Sendai virus C protein physically associates with Stat1.

BACKGROUND: The P/C gene of the Sendai virus (SeV), a member of the family Paramyxoviridae, encodes C protein, which plays a crucial role in counteracting the antiviral effect of interferon (IFN). The C protein blocks IFN signalling to prevent the activation of IFN stimulated genes. However, its underlying molecular mechanism remains to be defined. RESULTS: Signal transducer and activator of transcription 1 (Stat1) is a critical component of IFN-alpha/beta and IFN-gamma signalling. We found that both unphosphorylated Stat1 and tyrosine-phosphorylated (pY) Stat1 were present in a form of aberrant high molecular weight complexes (HMWCs) of over 2 MDa in infected cell extracts under low-salt conditions. Of recombinant vaccinia viruses carrying each SeV gene, only those expressing the C gene induced Stat1-HMWC. SeV infected cell extracts further displayed an in vitro ability to convert the pY-Stat1 homodimer to pY-Stat1-HMWC. This cell extract activity was not seen after removal of the C protein from the extracts. C protein was therefore involved in the formation of HMWCs. The HMWCs decomposed into smaller complexes in a high-salt buffer, and under this stringent (high-salt) condition, as well as a physiological (isotonic) condition, both unphosphorylated Stat1 and pY-Stat1 were co-precipitated with anti-C antibody. CONCLUSION: The C protein physically associates with Stat1. This suggests that SeV C protein directly targets Stat1 for inhibitory control on the transcriptional activation of IFN stimulated genes.

Role of intermolecular disulfide bonds of the organic solvent-stable PST-01 protease in its organic solvent stability.

The PST-01 protease is secreted by the organic solvent-tolerant microorganism Pseudomonas aeruginosa PST-01 and is stable in the presence of various organic solvents. Therefore, the PST-01 strain and the PST-01 protease are very useful for fermentation and reactions in the presence of organic solvents, respectively. The organic solvent-stable PST-01 protease has two disulfide bonds (between Cys-30 and Cys-58 and between Cys-270 and Cys-297) in its molecule. Mutant PST-01 proteases in which one or both of the disulfide bonds were deleted were constructed by site-directed mutagenesis, and the effect of the disulfide bonds on the activity and the various stabilities was investigated. The disulfide bond between Cys-270 and Cys-297 in the PST-01 protease was found to be essential for its activity. The disulfide bond between Cys-30 and Cys-58 played an important role in the organic solvent stability of the PST-01 protease.

Paramyxovirus accessory proteins as interferon antagonists.

A new role of the Paramyxovirus accessory proteins has been uncovered. The P gene of the subfamily Paramyxovirinae encodes accessory proteins including the V and/or C protein by means of pseudotemplated nucleotide addition (RNA editing) or by overlapping open reading frame. The Respirovirus (Sendai virus and human parainfluenza virus (hPIV)3) and Rubulavirus (simian virus (SV)5, SV41, mumps virus and hPIV2) circumvent the interferon (IFN) response by inhibiting IFN signaling. The responsible genes were mapped to the C gene for SeV and the V gene for rubulaviruses. On the other hand, wild type measles viruses isolated from clinical specimens suppress production of IFN, although responsible viral factors remain to be identified. Both human and bovine respiratory syncytial viruses (RSVs) counteract the antiviral effect of IFN with inhibiting neither IFN signaling nor IFN production. Bovine RSV NS1 and NS2 proteins cooperatively antagonize the antiviral effect of IFN. Studies on the molecular mechanism by which viruses circumvent the host IFN response will not only illustrate co-evolution of virus strategies of immune evasion but also provide basic information useful for engineering novel antiviral drugs as well as recombinant live vaccine.

Cloning and sequencing of a gene of organic solvent-stable protease secreted from Pseudomonas aeruginosa PST-01 and its expression in Escherichia coli.

A gene of organic solvent-stable protease (PST-01 protease) secreted by Pseudomonas aeruginosa PST-01 was cloned and its nucleotide was sequenced. The nucleotide sequence analysis revealed that the PST-01 protease was a pseudolysin, which was an elastase produced by P. aeruginosa and was well characterized by the previous investigators. The PST-01 protease produced in recombinant Escherichia coli was not secreted into the extracellular medium, but its proenzyme was released by the lysis of the cells and became a 33.1kDa mature enzyme autoproteolytically. Its characteristics including organic solvent stability were as same as those of the PST-01 protease secreted by P. aeruginosa PST-01.

Sendai virus blocks alpha interferon signaling to signal transducers and activators of transcription.

We demonstrate here that Sendai virus (SeV) blocks alpha interferon (IFN-alpha) signaling to signal transducers and activators of transcription (STATs) in HeLa cells. IFN-alpha-stimulated tyrosine phosphorylation of STATs and subsequent formation of the IFN-stimulated gene factor 3 transcription complex were inhibited in SeV-infected cells, resulting in inefficient induction of IFN-stimulated gene products. None of the components of the signaling pathway-type I IFN receptor subunits Jak1, Tyk2, Stat1, Stat2, and p48-was degraded. Moreover, tyrosine phosphorylation of Jak1 in response to IFN-alpha was unaffected at the early phase of infection, suggesting that oligomerization of the receptor subunits proceeded normally. In contrast to Jak1, IFN-alpha-stimulated tyrosine phosphorylation of Tyk2 was partially inhibited. Therefore, this partial inhibition of activation of Tyk2 probably contributes to the subsequent failure in the activation of STATs.

Knockout of the Sendai virus C gene eliminates the viral ability to prevent the interferon-alpha/beta-mediated responses.

Sendai virus (SeV) renders cells unresponsive to interferon (IFN)-alpha. To identify viral factors involved in this process, we examined whether recombinant SeVs, which could not express V protein, subsets of C proteins (C, C', Y1 and Y2) or any of four C proteins, retained the capability of impeding IFN-alpha-mediated responses. Among these viruses, only the 4C knockout virus completely lost the ability to suppress the induction of IFN-alpha-stimulated gene products and the subsequent establishment of an anti-viral state. These findings reveal crucial roles of the SeV C proteins in blocking IFN-alpha-mediated responses.

Replication-incompetent Sendai virus can suppress the antiviral action of type I interferon.

Altered baby hamster kidney (BHK-R) cells, which were established by serial passage of BHK cells in the presence of Sendai virus (SeV), allowed vesicular stomatitis virus (VSV) to replicate despite treatment with type I interferon (IFN). We have analyzed here mechanisms of the unresponsiveness to IFN. BHK-R cells cultured in the absence of SeV for 10 days under the conditions of no cell division (BHK-R10D) became sensitive to IFN. Studies on induction of unresponsiveness to IFN in BHK-R10D cells revealed that entry of SeV nucleocapsids into a cell was essential. Interestingly, even UV-inactivated SeV but not Newcastle disease virus was found to be able to confer resistance to IFN on HeLa or BHK cells as well as on BHK-R10D cells, suggesting that the IFN-resistance resulted from functions of SeV independent of replication of the viral genome but not from mutations of the cellular genome. Furthermore immunofluorescent experiments demonstrated that UV-inactivated SeV could rescue VSV replication from the antiviral action of IFN without expression of SeV antigens, confirming that the secondary transcription resulting in synthesis of large amounts of viral proteins was dispensable for the IFN-resistance. Thus we have revealed a unique strategy of SeV against the antiviral action of IFN.

Retention of bacterial lipopolysaccharide at the site of subcutaneous injection.

The tissue distribution of Klebsiella pneumoniae O3 lipopolysaccharide (KO3 LPS) was studied in mice injected subcutaneously (s.c.) or intraperitoneally (i.p.) with 125I-labeled KO3 LPS. Marked retention of KO3 LPS radioactivity could be found at the site of s.c. injection for several weeks. On the other hand, about 85% of the radioactivity rapidly disappeared from the peritoneal cavity within 6 h after i.p. injection. The long-term presence of KO3 LPS at the injection site was also supported by experiments with 51Cr-labeled KO3 LPS and immunoblotting and immunofluorescence staining methods. The R-form LPS lacking the O-specific polysaccharide chain of KO3 LPS and the lipid A fraction of KO3 LPS seemed to remain at the site in larger amounts and for longer times than KO3 LPS. There were no marked differences in the retention pattern at the injection site among KO3 LPS, Escherichia coli LPS, Salmonella typhosa LPS, and Salmonella enteritidis LPS. However, much less radioactivity accumulated in the livers and spleens of mice injected with either KO3 LPS or S. typhosa LPS compared with the other LPS preparations. It was suggested that retention of LPS at the site of s.c. injection may play an important role in the development of various biological actions of s.c. injected LPS.

Reversible susceptibility to natural cell-mediated cytotoxicity observed in altered BHK cells resistant to HVJ (Sendai virus) infection.

Altered baby hamster kidney (BHK-R) cells which were subcultured in the continuous presence of HVJ (hemagglutinating virus of Japan--the Sendai strain of parainfluenza 1 virus) showed a high susceptibility to natural cell-mediated cytotoxicity, although BHK-R cells are not transiently or persistently infected with HVJ but contain the restricted amount of sialic acid. By repeated subcultivation of BHK-R cells in growth medium free of HVJ, the sensitivity to natural killer cytotoxicity decreased to the level of normal BHK cells with a counter increase of cellular sialic acid, and the subsequent treatment of the cells with neuraminidase caused a loss of proper sialic acid residues, once again resulting in a significant enhancement of lysis by natural killer cells. In the BHK-R cell system which exhibits a reversible resistance to the interferon action, the enhancing effect induced by interferon on target cell susceptibility to natural killer activity became more pronounced in accord with the recovery of sensitivity to the antiviral action of interferon.

Effect of cytolytic infection on maintenance of resistance to HVJ (Sendai virus) in an altered BHK cell culture.

Altered baby hamster kidney (BHK-R) cells were serially cultured in the continuous presence of hemagglutinating virus of Japan (HVJ). These cells showed a distinct resistance to superinfection with the homologous HVJ. This resistance of BHK-R cells gradually disappeared after serial passages in the presence of ultraviolet-irradiated HVJ particles which lost infectivity but still preserved hemagglutinating and neuraminidase activities. When BHK-R cells were serially cultured in the presence of a temperature-sensitive mutant of HVJ at non-permissive temperature, the cells also lost the resistance. The resistance of BHK-R cells remained unchanged, even after prolonged incubation in virus-free maintenance medium under the conditions of no cell division. It was suggested that killing of virus-sensitive cells, which were generated during cell proliferation, was required for maintenance of the resistance.

Characterization of a porcine kidney cell line resistant to influenza virus infection.

A mutant cell line of porcine kidney cells that resists the cytopathic effect of influenza virus has been obtained and characterized. These cells, designated ESK-R, were originally obtained by prolonged cultivation of cells surviving influenza B/Kanagawa/73 virus infection. No infectious virus was recovered from ESK-R cells, and no evidence for the presence of virus antigens in the cells was demonstrated by immunofluorescent staining. ESK-R cells also showed a distinct resistance to various other strains of both types A and B influenza viruses. The growth of mumps, Sendai, or Newcastle disease virus was considerably restricted, but the cell line normally supported the replication of vesicular stomatitis virus. ESK-R cells were found to lack specific receptors for influenza virus as determined by fluorescence-activated cell sorter analyses. The membrane barrier of ESK-R cells was successfully overcome by nonspecific endocytosis of calcium-coprecipitated virus particles followed by production of an appreciable amount of progeny virus.

Reversible resistance to the antiviral action of interferon observed in altered BHK cells resistant to HVJ (Sendai virus) infection. Brief report.

Altered baby hamster kidney (BHK-R) cells which were subcultured in the continuous presence of HVJ (hemagglutinating virus of Japan--the Sendai strain of parainfluenza 1 virus) showed a resistance to the antiviral action of both type I and II interferons. No evidence for a direct inactivation of interferon molecules during incubation of BHK-R cells was obtained. After serial subculture of BHK-R cells in growth medium free of HVJ, surface membranes with the proper sialic acid residues were restored and the cells became susceptible to the interferon action. It is suggested that binding sites for interferons might be ranked above HVJ receptors in the "receptor gradient".

Trypsin-activated complex of human factor B with cobra venom factor (CVF), cleaving C3 and C5 and generating a lytic factor for unsensitized guinea pig erythrocytes. I. Generation of the activated complex.

A complex formed between cobra venom factor (CVF) and isolated human factor B (B) was found to be converted by trypsin to a stable enzyme, CVF-B which cleaved the third component (C3) and the fifth component (C5) of human complement. The formation of CVF-B by trypsin required divalent cations, whereas the formation of the lytic factor from human serum occurred even in the presence of EDTA. CVF-B purified by gel filtration could initiate the hemolysis of unsensitized guinea pig erythrocytes when incubated with human complement components C5 to C9 in 0.01 M EDTA buffer. C3 was not required for the lysis of guinea pig erythrocytes initiated by CVF-B because of the beta1C precipitation line formed between human serum and anti-beta1C antibody did not inhibit the hemolysis by CVF-B in agarose gel. Treatment of beta1C and beta1F globulins in whole human serum with CVF-B in the presence of 0.01 M EDTA converted them to components with higher mobilities on immunoelectrophoresis.

Trypsin-activated complex of human factor B with cobra venom factor (CVF), cleaving C3 and C5 and generating a lytic factor for unsensitized guinea pig erythrocytes. II. Physico-chemical characterization of the activated complex.

A complex, CVF-B, between cobra venom factor (CVF) and human factor B(B) showed weak, short-lived enzymatic activity against the third component of human complement (C3). Once activated with trypsin, it showed strong, stable activity against C3 and C5. CVF-B, an activated form of CVF-B complex, was not affected by the trypsin inhibitor, diisopropylfluorophosphate and neuraminidase. Heating at 56 C for 30 min completely destroyed its activity and heating at 50 C for 30 min destroyed approximately half its activity. The activity of DVF-B decrease markedly at pH 6.0 but was stable at pH 6.5 to 8.5. CVF-B lost 90% of its activity on reduction with 1 mM dithiothreitol, and was completely adsorbed on a cellulose acetate membrane. CVF-B was found to be a complex of CVF and glycine-rich gamma-glycoprotein, with a molecular weight of 340,000. The CVF-B molecule consisted of 4-polypeptide chains, 3 of which were derived from CVF and one from GGG. Hemolytically active CVF-B may be formed from 2 molecules with four-polypeptide chains linked by unknown bonds. Human, rat and guinea pig sera could react with CVF-B to generate a lytic factor. Human and sheep erythrocytes were not sensitive to the lytic factor generated by CVF-B, whereas liposomes prepared from their membrane lipids were equally sensitive to the lytic factor.