Characterization of VPg and the polyprotein processing of Cocksfoot mottle virus (genus Sobemovirus)

Abstract

The polyprotein of Cocksfoot mottle virus (CfMV ; genus Sobemovirus) is translated from two overlapping open reading frames (ORFs) 2a and 2b by a N1 ribosomal frameshifting mechanism.In this study, a 12 kDa protein was purified from viral RNA-derived samples that appears to correspond to the CfMV genome-linked protein (VPg). According to the determined N-terminal amino acid sequence, the VPg domain is located between the serine proteinase and replicase motifs and the N terminus of VPg is cleaved from the polyprotein between glutamic acid and asparagine residues. Western blot analysis of infected plant material showed that the polyprotein is processed at several additional sites. An antiserum against the ORF 2a product recognized six distinct proteins, whereas, of these, the VPg antiserum clearly recognized only a 24 kDa protein. This indicates that the fully processed 12 kDa VPg detected in viral RNA-derived samples is a minor product in infected plants. An antiserum against the ORF 2b product recognized a 58 kDa protein, which indicates that the fully processed replicase is entirely or almost entirely encoded by ORF 2b. The origin of the detected cleavage products and a proposed polyprotein processing model are discussed.

The Ryegrass mottle virus genome codes for a sobemovirus 3C-like serine protease and RNA-dependent RNA polymerase translated via −1 ribosomal frameshifting

“…2A), suggesting the formation of mature VPg and RdRP by proteolysis. The smallest possible RGMoV protein including the VPg consensus sequence (WAD) between E/N and E/S sites would be a 11.4 kDa protein, which is in a good agreement with the detected size (12 kDa) for VPg covalently bound to CfMV genomic RNA [12]. The AA sequence of RdRP contains only 4 putative E/T or E/S cleavage sites close to N-or C-termini.…”

“…An extreme long stretch of 774 nt has been detected in TRoV and a different start codon (CUG) has been suggested as a translation iniciation codon [4]. However, mature sobemovirus RdRPs are possibly formed as a result of proteolytic processing of corresponding polyproteins [10,12] and not as separate proteins translated from ATG.…”

“…As shown from investigation of the formation of mature VPg's and RdRP, sobemovirus 3C-like serine proteases cleave virus polyproteins between glutamic acid and threonine [10,13], asparagine [12], or serine residues [10]. Several putative E/T, E/N and E/S cleavage sites were identified also in RGMoV polyproteins ( Fig.…”

“Natively Unfolded” VPg Is Essential for Sesbania Mosaic Virus Serine Protease Activity

“…Mutational Analysis of VPg-Comparison of the VPg amino acid sequence between Sobemo-and Poleroviruses revealed a conserved W(A/G)D motif followed by a stretch of acidic amino acid residues (10). In addition to this conserved motif, the amino acid sequence of VPg in SeMV, Southern cowpea mosaic virus, and Southern bean mosaic virus-Ark contains a prolinerich sequence toward the C terminus.…”

P1 Protein of Cocksfoot Mottle Virus is Indispensable for the Systemic Spread of the Virus

“…The genome of sobemoviruses is a single-stranded positive sense polycistronic RNA molecule. The 5¢ end of viral RNA has a VPg and the 3¢ terminus lacks a poly(A) tail [2]. The genomic RNA of sobemoviruses expresses a P1 from the 5¢ ORF1 and a polyprotein (Pro-VPg-RdRp) precursor from the downstream ORF2 [3][4][5] via a leaky scanning mechanism [6].…”