A detailed kinetic analysis of the in vitro synthesis and processing of encephalomyocarditis virus products.
Translation of encephalomyocarditis virus RNA in rabbit reticulocyte lysates has been used to analyse the pathway of proteolytic processing of the primary translation products. A minimum of two distinct proteases is required to account for the results: one for the excision of the capsid precursor protein, A1, from the nascent polyprotein, and the other for all other cleavages including cleavage at the F/C junction. The excision of A1 is an extremely rapid reaction which occurs as soon as the cleavage site has been synthesised and is resistant to all the proteolytic inhibitors tested and to high temperature, characteristics which are more consistent with an intramolecular cleavage catalysed by a virus-coded protease than cleavage by an endogenous reticulocyte protease. Once excised, A1 remains stable until translation has reached the middle of the region of the genome coding for C, at which time a number of events occur in rapid succession: F is excised in its mature form probably via an intramolecular cleavage; a proteolytic activity capable of secondary processing of A1 to A, B, D1, alpha, gamma and epsilon appears; and a polypeptide of molecular weight about 32,000 appears. This protein (p32) originates from the N-terminal portion of C, and maps in the same position on the genome as p22, the protein previously identified as the virus-coded protease. Polypeptide p32 is derived from C by a single step cleavage generating E as the other product, a processing pathway at least as important, if not more important than the step-wise route via D as an intermediate. Since p32 first appeared at the same time as the start of secondary processing, whilst p22 was first detected much later, it is argued that at least the early stages of processing of the capsid precursor must have been carried out by p32 rather than p22.
D000818:Animals; D002213:Capsid / Q000235:genetics / Q000378:metabolism*; D002474:Cell-Free System; D004686:Encephalomyocarditis virus / Q000235:genetics / Q000378:metabolism*; D005814:Genes, Viral; D007700:Kinetics; D010447:Peptide Hydrolases / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D013696:Temperature; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*
D011498:Protein Precursors; D014764:Viral Proteins; D010447:Peptide Hydrolases