Sobemovirus RNA linked to VPg over a threonine residue

Abstract

Edited by Tamas DalmayKeywords: Southern bean mosaic virus Ryegrass mottle virus RNA linkage Phosphorylation Mass spectrometry Viral protein genome linked Protein RNA covalent bond a b s t r a c t Positive sense ssRNA virus genomes from several genera have a viral protein genome-linked (VPg) attached over a phosphodiester bond to the 5 0 end of the genome. The VPgs of Southern bean mosaic virus (SBMV) and Ryegrass mottle virus (RGMoV) were purified from virions and analyzed by mass spectrometry. SBMV VPg was determined to be linked to RNA through a threonine residue at position one, whereas RGMoV VPg was linked to RNA through a serine also at the first position. In addition, we identified the termini of the corresponding VPgs and discovered three and seven phosphorylation sites in SBMV and RGMoV VPgs, respectively. This is the first report on the use of threonine for linking RNA to VPg.

Protein-RNA linkage and posttranslational modifications of feline calicivirus and murine norovirus VPg proteins

“…Surprisingly for MNV the corresponding peptide was also detected without the RNA modification suggesting that RNA modification was lost during sample preparation. In addition we detected random aspartate and glutamate ethylation, methionine and tryptophan oxidations (Table 1 and data not shown), which are known to be generated in vitro during sample preparation (Stadtman & Levine, 2003;Xing et al, 2008;Olspert et al, 2011a) and were therefore not considered of biological relevance.…”

“…2-10 µg of total RNA was used per analysis. The samples were then dried under vacuum, purified with StageTips (Rappsilber, Mann & Ishihama Y, 2007) and analyzed by LC-MS/MS using an Agilent 1200 series nanoflow system (Agilent Technologies) connected to a LTQ Orbitrap massspectrometer (Thermo Electron) equipped with a nanoelectrospray ion source (Proxeon), as described previously (Olspert et al, 2011a). LTQ Orbitrap was operated in the data dependent mode with a full scan in the Orbitrap (mass range m/z 300-1900, resolution 60 000 at m/z 400, target value 1 Â 106 ions) followed by up to five MS/MS scans in the LTQ part of the instrument (normalized collision energy 35%, wideband activation enabled, target value 5000 ions).…”

Overview on Sobemoviruses and a Proposal for the Creation of the Family Sobemoviridae

“…The 5′UTR bounds covalently a viral genome-linked protein (VPg). The studies on the VPg-s of RYMV, CfMV, SBMV, and RGMoV identified a species-specific linkage between the 5′ phosphate group of the RNA and the hydroxyl group of the amino acid residue (tyrosine, serine or threonine) at the N-termini of VPg-s [ 58 , 59 ]. The RYMV VPg was shown to interact with eukaryotic translation initiation factor eIF(iso)4G [ 60 , 61 ].…”

Sesbania Mosaic Virus (SeMV) Infectious Clone: Possible Mechanism of 3′ and 5′ End Repair and Role of Polyprotein Processing in Viral Replication

“…Such an abnormal migration of VPg, due to post-translational modifications, has also been reported in other members [27], [28] of the genus Sobemovirus . Mass spectrometric analysis of CfMV, RYMV, SBMV and RGMoV VPgs isolated from native virus showed that they were phosphorylated and nucleotidylylated [28], [29]. The Pro-VPg and VPg bands could also be detected in native virus inoculated cotyledon leaf membrane fraction (data not shown).…”