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Sobemoviruses 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.

The agricultural importance of sea buckthorn (Hippophae rhamnoidesL.) is rapidly increasing. Several bacterial and fungal pathogens infecting sea buckthorn 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 sea buckthorn for the first time. Sequence analysis of the obtained viral genome revealed high similarity with sequences of several viruses belonging to the genusMarafivirus, especially olive latent virus 3 (OLV-3). The genome of the new virus is 6,989 nucleotides (nt) in length according to 5′ and 3′ rapid amplification of cDNA ends (RACE) (without polyA-tail), with 5′ and 3′ untranslated regions being 133 and 109 nt long, respectively. The viral genome encoded two open reading frames (ORFs). ORF1 encoded a polyprotein of 1,954 amino acids (aa) 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 a six nt and encoded the coat protein (CP). CP had typical signatures of minor (30.96 kDa) and major (21.18 kDa) forms. Both CP forms were cloned and expressed in a bacterial expression system, and 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. We suggest the newly discovered virus to be named as “Sea buckthorn marafivirus”, abbreviated as “SBuMV”.Author summarySea buckthorn is an exceptionally valuable plant that is currently widely cultivated as multipurpose horticultural species for food, pharmacology, cosmetics, and landscape conservation. Diseases and pests directly affect the cultivation of SBT. To date, several pests and diseases, mainly fungal and bacterial, but no viral, sea buckthorn have been reported. Identification of new pathogens would assist in the development of control strategies, and quarantine purposes and ensure sustainable sea buckthorn cultivation. Here, for the first time, we present a virus putatively infecting sea buckthorn. We had characterized its full genome, cloned and expressed minor and major forms of coat protein either individually or co-expressed. We also showed that major coat protein-derived virus-like particles self- assembled directly in the bacterial cells, and the majority of the expressed CPs were soluble. Our study suggests that the minor CP is not essential for the assembly of seemingly structurally intact viral particles, meaning that it can have other functions.

Blackcurrants (Ribes nigrum) are among the most important commercial berry crops in Latvia and, together with redcurrants and gooseberries, have a long history of local cultivation and breeding (Zu...

Virus-like particles (VLPs) are multisubunit self-assembly competent protein structures with identical or highly related overall structure to their corresponding native viruses. To construct a new filamentous VLP carrier, the coat protein (CP) gene from potato virus M (PVM) was amplified from infected potato plants, cloned, and expressed in Escherichia coli cells. As demonstrated by electron microscopy analysis, the PVM CP self-assembles into filamentous PVM-like particles, which are mostly 100-300 nm in length. Adding short Gly-Ser peptide at the C-terminus of the PVM, CP formed short VLPs, whereas peptide and protein A Z-domain fusions at the CP N-terminus retained its ability to form typical PVM VLPs. The PVM-derived VLP carrier accommodates up to 78 amino acid-long foreign sequences on its surface and can be produced in technologically significant amounts. PVM-like particles are stable at physiological conditions and also, apparently do not become disassembled in high salt and high pH solutions as well as in the presence of EDTA or reducing agents. Despite partial proteolytic processing of doubled Z-domain fused to PVM VLPs, the rabbit IgGs specifically bind to the particles, which demonstrates the functional activity and surface location of the Z-domain in the PVM VLP structure. Therefore, PVM VLPs may be recognized as powerful structural blocks for new human-made nanomaterials.