VPg Impact on Ryegrass Mottle Virus Serine-like 3C Protease Proteolysis and Structure.

Sobemoviruses encode serine-like 3C proteases (Pro) that participate in the processing and maturation of other virus-encoded proteins. Its cis and trans activity is mediated by the naturally unfolded virus-genome-linked protein (VPg). Nuclear magnetic resonance 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 ß2 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 Pro in cis activity is not regulated by VPg and that in trans, VPg can also mediate Pro in free form. Additionally, we observed Ca2+ and Zn2+ inhibitory effects on the Pro cleavage activity.

Identification and Full Genome Analysis of the First Putative Virus of Sea Buckthorn (Hippophae rhamnoides L.).

The agricultural importance of sea buckthorn (SBT; Hippophae rhamnoides L.) is rapidly increasing. Several bacterial and fungal pathogens infecting SBT have been identified and characterized; however, the viral pathogens are not yet known. In this study, we identified, isolated, and sequenced a virus from a wild plantation of SBT for the first time. Sequence analysis of the obtained viral genome revealed high similarity with several viruses belonging to the genus Marafivirus. The genome of the new virus is 6989 nucleotides (nt) in length according to 5', 3' RACE (without polyA-tail), with 5' and 3' 133 and 109 nt long untranslated regions, respectively. The viral genome encoded two open reading frames (ORFs). ORF1 encoded a polyprotein of 1954 amino acids with the characteristic marafivirus non-structural protein domains-methyltransferase, Salyut domain, papain-like cysteine protease, helicase, and RNA-dependent RNA polymerase. ORF1 was separated from ORF2 by 6 nt, encoding the coat protein (CP) with typical signatures of minor and major forms. Both CP forms were cloned and expressed in a bacterial expression system. Only the major CP was able to self-assemble into 30 nm virus-like particles that resembled the native virus, thus demonstrating that minor CP is not essential for virion assembly.