Expression and characterisation of the ryegrass mottle virus non-structural proteins

Abstract

Expression and characterisation of the ryegrass mottle virus non-structural proteins The Ryegrass mottle virus (RGMoV) single-stranded RNA genome is organised into four open reading frames (ORF) which encode several proteins: ORF1 encodes protein P1, ORF2a contains the membrane-associated 3C-like serine protease, genome-linked protein VPg and a P16 protein gene. ORF2b encodes replicase RdRP and the only structural protein, coat protein, is synthesised from ORF3. To obtain the non-structural proteins in preparative quantities and to characterise them, the corresponding RGMoV gene cDNAs were cloned in pET- and pColdI-derived expression vectors and overexpressed in several E. coli host cells. For protease and RdRP, the best expression system containing pColdI vector and E. coli WK6 strain was determined. VPg and P16 proteins were obtained from the pET- or pACYC- vectors and E. coli BL21 (DE3) host cells and purified using Ni-Sepharose affinity chromatography. Attempts to crystallize VPg and P16 were unsuccessful, possibly due to non-structured amino acid sequences in both protein structures. Methods based on bioinformatic analysis indicated that the entire VPg domain and the C-terminal part of the P16 contain unstructured amino acid stretches, which possibly prevented the formation of crystals.

VPg impact on Ryegrass mottle virus serine-like 3C protease proteolysis and structure

“…Proteolysis occurred at the N-terminal residues because the proteins were unable to bind to the IMAC column (Fig 1B ). A similar situation was previously observed in the case of SeMV Δ70Pro [32] and in our previous experiments [17]. However, the expressed RGMoV Pro sequence did not contain any confirmed Pro cleavage sites typically used by sobemoviruses [11].…”

“…This abnormal mobility has been reported previously [40]. A similar behavior of the P16 homolog has been reported earlier [17]. This unusual behavior of P16 in SDS-PAGE could be explained by its high isoelectric point (IP=9.26).…”

“…307043 from Japan (GenBank: EF091714; [4]) was used as the source of the DNA matrix. As sobemovirus Pro at its N-terminal part contains a TMD sequence, for soluble RGMoV Pro expression in E. coli [12], we created an N-terminally truncated Pro, which lacks the first 50 amino acids (aa) (Δ50Pro) and contained 6xH-tag (histidine) at the N-terminus originating from the chosen expression vector [17]. PCR product of Δ50Pro was first cloned into the The catalytic center Δ117Pro mutant (Δ117Pro cm ) was created using oligonucleotides SP-S/A-F and SP-S/A-R (S1 Table ), where S 159 (serine) was substituted with A by site direct mutagenesis, and using SP-NCE-HindIII-R and SP-AAV-NcoI-N6H-F (S1 Table ), cloning sites NcoI and HindIII were introduced for cloning into the pET28a(+) expression vector (pET-Δ117Pro cm -N6H).…”

“…These proteins adopt a rigid conformation stabilized in vivo upon interaction with natural substrates and are multifunctional [11]. Bioinformatics methods have unfolded the nature of VPgs of several sobemoviruses (SeMV, RGMoV, CfMV, RYMV, SBMV, and southern cowpea mosaic virus (SCPMV)), potyviruses-lettuce mosaic virus (LMV), potato virus Y (PVY), potato virus A (PVA), tobacco etch virus, turnip mosaic virus, bean yellow mosaic virus-and Caliciviridae family members-rabbit hemorrhagic disease virus (Lagovirus), vesicular exanthema of swine virus (Vesivirus), sapporo virus Manchester virus (Sapovirus), and Norwalk virus (Norovirus) [14,[16][17][18]. In VPgs of the RYMV group (to which RGMoV belongs), similar to potyvirus and Caliciviridae family members, the N-and C-termini are predicted to be disordered [16,18].…”

Near-Complete Genome Sequence of Ryegrass Mottle Virus from Irrigation Water in Ecuador

“…Mapping of deduplicated reads to the RGMoV assembled contig revealed an average coverage depth of 107.681Â. Five open reading frames (ORFs) on the positive strand, a 59 untranslated region (UTR) of 87 nucleotides, and a 39 UTR of 247 nucleotides were identified on the basis of the reference RGMoV genome, showing its typical organization (13,14).…”