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.

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.

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.

Antiviral action of interferon-beta on Newcastle disease virus: selectivity to the hemagglutinin-neuraminidase gene expression.

Interferon-beta (IFN-beta) strongly inhibited the expression of the hemagglutinin-neuraminidase (HN) gene of Newcastle disease virus (NDV), a paramyxovirus, in HeLa cells under the conditions where it did not affect the expression of the four upstream genes encoding the nucleocapsid protein, phosphoprotein, membrane protein and fusion protein. Even the downstream gene, encoding the large protein as well as the genome replication, appeared to be less susceptible to IFN-beta than the HN gene. This selective action of IFN-beta did not appear to be attributable to its well characterized antiviral mechanisms such as acceleration of RNA decay and translation inhibition. No similar down-regulation of a particular gene expression was found with another paramyxovirus, Sendai virus, or with a rhabdovirus, vesicular stomatitis virus, or seems to have been reported previously with any negative-strand RNA viruses. This new effect of IFN-beta thus suggests gene expression mechanism unique to NDV and may further lead to the discovery of a novel biochemical effect of IFN-beta.

A furin-defective cell line is able to process correctly the gp160 of human immunodeficiency virus type 1.

Furin, a subtilisin-like mammalian endoprotease, is thought to be responsible for the processing of many proprotein precursors of cellular and viral origin, including gp160 of human immunodeficiency virus type 1, which share the consensus processing site motif, Arg-X-Lys/Arg-Arg, for protease recognition (for reviews, see P. J. Barr, Cell 66:1-3, 1991, and Y. Nagai, Trends Microbiol. 1:81-87, 1993). To confirm and extend the concept that gp160 is processed by furin, we used here a cell line, LoVo, which was recently demonstrated to be furin defective. Unexpectedly, LoVo cells were found to process gp160 as efficiently as normal cell lines do, hence being able to fuse with CD4-expressing HeLa cells and to produce fully infectious virions. On the other hand, the same cell line was almost totally incapable of processing Newcastle disease virus fusion glycoprotein with a similar oligobasic cleavage recognition motif, providing a strong case for furin-mediated processing. Our present study thus raises a further need to search for and identify the proteinases involved in human immunodeficiency virus type 1 gp160 processing rather than supporting the notion that furin is responsible.

Assignment of disulfide bridges in the fusion glycoprotein of Sendai virus.

The mature fusion (F) glycoprotein of the paramyxovirus family consists of two disulfide-linked subunits, the N-terminal F2 and the C-terminal F1 subunits, and contains 10 cysteine residues which are highly conserved at specific positions. The high level of conservation strongly suggests that they are indeed disulfide linked and play important roles in the folding and functioning of the molecule. However, it has not even been clarified which cysteine residues link the F2 and F1 subunits. This report describes our assignment of the disulfide bridges in purified Sendai virus F glycoprotein by fragmentation of the polypeptide and isolation of cystine-containing peptides and determination of their N-terminal sequences. The data demonstrate that all of the 10 cysteine residues participate in disulfide bridges and that Cys-70, the only cysteine in F2, and Cys-199, the most upstream cysteine in F1, form the interchain bond. Of the remaining eight cysteine residues clustered near the transmembrane domain of F1, the specific bridges identified are Cys-338 to Cys-347 and Cys-362 to Cys-370. Although no exact pairings between the subsequent four residues were defined, it seems likely that the most downstream, Cys-424, is linked to Cys-394, Cys-399, or Cys-401. Thus, we conclude that the cysteine-rich domain indeed contributes to the formation of a bunched structure containing at least two tandem cystine loops.

p60v-src causes tyrosine phosphorylation and inactivation of the N-cadherin-catenin cell adhesion system.

Transformation of chick embryonic fibroblasts with Rous sarcoma virus strongly suppresses N-cadherin-mediated cell-cell adhesion, without inhibiting its expression. This suppression is correlated with tyrosine phosphorylation of N-cadherin and catenins, the cadherin-associated proteins, which are known to regulate cadherin function. Experiments with non-myristylation and temperature-sensitive mutants of RSV and with herbimycin A, a potent inhibitor of tyrosine kinases, suggest that both the suppression of cell adhesion and tyrosine phosphorylation of catenins are highly transformation-specific.

Mammalian subtilisin-related proteinases in cleavage activation of the paramyxovirus fusion glycoprotein: superiority of furin/PACE to PC2 or PC1/PC3.

The fusion glycoprotein precursor of Newcastle disease virus is ubiquitously cleaved in the constitutive secretory pathway if it possesses an oligobasic cleavage motif (RRQR/KR), whereas the precursor is refractory to cleavage if the motif is monobasic (GR/KQGR). We examined the cleavage activity of the mammalian subtilisin-related proteinases furin/PACE, PC2, and PC1/PC3, which are thought to be responsible for proprotein processing in either the constitutive (furin/PACE) or the regulated (PC2 and PC1/PC3) secretory pathway, for the viral precursors with different cleavage motifs. Only furin/PACE was fully capable of cleaving the precursors with the oligobasic motif. PC2 and PC1/PC3 were incapable or only partially capable of cleaving at this motif. None of the proteinases cleaved the monobasic motif. These results suggest involvement of furin/PACE in viral protein processing in the constitutive secretory pathway.

Expression of factor X and its significance for the determination of paramyxovirus tropism in the chick embryo.

Enveloped animal viruses usually possess a surface glycoprotein which mediates fusion between the viral envelope and host cell membrane, hence enabling the initiation of infection, and its biosynthesis often involves post-translational endoproteolytic activation of the inactive precursor by a host cell protease(s). Therefore, the protease distribution in the host must be critical for determining the viral tropism. We previously isolated from chick embryo a cogent candidate endoprotease of this kind for paramyxovirus infection, and demonstrated its identity with factor X (FX), a vitamin K-dependent serine protease in the prothrombin family which, in general, is synthesized in the liver and circulates as one of the plasma proteases essential for blood clotting. Here, we examined FX expression with specific cDNA and antibody probes in a series of embryonic tissues. Many tissues other than the liver expressed the specific mRNA but, in most instances, the translation products remained inactive zymogen forms. The enzymatically active FXa was detectable only in the allantoic fluid and amniotic fluid, and virus spreading was strictly confined to the tissues in direct contact with these FXa-containing fluids. Thus, the ectopically expressed FXa is probably the major host determinant of paramyxovirus tropism in ovo.

Isolation of factor Xa from chick embryo as the amniotic endoprotease responsible for paramyxovirus activation.

In chick embryo, certain paramyxoviruses mainly target the chorioallantois and the allantoamnion and show no extensive further spreading in the other organs. This has been explained by the possible presence of an endoprotease activating the viral fusion glycoprotein precursor in the allantoic and the amniotic fluid, and its absence in other places or organs. We previously isolated such an endoprotease from the allantoic fluid and demonstrated its identity with the clotting factor Xa. Exactly the same endoprotease by all the criteria including the N-terminal amino acid sequence was now isolated from the amniotic fluid. Thus, the factor Xa seems to be a major host determinant of the viral tropism in chick embryo.

Paramyxovirus tropism dependent on host proteases activating the viral fusion glycoprotein.

An essential step in paramyxovirus fusion (F) glycoprotein biosynthesis is the posttranslational endoproteolytic cleavage of the inactive precursor glycoprotein Fo by host cell proteases. When the Fo possesses a pair or a cluster of basic residues at the cleavage site, cleavage is catalyzed by a ubiquitous protease(s) and the infection is consequently pantropic. When the site is monobasic with a single arginine, cleavage is allowed to occur only by the enzyme(s) expressed in limited tissue types and the infection is localized there. We have isolated from chick embryo an example of the latter type of endoprotease specific for the single arginine motif and demonstrate its identity with the clotting factor Xa. The ectopic expression of the FXa appeared to be the sole determinant for the viral tropism in chick embryo. The latter type of protease specific for a paired or multiple basic cleavage motif have neither been identified nor characterized extensively. We show here that this cleavage can be induced by the yeast KEX2 protease, a unique subtilisin-like serine protease, responsible for pro factor processing at the paired basic sites.

Primary structure of the virus activating protease from chick embryo. Its identity with the blood clotting factor Xa.

Host cell proteases activating para- and orthomyxovirus fusion glycoprotein precursors play a crucial role in determining the viral tropism in infected organisms. We previously isolated such an endoprotease from the allantoic fluid of chick embryo and showed its close similarity to the activated form of blood clotting factor X (FXa) by partial amino acid sequencing. In this report, we have cloned and sequenced a cDNA of the protease, and show that it is encoded in a single gene as a preproform with all the functional and structural domains known to be characteristic of bovine or human FX, establishing the identity between the protease and FXa.

Analysis of the protective effect of the haemagglutinin-neuraminidase protein in Newcastle disease virus infection.

The role of immune responses to haemagglutinin-neuraminidase (HN) protein in protection against a Newcastle disease virus (NDV) infection was investigated using a recombinant vaccinia virus expressing HN (HN-RVV). Live HN-RVV replicated in chickens and completely protected them from lethal infection with virulent NDV. Inactivated HN-RVV also protected chickens when administered with adjuvant but not when administered without adjuvant. However, large amounts of the inactivated HN-RVV (100-fold excess) without adjuvant provided protection. Specific antibodies against the HN protein of NDV were detected in sera from survivors but not from dying birds. However, the kinetics of antibody responses in chickens inoculated with live HN-RVV and inactivated HN-RVV were considerably different. These results clearly confirm that immune response(s) solely to the HN protein of NDV can provide chickens with protection against NDV challenge, and show that the presence of antibodies to the HN protein correlates significantly with the protection from NDV infection at least in HN-immunized chickens.

An endoprotease homologous to the blood clotting factor X as a determinant of viral tropism in chick embryo.

Host cell proteases responsible for activation of viral fusion glycoproteins are an important determinant for spread and tropism of various animal viruses. Exemplifying such proteases for the first time, we isolated an endoprotease from chick embryo, that activates para- and orthomyxovirus fusion glycoproteins by cleaving their precursor proteins at a specific, single arginine site. The protease is a calcium dependent serine protease consisting of two subunits, the 33 kd catalytic chain and the 23 kd chain possibly required for Ca2+ binding, and was found to be highly homologous, if not identical, to the blood clotting factor X(FX), a member of the prothrombin family. Its high efficiency and specificity in cleavage reactions was attributable to the properties characteristic of FX. Its role in vivo was strongly supported by cleavage inhibition in ovo highly selective for this virus group with a specific peptide inhibitor against FX.

Growth restriction of negative-strand-RNA viruses in a rat 3Y1 cell line.

A rat fibroblast cell line, 3Y1 is nonpermissive for infection by several negative-strand-RNA viruses including influenza virus A, Sendai virus, Newcastle disease virus and vesicular stomatitis virus (VSV), but not refractory to that of a positive-strand-RNA virus, Sindbis virus. To elucidate the mechanism of the restricted viral growth, we compared the replication pattern of VSV in 3Y1 cells and in baby hamster kidney cells, which are fully permissive for those viruses. The results indicated that the restriction was imposed predominantly on the transcription of the viral RNA. The subsequent steps such as protein synthesis and nucleocapsid assembly appeared to occur normally, although these levels remained low due to the restricted transcription. Virus attachment onto, and penetration into, 3Y1 cells were also not restricted.

Evaluation of the antiviral effect of synthetic oligopeptides whose sequences are derived from paramyxovirus F1 N termini.

We examined the antiviral effects of three oligopeptides, carbobenzoxy(Z)-D-Phe-Ile-Gly, Z-D-Leu-Ile-Gly and Z-D-Phe-Phe-Gly, which mimic the N-terminal regions of F1 glycoproteins of two Newcastle disease virus strains (Miyadera and D26) and Sendai virus, respectively. Only one of these peptides, Z-D-Phe-Phe-Gly, significantly and with a similar potency inhibited viruses of homologous and heterologous F1 N-terminal sequences, suggesting no strict sequence requirement for inhibition. Furthermore, the enveloped RNA viruses of several different families showed essentially the same sensitivity to the three peptides as the paramyxoviruses, while a non-enveloped RNA virus was not susceptible to any of them. In addition, the Z-D-Phe-Phe-Gly peptides was effective only when the virus particles had been pretreated before infection.