Stacking interactions of W271 and H275 of SeMV serine protease with W43 of natively unfolded VPg confer catalytic activity to protease

Abstract

N-terminal serine protease domain of Sesbania mosaic virus polyprotein, requires fused VPg for its activity. W43 of VPg mediates aromatic stacking interactions (characterized by 230 nm positive CD peak) with protease. A stretch of aromatic residues (F269, W271, Y315, and Y319) exposed in the protease domain were mutated to identify the interacting partner of W43. W271A Protease-VPg mutant showed absence of cleavage activity both in vivo and in trans, with concomitant loss of the 230 nm CD peak. F269A Protease-VPg mutant was partially active. Mutations of the tyrosines did not result in loss of protease activity or the CD peak. Interestingly, H275, though not a part of the exposed aromatic stretch, was shown to be essential for protease activity and contributed significantly to the CD peak. Hence, we conclude that W271 and H275 of the protease domain mediate aromatic stacking interactions with W43 of VPg thereby rendering the protease active.

Interaction of Sesbania Mosaic Virus Movement Protein with VPg and P10: Implication to Specificity of Genome Recognition

“…The structure and the assembly of the virus have been described earlier [18], [19], [20], [21]. The Pro, RdRp, VPg and the P8 domains have been functionally characterised and the latter two are shown to be natively unfolded [22], [23], [24], [25]. In an earlier study SeMV MP was shown to interact with SeMV CP via the N terminal domain [26].…”

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

“…Loss of infectivity upon mutation of E132 suggests that cleavage at this site by the protease domain is important for release of NΔ132Pro-VPg from membrane. This NΔ132Pro-VPg might perform proteolytic functions in trans as shown earlier [11], [15], [26] and these trans functions also are crucial for replication/CP accumulation. In Potato leafroll virus (genus Polerovirus ), a similar cleavage site was identified and it was proposed that release of protease domain from the membrane may have a regulatory role [36].…”

“…Earlier studies have shown that mutation of cleavage site E325 in the ΔN70 polyprotein 2a results in accumulation of ΔN70Protease-VPg, which is an active form of the protease [11], [15]. Further in vitro studies also showed ΔN70Protease-VPg could act in trans and could cleave the polyprotein 2a at E325 and E402 positions [11], [15], [26]. A significant accumulation of full length Pro-VPg was also observed in membrane fractions of SeMV icDNA infiltrated leaf samples (Fig.…”

“…In addition, bands with molecular weight ranging from 12–17 kDa were also observed with a prominent band at 16 kDa. It may be noted that the expected molecular mass of the VPg is 9 kDa and the E.coli expressed VPg does not move abnormally on SDS-PAGE [15], [26]. This observed abnormal mobility of VPg could be due to post-translational modifications of VPg in planta .…”

Intrinsically disordered proteins of viruses: Involvement in the mechanism of cell regulation and pathogenesis

“…262,[273][274][275][276] The computational analysis showed that functionally important disordered VPg representative of viral diversity includes four members of the Caliciviridae family, six potyviruses and six sobemoviruses. 276 The disordered VPg components associated with the regulation of enzymatic activity in different viruses 273,277 in addition to performing specific regulation and transportation of viral RNA from one cell to another. 278…”