analyses of two different cell-types were performed. An epithelial (293SLAM; a 293 cell line stably expressing SLAM) and lymphoid (COBL-a) cell line were inoculated with purified wild-type MV. Microarray analyses revealed significant differences in the regulation of cellular gene expression between these two different cells. In 293SLAM cells, upregulation of genes involved in the antiviral response was rapidly induced; in the later stages of infection, this was followed by regulation of many genes across a broad range of functional categories. On the other hand, in COBL-a cells, only a limited set of gene expression profiles was modulated after MV infection. Since it was reported that V protein of MV inhibited the IFN signaling pathway, we performed a microarray analysis using V knockout MV to evaluate V protein''s effect on cellular gene expression. The V knockout MV displayed a similar profile to that of parental MV. In particular, in COBL-a cells infected with the virus, no alteration of cellular gene expression, including IFN signaling, was observed. Furthermore, IFN signaling analyzed in vitro was completely suppressed by MV infection in the COBL-a cells. These results reveal that MV induces different cellular responses in a cell-type specific manner. Microarray analyses will provide us useful information about potential mechanisms of MV pathogenesis. [Copyright &y& Elsevier]

Abstract: The C and V proteins of the measles virus (MV) have been shown to block the signaling of type I and II interferon (IFN-α/β and IFN-γ). The relative contribution of the C and V proteins to the inhibition of IFN signaling and the extent to which this activity differs in attenuated or wild-type strains of MV remains undefined. This study presents a comparison of the IFN-antagonist activities of C and V proteins from four attenuated and two wild-type strains of MV. The V proteins were more potent inhibitors of IFN-inducible reporter gene expression than the C proteins, and this effect was unrelated to whether the protein originated from an attenuated or wild-type strain. The results also demonstrated the importance of the tyrosine at position 110 in the inhibition of IFN-α/β and IFN-γ signaling by the V protein, and identified a non-recombinant MV expressing a V protein that was impaired due to a mutation at this residue. [Copyright &y& Elsevier]

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]

Abstract: Our previous results have shown that some residues of V protein-specific domain in human parainfluenza virus type 2 (hPIV2) are essential not only for STAT protein degradation but also for promoting virus growth. Here, we demonstrated that the virus growth of these recombinant hPIV2s (rPIV2) expressing mutated V proteins were improved in HeLa cell transiently expressing the wild-type V protein, but not in the cells constitutively expressing it. Consequently, we identified the region of the V protein that is essential for its oligomerization and for complex formation with NP protein. We also identified a host protein, AlP1/Alix, involved in apoptosis and efficient budding of several enveloped viruses as an interacting partner of the V and NP proteins. Depletion of AIP1/Alix by small interfering RNA suppressed virus growth. These data suggest that the conserved carboxyl terminus of the V protein plays an important role in virus growth. [Copyright &y& Elsevier]

Abstract: The present study was undertaken to clarify the role of Sendai virus (SeV) V protein, which has been shown to downregulate IFN-β induction through inhibition of IRF-3 activation, in viral pathogenesis. Mice infected with rSeV mutants, deficient in V expression or expressing V lacking the C-terminus, had several-fold higher IFN activity levels in the lungs than those in wild-type virus-infected mice, and the mutant viruses were rapidly excluded from the lung from the early phase of infection before induction of acquired immunity. In addition, the unique early clearance of the mutants did not occur in IRF-3 knockout (KO) mice. However, high titers of IFN were detected even in the infected KO mice. Furthermore, early clearance of the mutant viruses was also observed in IFN signaling-deficient mice, IFN-α/β receptor KO mice and STAT1 KO mice. These results indicate that SeV V protein counteracts IRF-3-mediated innate antiviral immunity for efficient virus replication and pathogenesis in mice, but it is not IFN. [Copyright &y& Elsevier]

Abstract: Measles virus V protein is a Cys-rich polypeptide that is dispensable for virus propagation in continuous cell lines, but necessary for efficient viral replication in animals. Those functions modulating virus propagation in vivo are not understood completely, although V protein is known to interfere with the host interferon response and control of viral gene expression. The ability to modulate gene expression was investigated further with a minireplicon transient expression system in which V protein was found to repress reporter activity. Two regions of the polypeptide contributed to this repressive effect including the carboxy-terminus and a region conserved in morbillivirus V proteins located between amino acids 110–131, whereas domains known to mediate the interaction between V and the nucleocapsid (N) protein were not essential. Accumulation of encapsidated minigenome in transfected cells was inhibited by V protein suggesting that it acted as a repressor of genome replication thereby limiting availability of template for reporter gene mRNA transcription. [Copyright &y& Elsevier]

Abstract: Measles virus V protein represses genome replication through a poorly understood mechanism, which led us to investigate whether V protein might be an RNA-binding modulatory factor. Recombinant V protein, expressed from transfected HEp-2 cells or E. coli, formed protein–RNA complexes with poly-guanosine (poly-G) or poly-U linked to agarose beads. RNA binding was not exclusive to ribonucleotide homopolymers as complex formation between V protein and an RNA molecule equivalent to the 3′ terminal 107 bases of the measles virus genome was observed with an electrophoretic mobility shift assay (EMSA). The interaction with poly-G was used to further examine the RNA binding properties of V demonstrating that protein–RNA complex formation was dependent upon the unique Cys-rich carboxy terminus, a region also required to induce maximal repression of minireplicon-encoded reporter gene expression in transient assays. Surprisingly, two mutant proteins that contained Cys-to-Ala substitutions in the C-terminus were found to retain their ability to bind poly-G binding and repress minireplicon reporter gene expression indicating that neither activity was dependent on the integrity of all 7 C-terminal Cys residues. Additional genetic analysis revealed that amino acids 238–266 were necessary for efficient RNA binding and overlapped with residues (238–278) required for maximal repression induced by the C-terminal domain. In addition, a 10 amino acid deletion was identified (residues 238–247) that blocked RNA binding and repression indicating that these two activities were related. [Copyright &y& Elsevier]

We have shown that the P and V proteins of human parainfluenza virus type 2 (hPIV-2) bind to genomic RNA by using Northwestern blot analysis. To identify the RNA-binding regions on the P and V proteins, we used a set of deletion mutants produced in Escherichia coli. One region required for the RNA-binding was found in the P–V common domain (aa 1–82). Others were found in the P protein-specific region (aa 249–354) and the V protein-specific region (aa 176–225). In addition, we have shown that substitutions of some basic residues with alanines in these regions abrogate RNA-binding by the P or V proteins. Intriguingly, the P and V proteins of hPIV2 can selectively bind to the viral RNA under our experimental conditions. [ABSTRACT FROM AUTHOR]

We here report a molecular basis for downregulation of interferon (IFN)-β production by V and C proteins of Sendai virus (SeV). The infection of HeLa cells with SeV poorly induced IFN-β even if the expression of C/C′ was disrupted. In contrast, when the expression of C/C′/Y1/Y2 or V/W was disrupted, SeV infection strongly induced IFN-β production and significantly activated the interferon regulatory factor (IRF)-3 pathway. The independent expression of C or V inhibited the double-stranded (ds) RNA- or Newcastle disease virus (NDV)-induced activation of IRF-3 and NF-κB, as well as the IFN-β promoter. This inhibitory effect was also observed when Y1, Y2, or a C-terminal half fragment (aa 85–204) of C was independently expressed. Phosphorylation and homodimer formation of IRF-3 were suppressed not only in cells infected with SeV capable of expressing both C/C′/Y1/Y2 (or Y1/Y2) and V/W, but also in HeLa cells constitutively expressing Y1. These results suggest that C, Y1, Y2, and V block signaling pathways leading to IRF-3 activation to downregulate IFN-β production. [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]

Sendai virus V protein is not essential for virus replication in cultured cells but is essential for efficient virus replication and pathogenesis in mice, indicating that the V protein has a luxury function to facilitate virus propagation in mice. This was discovered in the Z strain, an egg-adapted avirulent laboratory strain. In the present study, we reexamined the function of Sendai virus V protein by generating a V-knockout Sendai virus derived from the Hamamatsu strain, a virulent field isolate, which is an appropriate model for studying the natural course of Sendai virus infection in mice. We unexpectedly found that the V-knockout virus propagated efficiently in mice and was as virulent as the wild-type virus. Switching of the functionally important V unique region demonstrated that this region of the Hamamatsu strain was also functional in a Z strain background. It thus appears that the V protein is nonsense in a field isolate of Sendai virus. However, the V protein was required for virus growth and pathogenesis of the Hamamatsu strain in mice when the virulence of the virus was attenuated by introducing mutations that had been found in an egg-adapted, avirulent virus. The V protein therefore seems to be potentially functional in the highly virulent Hamamatsu strain and to be prominent if virus replication is restricted. [Copyright &y& Elsevier]

Measles virus (MV), a member of the family Paramyxoviridae, encodes C and V non-structural proteins. To clarify the functions of MV C and V proteins, HeLa cell lines constitutively expressing C or V protein were established. We found that expression of V protein inhibited interferon (IFN)-α/β signaling but not IFN-γ signaling. C protein had no inhibitory effect on IFN signaling in our experimental condition. Degradation of selective signal transducers and activators of transcription (STAT) proteins was not observed in HeLa cells expressing V protein. In contrast, tyrosine phosphorylation of both STAT1 and STAT2 was inhibited in these cells after IFN-β stimulation. [Copyright &y& Elsevier]

Signal transducer and activator of transcription (STAT) proteins are normally long-lived, but infection with certain Paramyxoviruses results in efficient loss of IFN-responsive STAT1 or STAT2. Expression of a virus-encoded protein called “V” is sufficient to mediate the destruction of STAT proteins. STAT degradation is blocked by proteasome inhibitors, strongly implicating the ubiquitin (Ub)-proteasome targeting system. We demonstrate that cellular expression of V proteins from simian virus 5 (SV5) and type II human parainfluenza virus (HPIV2) induces polyubiquitylation of STAT1 and STAT2 targets. In vitro, the V proteins catalyze Ub transfer in an ATP-dependent process that requires both Ub-activating (E1) and Ub-conjugating (E2) activities. Furthermore, SV5 and HPIV2 V-interacting protein partners were isolated by affinity purification from human cells and reveal a complex of associated cellular proteins. This complex includes both STAT1 and STAT2, and the damaged DNA binding protein, DDB1. In addition, a protein related to a family of cellular Ub ligase complex subunits, cullin 4A (Cul4A), associated with the V proteins. The roles of both DDB1 and Cul4A in STAT1 degradation by SV5 infection were analyzed using small interfering RNAs. These findings demonstrate the assembly of a V-dependent degradation complex that includes STAT1, STAT2, DDB1, and Cul4A. In agreement with prior nomenclature on SCF-type cellular E3 enzymes, we refer to this complex as VDC. [Copyright &y& Elsevier]

The V protein of Sendai virus (SeV) is nonessential for virus replication in cell culture but indispensable for viral pathogenicity in mice. At the C terminus of the V protein, there are amino acid residues conserved among the members of the Paramyxovinae subfamily that are clustered in three regions: region I, just downstream of the RNA editing site; and regions II and III, cysteine-rich zinc-finger-like regions. In the present study, we introduced mutations into the conserved amino acids and generated nine mutant viruses. All of the viruses had impaired virus replication in mouse lungs and attenuated virulence in mice. Furthermore, the C-terminal polypeptides fused with glutathione-S-transferase with a mutation in region I, II, or III all had impaired Zn binding in a 65Zn-binding assay in solution. These results demonstrate that the conserved amino acids are important for V protein function, probably via protein conformation dependent on Zn binding. One mutant, SeV V-H318N, had inefficient RNA editing, indicating that the nucleotide that is a part of the codon encoding histidine at position 318 is conserved for the RNA editing machinery. In addition, to determine the function of the C-terminal extension of the V protein, which is not translated in recent virulent field isolates, a translational stop codon was introduced to generate the corresponding short V protein. The mutant virus showed similar virus propagation and pathogenicity, indicating that C-terminal extension of the V protein is not relevant to virus pathogenesis. [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]