Processing of SeMV polyproteins revisited

Abstract

Processing of Sesbania mosaic virus (SeMV) polyprotein 2a and 2ab was reanalyzed in the view of the new genome organization of sobemoviruses. Polyprotein 2a when expressed in E. coli, from the new cDNA clone, got cleaved at the earlier identified sites E325-T326, E402-T403 and E498-S499 to release protease, VPg, P10 and P8, respectively. Additionally, a novel cleavage was identified within the protease domain at position E132-S133, which was found to be essential for efficient polyprotein processing. Products, corresponding to cleavages identified in E. coli, were also detected in infected Sesbania leaves. Interestingly, though the sites are exactly the same in polyprotein 2ab, it got cleaved between Protease-VPg but not between VPg-RdRp. This indicates to a differential cleavage preference, governed probably by the conformation of 2ab. Also, the studies revealed that, in SeMV, processing is regulated by mode of cleavage and context of the cleavage site.

Protein-RNA linkage and post-translational modifications of two sobemovirus VPgs

“…The data determined the CfMV VPg C-terminal processing site E 396 /T 397 upstream of the 21 PRF signal. The C terminus of the CfMV VPg is in accordance with the experimentally demonstrated C terminus of the VPg of SeMV (Nair & Savithri, 2010). The previously described molecular mass of 12 kDa was more likely a result of shift in mobility in SDS-PAGE caused by the acidic nature of the VPg protein (pI~4).…”

“…The VPgs of sobemoviruses are translated as part of the polyprotein and cleaved by the viral protease (Nair & Savithri, 2010;van der Wilk et al, 1998). In contrast to potyviruses, the polyprotein processing and VPg maturation of sobemoviruses is poorly described.…”

“…In contrast to potyviruses, the polyprotein processing and VPg maturation of sobemoviruses is poorly described. The specificity of the sobemoviral protease has been proposed as Q, E/T, S, N (Gorbalenya et al, 1988;Mäkinen et al, 2000;Nair & Savithri, 2010;van der Wilk et al, 1998), based on the fact that many different cleavage sites can be predicted for the N and C termini of sobemovirus VPgs. For several sobemoviruses -CfMV, RYMV, Southern bean mosaic virus (SBMV) and Sesbania mosaic virus (SeMV) -the N terminus of VPg has been mapped (Hébrard et al, 2008;Mäkinen et al, 2000;Nair & Savithri, 2010;van der Wilk et al, 1998), while the C terminus of VPg has so far been experimentally proven for only SeMV (Nair & Savithri, 2010).…”

Cymbidium chlorotic mosaic virus, a new sobemovirus isolated from a spring orchid (Cymbidium goeringii) in Japan

“…S2). This domain contains 384 WAE 386 and a short E/D sequence ( 389 EDE 391 ) instead of the relatively conserved W(A/G)D motif and the E/D-rich region, respectively [17,22]. In addition, a proteolytic cleavage site located between the N-terminal region and protease domain of sobemovirus P2a proteins [17,22] is also conserved in the sequence of CyCMV P2a ( Fig.…”

“…A conserved 3181 ACAAA 3185 sequence, which is identical to the and P2a-P2b. The 2a polyprotein is predicted to encode five mature proteins [17]: p14 (membrane anchor), Pro (serine protease), VPg (viral protein, genome-linked), p10 (unknown function), and p6 (unknown function). The locations of putative cleavage sites by the viral proteinases are indicated as dashed vertical lines in the coding region with the peptide sequence at each site (E/A, E/T and E/N).…”