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Identification of the cleavage site recognized by the turnip yellow mosaic virus protease
K L Bransom1, S E Wallace, T W Dreher
1Department of Agricultural Chemistry, Oregon State University, Corvallis 97331-7301, USA.
Abstract:
The noncapsid protein expressed from ORF-206 of turnip yellow mosaic virus (TYMV) is autocatalytically processed by a papain-like protease, producing N-terminal 150-kDa and C-terminal 70-kDa proteins. By introducing two methionine residues near the N-terminus of the 70-kDa protein, we have obtained N-terminal amino acid sequence of that protein produced from [35S]methionine-labeled in vitro translations. The introduction of methionine residues was demonstrated to not interfere with viral replication or proteolysis, as assayed by inoculating mutant RNA transcripts onto whole plants and protoplasts, as well as by translating the RNAs in a rabbit reticulocyte lysate. This has allowed us to determine that the TYMV protease cleaves between alanine1259 and threonine1260 of the precursor protein p206, yielding proteins of calculated Mr 140,618 and 66,037, which will be referred to henceforth as p141 and p66, respectively. The sequence context around the cleavage site is LNGA/TP.
Insights
Turnip yellow mosaic virus (TYMV) noncapsid protein undergoes autocatalytic processing. Researchers pinpointed the precise cleavage site, identifying the resulting p141 and p66 proteins.
Area of Science:
- Plant virology
- Molecular biology
- Protease biochemistry
Background:
- The noncapsid protein of Turnip Yellow Mosaic Virus (TYMV) is processed by an intrinsic protease.
- Understanding this proteolytic cleavage is crucial for viral replication mechanisms.
Purpose of the Study:
- To precisely determine the cleavage site of the TYMV noncapsid protein.
- To identify the N-terminal amino acid sequence of the C-terminal processing product.
Main Methods:
- Introduction of methionine residues into the 70-kDa protein for radiolabeling.
- [35S]methionine-labeled in vitro translation.
- Analysis of viral replication and proteolysis in plants and protoplasts.
- Determination of N-terminal amino acid sequence.
Main Results:
- The TYMV protease cleaves the p206 precursor protein between alanine1259 and threonine1260.
- This cleavage yields N-terminal p141 (Mr 140,618) and C-terminal p66 (Mr 66,037) proteins.
- The sequence context at the cleavage site is Leu-Asn-Gly-Ala/Thr-Pro.
Conclusions:
- The precise cleavage site and resulting protein products (p141 and p66) of TYMV noncapsid protein processing have been identified.
- The introduced methionine residues did not affect viral replication or proteolysis, validating the experimental approach.