Virus-induced Protein Research Articles

Viperin is induced following toll-like receptor 3 (TLR3) ligation and has a virus-responsive function in human trophoblast cells

IntroductionViperin, a virus-inducible antiviral protein, has been reported to inhibit the replication of a variety of viruses. However, its expression and function in trophoblast cells remains unclear. Toll-like receptor 3 (TLR3) is a key component of the innate immune system that recognizes viral double-stranded RNA and triggers immune reactions by producing type I interferon. We hypothesized that viperin inhibits the replication of human cytomegalovirus (HCMV) in trophoblast cells. MethodsIn situ examinations of viperin expression was conducted in the human first-trimester placenta by immunohistochemical staining. Using a human trophoblast cell culture system, we studied the effect of TLR-3 ligation on viperin expression by treating trophoblasts with polyinosinic-polycytidylic acid [Poly (I: C)] (a synthetic double-stranded RNA, which mimics viral RNA). Viperin mRNA and protein expression were evaluated by real-time PCR and Western blot analysis. In a HCMV infected Swan 71 cell model, HCMV immediate early 1 (IE1) protein mRNA expression was evaluated by real-time PCR after viperin RNA interference. ResultsViperin was localized in trophoblast cells. Poly (I: C) induced viperin expression in a dosage and time-dependent manner. Blocking of TLR3 signaling by neutralizing antibody against IFN-β abolished the stimulation of viperin expression. After HCMV infection, expression of viperin mRNA and protein was unregulated. HCMV IE1 mRNA expression was significantly inhibited by viperin RNA interference. DiscussionViperin is a virus-responsive protein that is constitutively expressed in human trophoblast cells. However, contrary to our hypothesis, viperin facilitates HCMV replication post infection.

Inhibition of reactive oxygen species production ameliorates inflammation induced by influenza A viruses via upregulation of SOCS1 and SOCS3.

Highly pathogenic avian influenza virus infection is associated with severe mortality in both humans and poultry. The mechanisms of disease pathogenesis and immunity are poorly understood although recent evidence suggests that cytokine/chemokine dysregulation contributes to disease severity following H5N1 infection. Influenza A virus infection causes a rapid influx of inflammatory cells, resulting in increased reactive oxygen species production, cytokine expression, and acute lung injury. Proinflammatory stimuli are known to induce intracellular reactive oxygen species by activating NADPH oxidase activity. We therefore hypothesized that inhibition of this activity would restore host cytokine homeostasis following avian influenza virus infection. A panel of airway epithelial and immune cells from mammalian and avian species were infected with A/Puerto Rico/8/1934 H1N1 virus, low-pathogenicity avian influenza H5N3 virus (A/duck/Victoria/0305-2/2012), highly pathogenic avian influenza H5N1 virus (A/chicken/Vietnam/0008/2004), or low-pathogenicity avian influenza H7N9 virus (A/Anhui/1/2013). Quantitative real-time reverse transcriptase PCR showed that H5N1 and H7N9 viruses significantly stimulated cytokine (interleukin-6, beta interferon, CXCL10, and CCL5) production. Among the influenza-induced cytokines, CCL5 was identified as a potential marker for overactive immunity. Apocynin, a Nox2 inhibitor, inhibited influenza-induced cytokines and reactive oxygen species production, although viral replication was not significantly altered in vitro. Interestingly, apocynin treatment significantly increased influenza virus-induced mRNA and protein expression of SOCS1 and SOCS3, enhancing negative regulation of cytokine signaling. These findings suggest that apocynin or its derivatives (targeting host responses) could be used in combination with antiviral strategies (targeting viruses) as therapeutic agents to ameliorate disease severity in susceptible species. Highly pathogenic avian influenza virus infection causes severe morbidity and mortality in both humans and poultry. Wide-spread antiviral resistance necessitates the need for the development of additional novel therapeutic measures to modulate overactive host immune responses after infection. Disease severity following avian influenza virus infection can be attributed in part to hyperinduction of inflammatory mediators such as cytokines, chemokines, and reactive oxygen species. Our study shows that highly pathogenic avian influenza H5N1 virus and low-pathogenicity avian influenza H7N9 virus (both associated with human fatalities) promote inactivation of FoxO3 and downregulation of the TAM receptor tyrosine kinase, Tyro3, leading to augmentation of the inflammatory cytokine response. Inhibition of influenza-induced reactive oxygen species with apocynin activated FoxO3 and stimulated SOCS1 and SOCS3 proteins, restoring cytokine homeostasis. We conclude that modulation of host immune responses with antioxidant and/or anti-inflammatory agents in combination with antiviral therapy may have important therapeutic benefits.

Human hepatitis B virus surface and e antigens inhibit major vault protein signaling in interferon induction pathways

We previously demonstrated that major vault protein (MVP) is a novel virus-induced host factor and its expression upregulates type-I interferon production, leading to cellular antiviral response. However, it remains unclear whether the antiviral function of MVP is impaired during hepatitis B virus (HBV) infection and what mechanisms are involved. Therefore, the aim of this study was to assess whether HBV can alter MVP expression despite the lack of type-I IFN induction and shed light on the underlying mechanisms HBV utilizes to evade host innate immune response. The ability of HBV surface and e antigens to inhibit MVP signaling in interferon induction pathways was evaluated by co-immunoprecipitation, immunofluorescence, quantitative RT-PCR, Western blot and reporter assays. In our current study, we found high levels of MVP in peripheral blood mononuclear cells, sera, and liver tissue from HBV-infected patients relative to healthy individuals. We determined that MVP intracellularly associates with MyD88, an adapter protein involved in virus-triggered induction of type-I IFN. Protein truncation analysis revealed that the middle domain of MVP (amino acid residues 310-620) was essential for MyD88 binding. Conversely, HBV inhibited MVP-induced type-I IFN production by suppressing MVP/MyD88 interaction. HBV antigens, both HBsAg and HBeAg, suppressed this interaction by competitively binding to the essential MyD88 binding region of MVP and limiting downstream IFN signaling. MVP is a virus-induced protein capable of binding with MyD88 leading to type-I IFN production. HBV may evade an immune response by disrupting this interaction and limiting type-I IFN antiviral activity.

Extracellular virion proteins of two Chinese CyHV-3/KHV isolates, and identification of two novel envelope proteins

Proteins in extracellular virions of two Chinese cyprinid herpesvirus-3/koi herpesvirus (CyHV-3/KHV) isolates were identified through one-dimensional gel-based matrix-assisted laser desorption/ionization tandem time of flight mass spectrometry in combination with liquid chromatography tandem mass spectrometry (LC ESI–MS/MS). A total of 43 viral proteins were identified, seven of which (pORF62, 68, 43, 51, 92, 84, and 72) were characterized as marked characteristic viral proteins in CyHV-3/KHV virions and six of which (pORF11, 27, 83, 91, 106 and 116) were not detected previously. Of the newly identified proteins, pORF83 was identified as a novel viral minor envelope protein by Western blot analysis and indirect immunofluorescence assay. Another 27 cellular proteins were validated by LC ESI–MS/MS to be involved in purified extracellular CyHV-3/KHV virions, among which a highly abundant, virus-inducible stress protein was shown and characterized as a cell-derived envelope protein in CyHV-3/KHV virions. Our study extends the number of known CyHV-3/KHV virion-associated viral proteins from 40 to 46 and identifies a novel viral envelope protein as well as a cellular envelope protein. The present findings provide some new insights for better understanding CyHV-3/KHV.

Regulated IRE1-dependent decay pathway is activated during Japanese encephalitis virus-induced unfolded protein response and benefits viral replication

Japanese encephalitis virus (JEV) infection-induced encephalitis causes extensive death or long-term neurological damage, especially among children, in south and south-east Asia. Infection of mammalian cells has shown induction of an unfolded protein response (UPR), presumably leading to programmed cell death or apoptosis of the host cells. UPR, a cellular response to accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) lumen, is initiated by three ER-lumen-resident sensors (PERK, IRE1 and ATF6), and involves transcriptional and translational regulation of the expression of several genes. The sensor IRE1 possesses an intrinsic RNase activity, activated through homo-dimerization and autophosphorylation during UPR. Activated IRE1 performs cytoplasmic cleavage of Xbp1u transcripts, thus facilitating synthesis of XBP1S transcription factor, in addition to cleavage of a cohort of cellular transcripts, the later initiating the regulated IRE1-dependent decay (RIDD) pathway. In this study, we report the initiation of the RIDD pathway in JEV-infected mouse neuroblastoma cells (Neuro2a) and its effect on viral infection. Activation of the RIDD pathway led to degradation of known mouse cell target transcripts without showing any effect on JEV RNA despite the fact that both when biochemically purified showed significant enrichment in ER membrane-enriched fractions. Additionally, inhibition of the IRE1 RNase activity by STF083010, a specific drug, diminished viral protein levels and reduced the titre of the virus produced from infected Neuro2a cells. The results present evidence for the first report of a beneficial effect of RIDD activation on the viral life cycle.

Negative Regulation of Nmi on Virus-Triggered Type I IFN Production by Targeting IRF7

Viral infection causes host cells to produce type I IFNs, which play a critical role in viral clearance. IFN regulatory factor (IRF) 7 is the master regulator of type I IFN-dependent immune responses. In this article, we report that N-Myc and STATs interactor (Nmi), a Sendai virus-inducible protein, interacted with IRF7 and inhibited virus-triggered type I IFN production. The overexpression of Nmi inhibited the Sendai virus-triggered induction of type I IFNs, whereas the knockdown of Nmi promoted IFN production. Furthermore, the enhanced production of IFNs resulting from Nmi knockdown was sufficient to protect cells from infection by vesicular stomatitis virus. In addition, Nmi was found to promote the K48-linked ubiquitination of IRF7 and the proteasome-dependent degradation of this protein. Finally, an impairment of antiviral responses is also detectable in Nmi-transgenic mice. These findings suggest that Nmi is a negative regulator of the virus-triggered induction of type I IFNs that targets IRF7.

Proteomic analysis on the leaves of TaBTF3 gene virus-induced silenced wheat plants may reveal its regulatory mechanism

Basic transcription factor 3 (BTF3) is involved in the transcriptional initiation of RNA polymerase II and is also associated with apoptosis. In this study, virus-induced gene silencing of TaBTF3 caused severe viral symptoms in wheat seedlings, which then displayed stunted growth, reduced height, and decreased total fresh and dry weights. A proteomic approach was further used to identify the protein species showing differential abundance between the TaBTF3 virus-induced gene silenced wheat plants and the barley stripe mosaic virus-induced gene silencing green fluorescent protein transgenic wheat plants (control) with the objective of exploring its regulatory mechanism in higher plants. Using two-dimensional electrophoresis technologies, 59 protein spots showed significant changes, of which 54 were successfully identified by tandem mass spectrometry with matrix-assisted laser desorption/ionization-time of flight spectrometry. Analysis of protein abundance revealed that the differential protein species were associated with signal transduction, stress defense, photosynthesis, carbohydrate metabolism, and protein metabolism, and were mostly localized in both chloroplasts and mitochondria. Furthermore, the BTF3-responsive protein interaction network revealed 20 key protein species, most of which are regulated by abscisic acid, ethane, or oxidative stress. This suggested that changes of these protein species could be critical in the BTF3 pathway. Basic transcription factor 3 (BTF3), the β-subunit of NAC, has originally been identified as a basic transcription factor that is both involved in the transcriptional initiation of RNA polymerase II and associated with diverse biological functions. Reports on BTF3 mainly focus in animals, however, there has been limited molecular information about BTF3 in higher plants so far. In previous studies, we first isolated the TaBTF3 gene from common wheat (Triticum aestivum L.) and obtained silenced transgenic wheat seedlings using the VIGS method. In TaBTF3-silenced transgenic wheat plants, the structure of the wheat mesophyll cell was seriously damaged and transcripts of the chloroplast- and mitochondrial-encoded genes were significantly reduced. These results suggested that the TaBTF3 gene may be involved in regulating the growth and development of wheat seedlings. However, the induced or related genes by TaBTF3 have not been identified. The significance of this study is to first identify many protein species with the altered abundance between the TaBTF3 virus-induced silencing wheat plants and the BSMV-VIGS GFP transgenic wheat plants (control) using the proteomic approach. In addition, 20 of these identified protein species which might play critical roles in the BTF3 interaction network are identified using protein interaction network. These results help to further explore the molecular mechanism of BTF3 in higher plants.

Virions proteins of an RSIV-type megalocytivirus from spotted knifejaw Oplegnathus punctatus (SKIV-ZJ07)

Megalocytiviruses have three main genotypes, which are represented by ISKNV, RSIV, and TRBIV. To date, the virion-associated proteins of RSIV and TRBIV are still unknown. The spotted knifejaw iridovirus (SKIV) is a newly characterized RSIV-type megalocytivirus. In this study, the virion-associated proteins of SKIV were identified by systemic one-dimensional gel electrophoresis-based proteomic approaches. A total of 49 viral proteins and 33 cellular proteins were associated with the SKIV virions by LC MS/MS, including 18 highly abundant structural proteins that were detected by MALDI TOF/TOF-MS. One highly abundant structural protein of interest was identified as the virus-inducible stress protein (VISP) and further characterized as an envelope protein. However, knockdown of mVISP by siRNA method showed no effect in virion production. The current study is the first to present detailed information on the virion-associated proteins of an RSIV-type megalocytivirus and to identify a novel cellular envelope protein of this virus.

Intrahepatic Expression of Programmed Death-1 and its Ligands in Patients with HBV-Related Acute-on-Chronic Liver Failure

Hepatitis B virus (HBV) infection is a major public health problem, and HBV-related acute-on-chronic liver failure (HBV-ACLF) has an extremely poor prognosis due to a lack of understanding of pathogenesis as well as a lack of effective treatments. Signals from the inhibitory receptor programmed death-1 (PD-1) have been demonstrated to be involved in regulating the pathogenesis of infectious diseases. However, the expression of PD-1 and its ligands in HBV-ACLF patients has yet to be evaluated. In this study, the expression of PD-1 and its ligands, PD-L1 and PD-L2, in liver biopsies from HBV-ACLF as well as chronic hepatitis B (CHB) patients were analyzed by immunohistochemistry. The results showed that all three molecules were observed in the HBV-ACLF samples and their levels were significantly higher than they were in CHB. Immunofluorescence double-staining showed that PD-1 was found on CD3(+), CD8(+) T cells, CD56(+) NK cells, CD68(+) macrophages, CK-18(+) epithelial cells, and CD16(+) monocytes. The PD-L1 expression was observed on all cell types detected and the PD-L2 was chiefly on CK-18(+) epithelial cells and CD31(+) endothelial cells. Interestingly, high levels of virus-induced procoagulant molecule fibrinogen-like protein 2 (FGL2) were observed in liver sections from HBV-ACLF, and PD-L1 and PD-L2 expression was also observed on FGL2(+) cells in these patients. Our combined results suggest that the expression of PD-L1 and PD-L2 may be biomarkers to identify and diagnose ACLF, and a clear understanding of their functional roles should further elucidate the pathogenesis of this disease.

Hepatitis B Virus-Induced Calreticulin Protein Is Involved in IFN Resistance

IFN-α is a widely used treatment for hepatitis B virus (HBV) infection, and IFN resistance caused by viral and/or host factors is currently a challenging clinical problem. A better understanding of the molecular mechanisms underlying IFN immunotherapy in the treatment of viral infection would be very beneficial clinically and is of immense clinical importance. Calreticulin (CRT) is an endoplasmic reticulum luminal calcium-binding chaperone that is involved in the regulation of calcium homoeostasis, the folding of newly synthesized proteins, and many other cellular functions. However, little is known about the role of CRT in HBV infection. In this study, we observed high levels of CRT expression in the sera and PBMCs of patients with HBV relative to those of healthy individuals. HBV upregulated the expression of CRT at the transcriptional level. Further investigation showed that HBV-induced CRT enhanced HBV replication by antagonizing the IFN pathway. CRT suppressed the production of endogenous IFN-α by reducing the nuclear translocation of IFN regulatory factor-7 but not IFN regulatory factor-3. Furthermore, CRT also suppressed the antiviral activity of IFN-α by inhibiting the phosphorylation of STAT1 and decreasing the expression of two IFN-α downstream effectors, protein kinase R and 2',5'-oligoadenylate synthetase. Our results offer new insights into the pathogenesis of HBV infection and may provide potential targets for anti-HBV therapy.

Guanylate binding protein (GBP) 4 negatively regulates virus induced type I interferon and antiviral response by targeting interferon regulatory factor (IRF) 7 (168.30)

Abstract In this study, we identify GBP4 as a virus-induced protein interacting with IRF7, which is the master regulator in virus triggered induction of type I IFNs. Overexpression of GBP4 inhibits virus triggered activation of IRF7 dependent signaling, but has no effect on NF-kB signaling, whereas the knockdown of GBP4 has opposite effects. Consistently, the supernatant from sendai virus (SeV) infected cells in which GBP4 has been silenced inhibits the replication of vesicular stomatitis virus (VSV) more efficiently. Competitive coimmunoprecipitation experiments indicate that GBP4 disrupts the interactions between TRAF6 and IRF7, resulting in the impaired virus induced IRF7 phosphorylation. Collectively, our results suggest that GBP4 is a negative regulator of virus triggered type I interferon production, and it is identified as a novel protein targeting IRF7 and inhibiting its function.

Antibody that Targets Virus-Induced Protein Safe Enough for Phase II Trial

Antibody that Targets Virus-Induced Protein Safe Enough for Phase II Trial

Guanylate Binding Protein 4 Negatively Regulates Virus-Induced Type I IFN and Antiviral Response by Targeting IFN Regulatory Factor 7

IRF7 is known as the master regulator in virus-triggered induction of type I IFNs (IFN-I). In this study, we identify GBP4 virus-induced protein interacting with IRF7 as a negative regulator for IFN-I response. Overexpression of GBP4 inhibits virus-triggered activation of IRF7-dependent signaling, but has no effect on NF-κB signaling, whereas the knockdown of GBP4 has opposite effects. Furthermore, the supernatant from Sendai virus-infected cells in which GBP4 have been silenced inhibits the replication of vesicular stomatitis virus more efficiently. Competitive coimmunoprecipitation experiments indicate that overexpression of GBP4 disrupts the interactions between TRAF6 and IRF7, resulting in impaired TRAF6-mediated IRF7 ubiquitination. Our results suggest that GBP4 is a negative regulator of virus-triggered IFN-I production, and it is identified as a novel protein targeting IRF7 and inhibiting its function.

Interleukin-35 Mediates Mucosal Immune Responses That Protect Against T-Cell–Dependent Colitis

The soluble hematopoietin receptor Epstein-Barr virus-induced protein (EBI)-3 is an immune regulator that has been associated with the pathogenesis of inflammatory bowel disease. However, the concept that EBI3 is part of an interleukin (IL)-27 heterodimer that mediates chronic inflammatory and autoimmune diseases has been challenged by the description of IL-35, a bioactive cytokine comprising EBI3 and IL-12 p35. We investigated the roles of IL-27 and IL-35 in chronic inflammation of the intestine. We analyzed EBI3-deficient mice and IL-27p28-deficient mice with spontaneous or T-cell transfer-induced colitis and compared outcomes with wild-type mice (controls). We constructed vectors that express EBI3 covalently linked to the IL-12p35 chain (recombinant [r]IL-35). Intestines of EBI3-deficient mice had increased pathologic features of colitis, compared with IL-27p28-deficient or control mice; they also had shorter survival times, indicating that IL-35, rather than IL-27, protects the intestine from immune responses in mice. The mucosa of EBI3-deficient mice accumulated subsets of activated CD4+ T cells that produced T-helper (Th)1 and Th17 cytokines. Adoptive transfer of these T cells induced colitis in RAG-deficient mice. The rIL-35 significantly reduced the development of several forms of experimental colitis and reduced levels of markers of Th1 and Th17 cells. IL-35 controls the development of T-cell-dependent colitis in mice. It might be developed as a therapeutic target for patients with chronic intestinal inflammation.

Efficiency of VIGS and gene expression in a novel bipartite potexvirus vector delivery system as a function of strength of TGB1 silencing suppression

We have developed plant virus-based vectors for virus-induced gene silencing (VIGS) and protein expression, based on Alternanthera mosaic virus (AltMV), for infection of a wide range of host plants including Nicotiana benthamiana and Arabidopsis thaliana by either mechanical inoculation of in vitro transcripts or via agroinfiltration. In vivo transcripts produced by co-agroinfiltration of bacteriophage T7 RNA polymerase resulted in T7-driven AltMV infection from a binary vector in the absence of the Cauliflower mosaic virus 35S promoter. An artificial bipartite viral vector delivery system was created by separating the AltMV RNA-dependent RNA polymerase and Triple Gene Block (TGB)123-Coat protein (CP) coding regions into two constructs each bearing the AltMV 5' and 3' non-coding regions, which recombined in planta to generate a full-length AltMV genome. Substitution of TGB1 L(88)P, and equivalent changes in other potexvirus TGB1 proteins, affected RNA silencing suppression efficacy and suitability of the vectors from protein expression to VIGS.

Phylogenetic studies of sinipercid fish (Perciformes: Sinipercidae) based on multiple genes, with first application of an immune-related gene, the virus-induced protein (viperin) gene

The sinipercid fish represent a group of 12 species of freshwater percoid fish endemic to East Asia. To date published morphological and molecular phylogenetics hypotheses of sinipercid fish are part congruent, and there are some areas of significant disagreement with respect to species relationships. The present study used separate and combined methods to analyze 7307 bp of data from three mitochondrial genes (cyt b, CO1 and 16S rRNA; ≈2312 bp) and three nuclear genes (viperin, the first two introns of S7 ribosomal protein gene; ≈4995 bp) for the attempts to estimate the relationships among sinipercids and to assess the phylogenetic utility of these markers. Phylogenetic trees were reconstructed using maximum parsimony, maximum likelihood and partitioned Bayesian analyses. Despite the detection of significant heterogeneity of phylogenetic signal between the mitochondrial and nuclear partitions, the combined data analysis represented the best-supported topology of all data. The sinipercid fish form a monophyletic group with two distinct clades, one corresponding to the genus Siniperca and the other to Coreoperca. Coreoperca whiteheadi is the sister taxon to Coreoperca herzi plus Coreoperca kawamebari. In the Siniperca, Siniperca undulata is the sister taxon to the other members of Siniperca, within the subclade containing the other members of the genus, Siniperca chuatsi and Siniperca kneri are sister species, next joined by Siniperca obscura, Siniperca roulei, Siniperca scherzeri and finally by Siniperca fortis. The potential utilities of six different genes for phylogenetic resolution of closely related sinipercid species were also evaluated, with special interest in that of the novel virus-induced protein (viperin) gene.

Humoral anti-proteasomal autoimmunity in dilated cardiomyopathy

Virus-induced chronic inflammation, autoimmune processes and impaired protein quality control may be involved in the pathogenesis of dilated cardiomyopathy (DCM). The ubiquitin-proteasome system is important in the modulation of inflammatory processes and the immune response. Proteasomes were identified as targets of a humoral autoimmune response in systemic inflammatory diseases, which provoked us to investigate anti-proteasomal immunity in DCM in detail: a total of 90 DCM patients with impaired left-ventricular function (LVEF < or = 45%) were enrolled in this study. Autoimmune response to cardiac proteasomes was found to be enhanced in DCM patients, revealing the detection of predominantly alpha subunits of the 20S proteasome complex. Proteasome antibody (ProtAb) levels were found to be particularly enhanced at stages of advanced heart failure: moderately decreased LVEF and considerably increased NT-pro BNP levels were observed in DCM patients who tested positive for ProtAb (P < 0.05). A linear regression model suggested a link between the detection of cardiotropic viruses in endomyocardial biopsies and anti-proteasomal immunity (P < 0.01). Likewise, ProtAb levels were enhanced in a murine model of chronic enterovirus myocarditis. Our data also point to a potential interaction of ProtAb with the cell surface: ProtAb exerted negative inotropic effects in field-stimulated cardiomyocytes. In conclusion, humoral autoreactive anti-proteasome immune responses appear to be enhanced in DCM. Viral infection of the myocardium may be linked to the induction of anti-proteasomal immunity in DCM.

ISG56 is a negative-feedback regulator of virus-triggered signaling and cellular antiviral response

IFN-stimulated gene 56 (ISG56) is one of the first identified proteins induced by viruses and type I IFNs. In this study, we identified ISG56 as a virus-induced protein associated with MITA, an adapter protein involved in virus-triggered induction of type I IFNs. Overexpression of ISG56 inhibited Sendai virus-triggered activation of IRF3, NF-kappaB, and the IFN-beta promoter, whereas knockdown of ISG56 had opposite effects. Consistently, overexpression of ISG56 reversed cytoplasmic poly(I:C)-induced inhibition of vesicular stomatitis virus (VSV) replication, whereas knockdown of ISG56 inhibited VSV replication. Competitive coimmunoprecipitation experiments indicated that ISG56 disrupted the interactions between MITA and VISA or TBK1, two components in the virus-triggered IFN signaling pathways. These results suggest that ISG56 is a mediator of negative-feedback regulation of virus-triggered induction of type I IFNs and cellular antiviral responses.

The Degree of Folding Instability of the Envelope Protein of a Neurovirulent Murine Retrovirus Correlates with the Severity of the Neurological Disease

A small group of ecotropic murine retroviruses cause a spongiform neurodegenerative disease manifested by tremor, paralysis, and wasting. The neurovirulence of these viruses has long been known to be determined by the sequence of the viral envelope protein, although the nature of the neurotoxicity remains to be clarified. Studies on the neurovirulent viruses FrCas(NC) and Moloney murine leukemia virus ts1 indicate that the nascent envelope protein misfolds, is retained in the endoplasmic reticulum (ER), and induces an unfolded protein response. In the present study we constructed a series of viruses with chimeric envelope genes containing segments from virulent and avirulent retroviruses. Each of the viruses studied was highly neuroinvasive but differed in the severity of the neurological disease they induced. Only viruses that contained the receptor-binding domain (RBD) of the neurovirulent virus induced neurological disease. Likewise, only viruses containing the RBD of the neurovirulent virus exhibited increased binding of the ER chaperone BiP to the envelope precursor protein and induced the unfolded protein response. Thus, the RBD determined both neurovirulence and folding instability. Among viruses carrying the neurovirulent RBD, the severity of the disease was increased when envelope sequences from the neurovirulent virus outside the RBD were also present. Interestingly, these sequences appeared to further increase the degree of folding instability (BiP binding) of the viral envelope protein. These results provide strong support for the hypothesis that this spongiform neurodegenerative disease represents a virus-induced protein folding disorder.

Cloning and characterization of VIGG, a novel virus-induced grapevine protein, correlated with fruit quality

We report here the identification and characterization of VIGG, a novel virus-induced grapevine protein. Analysis of VIGG expression in grapevine demonstrated that VIGG was constitutively expressed in leaves and stems in virus-infected grapevine, and that VIGG expression was induced by grapevine virus A (GVA) infection, but not by infection with other viruses. The virus-induced expression profile of VIGG was supported by the finding that virus-free meristem cultures prepared from virus-infected grapevines did not express VIGG. An experiment using GFP-VIGG fusion protein demonstrated that VIGG might be localized in or around the endoplasmic reticulum (ER). Treatment of grapevine cells with ER stress inducers resulted in the induction of VIGG expression. Berries from VIGG-expressing grapevines had higher organic acid and phenolic contents than those from control grapevines that did not express VIGG. Interestingly, fruit composition of a grapevine that was simultaneously infected by GVA and grapevine virus B (GVB), which did not express VIGG, was significantly different from that of GVA-infected grapevines expressing VIGG, suggesting that the effector of fruit composition alteration might be VIGG expression, but not GVA infection. Taken together, VIGG expression might suppress the decrease in organic acid content and increase phenol content in berries. Further investigation of the biological function of VIGG is expected to provide new information on the fruit quality of grapevines.