Leader polypeptides encoded in the 5′-region of the encephalomyocarditis virus genome

Abstract

The synthesis of encephalomyocarditis (EMC)virus proteins, like proteins of other picornaviruses, is initiated at a site located in the 5’-adjacent region of the viral genome; the product of this synthesis is represented by a giant polypeptide chain, polyprotein, which contains all the amino acid sequences present in the structural and non-structural viral proteins; the final products of translation, ‘mature’ proteins, are generated by sequential proteolytic cleavages of the polyprotein molecule starting while the latter is in the nascent form [I]. Using a variety of techniques, cleavage maps of the EMC virus polyprotein were constructed, which showed the order of different polypeptide chains relative to the N-terminus of the polyprotein molecule [ 11. Most of the details of these maps are now generally accepted, but some points remain unclear or disputable. One of these important points concerns the uppermost N-terminal region of the polyprotein: which particular polypeptide is the first to be translated? The usually considered candidate is the precursor of capsid proteins, polypeptide A, which is assumed to share the N-terminal amino acid sequence with its cleavage products, polypeptides B and Dl . This view was supported by the finding that, upon in vitro translation of EMC virus RNA, formyl[35S]Met-tRNAyet could donate a labelled formylmethionyl residue to the polypeptides (by inference, to their N-terminal position) that comigrated with polypeptides A, B and possibly, Dl [2]. On the other hand, earlier data obtained with the same labelled substrate but using less efficient cell-free translation systerns suggested that formyl-methionine might be incorporated in peptides other than those corresponding to the capsid proteins [3-51. We have attempted to solve the controversy and show here that: (i) The initiator amino acid sequences, as defined by the presence of formyl-methionyl residue, are contained in a precursor polypeptide, preA; (ii) Upon a short treatment of preA with a preparation of EMC virus-specific protease [6], these initiator sequences are cleaved off and emerge in two low-M, closely-related polypeptides, p14 and p12; (iii) After the removal of p 14 (p 12) the remaining moiety of preA corresponds to the precursor of capsid proteins, polypeptide A; (iv) preA, p 14 and p 12 appear to share an N-terminal amino acid sequence. These data suggest that all 3 latter polypeptides are initiated at a common single site on the viral genome and that p14 (p 12) should occupy the N-terminal position on the EMC virus polyprotein cleavage map followed by polypeptide A, a precursor of the capsid proteins.