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Stimulation of Sendai virus C' protein synthesis by cycloheximide
1Department of Immunology/Microbiology, Rush Medical College, Chicago, IL 60612, U.S.A.
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.
Insights
Sendai virus P/C mRNA translation yields multiple proteins. Low cycloheximide levels surprisingly boost C
Area of Science:
- Virology
- Molecular Biology
- Protein Synthesis
Background:
- Polycistronic mRNA translation in viruses is complex.
- Sendai virus P/C mRNA encodes five proteins (P, C', C, Y1, Y2) from overlapping reading frames.
- Mechanisms governing differential protein synthesis from this mRNA are not fully understood.
Purpose of the Study:
- To investigate the translation initiation mechanism of the C' protein from Sendai virus P/C mRNA.
- To elucidate the regulatory factors influencing the synthesis of P, C, and C' proteins.
- To explore the potential conformational changes of P/C mRNA during translation.
Main Methods:
- Utilized cycloheximide (CHX), anisomycin, pactamycin, and puromycin to study translation inhibition and initiation.
- Introduced mutations in the ACG start site for C' synthesis.
- Exposed infected cells to hypotonic growth medium to observe effects on protein synthesis.
Main Results:
- C' protein synthesis was resistant to elongation inhibitors (CHX, anisomycin) but sensitive to initiation inhibitors and chain terminators.
- Low CHX concentrations (<30 microg/ml) significantly stimulated C' synthesis while decreasing P and C protein synthesis by over 95%.
- CHX-stimulated C' synthesis was independent of the ACG start codon and also observed under hypotonic conditions.
Conclusions:
- Sendai virus P/C mRNA may adopt at least two reversible conformations, favoring either P/C or C' protein synthesis.
- Low CHX concentrations or hypotonic stress may induce a conformational shift favoring C' protein production.
- This suggests a novel viral mechanism for expressing both non-stress-related (P, C) and stress-related (C') proteins from a single mRNA.