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A regulatory role for the 32K protein in proteolytic processing of cowpea mosaic virus polyproteins
S A Peters1, W G Voorhorst, J Wery
1Department of Molecular Biology, Agricultural University, Wageningen, The Netherlands.
Abstract:
We have studied the regulation of proteolytic processing of the polyproteins encoded by cowpea mosaic virus M-RNA and B-RNA. For that purpose mutations were introduced in full-length cDNA clones of these RNAs. RNA transcripts were translated in rabbit reticulocyte lysate and the effect of mutations on the processing was analysed. These studies revealed that the 32K protein is released from the 200K B-polyprotein by an intramolecular cleavage and remains associated with the 170K protein, probably by interaction with the 58K domain of the 170K protein. In this complex the conformation of the 170K protein is such that further cleavages are very slow. This complex carries out the processing of the Gln/Met site in the M-polyprotein. The 170K protein produced by a B-RNA mutant that lacks the 32K coding region was efficiently processed into 110K, 87K, 84K, 60K, 58K and 24K cleavage products. Thus, the 32K protein regulates the B-polyprotein processing by slowing it down and, on the other hand, enhances trans cleavage of M-polyproteins at a Gln/Met site.
Insights
Cowpea mosaic virus 32K protein regulates viral polyprotein processing. It slows B-RNA polyprotein cleavage while enhancing M-RNA polyprotein processing at specific sites.
Area of Science:
- Molecular Virology
- Plant Pathology
Background:
- Cowpea mosaic virus (CpMV) replication relies on proteolytic processing of viral polyproteins.
- Understanding the regulation of this processing is crucial for viral replication studies.
Purpose of the Study:
- To investigate the role of the 32K protein in the proteolytic processing of CpMV M-RNA and B-RNA encoded polyproteins.
- To elucidate the mechanism by which the 32K protein influences polyprotein processing.
Main Methods:
- Introduction of mutations into full-length cDNA clones of CpMV M-RNA and B-RNA.
- In vitro translation of RNA transcripts using rabbit reticulocyte lysate.
- Analysis of polyprotein processing products to determine the effect of mutations.
Main Results:
- The 32K protein, released from the B-polyprotein, forms a complex with the 170K protein, slowing down B-polyprotein cleavage.
- This 32K-170K protein complex facilitates the processing of the M-polyprotein at the Gln/Met site.
- A B-RNA mutant lacking the 32K protein showed highly efficient processing of the 170K protein into multiple smaller products.
Conclusions:
- The 32K protein acts as a key regulator of CpMV polyprotein processing.
- It modulates the rate of B-polyprotein processing and enhances M-polyprotein processing.
- This regulatory mechanism is essential for the efficient replication of Cowpea mosaic virus.