Downloaded from www.microbiologyresearch.org by IP: 54.157.140.51 On: Mon, 04 Apr 2016 08:20:19 Journal of General Virology (1997), 78, 13–21. Printed in Great Britain ........................................................................................................................................................................................................................................................................................... The cleavage activities of aphthovirus and cardiovirus 2A proteins Michelle L. L. Donnelly, 1 David Gani, 1 Mike Flint, 2 Susan Monaghan 2 and Martin D. Ryan 3 1 School of Chemistry, University of St Andrews, Purdie Building, North Haugh, St Andrews KY16 9ST, UK 2 Department of Biochemistry, School of Biology and Medical Sciences, University of St Andrews, Irvine Building, North Street, St Andrews KY16 9AL, UK 3 BBSRC Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK The primary 2A/2B polyprotein cleavage of aphtho- and cardioviruses is mediated by their 2A proteins cleaving C-terminally. Whilst the aphthovirus 2A region is only 16 aa (possibly 18 aa) long, the cardiovirus 2A protein is some 150 aa. We have previously shown that foot-and-mouth disease virus (FMDV) 2A is able to mediate cleavage in an artificial (chloramphenicol acetyltransferase/FMDV 2A/β- glucuronidase [CAT-2A-GUS]) polyprotein system devoid of any other FMDV sequences with high ( 85%), although not complete, cleavage. In this paper we show that insertion of upstream FMDV capsid protein 1D sequences increases the activity. In addition, we have demonstrated that the cardio- virus Theiler’s murine encephalomyelitis virus (TME) 2A protein, when linked to GUS in a single ORF, is able to cleave at its own C terminus with high Introduction Picornavirus genomes contain a single, long, open reading frame encoding a polyprotein of some 225 kDa, although full- length translation products are not normally observed due to extremely rapid ‘ primary’ intramolecular cleavages (in cis) mediated by virus encoded proteinases. Similarities between cellular serine proteinases and both the 2A (entero-, rhino- viruses) and 3C proteinases (all picornaviruses) can be demonstrated by sequence alignments (Gorbalenya et al., 1986 ; Bazan & Fletterick, 1988), or by structural analyses (Allaire et al., 1994 ; Matthews et al., 1994). In the case of the entero- and rhinoviruses, a primary cleavage occurs between the P1 capsid protein precursor and the replicative domains of the polyprotein (P2, P3 ; Fig. 1). This cleavage is mediated by Author for correspondence : Martin D. Ryan. Fax 44 1334 463400. e-mail martin.ryanst-and.ac.uk efficiency – if not completely. The C-terminal 19 aa of TME 2A, together with the N-terminal proline residue of protein 2B, were inserted into the CAT/GUS artificial polyprotein system (in a single ORF). This recombinant [CAT-ΔTME2A-GUS] poly- protein was able to mediate cleavage with high ( 85%) efficiency – directly comparable to the activity observed when FMDV 2A was inserted. A similar insertion into [CAT-GUS] of the C-terminal 19 aa of the cardiovirus encephalomyocarditis virus (EMC) 2A, together with the N-terminal proline residue of protein 2B, produced a [CAT-ΔEMC2A- GUS] polyprotein which also mediated cleavage at  85%. Analysis of the products of expression of these artificial polyproteins in a prokaryotic translation system did not, apparently, reveal any GUS cleavage product. a virus encoded proteinase (2A pro ), of some 17 kDa, cleaving at its own N terminus (Toyoda et al., 1976 ; Sommergruber et al., 1989) and recently demonstrated to be a zinc-containing enzyme (Sommergruber et al., 1994). In contrast, the aphtho-, or foot-and-mouth disease viruses (FMDV) and cardiovirus polyproteins undergo a primary polyprotein cleavage at the C terminus of the 2A region between the capsid protein precursor [(P1–2A) – aphthoviruses ; (L-P1–2A) – cardioviruses] and 2BC and P3 (Fig. 1). Precursors spanning the 2A2B cleavage site are not detected during native polyprotein processing. Whilst the cardiovirus 2A protein ( 15 kDa) is comparable in size to the 2A proteinases of the entero- and rhinovirus groups, no sequence similarity is observed. Although 2A proteins are highly conserved amongst Theiler’s murine encephalomyelitis (TME) viruses and amongst encephalomycarditis (EMC) viruses, only the C-terminal region is highly conserved across the cardiovirus group. The C-terminal region of cardiovirus 2A is, however, highly similar to the much shorter 2A region of 0001-4221  1997 SGM BD