Tripartite motif 2b (trim2b) restricts spring viremia of carp virus by degrading viral proteins and negative regulators NLRP12-like receptors.

Tripartite motif (TRIM) proteins are involved in different cellular functions, including regulating virus infection. In teleosts, two orthologous genes of mammalian TRIM2 are identified. However, the functions and molecular mechanisms of piscine TRIM2 remain unclear. Here, we show that trim2b-knockout zebrafish are more susceptible to spring viremia of carp virus (SVCV) infection than wild-type zebrafish. Transcriptomic analysis demonstrates that NOD-like receptor (NLR), but not RIG-I-like receptor (RLR), signaling pathway is significantly enriched in the trim2b-knockout zebrafish. In vitro, overexpression of Trim2b fails to degrade RLRs and those key proteins involved in the RLR signaling pathway but does for negative regulators NLRP12-like proteins. Zebrafish Trim2b degrades NLRP12-like proteins through its NHL_TRIM2_like and IG_FLMN domains in a ubiquitin-proteasome degradation pathway. SVCV-N and SVCV-G proteins are also degraded by NHL_TRIM2_like domains, and the degradation pathway is an autophagy lysosomal pathway. Moreover, zebrafish Trim2b can interfere with the binding between NLRP12-like protein and SVCV viral RNA and can completely block the negative regulation of NLRP12-like protein on SVCV infection. Taken together, our data demonstrate that the mechanism of action of zebrafish trim2b against SVCV infection is through targeting the degradation of host-negative regulators NLRP12-like receptors and viral SVCV-N/SVCV-G genes.IMPORTANCESpring viremia of carp virus (SVCV) is a lethal freshwater pathogen that causes high mortality in cyprinid fish. In the present study, we identified zebrafish trim2b, NLRP12-L1, and NLRP12-L2 as potential pattern recognition receptors (PRRs) for sensing and binding viral RNA. Zebrafish trim2b functions as a positive regulator; however, NLRP12-L1 and NLRP12-L2 function as negative regulators during SVCV infection. Furthermore, we find that zebrafish trim2b decreases host lethality in two manners. First, zebrafish Trim2b promotes protein degradations of negative regulators NLRP12-L1 and NLRP12-L2 by enhancing K48-linked ubiquitination and decreasing K63-linked ubiquitination. Second, zebrafish trim2b targets viral RNAs for degradation. Therefore, this study reveals a special antiviral mechanism in lower vertebrates.

FIP200 restricts RNA virus infection by facilitating RIG-I activation.

Retinoic acid-inducible gene I (RIG-I) senses viral RNA and instigates an innate immune signaling cascade to induce type I interferon expression. Currently, the regulatory mechanisms controlling RIG-I activation remain to be fully elucidated. Here we show that the FAK family kinase-interacting protein of 200 kDa (FIP200) facilitates RIG-I activation. FIP200 deficiency impaired RIG-I signaling and increased host susceptibility to RNA virus infection. In vivo studies further demonstrated FIP200 knockout mice were more susceptible to RNA virus infection due to the reduced innate immune response. Mechanistic studies revealed that FIP200 competed with the helicase domain of RIG-I for interaction with the two tandem caspase activation and recruitment domains (2CARD), thereby facilitating the release of 2CARD from the suppression status. Furthermore, FIP200 formed a dimer and facilitated 2CARD oligomerization, thereby promoting RIG-I activation. Taken together, our study defines FIP200 as an innate immune signaling molecule that positively regulates RIG-I activation.

Peripheral Injection of Tim-3 Antibody Attenuates VSV Encephalitis by Enhancing MHC-I Presentation.

Viral encephalitis is the most common cause of encephalitis. It is responsible for high morbidity rates, permanent neurological sequelae, and even high mortality rates. The host immune response plays a critical role in preventing or clearing invading pathogens, especially when effective antiviral treatment is lacking. However, due to blockade of the blood-brain barrier, it remains unclear how peripheral immune cells contribute to the fight against intracerebral viruses. Here, we report that peripheral injection of an antibody against human Tim-3, an immune checkpoint inhibitor widely expressed on immune cells, markedly attenuated vesicular stomatitis virus (VSV) encephalitis, marked by decreased mortality and improved neuroethology in mice. Peripheral injection of Tim-3 antibody enhanced the recruitment of immune cells to the brain, increased the expression of major histocompatibility complex-I (MHC-I) on macrophages, and as a result, promoted the activation of VSV-specific CD8+ T cells. Depletion of macrophages abolished the peripheral injection-mediated protection against VSV encephalitis. Notably, for the first time, we found a novel post-translational modification of MHC-I by Tim-3, wherein, by enhancing the expression of MARCH9, Tim-3 promoted the proteasome-dependent degradation of MHC-I via K48-linked ubiquitination in macrophages. These results provide insights into the immune response against intracranial infections; thus, manipulating the peripheral immune cells with Tim-3 antibody to fight viruses in the brain may have potential applications for combating viral encephalitis.

Transcriptome profiling in head kidney of rainbow trout (Oncorhynchus mykiss) after infection with the low-virulent Nagano genotype of infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) causes clinical diseases and mortality in a wide variety of salmonid species. Here, we studied transcriptional responses in rainbow trout infected by the IHNV-Nagano strain isolated in Korea. RNA-seq-based transcriptome analysis of head kidney tissues cataloged differentially expressed genes. Enrichment analysis of gene ontology annotations was performed, and a total of fifteen biological process terms were commonly identified at all time points. In the Kyoto Encyclopedia of Genes and Genomes pathway analysis, pathogen recognition receptor (PRR) signaling pathways such as the retinoic-acid-inducible gene-I-like receptor signaling pathway and the Toll-like receptor signaling pathway were identified at all time points. The nucleotide-binding oligomerization-domain-like receptor signaling pathway and cytosolic DNA-sensing pathway were identified at days 1 and 3. Protein-protein interaction network and centrality analyses revealed that the immune system, signaling molecules, and interaction pathways were upregulated at days 1 and 3, with the highest centrality of tumor necrosis factor. Cancer, cellular community, and endocrine system pathways were downregulated, with the highest centrality of fibronectin 1 at day 5. STAT1 was upregulated from days 1 to 5 with a high centrality. The reproducibility and repeatability of the transcriptome analysis were validated by RT-qPCR. IHNV-Nagano infection dynamically changed the transcriptome profiles in the head kidney of rainbow trout and induced a defense mechanism by regulating the immune and inflammatory pathways through PRR signaling at an early stage. Downregulated pathways involved in extracellular matrix formation and focal adhesion at day 5 indicated the possible failure of wound healing, which is important in the pathogenesis of IHNV infection.

Murine GFP-Mx1 forms nuclear condensates and associates with cytoplasmic intermediate filaments: Novel antiviral activity against VSV.

Type I and III interferons induce expression of the "myxovirus resistance proteins" MxA in human cells and its ortholog Mx1 in murine cells. Human MxA forms cytoplasmic structures, whereas murine Mx1 forms nuclear bodies. Whereas both HuMxA and MuMx1 are antiviral toward influenza A virus (FLUAV) (an orthomyxovirus), only HuMxA is considered antiviral toward vesicular stomatitis virus (VSV) (a rhabdovirus). We previously reported that the cytoplasmic human GFP-MxA structures were phase-separated membraneless organelles ("biomolecular condensates"). In the present study, we investigated whether nuclear murine Mx1 structures might also represent phase-separated biomolecular condensates. The transient expression of murine GFP-Mx1 in human Huh7 hepatoma, human Mich-2H6 melanoma, and murine NIH 3T3 cells led to the appearance of Mx1 nuclear bodies. These GFP-MuMx1 nuclear bodies were rapidly disassembled by exposing cells to 1,6-hexanediol (5%, w/v), or to hypotonic buffer (40-50 mosm), consistent with properties of membraneless phase-separated condensates. Fluorescence recovery after photobleaching (FRAP) assays revealed that the GFP-MuMx1 nuclear bodies upon photobleaching showed a slow partial recovery (mobile fraction: ∼18%) suggestive of a gel-like consistency. Surprisingly, expression of GFP-MuMx1 in Huh7 cells also led to the appearance of GFP-MuMx1 in 20-30% of transfected cells in a novel cytoplasmic giantin-based intermediate filament meshwork and in cytoplasmic bodies. Remarkably, Huh7 cells with cytoplasmic murine GFP-MuMx1 filaments, but not those with only nuclear bodies, showed antiviral activity toward VSV. Thus, GFP-MuMx1 nuclear bodies comprised phase-separated condensates. Unexpectedly, GFP-MuMx1 in Huh7 cells also associated with cytoplasmic giantin-based intermediate filaments, and such cells showed antiviral activity toward VSV.

Lyssavirus P-protein selectively targets STAT3-STAT1 heterodimers to modulate cytokine signalling.

Many viruses target signal transducer and activator of transcription (STAT) 1 to antagonise antiviral interferon signalling, but targeting of STAT3, a pleiotropic molecule that mediates signalling by diverse cytokines, is poorly understood. Here, using lyssavirus infection, quantitative live cell imaging, innate immune signalling and protein interaction assays, and complementation/depletion of STAT expression, we show that STAT3 antagonism is conserved among P-proteins of diverse pathogenic lyssaviruses and correlates with pathogenesis. Importantly, P-protein targeting of STAT3 involves a highly selective mechanism whereby P-protein antagonises cytokine-activated STAT3-STAT1 heterodimers, but not STAT3 homodimers. RT-qPCR and reporter gene assays indicate that this results in specific modulation of interleukin-6-dependent pathways, effecting differential antagonism of target genes. These data provide novel insights into mechanisms by which viruses can modulate cellular function to support infection through discriminatory targeting of immune signalling complexes. The findings also highlight the potential application of selective interferon-antagonists as tools to delineate signalling by particular STAT complexes, significant not only to pathogen-host interactions but also cell physiology, development and cancer.

Chandipura Virus Utilizes the Prosurvival Function of RelA NF-κB for Its Propagation.

Chandipura virus (CHPV), a cytoplasmic RNA virus, has been implicated in several outbreaks of acute encephalitis in India. Despite the relevance of CHPV to human health, how the virus interacts with the host signaling machinery remains obscure. In response to viral infections, mammalian cells activate RelA/NF-κB heterodimers, which induce genes encoding interferon beta (IFN-ß) and other immune mediators. Therefore, RelA is generally considered to be an antiviral transcription factor. However, RelA activates a wide spectrum of genes in physiological settings, and there is a paucity of direct genetic evidence substantiating antiviral RelA functions. Using mouse embryonic fibroblasts, we genetically dissected the role of RelA in CHPV pathogenesis. We found that CHPV indeed activated RelA and that RelA deficiency abrogated the expression of IFN-ß in response to virus infections. Unexpectedly, infection of Rela-/- fibroblasts led to a decreased CHPV yield. Our investigation clarified that RelA-dependent synthesis of prosurvival factors restrained infection-inflicted cell death and that exacerbated cell death processes prevented multiplication of CHPV in RelA-deficient cells. Chikungunya virus, a cytopathic RNA virus associated also with epidemics, required RelA, and Japanese encephalitis virus, which produced relatively minor cytopathic effects in fibroblasts, circumvented the need of RelA for their propagation. In sum, we documented a proviral function of the pleiotropic factor RelA linked to its prosurvival properties. RelA promoted the growth of cytopathic RNA viruses by extending the life span of infected cells, which serve as the replicative niche of intracellular pathogens. We argue that our finding bears significance for understanding host-virus interactions and may have implications for antiviral therapeutic regimes.IMPORTANCE RelA/NF-κB participates in a wide spectrum of physiological processes, including shaping immune responses against invading pathogens. In virus-infected cells, RelA typically induces the expression of IFN-ß, which restrains viral propagation in neighboring cells involving paracrine mechanisms. Our study suggested that RelA might also play a proviral role. A cell-autonomous RelA activity amplified the yield of Chandipura virus, a cytopathic RNA virus associated with human epidemics, by extending the life span of infected cells. Our finding necessitates a substantial revision of our understanding of host-virus interactions and indicates a dual role of NF-κB signaling during the course of RNA virus infections. Our study also bears significance for therapeutic regimes which alter NF-κB activities while alleviating the viral load.

The stress granule protein G3BP1 binds viral dsRNA and RIG-I to enhance interferon-ß response.

RIG-I senses viral RNA in the cytosol and initiates host innate immune response by triggering the production of type 1 interferon. A recent RNAi knockdown screen yielded close to hundred host genes whose products affected viral RNA-induced IFN-ß production and highlighted the complexity of the antiviral response. The stress granule protein G3BP1, known to arrest mRNA translation, was identified as a regulator of RIG-I-induced IFN-ß production. How G3BP1 functions in RIG-I signaling is not known, however. Here, we overexpress G3BP1 with RIG-I in HEK293T cells and found that G3BP1 significantly enhances RIG-I-induced ifn-b mRNA synthesis. More importantly, we demonstrate that G3BP1 binds RIG-I and that this interaction involves the C-terminal RGG domain of G3BP1. Confocal microscopy studies also show G3BP1 co-localization with RIG-I and with infecting vesicular stomatitis virus in Cos-7 cells. Interestingly, immunoprecipitation studies using biotin-labeled viral dsRNA or poly(I·C) and cell lysate-derived or in vitro translated G3BP1 indicated that G3BP1 could directly bind these substrates and again via its RGG domain. Computational modeling further revealed a juxtaposed interaction between G3BP1 RGG and RIG-I RNA-binding domains. Together, our data reveal G3BP1 as a critical component of RIG-I signaling and possibly acting as a co-sensor to promote RIG-I recognition of pathogenic RNA.

Glutamine starvation inhibits snakehead vesiculovirus replication via inducing autophagy associated with the disturbance of endogenous glutathione pool.

Autophagy is a degradation cellular process which also plays an important role in virus infection. Glutamine is an essential substrate for the synthesis of glutathione which is the most abundant thiol-containing compound within the cells and plays a key role in the antioxidant defense and intracellular signaling. There is an endogenous cellular glutathione pool which consists of two forms of glutathione, i.e. the reduced form (GSH) and the oxidized form (GSSG). GSH serves as an intracellular antioxidant to maintain cellular redox homeostasis by scavenging free radicals and other reactive oxygen species (ROS) which can lead to autophagy. Under physiological conditions, the concentration of GSSG is only about 1% of total glutathione, while stress condition can result in a transient increase of GSSG. In our previous report, we showed that the replication of snakehead fish vesiculovirus (SHVV) was significant inhibited in SSN-1 cells cultured in the glutamine-starvation medium, however the underlying mechanism remains enigmatic. Here, we revealed that the addition of L-Buthionine-sulfoximine (BSO), a specific inhibitor of the GSH synthesis, could decrease the γ-glutamate-cysteine ligase (GCL) activity and GSH levels, resulting in autophagy and significantly inhibition of the replication of SHVV in SSN-1 cells cultured in the complete medium. On the other hand, the replication of SHVV was rescued and the autophagy was inhibited in the SSN-1 cells cultured in the glutamine-starvation medium supplemented with additional GSH. Furthermore, the inhibition of the synthesis of GSH had not significantly affected the generation of reactive oxygen species (ROS). However, it significantly decreased level of GSH and enhanced the level of GSSG, resulting in the decrease of the value of GSH/GSSG, indicating that it promoted the cellular oxidative stress. Overall, the present study demonstrated that glutamine starvation impaired the replication of SHVV in SSN-1 cells via inducing autophagy associated with the disturbance of the endogenous glutathione pool.

NV Proteins of Fish Novirhabdovirus Recruit Cellular PPM1Bb Protein Phosphatase and Antagonize RIG-I-Mediated IFN Induction.

Non virion (NV) protein expression is critical for fish Novirhabdovirus, viral hemorrhagic septicemia virus (VHSV) and infectious hematopoietic necrosis virus (IHNV), in vivo pathogenesis. However, the mechanism by which NV promotes the viral replication is still unclear. We developed an approach based on reverse genetics and interactomic and identified several NV-associated cellular partners underlying cellular pathways as potential viral targets. Among these cell partners, we showed that NV proteins specifically interact with a protein phosphatase, Mg2+/Mn2+-dependent, 1Bb (PPM1Bb) and recruit it in the close vicinity of mitochondria, a subcellular compartment important for retinoic acid-inducible gene-I- (RIG-I)-mediated interferon induction pathway. PPM1B proteins belong to the PP2C family of serine/threonine (Ser/Thr) protein phosphatase and have recently been shown to negatively regulate the host antiviral response via dephosphorylating Traf family member-associated NF-κB activator (TANK)-binding kinase 1 (TBK1). We demonstrated that NV proteins and PPM1Bb counteract RIG-I- and TBK1-dependent interferon (IFN) and IFN-stimulated gene promoter induction in fish cells and, hence, the establishment of an antiviral state. Furthermore, the expression of VHSV NV strongly reduced TBK1 phosphorylation and thus its activation. Our findings provide evidence for a previously undescribed mechanism by which a viral protein recruits PPM1Bb protein phosphatase to subvert innate immune recognition.

IRTKS negatively regulates antiviral immunity through PCBP2 sumoylation-mediated MAVS degradation.

RNA virus infection is recognized by the RIG-I family of receptors that activate the mitochondrial adaptor MAVS, leading to the clearance of viruses. Antiviral signalling activation requires strict modulation to avoid damage to the host from exacerbated inflammation. Insulin receptor tyrosine kinase substrate (IRTKS) participates in actin bundling and insulin signalling and its deficiency causes insulin resistance. However, whether IRTKS is involved in the regulation of innate immunity remains elusive. Here we show that IRTKS deficiency causes enhanced innate immune responses against RNA viruses. IRTKS-mediated suppression of antiviral responses depends on the RIG-I-MAVS signalling pathway. IRTKS recruits the E2 ligase Ubc9 to sumoylate PCBP2 in the nucleus, which causes its cytoplasmic translocation during viral infection. The sumoylated PCBP2 associates with MAVS to initiate its degradation, leading to downregulation of antiviral responses. Thus, IRTKS functions as a negative modulator of excessive inflammation.

Transcriptome analysis of epithelioma papulosum cyprini cells after SVCV infection.

BACKGROUND: Spring viraemia of carp virus (SVCV) has been identified as the causative agent of spring viraemia of carp (SVC) and it has caused significant losses in the cultured common carp (Cyprinus carpio) industry. The molecular mechanisms that underlie the pathogenesis of the disease remain poorly understood. In this study, deep RNA sequencing was used to analyse the transcriptome and gene expression profile of EPC cells at progressive times after SVCV infection. This study addressed the complexity of virus-cell interactions and added knowledge that may help to understand SVCV. RESULTS: A total of 33,849,764 clean data from 36,000,000 sequence reads, with a mean read length 100 bp, were obtained. These raw data were assembled into 88,772 contigs. Of these contigs, 19,642 and 25,966 had significant hits to the NR and Uniprot databases where they matched 17,642 and 13,351 unique protein accessions, respectively. At 24 h post SVCV infection (1.0 MOI), a total of 623 genes were differentially expressed in EPC cells compared to non-infected cells, including 288 up-regulated genes and 335 down-regulated genes. These regulated genes were primarily involved in pathways of apoptosis, oxidative stress and the interferon system, all of which may be involved in viral pathogenesis. In addition, 8 differentially expressed genes (DEGs) were validated by quantitative PCR. CONCLUSIONS: Our findings demonstrate previously unrecognised changes in gene transcription that are associated with SVCV infection in vitro, and many potential cascades identified in the study clearly warrant further experimental investigation. Our data provide new clues to the mechanism of viral susceptibility in EPC cells.

Mitogen-activated protein kinase-mediated licensing of interferon regulatory factor 3/7 reinforces the cell response to virus.

The induction of the intrinsic antiviral defense in mammals relies on the accumulation of foreign genetic material. As such, complete engagement of this response is limited to replication-competent viruses. Interferon regulatory factors (IRFs) are mediators of this defense with shared enhancer elements but display a spectrum of transcriptional potential. Here we describe a mechanism designed to enhance this response should a pathogen not be successfully inhibited. We find that activation of IRF7 results in the induction of MAP3K8 and restructuring of the antiviral transcriptome. MAP3K8 mediates the phosphorylation and repression of IRF3 homodimers to promote greater transcriptional activity through utilization of IRF3:IRF7 heterodimers. Among the genes influenced by the MAP3K8/IRF7 signaling axis are members of the SP100 gene family that serve as general transcriptional enhancers of the antiviral defense. We propose that this feed forward loop serves to reinforce the cellular response and is reserved for imminent threats to the host.

Two types of TNF-α exist in teleost fish: phylogeny, expression, and bioactivity analysis of type-II TNF-α3 in rainbow trout Oncorhynchus mykiss.

TNF-α is a cytokine involved in systemic inflammation and regulation of immune cells. It is produced chiefly by activated macrophages as a membrane or secreted form. In rainbow trout, two TNF-α molecules were described previously. In this article, we report a third TNF-α (TNF-α3) that has only low identities to known trout molecules. Phylogenetic tree and synteny analyses of trout and other fish species suggest that two types (named I and II) of TNF-α exist in teleost fish. The fish type-II TNF-α has a short stalk that may impact on its enzymatic release or restrict it to a membrane-bound form. The constitutive expression of trout TNF-α3 was generally lower than the other two genes in tissues and cell lines, with the exception of the macrophage RTS-11 cell line, in which expression was higher. Expression of all three TNF-α isoforms could be modulated by crude LPS, peptidoglycan, polyinosinic:polycytidylic acid, and rIFN-γ in cell lines and primary macrophages, as well as by bacterial and viral infections. TNF-α3 is the most responsive gene at early time points post-LPS stimulation and can be highly induced by the T cell-stimulant PHA, suggesting it is a particularly important TNF-α isoform. rTNF-α3 produced in CHO cells was bioactive in different cell lines and primary macrophages. In the latter, it induced the expression of proinflammatory cytokines (IL-1ß, IL-6, IL-8, IL-17C, and TNF-αs), negative regulators (SOCS1-3, TGF-ß1b), antimicrobial peptides (cathelicidin-1 and hepcidin), and the macrophage growth factor IL-34, verifying its key role in the inflammatory cytokine network and macrophage biology of fish.

Chandipura virus induces neuronal death through Fas-mediated extrinsic apoptotic pathway.

Chandipura virus (CHPV; genus Vesiculovirus, family Rhabdoviridae) is an emerging tropical pathogen with a case fatality rate of 55 to 75% that predominantly affects children in the age group of 2 to 16 years. Although it has been established as a neurotropic virus causing encephalitis, the molecular pathology leading to neuronal death is unknown. The present study elucidates for the first time the mechanism of cell death in neurons after CHPV infection that answers the basic cause of CHPV-mediated neurodegeneration. Through various cell death assays in vitro and in vivo, a relationship between viral replication within neuron and neuronal apoptosis has been established. We report that expression of CHPV phosphoprotein increases up to 6 h postinfection and diminishes thereafter in neuronal cell lines, signifying the replicative phase of CHPV. Various analyses conducted during the investigation established that CHPV-infected neurons are undergoing apoptosis through an extrinsic pathway mediated through the Fas-associated death domain (FADD) following activation of caspase-8 and -3 and prominent cleavage of poly(ADP-ribose) polymerase (PARP). Knocking down the expression of caspase-3, the final executioner of apoptosis, in a neuronal cell line by endoribonuclease-prepared small interfering RNA (siRNA) validated its pivotal role in CHPV-mediated neurodegeneration by showing reduction in apoptosis after CHPV infection.

SARM is required for neuronal injury and cytokine production in response to central nervous system viral infection.

Four of the five members of the Toll/IL-1R domain-containing adaptor family are required for signaling downstream of TLRs, promoting innate immune responses against different pathogens. However, the role of the fifth member of this family, sterile α and Toll/IL-1R domain-containing 1 (SARM), is unclear. SARM is expressed primarily in the CNS where it is required for axonal death. Studies in Caenorhabditis elegans have also shown a role for SARM in innate immunity. To clarify the role of mammalian SARM in innate immunity, we infected SARM(-/-) mice with a number of bacterial and viral pathogens. SARM(-/-) mice show normal responses to Listeria monocytogenes, Mycobacterium tuberculosis, and influenza virus, but show dramatic protection from death after CNS infection with vesicular stomatitis virus. Protection correlates with reduced CNS injury and cytokine production by nonhematopoietic cells, suggesting that SARM is a positive regulator of cytokine production. Neurons and microglia are the predominant source of cytokines in vivo, supporting a role for SARM as a link between neuronal injury and innate immunity.

Proteomic analysis of epithelioma papulosum cyprini cells infected with spring viremia of carp virus.

Spring viremia of carp (SVC), caused by spring viremia of carp virus (SVCV) is an important disease due to its drastic effects on carp fisheries in many countries. To better understand molecular responses to SVCV infection, two dimensional electrophoresis (2-DE) and MALDI-TOF/TOF were performed to investigate altered proteins in epithelioma papulosum cyprini cells (EPCs). Differentially expressed proteins in mock-infected EPCs and SVCV-infected EPCs were compared. A total of 54 differentially expressed spots were successfully identified (33 up-regulated spots and 21 down-regulated spots) which include cytoskeleton proteins, macromolecular biosynthesis-associated proteins, stress response proteins, signal transduction proteins, energy metabolism, and ubiquitin proteasome pathway-associated proteins. Moreover, 7 corresponding genes of the differentially expressed proteins were quantified using real time RT-PCR to examine their transcriptional profiles. The presence of four selected cellular proteins (beta-actin, gamma1-actin, heat shock cognate 71 kDa protein and annexin A2) associated with the spring viremia of carp virus (SVCV) particles was validated by Western blot assay. This study provides dynamic and useful protein-related information to further understand the underlying pathogenesis of SVCV infection.

FoxO1 negatively regulates cellular antiviral response by promoting degradation of IRF3.

Viral infection causes activation of the transcription factor IRF3, which is critical for production of type I interferons (IFNs) and innate antiviral immune response. How virus-induced type I IFN signaling is controlled is not fully understood. Here we identified the transcription factor FoxO1 as a negative regulator for virus-triggered IFN-ß induction. Overexpression of FoxO1 inhibited virus-triggered ISRE activation, IFN-ß induction as well as cellular antiviral response, whereas knockdown of FoxO1 had opposite effects. FoxO1 interacted with IRF3 in a viral infection-dependent manner and promoted K48-linked polyubiquitination and degradation of IRF3 in the cytosol. Furthermore, FoxO1-mediated degradation of IRF3 was independent of the known E3 ubiquitin ligases for IRF3, including RBCK1 and RAUL. Our findings thus suggest that FoxO1 negatively regulates cellular antiviral response by promoting IRF3 ubiquitination and degradation, providing a previously unknown mechanism for control of type I IFN induction and cellular antiviral response.

Viral-induced encephalitis initiates distinct and functional CD103+ CD11b+ brain dendritic cell populations within the olfactory bulb.

Dendritic cells (DC) are antigen-presenting cells found in both lymphoid and nonlymphoid organs, including the brain (bDC) of Cd11c/eyfp transgenic C57BL/6 mice. Using an intranasal vesicular stomatitis virus infection, we demonstrated that EYFP(+) cells amass in areas associated with viral antigens, take on an activated morphology, and project their processes into infected neuronal tissue within the olfactory bulb. These bDC separated into three EYFP(+) CD45(+) CD11b(+) populations, all but one being able to functionally promote both T lymphocyte proliferation and T(H)1 cytokine production. One population was shown to emanate from the brain and a second population was peripherally derived. The third population was of indeterminate origin, being both radiosensitive and not replenished by donor bone marrow. Finally, each EYFP(+) population contained CD11b(+) CD103(+) subpopulations and could be distinguished in terms of CD115, Gr-1, and Ly-6C expression, highlighting mucosal and monocyte-derived DC lineages.

A nuclear localization of the infectious haematopoietic necrosis virus NV protein is necessary for optimal viral growth.

The nonvirion (NV) protein of infectious hematopoietic necrosis virus (IHNV) has been previously reported to be essential for efficient growth and pathogenicity of IHNV. However, little is known about the mechanism by which the NV supports the viral growth. In this study, cellular localization of NV and its role in IHNV growth in host cells was investigated. Through transient transfection in RTG-2 cells of NV fused to green fluorescent protein (GFP), a nuclear localization of NV was demonstrated. Deletion analyses showed that the (32)EGDL(35) residues were essential for nuclear localization of NV protein, and fusion of these 4 amino acids to GFP directed its transport to the nucleus. We generated a recombinant IHNV, rIHNV-NV-ΔEGDL in which the (32)EGDL(35) was deleted from the NV. rIHNVs with wild-type NV (rIHNV-NV) or with the NV gene replaced with GFP (rIHNV-ΔNV-GFP) were used as controls. RTG-2 cells infected with rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL yielded 12- and 5-fold less infectious virion, respectively, than wild type rIHNV-infected cells at 48 h post-infection (p.i.). While treatment with poly I∶C at 24 h p.i. did not inhibit replication of wild-type rIHNVs, replication rates of rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL were inhibited by poly I∶C. In addition, both rIHNV-ΔNV and rIHNV-NV-ΔEGDL induced higher levels of expressions of both IFN1 and Mx1 than wild-type rIHNV. These data suggest that the IHNV NV may support the growth of IHNV through inhibition of the INF system and the amino acid residues of (32)EGDL(35) responsible for nuclear localization are important for the inhibitory activity of NV.