Abstract
Strawberry mottle virus (SMoV, family Secoviridae, order Picornavirales) is one of several viruses found in association with strawberry decline disease in Eastern Canada. The SMoV genome consists of two positive-sense single-stranded RNAs, each encoding one large polyprotein. The RNA1 polyprotein (P1) includes the domains for a putative helicase, a VPg, a 3C-like cysteine protease and an RNA-dependent RNA polymerase at its C-terminus, and one or two protein domains at its N-terminus. The RNA2 polyprotein (P2) is predicted to contain the domains for a movement protein (MP) and one or several coat proteins at its N-terminus, and one or more additional domains for proteins of unknown function at its C-terminus. The RNA1-encoded 3C-like protease is presumed to cleave the two polyproteins in cis (P1) and in trans (P2). Using in vitro processing assays, we systematically scanned the two polyproteins for cleavage sites recognized by this protease. We identified five cis-cleavage sites in P1, with cleavage between the putative helicase and VPg domains being the most efficient. The presence of six protein domains in the SMoV P1, including two upstream of the putative helicase domain, is a feature shared with nepoviruses but not with comoviruses. Results from trans-cleavage assays indicate that the RNA1-encoded 3C-like protease recognized a single cleavage site, which was between the predicted MP and coat protein domains in the P2 polyprotein. The cleavage site consensus sequence for the SMoV 3C-like protease is AxE (E or Q)/(G or S).
Highlights
Strawberry decline disease has emerged as a significant problem for strawberry production in Eastern Canada and is likely caused by the synergistic effects of mixed virus infections
Based on alignments of P1 polyproteins amongst Canadian Strawberry mottle virus (SMoV) isolates and with related secovirids, putative cleavage sites were previously predicted including Q465/G, Q964/G, Q989/G, and Q1220/G (Figure 1, numbering correspond to the amino acid position starting from the beginning of the polyprotein) (Thompson et al, 2002; Bhagwat et al, 2016)
A few smaller minor bands were observed but were likely not due to a specific proteolytic event directed by the 3C-like protease, since they were observed in the Pronull derivative (Figure 2B, compare lanes 1 and 3)
Summary
Strawberry decline disease has emerged as a significant problem for strawberry production in Eastern Canada and is likely caused by the synergistic effects of mixed virus infections. The species Strawberry mottle virus has been classified within the family Secoviridae (order Picornavirales) but is currently not assigned to a specific genera, mostly because its genomic organization has not yet been clarified (Sanfacon et al, 2011; Sanfacon, 2015). The specificity for a glutamine (Q) or glutamate (E) at the −1 position of the cleavage site is conferred by the conserved histidine in the substrate-binding pocket of the protease, which is present in the SMoV protease (Bazan and Fletterick, 1988; Allaire et al, 1994; Sanfacon et al, 2011)
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