At the interface of host-virus interactions: cleavage of plant proteins by a potyvirus protease and potential applications for novel antiviral strategies.

Potyviruses constitute a large and economically important group of plant positive-strand
RNA viruses. The potyvirus NIa protease (NIa-Pro) is a chymotrypsin-like cysteine protease
closely related to the 3C protease of human and animal picornaviruses. Like the 3C protease, NIa cleaves the viral polyprotein at multiple sites. The picornavirus 3C protease also cleaves hundreds of host proteins to facilitate its infection. However, whether or not the NIa protease also targets plant proteins was not known. To address this question, we used a bioinformatic approach to identify putative NIa cleavage sites in the plant proteome. We focused on the NIa proteases from plum pox virus (PPV) and turnip mosaic virus (TuMV), for which consensus cleavage site sequences are similar and well-characterized. Regular expression search using the consensus cleavage site sequence [EQN]xVxH[QE]↓[SGTA] for the PPV NIa protease allowed the identification of 90-94 putative cleavage events in the proteomes of Arabidopsis thaliana, Nicotiana benthamiana and Prunus persica. Using in vitro processing assays, we confirmed cleavage of 11 host proteins by purified PPV and TuMV NIa proteases. To examine cleavage in infected cells, we transiently expressed epitope-tagged versions of candidate plant proteins and simultaneously launched virus infection from a TuMV infectious clone. Using this method, we demonstrate in vivo cleavage of AtEML2 (At5g06780), an Emsy-like protein belonging to a family of histone readers known to be involved in pathogen resistance and AtUFD1 (At4g15420), a ubiquitin fusion degradation-like protein probably involved in the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway. AtUFD1 also includes a TRAF-like domain, a domain family which has been associated with the regulation of plant immune responses. Cleavage of AtEML2 and AtUFD1 was also observed in plant cells that co-express the PPV or TuMV NIa-Pros. Mutation of the predicted cleavage sites prevented the processing of these host proteins. Using a differential centrifugation fractionation method, we investigated the subcellular localization of the uncleaved proteins and of the cleaved fragments. Uncleaved AtEML2 protein was found predominantly in nuclear-enriched fractions while the N- and C-terminal cleaved fragments accumulated in cytoplasmic-enriched fractions. Full-length AtUFD1 and the cleaved AtUFD1 fragments were detected predominantly in cytoplasmic-enriched fractions. These results suggest that NIa-Pro is able to cleave plant proteins at diverse subcellular localization in infected cells, consistent with the known association of mature or precursor NIa-Pro with the nucleus, cytoplasm and
ER membranes. We are now examining the biological impact of these cleavage events. It
is anticipated that mutation of the NIa-Pro cleavage sites to prevent cleavage of the identified
plant proteins could result in increased resistance to potyvirus infection.