Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Mon, 07 Jan 2019 21:45:01 Mechanistic divergence between P1 proteases of the family Potyviridae Bernardo Rodamilans, Adria ´ n Valli3 and Juan Antonio Garcı ´a Correspondence Bernardo Rodamilans brodamilans@cnb.csic.es Juan Antonio Garcı ´a jagarcia@cnb.csic.es Received 19 December 2012 Accepted 3 February 2013 Centro Nacional de Biotecnologı ´a-CSIC, Campus Universidad Auto ´ noma de Madrid, 28049 Madrid, Spain P1a and P1b are two serine proteases of Cucumber vein yellowing virus (an ipomovirus). They belong to the group of P1 factors present at the N terminus of the polyproteins of most members of the family Potyviridae. The present work compares the protease activities of P1a and P1b in different experimental systems. The findings made regarding how these two proteases work, such as the requirement for a host factor by P1a but not by P1b, underscore important differences in their catalytic activity that point towards their undergoing divergent evolution involving the acquisition of mechanistic variations. The expression of several truncated forms of P1b in bacteria and in planta helped define the protease domain of P1b, along with other important features such as its apparently in cis mode of action. Recent phylogenetic data, together with the present results, allow an appealing hypothesis to be proposed regarding P1 evolution and its involvement in potyvirid speciation. INTRODUCTION Cucumber vein yellowing virus (CVYV) is an ipomovirus that infects cucurbit plants, causing economic losses in Asia, Africa and Europe. Infected plants may remain symptomless or present a variety of external features ranging from vein clearing and chlorosis to general necrosis and death (OEPP/EPPO, 2005). CVYV belongs to the family Potyviridae, whose members show a conserved organization of the genome and of their structural and non-structural proteins (Shukla et al., 1998). The N- terminal part of the polyprotein of CVYV, however, is quite different to that of other members of Potyviridae in that it lacks a coding sequence for HCPro, an RNA- silencing suppressor (RSS) with cysteine protease activity (Janssen et al., 2005). Rather, it has an extra copy of serine protease P1, making a P1a–P1b tandem at the N terminus of the polyprotein (Valli et al., 2006). The second cistron (P1b), which may have appeared through gene duplication or interspecific RNA recombination (Valli et al., 2007), is also present in Squash vein yellowing virus (another ipomovirus) (Li et al., 2008). Sequence comparisons have shown that P1a in both these two viruses is similar to the typical P1 of members of the genus Potyvirus, whereas P1b aligns with the P1 proteins of other ipomoviruses, as well as with those belonging to tritimoviruses and poaceviruses (Abraham et al., 2012; Giner et al., 2010). P1b has been described as an RSS that specifically sequesters 21 nt small RNAs (Valli et al., 2011), and this function is independent of its protease activity (Valli et al., 2008). It therefore at least partially assumes the role of HCPro, the usual RSS of potyviruses, in CVYV. It has also been shown that P1b can replace HCPro of the chimerical potyvirus Plum pox virus (PPV), investing it with full infective functionality (Carbonell et al., 2011). HCPro is a cysteine protease with functions including viral defence, genome amplification, movement and aphid transmission (Anandalakshmi et al., 1998; Berger et al., 1989; Brigneti et al., 1998; Cronin et al., 1995; Kasschau & Carrington, 1998; Kasschau et al., 1997; Rojas et al., 1997). It works in cis to release its C-end from the potyviral polyprotein. In Turnip mosaic virus (TuMV), the structure of its protease domain has been solved by X-ray crystallography, providing an important insight into its unique folding and catalysis mechanism (Guo et al., 2011). The P1 protein of potyviruses, however, is a serine protease with no clearly defined function besides its proteolytic activity (Rohoz ˇkova ´ & Navra ´til, 2011), although there is evidence that it might be involved in host range definition, genome amplification and RNA-silencing reinforcement, among other activities (Salvador et al., 2008; Verchot & Carrington, 1995). Interestingly, P1 needs a factor from the plant to release itself from HCPro; no such factor would appear to be required for HCPro protease activity (Carrington et al., 1990; Thornbury et al., 1993; Verchot et al., 1992). The two P1 proteins of CVYV, P1a (525 aa) and P1b (318 aa), show proteolytic activity in plants and bear the catalytic triad formed by histidine, aspartic and serine that is characteristic of the serine protease family (Rohoz ˇkova ´& 3Present address: Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, UK. Supplementary material is available with the online version of this paper. Journal of General Virology (2013), 94, 1407–1414 DOI 10.1099/vir.0.050781-0 050781 G 2013 SGM Printed in Great Britain 1407