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Stimulation of Sendai virus C' protein synthesis by cycloheximide.

ABSTRACT: The polycistronic Sendai virus P/C mRNA is translated into five proteins (P, C', C, Y1 and Y2) from distinct start sites in virus-infected cells. The translation mechanism(s) of these proteins from two overlapping open reading frames in the P/C mRNA are poorly understood [Gupta, Ono and Xu (1996) Biochemistry 35, 1223-1231]. While investigating the initiation mechanism of C' from an ACG start site, we found that C' synthesis was resistant to inhibitors of peptide chain elongation such as cycloheximide (CHX) and anisomycin, but not to pactamycin (an inhibitor of chain initiation) or puromycin (a peptide chain terminator). Moreover, low levels (less than 30 microg/ml) of CHX significantly stimulated C' synthesis. Whereas C' synthesis was stimulated, synthesis of the P and C proteins, which are translated from the same mRNA, decreased by more than 95%. Stimulation of C' synthesis by CHX is not related to its initiation at an ACG codon. Mutation of ACG to alternative start sites had no effect on the CHX-stimulated C' synthesis. Similarly, C' synthesis was preferentially stimulated when Sendai virus-infected cells were exposed to hypotonic growth medium. These results suggest that the P/C mRNA may exist in at least two reversible conformations: whereas one conformation allows synthesis of the P and C proteins, the alternative conformation allows synthesis of the C' protein. It might be that low concentrations of CHX somehow increase the alternative conformation, which increases C' synthesis. The C' protein synthesis is reminiscent of the synthesis of stress-related proteins. Perhaps Sendai virus has evolved a novel mechanism to express both non-stress-related and stress-related proteins from the same mRNA.

SUBMITTER: Gupta KC

PROVIDER: S-EPMC1218139 | biostudies-other | 1997 Feb

REPOSITORIES: biostudies-other

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Project description:UNLABELLED:Sendai virus (SeV) C protein inhibits the signal transduction pathways of interferon alpha/beta (IFN-?/?) and IFN-? by binding to the N-terminal domain of STAT1 (STAT1ND), thereby allowing SeV to escape from host innate immunity. Here we determined the crystal structure of STAT1ND associated with the C-terminal half of the C protein (Y3 [amino acids 99 to 204]) at a resolution of 2.0 Å. This showed that two molecules of Y3 symmetrically bind to each niche created between two molecules of the STAT1ND dimer. Molecular modeling suggested that an antiparallel form of the full-length STAT1 dimer can bind only one Y3 molecule and that a parallel form can bind two Y3 molecules. Affinity analysis demonstrated anticooperative binding of two Y3 molecules with the STAT1 dimer, which is consistent with the hypothetical model that the second Y3 molecule can only target the STAT1 dimer in a parallel form. STAT1 with excess amounts of Y3 was prone to inhibit the dephosphorylation at Tyr(701) by a phosphatase. In an electrophoretic mobility shift assay, tyrosine-phosphorylated STAT1 (pY-STAT1) with Y3 associated with the ?-activated sequence, probably as high-molecular-weight complexes (HMWCs), which may account for partial inhibition of a reporter assay from IFN-? by Y3. Our study suggests that the full-length C protein interferes with the domain arrangement of the STAT1 dimer, leading to the accumulation of pY-STAT1 and the formation of HMWCs. In addition, we discuss the mechanism by which phosphorylation of STAT2 is inhibited in the presence of the C protein after stimulation by IFN-?/?. IMPORTANCE:Sendai virus, a paramyxovirus that causes respiratory diseases in rodents, possesses the C protein, which inhibits the signal transduction pathways of interferon alpha/beta (IFN-?/?) and IFN-? by binding to the transcription factor STAT1. In virus-infected cells, phosphorylation of STAT1 at the Tyr(701) residue is potently enhanced, although transcription by STAT1 is inert. Here, we determined the crystal structure of the N-terminal domain of STAT1 associated with the C-terminal half of the C protein. Molecular modeling and experiments suggested that the two C proteins bind to and stabilize the parallel form of the STAT1 dimer, which are likely to be phosphorylated at Tyr(701), further inducing high-molecular-weight complex formation and inhibition of transcription by IFN-?. We also discuss the possible mechanism of inhibition of the IFN-?/? pathways by the C protein. This is the first structural report of the C protein, suggesting a mechanism of evasion of the paramyxovirus from innate immunity.

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Stimulation of Sendai virus C' protein synthesis by cycloheximide.

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