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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.

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.

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.

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.

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-V(ko)) 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-V(ko), unlike WT HPIV2, strongly induced IFN-beta and permitted IFN signaling, leading to establishment of a robust antiviral state. rHPIV2-V(ko) infection induced extensive syncytia and cytopathicity that was due to both apoptosis and necrosis. Replication of rHPIV2-V(ko) 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-V(ko) is over-attenuated for use as a live attenuated vaccine.

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.

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.

We here report a molecular basis for downregulation of interferon (IFN)-beta production by V and C proteins of Sendai virus (SeV). The infection of HeLa cells with SeV poorly induced IFN-beta 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-beta 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-kappa B, as well as the IFN-beta 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-beta production.

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.

The present study was undertaken to clarify the role of Sendai virus (SeV) V protein, which has been shown to downregulate IFN-beta 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-alpha/beta 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.