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Polyprotein processing of Theiler's murine encephalomyelitis virus
R P Roos1, W P Kong, B L Semler
1Department of Neurology, University of Chicago Medical Center, Illinois 60637.
Abstract:
To investigate polyprotein processing of Theiler's murine encephalomyelitis viruses, we analyzed in vitro translation reactions programmed by in vitro-derived transcripts from an infectious full-length cDNA clone of the DA strain of Theiler's virus. To help identify the proteinases that carried out the processing, we modified the DA cDNA clone transcription template by linearization with different restriction endonucleases that generate templates of different lengths or by constructing linker insertion or deletion mutations or both in putative proteinase-coding regions. Protein 3C carried out most of the cleavages of the polyprotein, as is true for the other picornaviruses that have been studied. A second proteinase also appeared active at the LP12A-2B junction. A protein of slightly faster mobility than the leader protein was seen with translation of transcripts derived from DA cDNA but not GDVII cDNA. This protein may be synthesized from an alternative initiation site in the DA leader-coding region out of phase with the polyprotein reading frame. Our findings are relevant to ongoing investigations of the abnormal virus expression seen in DA virus late demyelinating disease, since polyprotein processing is critical in regulating picornaviral gene expression.
Insights
Theiler's murine encephalomyelitis virus polyprotein processing was studied using in vitro translation. Protein 3C performed most cleavages, with a second proteinase active at a specific junction, impacting viral gene expression.
Area of Science:
- Virology
- Molecular Biology
- Biochemistry
Background:
- Theiler's murine encephalomyelitis viruses (TMEV) are significant pathogens.
- Understanding viral polyprotein processing is crucial for TMEV pathogenesis.
- Previous studies on picornaviruses highlight the role of specific proteinases.
Purpose of the Study:
- To investigate the polyprotein processing mechanisms of Theiler's murine encephalomyelitis viruses (TMEV).
- To identify the specific proteinases responsible for TMEV polyprotein cleavage.
- To explore potential alternative translation initiation sites in TMEV DA strain.
Main Methods:
- In vitro translation reactions using transcripts from infectious full-length TMEV DA strain cDNA clones.
- Modification of cDNA templates via restriction endonuclease linearization, linker insertion, or deletion mutations.
- Analysis of protein products to identify cleavage sites and active proteinases.
Main Results:
- Protein 3C was identified as the primary proteinase responsible for most polyprotein cleavages in TMEV.
- A second, distinct proteinase activity was observed at the L-P1-2A junction.
- An alternative translation product, potentially from an out-of-frame initiation site, was detected in DA strain transcripts.
Conclusions:
- Protein 3C plays a central role in TMEV polyprotein processing, consistent with other picornaviruses.
- The identified second proteinase activity may contribute to specific cleavage events.
- Alternative translation initiation could influence TMEV gene expression and potentially disease pathogenesis.