Noriko Yokosawa

Measles virus suppresses interferon-α signaling pathway: suppression of Jak1 phosphorylation and association of viral accessory proteins, C and V, with interferon-α receptor complex

Abstract To establish infections, viruses use various strategies to suppress the host defense mechanism, such as interferon (IFN)-induced antiviral state. We found that cells infected with a wild strain of measles virus (MeV) displayed nearly complete suppression of IFN-α-induced antiviral state, but not IFN-γ-induced state. This phenomenon is due to the suppression of IFN-α-inducible gene expression at a transcriptional level. In the IFN-α signal transduction pathway, Jak1 phosphorylation induced by IFN-α is dramatically suppressed in MeV-infected cells; however, phosphorylation induced by IFN-γ is not. We performed immunoprecipitation experiments using antibodies against type 1 IFN receptor chain 1 (INFAR1) and antibody against RACK1, which is reported to be a scaffold protein interacting with type I IFN receptor chain 2 and STAT1. These experiments indicated that IFNAR1 forms a complex containing the MeV-accessory proteins C and V, RACK1, and STAT1 in MeV-infected cells but not in uninfected cells. Composition of this complex in the infected cells altered little by IFN-α treatment. These results indicate that MeV suppresses the IFN-α, but not IFN-γ, signaling pathway by inhibition of Jak1 phosphorylation. Our data suggest that functional disorder of the type I IFN receptor complex is due to “freezing” of the receptor through its association with the C and/or V proteins of MeV.

Induction of suppressor of cytokine signaling-3 by herpes simplex virus type 1 contributes to inhibition of the interferon signaling pathway.

ABSTRACT We showed previously that herpes simplex virus type 1 (HSV-1) suppresses the interferon (IFN) signaling pathway during the early infection stage in the human amnion cell line FL. HSV-1 inhibits the IFN-induced phosphorylation of Janus kinases (JAK) in infected FL cells. In the present study, we showed that the suppressor of cytokine signaling-3 (SOCS3), a host negative regulator of the JAK/STAT pathway, is rapidly induced in FL cells after HSV-1 infection. Maximal levels of SOCS3 protein were detected at around 1 to 2 h after infection. This is consistent with the occurrence of HSV-1-mediated inhibition of IFN-induced JAK phosphorylation. The HSV-1 wild-type strain VR3 induced SOCS3 more efficiently than did mutants that are defective in UL41 or UL13 and that are hyperresponsive to IFN. Induction of the IRF-7 protein and transcriptional activation of IFN-α4, which occur in a JAK/STAT pathway-dependent manner, were poorly induced by VR3 but efficiently induced by the mutant viruses. In contrast, phosphorylation of IRF-3 and transcriptional activation of IFN-β, which are JAK/STAT pathway-independent process, were equally well induced by the wild-type strain and the mutants. In conclusion, the SOCS3 protein appears to be mainly responsible for the suppression of IFN signaling and IFN production that occurs during HSV-1 infection.

C-Terminal Region of STAT-1α Is Not Necessary for Its Ubiquitination and Degradation Caused by Mumps Virus V Protein

ABSTRACT Constitutive levels of production of STAT-1 were reduced by 10 h postinfection (p.i.) and significantly lost by 24 h p.i. in FL cells acutely infected with mumps virus (MuV). This result was consistent with that observed in previous studies and experiments with cells persistently infected with MuV (FLMT cells). There was a marked decrease in the amount of STAT-1 in cells expressing MuV accessory protein V (MuV-V). Furthermore, single amino acid substitutions in the Cys-rich region of V protein (Vc189a, Vc207a, and Vc214a) showed that each cysteine residue plays an important role in the decrease in STAT-1 production, but substitution of a histidine residue at amino acid position 203 had no effect. These events and the resultant suppression of the alpha interferon (IFN-α) response were confirmed by a luciferase reporter gene assay with five tandem repeats of the IFN-α-stimulated response element as an enhancer element of the firely luciferase gene. STAT-1 production was restored and detectable in FLMT cells treated with a proteosome inhibitor, such as MG132 or lactacystin. In the presence of MG132, ubiquitination of STAT-1 and the interaction of MuV-V with STAT-1 were demonstrated in FLMT cells by immunoprecipitation with anti-STAT-1 antibody. The same results for the interaction and ubiquitination were obtained in experiments with an expression vector for a C-terminal deletion mutant of STAT-1. The truncated STAT-1 molecules were degraded in the presence of MuV-V. Therefore, the C-terminal region (transcriptional activation and Src homology 2 domains) of STAT-1 is not necessary for its degradation caused by MuV-V. Our data suggest that MuV-V promotes ubiquitination and degradation of STAT-1.

Association of Mumps Virus V Protein with RACK1 Results in Dissociation of STAT-1 from the Alpha Interferon Receptor Complex

ABSTRACT It has been reported that mumps virus protein V or the C-terminal Cys-rich region of protein V (Vsp) is associated with blocking of the interferon (IFN) signal transduction pathway through a decrease in STAT-1 production. The intracellular target of the V protein was investigated by using a two-hybrid screening system with Vsp as bait. Full-length V protein and Vsp were able to bind to RACK1, and the interaction did not require two WD domains, WD1 and WD2, in RACK1. A significant interaction between V protein and RACK1 was also demonstrated in cells persistently infected with mumps virus (FLMT cells), and the formation of the complex was not affected by treatment with IFN. On the other hand, in uninfected cells, STAT-1 was associated with the long form of the β subunit of the alpha IFN receptor, and this association was mediated by the function of RACK1 as an adaptor protein. Immunoprecipitation and glutathione S-transferase pull-down experiments revealed that the association of RACK1 or mumps virus V protein with the IFN receptor was undetectable in mumps virus-infected cells. Furthermore, RACK1 interacted with mumps virus V protein with a higher affinity than STAT-1 did. Therefore, it is suggested that mumps virus V protein has the ability to interact strongly with RACK1 and consequently to bring about the disruption of the complex formed from STAT-1, RACK1, and the IFN receptor.

Poor induction of interferon-induced 2′,5′-oligoadenylate synthetase (2–5 AS) in cells persistently infected with mumps virus is caused by decrease of STAT-1α

SummaryPoor induction of interferon-induced 2′,5′-oligoadenylate synthetase (2-5AS) has been demonstrated in cells persistently infected with mumps virus as compared with uninfected cells. As for the number of interferon (IFN) receptors and the level of IFN regulatory factors (IRF-1 and IRF-2) mRNAs, there was little difference between them. Therefore, it is suggested that the IFN-α signaling system is ineffective in the persistently infected cells. Components of IFN-stimulating gene factor 3α (ISGF-3α), STAT-1α (p91) and STAT-2 (p113), were investigated in human amnion (FL), human nasopharyngeal cancer (KB), human T-lymphoid (HUT 78), and human B-lymphoid (Akata) cells persistently infected with mumps virus. STAT-1α, but not STAT-2, disappeared in these persistently infected cells, and this factor was not restored by treatment of these cells with IFN. However, no difference was observed between the levels of STAT-1α mRNA transcript in persistently infected and uninfected control cells. It is reason- able to infer that the poor induction of 2-5AS activity is due to the decrease of STAT-1α in correlation with the IFN-signal transduction pathway. Furthermore, induction of other IFN-stimulated genes (ds-RNA activated protein kinase, PKR, and MxA protein) was also reduced in the cells persistently infected with mumps virus.

The complete nucleotide sequence of the gene coding for botulinum type C1 toxin in the C-ST phage genome

Two DNA fragments, 3 kbp and 7.8kbp, which encode the type C1 botulinum neurotoxin gene, were obtained from toxigenic bacteriophage DNA by treatment with a restriction enzyme. They were cloned into the plasmid vectors for nucleotide sequence determination. The nucleotide sequence contained a single open reading frame coding for 1,291 amino acids corresponding to a polypeptide with a molecular weight of 149,000. The amino acid sequence of the C1 toxin has a few regions highly homologous with tetanus toxin.

The complete nucleotide sequence of the gene encoding the nontoxic component of Clostridium botulinum type E progenitor toxin

We have analysed the genes borne on a 6.0 kb HindIII fragment cloned from the chromosome of Clostridium botulinum type E strain Mashike. This fragment, cloned within plasmid pU9EMH, contains part of the structural gene for botulinum toxin type E neurotoxin as well as the entire structural gene for a nontoxic component of botulinum type E progenitor neurotoxin gene, ent-120. ent-120 is transcribed in the same direction as the neurotoxin gene and consists of one open reading frame encoding 1162 amino acid residues. Western blotting with anti-nontoxic component sera demonstrates that ent-120 encodes a protein of 120 kDa which forms part of the nontoxic component. ent-120 is homologous to an analogous gene found in botulinum type C strains (69.3% identity at the nucleotide level and 56.1% at the amino acid level). Two stretches of amino acids at the N-terminus of the ent-120 protein are highly homologous to amino acid sequences within the type E neurotoxin. The stop codon of the ent-120 gene is situated 27 nucleotides upstream from the start codon of the neurotoxin gene.

Measles virus P protein suppresses Toll-like receptor signal through up-regulation of ubiquitin-modifying enzyme A20

We recently reported that the activation of NF‐κB and AP‐1 was suppressed in monocytes infected with measles virus, but not in infected epithelial cells. This cell‐type‐specific suppression of the inflammatory re‐sponse represents a potential for measles virus to evade host immune system. In the current study, we examined the suppression mechanism of lipopolysaccharide (LPS)‐induced, namely Toll‐like receptor 4 (TLR4)‐mediated, activation of NF‐κB and AP‐1 in measles virus‐infected monocytic cells. In the infected cells, LPS treatment failed to induce the formation of active protein kinase complex containing TAK1, TAB2 and tumor necrosis factor receptor‐associated factor 6 (TRAF6), dissociate from TLR complexes containing Interleukin‐1 receptor‐associated kinase 1 (IRAK1). Ubiquitin‐modifying enzyme A20, which is a host negative feedback regulator of NF‐κB, was dramatically up‐regulated in infected monocytic cells, but not in infected epithelial cells. Suppression of A20 expression by siRNA restored LPS‐induced signaling in infected cells. Measles virus phosphoprotein (P protein) expression was necessary and sufficient for the induction of A20. P protein interacted indirectly with a negative regulatory motif in the A20 gene promoter, and released the suppression of A20 transcription, independent of the activation of NF‐κB.— Yokota, S., Okabayashi, T., Yokosawa, N., Fujii, N. Measles virus P protein suppresses Toll‐like receptor signal through up‐regulation of ubiquitin‐modi‐fying enzyme A20. FASEB J. 22, 74–83 (2008)

Cytokine regulation in SARS coronavirus infection compared to other respiratory virus infections

The pathogenesis of severe acute respiratory syndrome (SARS) is poorly understood and cytokine dysregulation has been suggested as one relevant mechanism to be explored. We compared the cytokine profile in Caco2 cells after infection of SARS coronavirus (SARS‐CoV) with other respiratory viruses including respiratory syncytial virus (RSV), influenza A virus (FluAV), and human parainfluenza virus type 2 (hPIV2). Interferon (IFN) system (production and response) was not suppressed by SARS‐CoV infection. Therefore, SARS‐CoV replication was suppressed by pretreatment with IFN. SARS‐CoV and RSV induced high levels of IL‐6 and RANTES compared with FluAV and hPIV2. Induction level of suppressor of cytokine signaling‐3 (SOCS3) by SARS‐CoV was significantly lower than that by RSV in spite of the significant production of IL‐6. Toll‐like receptors 4 and 9, which correlate with the induction of inflammatory response, were upregulated by SARS‐CoV infection. Collectively, overinduction of inflammatory cytokine and dysregulation of cytokine signaling may contribute to the immunopathology associated with “severe” inflammation in SARS. J. Med. Virol. 78:417–424, 2006.

The complete nucleotide sequence of the gene coding for the nontoxic-nonhemagglutinin component of Clostridium botulinum type C progenitor toxin

The structural gene for a nontoxic-nonhemagglutinin component of Clostridium botulinum type C progenitor toxin was found to exist on a 7.8 kb DNA fragment obtained from a type C phage DNA. The gene existed between the neurotoxin and hemagglutinin genes, and consisted of an 3588 bp open reading frame (1196 amino acid residues). It was speculated that this gene and the neurotoxin gene were transcribed by the same mRNA (polycistronic transcription) in C. botulinum organisms.