Mst1 shuts off cytosolic antiviral defense through IRF3 phosphorylation.

Cytosolic RNA/DNA sensing elicits primary defense against viral pathogens. Interferon regulatory factor 3 (IRF3), a key signal mediator/transcriptional factor of the antiviral-sensing pathway, is indispensible for interferon production and antiviral defense. However, how the status of IRF3 activation is controlled remains elusive. Through a functional screen of the human kinome, we found that mammalian sterile 20-like kinase 1 (Mst1), but not Mst2, profoundly inhibited cytosolic nucleic acid sensing. Mst1 associated with IRF3 and directly phosphorylated IRF3 at Thr75 and Thr253. This Mst1-mediated phosphorylation abolished activated IRF3 homodimerization, its occupancy on chromatin, and subsequent IRF3-mediated transcriptional responses. In addition, Mst1 also impeded virus-induced activation of TANK-binding kinase 1 (TBK1), further attenuating IRF3 activation. As a result, Mst1 depletion or ablation enabled an enhanced antiviral response and defense in cells and mice. Therefore, the identification of Mst1 as a novel physiological negative regulator of IRF3 activation provides mechanistic insights into innate antiviral defense and potential antiviral prevention strategies.

Down-regulation of heme oxygenase-1 by SVCV infection.

Heme oxygenase (HO-1) is a cytoprotective enzyme that plays a critical role in defending the body against oxidant-induced injury during inflammatory processes. In mammalian systems, viral infection or antigen expression can down-regulate the expression of HO-1. In turn, the induction of HO-1 or overexpression of HO-1 results in potent and direct antiviral activity that targets the replication of several mammalian viruses. In this study, the HO-1 gene of Cyprinus carpio was cloned, and the expression profile of HO-1 was investigated during spring viremia of carp virus (SVCV) infection. The results demonstrate that the expression of HO-1 was down-regulated during SVCV infection in the EPC cells and in common carp. These results indicated that SVCV infection could induce host oxidative stress, which may contribute to tissue injury in affect fish.

Identification and characterization of common carp (Cyprinus carpio L.) granzyme A/K, a cytotoxic cell granule-associated serine protease.

Granzyme (Gzm) is an important member of serine protease family, and key component in the specific and non-specific cell-mediated cytotoxicity. Partial GzmA/K cDNA sequence of common carp (Cyprinus carpio L.) was isolated from thymus cDNA library by the method of suppression subtractive hybridization (SSH). Subsequently, the full length cDNA of carp GzmA/K was obtained by means of 3' RACE and 5' RACE, respectively. The full length cDNA of carp GzmA/K was 1053 bp, consisting of a 5'-terminal untranslated region (UTR) of 65 bp, a 3'-terminal UTR of 214 bp, and an open reading frame of 774 bp. Amino acid sequence analysis indicated the existence of a signal peptide, eight consensus cysteine residues, one conserved IIGG motif and three conserved residues as central elements of the GzmA/K active site. Carp GzmA/K shared 36% and 39% amino acid identity to human GzmA and K, respectively, and was phylogenetically related to the granzyme A and K subgroups. Then, a genomic DNA, which covers the promoter region and entire coding region of carp GzmA/K, was obtained by PCR. In the 5.4 k-long genomic sequence, five exons and four introns were identified. Real-time RT-PCR analysis showed that carp GzmA/K transcript was predominantly detected in the immune-related tissues, and after SVCV infection, was up-regulated in most immune-related tissues in a time-dependent manner. Real-time RT-PCR results also showed that carp GzmA/K transcript was up-regulated in thymus tissue of GH transgenic carp. These results will help to understand the molecular characterization and the potential role of teleost GzmA/K, a cytotoxic cell granule-associated serine protease.

RNA adenosine deaminase ADAR1 deficiency leads to increased activation of protein kinase PKR and reduced vesicular stomatitis virus growth following interferon treatment.

Two size forms of ADAR1 adenosine deaminase are known, one constitutively expressed (p110) and the other interferon (IFN)-induced (p150). To test the role of ADAR1 in viral infection, HeLa cells with ADAR1 stably knocked down and 293 cells overexpressing ADAR1 were utilized. Overexpression of p150 ADAR1 had no significant effect on the yield of vesicular stomatitis virus. Likewise, reduction of p110 and p150 ADAR1 proteins to less than approximately 10 to 15% of parental levels (ADAR1-deficient) had no significant effect on VSV growth in the absence of IFN treatment. However, inhibition of virus growth following IFN treatment was approximately 1 log(10) further reduced compared to ADAR1-sufficient cells. The level of phosphorylated protein kinase PKR was increased in ADAR1-deficient cells compared to ADAR1-sufficient cells following IFN treatment, regardless of viral infection. These results suggest that ADAR1 suppresses activation of PKR and inhibition of VSV growth in response to IFN treatment.

An immune responsive complement factor D/adipsin and kallikrein-like serine protease (PoDAK) from the olive flounder Paralichthys olivaceus.

The cDNA encoding of a complement factor D/adipsin and kallikrein-like serine protease, designated PoDAK, was isolated from the olive flounder Paralichthys olivaceus. PoDAK cDNA encodes a polypeptide with 277 amino acids containing conserved catalytic triad residues of serine proteases. The amino acid sequence of PoDAK showed high similarity to the kallikrein-like protein of medaka, mammalian adipsin/complement factor D and tissue kallikrein homolog, KT-14 of trout, complement factor D of zebrafish, and shared 31.6-36.8% homology with complement factor D/adipsin known from other species, including mammals. Phylogenetic analysis revealed that PoDAK clustered with the kallikrein-like protein of medaka and mammalian adipsin/complement factor D and tissue kallikrein homolog KT-14 of trout. The expression of PoDAK mRNA was high in the gills and heart, moderate in muscle, liver, intestine, stomach, kidney, and spleen of healthy flounder, and increased in the kidney, liver, and spleen of flounder challenged by the viral hemorrhagic septicemia virus (VHSV) or Streptococcus iniae. In situ hybridization confirmed that PoDAK mRNA is localized in the kidney and heart of individuals infected with VHSV. Further investigations are needed to clarify the function of PoDAK in vivo and in vitro.

NK-like and oxidative burst activities are the main early cellular innate immune responses activated after virus inoculation in reservoir fish.

Viral diseases are a major problem in fish farming and a deeper knowledge of the immunological mechanisms playing a part in the antiviral defence is still important. Moreover, fish farming practices (high densities, new areas of culture and egg/larvae/adult transport) are significantly increasing the spread of viruses and the number of susceptible or reservoir fish species. In this last point, no studies have focused on the immunological mechanisms playing a part in the antiviral responses in reservoir and non-susceptible fish species. Thus, we have evaluated the very early innate immune responses of gilthead seabream (Sparus aurata) to the virus causing viral haemorrhagic septicaemia (VHSV) in salmonids since this virus has been found in seabream and neighbouring farmed marine fish species acting as a viral reservoir. The virus was detected in liver, head-kidney, spleen and peritoneal cavity suggesting that the virus reached these tissues but did not replicate as viral expression was almost absent by 72 h post-inoculation. Interestingly, VHSV provoked an influx of leucocytes to the peritoneal cavity and a redistribution of peritoneal exudate (PELs) and head-kidney (HKLs) leucocytes and their innate immune responses (non-specific cytotoxic (NCC or NK-like) activity, phagocytosis, reactive oxygen intermediate (ROI) production and myeloperoxidase (MPO) activity) were generally increased demonstrating that the immune system is activated and involved in the clearance of the virus. Strikingly, NK-like, ROI and MPO were the most enhanced by the presence of VHSV in both PELs and HKLs suggesting that these early innate immune events are crucial during early viral infection stages in non-susceptible or reservoir species. Differences in the immunological mechanisms between susceptible and reservoir species and with other particulate antigens are discussed.

Loss of DExD/H box RNA helicase LGP2 manifests disparate antiviral responses.

The DExD/H box RNA helicase retinoic acid-inducible gene I (RIG-I) and the melanoma differentiation-associated gene 5 (MDA5) are key intracellular receptors that recognize virus infection to produce type I IFN. A third helicase gene, Lgp2, is homologous to Rig-I and Mda5 but lacks a caspase activation and recruitment domain. We generated Lgp2-deficient mice and report that the loss of this gene greatly sensitizes cells to cytosolic polyinosinic/polycytidylic acid-mediated induction of type I IFN. However, negative feedback inhibition of IFN-beta transcription was found to be normal in the absence of LGP2, indicating that LGP2 is not the primary negative regulator of type I IFN production. Our data further indicate that Lgp2-/- mice exhibited resistance to lethal vesicular stomatitis virus infection, a virus whose replicative RNA intermediates are recognized specifically by RIG-I rather than by MDA5 to trigger the production of type I IFN. However, mice lacking LGP2 were observed to exhibit a defect in type I IFN production in response to infection by the encephalomyocarditis virus, the replication of which activates MDA5-dependent innate immune responses. Collectively, our data indicate a disparate regulatory role for LGP2 in the triggering of innate immune signaling pathways following RNA virus infection.

Molecular characterisation and inductive expression of a fish protein arginine methyltransferase 1 gene in response to virus infection.

Protein arginine methyltransferase 1 (PRMT1) is currently thought as an effector to regulate interferon (IFN) signalling. Here Paralichthys olivaceus PRMT1 (PoPRMT1) gene was identified as a virally induced gene from UV-inactivated Scophthalmus maximus Rhabdovirus (SMRV)-infected flounder embryonic cells (FEC). PoPMRT1 encodes a 341-amino-acid protein that shares the conserved domains including post-I, motif I, II and III. Homology comparisons show that the putative PoPMRT1 protein is the closest to zebrafish PMRT1 and belongs to type I PRMT family (including PRMT1, PRMT2, PRMT3, PRMT4, PRMT6, PRMT8). Expression analyses revealed an extensive distribution of PoPMRT1 in all tested tissues of flounder. In vitro induction of PoPRMT1 was determined in UV-inactivated SMRV-infected FEC cells, and under the same conditions, flounder Mx was also transcriptionally up-regulated, indicating that an IFN response might be triggered. Additionally, live SMRV infection of flounders induced an increased expression of PoPRMT1 mRNA and protein significantly in spleen, and to a lesser extent in head kidney and intestine. Immunofluorescence analysis revealed a major cyptoplasmic distribution of PoPRMT1 in normal FEC but an obvious increase occurred in nucleus in response to UV-inactivated SMRV. This is the first report on in vitro and in vivo expression of fish PRMT1 by virus infection, suggesting that PoPRMT1 might be implicated in flounder antiviral immune response.

MEK kinase 1 is a negative regulator of virus-specific CD8(+) T cells.

MEK kinase 1 (MEKK1) is a potent JNK-activating kinase, a regulator of T helper cell differentiation, cytokine production and proliferation in vitro. Using mice deficient for MEKK1 activity (Mekk1(DeltaKD)) exclusively in their hematopoietic system, we show that MEKK1 has a negative regulatory role in the generation of a virus-specific immune response. Mekk1(DeltaKD) mice challenged with vesicular stomatitis virus (VSV) showed a fourfold increase in splenic CD8(+) T cell numbers. In contrast, the number of splenic T cells in infected WT mice was only marginally increased. The CD8(+) T cell expansion in Mekk1(DeltaKD) mice following VSV infection is virus-specific and the frequency of virus-specific T cells is significantly higher (more than threefold) in Mekk1(DeltaKD) as compared to WT animals. Moreover, the hyper-expansion of T cells seen in Mekk1(DeltaKD) mice after VSV infection is a result of increased proliferation, since a significantly higher percentage of virus-specific Mekk1(DeltaKD) CD8(+) T cells incorporated BrdU as compared to virus-specific WT CD8(+) T cells. In contrast, similar levels of apoptosis were detected in Mekk1(DeltaKD) and WT T cells following VSV infection. These results strongly suggest that MEKK1 plays a negative regulatory role in the expansion of virus-specific CD8(+) T cells in vivo.

Contaminants as viral cofactors: assessing indirect population effects.

Current toxicological methods often miss contaminant effects, particularly when immune suppression is involved. The failure to recognize and evaluate indirect and sublethal effects severely limits the applicability of those methods at the population level. In this study, the Vitality model is used to evaluate the population level effects of a contaminant exerting only indirect, sublethal effects at the individual level. Juvenile rainbow trout (Oncorhynchus mykiss) were injected with 2.5 or 10.0 mg/kg doses of the model CYP1A inducer, beta-naphthoflavone (BNF) as a pre-stressor, then exposed to a challenge dose of 10(2) or 10(4) pfu/fish of infectious hematopoietic necrosis virus (IHNV), an important viral pathogen of salmonids in North America. At the end of the 28-d challenge, the mortality data were processed according to the Vitality model which indicated that the correlation between the average rate of vitality loss and the pre-stressor dose was strong: R2=0.9944. Average time to death and cumulative mortality were dependent on the BNF dose, while no significant difference between the two viral dosages was shown, implying that the history of the organism at the time of stressor exposure is an important factor in determining the virulence or toxicity of the stressor. The conceptual framework of this model permits a smoother transfer of results to a more complex stratum, namely the population level, which allows the immunosuppressive results generated by doses of a CYP1A inducer that more accurately represent the effects elicited by environmentally-relevant contaminant concentrations to be extrapolated to target populations. The indirect effects of other environmental contaminants with similar biotransformation pathways, such as polycyclic aromatic hydrocarbons (PAH), could be assessed and quantified with this model and the results applied to a more complex biological hierarchy.

Selective inhibition of COX-2 is beneficial to mice infected intranasally with VSV.

Cyclooxygenase (COX) is the key enzyme for prostaglandin (PG) synthesis. PGs are mediators of many critical physiological and inflammatory responses. There are two isoforms, COX-1 and COX-2, both of which are constitutively expressed in the central nervous system (CNS). Studies have shown that COX-1 and COX-2 are involved in physiological and pathological conditions of the brain. However, little is known about the role(s) of COX in the host defense system against a viral infection in the CNS. In this report, we used Vesicular Stomatitis Virus (VSV) induced acute encephalitis to distinguish between the contribution(s) of the two isoforms. COX-2 activity was inhibited with a COX-2 selective drug, celecoxib (Celebrex), and COX-1 was antagonized with SC560. We found that inhibition of COX-2 led to decreased viral titers, while COX-1 antagonism did not have the same effect at day 1 post infection. 5-lipooxygenase (5-LO) expression and neutrophil recruitment in the CNS were increased in celecoxib-inhibited mice. Furthermore, mice treated with celecoxib expressed more Nitric Oxide Synthase-1 (NOS-1), a crucial component of the innate immune system in the restriction of VSV propagation. The expression of type 1 cytokines, IFN-gamma and IL-12, were also increased in celecoxib-treated mice.

Regulation of the BBB during viral encephalitis: roles of IL-12 and NOS.

Intranasal infection of mice by Vesicular Stomatitis Virus (VSV) often leads to breakdown of the blood-brain barrier (BBB). The role of Interleukin 12 (IL-12) and nitric oxide synthase (NOS) was examined here. Wild-type (WT), NOS-1 knockout (KO), and NOS-3 KO mice were infected with VSV and treated with either IL-12 or medium. IL-12 treatment of uninfected hosts did not result in pathology. In contrast with WT and NOS-1 KO mice, where extensive gross and ultrastructural correlation of BBB breakdown were evident following infection, in NOS-3 KO mice, integrity of the BBB was observed. Thus NOS-3 activity in astrocytes, endothelial cells, or ependymal cells may play an essential role in regulating the BBB.

Activation of type III nitric oxide synthase in astrocytes following a neurotropic viral infection.

Type III nitric oxide synthase (type III NOS), also known as endothelial cell nitric oxide synthase (eNOS or ecNOS or NOS-3), is a constitutively expressed, calcium- and calmodulin-dependent, isoform of NOS. Its expression has been localized to endothelial cells and a subset of neurons in the brain. We report here that resident astrocytes of the central nervous system (CNS) of mice express type III NOS. Following an experimental neurotropic viral infection, the expression of type III NOS on reactive astrocytes increases substantially, predominantly in virally infected regions of the brain. This upregulation of type III NOS expression is also evident following cytokine treatment in vitro. The intraperitoneal (i.p.) administration of IL-12, a potent activator of IFN-gamma and TNF-alpha production, results in a substantial increase in type III NOS immunoreactivity in astrocytes. Cytokine-mediated activation of type III NOS is observed in vitro following exposure of a C6 glioma cells, which constitutively express type III NOS, to IL-12, IFN-gamma, and TNF-alpha treatment. We conclude that astrocytes of the murine CNS express type III NOS, which may be positively regulated by a number of cytokines following viral infection. Type III NOS expression by astrocytes represents a novel source of nitric oxide in the brain. It may be important in regulating perfusion and maintaining the blood-brain barrier. Given the intimate association of astrocytes with endothelial cells and neurons, increased activity of type III NOS following viral infection may be beneficial in inhibition of viral infection in neighboring cells.

IL-12 promotes enhanced recovery from vesicular stomatitis virus infection of the central nervous system.

To investigate the role of a cytokine in host defense against the vesicular stomatitis virus (VSV) infection of the central nervous system (CNS), IL-12 was injected i.p. into groups of 10 BALB/c mice on days -1, 0, 1, 2, and 3 postinfection. Four days postinfection, mice were examined. IL-12 strongly enhanced immunity to VSV infection in the CNS as demonstrated by 1) decreased VSV titers in brain homogenate of IL-12-injected mice compared with those of controls; 2) increased expression of inducible nitric oxide synthase in the CNS; 3) enhanced expression of both MHC class I and class II Ags in the CNS; 4) increased T cell infiltration in the CNS, especially in the olfactory bulb; and 5) diminished VSV-induced apoptosis in olfactory bulb. No detrimental effect was observed even with the 200 ng/mouse dose of IL-12. Protective effects of IL-12 were dose dependent. Collectively, these results demonstrate that exogenously added IL-12, even when injected peripherally, significantly enhances recovery from VSV infection of the CNS.