Kalnciema, I. | Search bioRxiv

bioRxiv2023

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). 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 {beta}2 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 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 active in cis and in trans, where only in trans activity 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 the in trans activity 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.

microbiology↗

bioRxiv2022

Identification and full genome analysis of the first putative virus of sea buckthorn (Hippophae rhamnoides L.)

The agricultural importance of sea buckthorn (Hippophae rhamnoides L.) 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 genus Marafivirus, 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.

molecular biology↗