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Initiation of picornavirus protein synthesis in ascites cell extracts
Abstract:
The current model of picornavirus protein formation implies that initiation of protein synthesis occurs at a single site on the viral RNA, and that the large polypeptide formed is later cleaved. A direct test of this model was made in vitro by studying the incorporation of [(35)S]methionine from rabbit liver Met-tRNA(M) (Met) and fMet-tRNA(F) (Met) into encephalomyocarditis virus RNA-coded proteins in extracts of Ehrlich ascites cells. The incorporation of N-formylmethionine was complete within 5 min, while utilization of Met-tRNA(M) (Met) continued for 20 min. Tryptic digests of [(35)S]methionine-labeled products from Met-tRNA(M) (Met) analyzed by anion-exchange chromatography yielded more than 30 peptides, as compared to about 15 [(35)S]methionine-labeled peptides from purified encephalomyocarditis virus. In contrast, products labeled with fMet-tRNA(F) (Met) yielded one major (26)S-labeled tryptic peptide. The N-terminal location of methionine in this peptide was verified by Edman degradation. One predominant N-terminal tryptic peptide was also obtained with fMet-tRNA(F) (Met) when mouse Elberfeld and mengo-virus RNAs were used as messengers. On the basis of N-terminal compared with internal labeling of the products, no evidence for in vitro post-translational cleavage was found. The results are consistent with a single initiation site for synthesis of picornavirus proteins.
Insights
This study investigated picornavirus protein synthesis initiation. Results show a single initiation site, challenging models suggesting multiple sites and post-translational cleavage for encephalomyocarditis virus protein formation.
Area of Science:
- Molecular Biology
- Virology
- Protein Synthesis
Background:
- Picornavirus protein synthesis models propose a single initiation site followed by cleavage.
- Investigating the precise mechanism of viral protein formation is crucial for understanding viral replication.
Purpose of the Study:
- To directly test the in vitro model of picornavirus protein synthesis.
- To determine if encephalomyocarditis virus (EMCV) protein synthesis initiates at a single site.
- To investigate potential post-translational cleavage events.
Main Methods:
- In vitro protein synthesis using rabbit liver Met-tRNA(M) (Met) and fMet-tRNA(F) (Met) with EMCV RNA in Ehrlich ascites cell extracts.
- Analysis of [(35)S]methionine incorporation patterns.
- Peptide mapping of tryptic digests using anion-exchange chromatography.
- Edman degradation to verify N-terminal methionine placement.
Main Results:
- N-formylmethionine incorporation was rapid (5 min), while Met-tRNA(M) (Met) utilization was prolonged (20 min).
- Labeling with fMet-tRNA(F) (Met) yielded a single major N-terminal tryptic peptide, confirmed by Edman degradation.
- Labeling with Met-tRNA(M) (Met) produced over 30 peptides, unlike the ~15 from purified EMCV.
- Similar results were observed with mouse Elberfeld and mengo-virus RNAs, indicating a conserved mechanism.
Conclusions:
- The findings support a single initiation site for picornavirus protein synthesis.
- No evidence for in vitro post-translational cleavage was found, contradicting some models.
- This research clarifies the fundamental process of viral protein production.