Abstract
AbstractSobemoviruses encode serine-like 3C proteases (Pro) that participate in the processing and maturation of other virus-encoded proteins. Itscisandtransactivity is mediated by the naturally unfolded virus-genome-linked protein (VPg). NMR studies show a Pro-VPg complex interaction and VPg tertiary structure; however, information regarding structural changes of the Pro-VPg complex during interaction is lacking. Here, we solved a full Pro-VPg 3D structure of ryegrass mottle virus (RGMoV) that demonstrates the structural changes in three different conformations due to VPg interaction with Pro. We identified a unique site of VPg interaction with Pro that was not observed in other sobemoviruses and observed different conformations of the Pro β2 barrel. This is the first report of a full plant Pro crystal structure with its VPg cofactor. We also confirmed the existence of an unusual previously unmapped cleavage site for sobemovirus Pro in the transmembrane domain: E/A. We demonstrated that RGMoV Proin cisactivity is not regulated by VPg and thatin trans, VPg can also mediate Pro in free form. Additionally, we observed Ca2+and Zn2+inhibitory activities on the Pro cleavage activity.Author summaryThe gRNA of sobemoviruses encodes two polyproteins that are processed by a serine protease. We found that in the bacterial expression system, Pro is activein cisandin trans, where onlyin transactivity is mediated by VPg not only in the fusion form with Pro but also in the free form. Here, we present structural changes in the catalytic and substrate-binding sites of Pro caused by VPg, which can explain thein transactivity and structure of sobemovirus VPg C-terminal peptide. In addition, we confirmed a new cleavage site not previously characterized in sobemoviruses. Additionally, Ca2+and Zn2+decreased Pro cleavage activity. This information could provide a better understanding of a serine protease and their proteolytic mechanisms during viral protein maturation.
Publisher
Cold Spring Harbor Laboratory