Mutagenic analysis of Potato Virus X movement protein (TGBp1) and the coat protein (CP): in vitro TGBp1–CP binding and viral RNA translation activation

Abstract

Previously, we have shown that encapsidated Potato virus X (PVX) RNA was non-translatable in vitro, but could be converted into a translatable form by binding of the PVX movement protein TGBp1 to one end of the virion or by coat protein (CP) phosphorylation. Here, a mutagenic analysis of PVX CP and TGBp1 was used to identify the regions involved in TGBp1-CP binding and translational activation of PVX RNA by TGBp1. It was found that the C-terminal (C-ter) 10/18 amino acids region was not essential for virus-like particle (VP) assembly from CP and RNA. However, the VPs assembled from the CP lacking C-ter 10/18 amino acids were incapable of TGBp1 binding and being translationally activated. It was suggested that the 10-amino-acid C-ter regions of protein subunits located at one end of a polar helical PVX particle contain a domain accessible to TGBp1 binding and PVX remodelling. The non-translatable particles assembled from the C-ter mutant CP could be converted into a translatable form by CP phosphorylation. The TGBp1-CP binding activity was preserved unless a conservative motif IV was removed from TGBp1. By contrast, TGBp1-dependent activation of PVX RNA translation was abolished by deletions of various NTPase/helicase conservative motifs and their combinations. The motif IV might be essential for TGBp1-CP binding, but insufficient for PVX RNA translation activation. The evidence to discriminate between these two events, i.e. TGBp1 binding to the CP-helix and TGBp1-dependent RNA translation activation, is discussed.

Mixed Infections ofPepino Mosaic VirusStrains Modulate the Evolutionary Dynamics of this Emergent Virus

“…We found few codons under significant purifying selection, and most of them were in the TGBp1 and CP genes. The TGP1 codons under purifying selection in the PepMV-EU subpopulation (R109, T160, and S183) appear to be located in the NTPase/helicase domain, which contains a conserved motif necessary for CP-TGBp1 interaction in Potato virus X (46,79). Most of the sites in TGBp2 and TGBp3 were selectively neutral and likely being transiently present in the populations.…”

The TGB1 Movement Protein ofPotato virus XReorganizes Actin and Endomembranes into the X-Body, a Viral Replication Factory

“…Thus, the organization of PVX VRCs into the X-body, while not absolutely required for encapsidation, may function to increase the efficiency of vRNA packaging in a natural infection. Additionally, potexviral CP mutants defective for interaction with TGB1 can encapsidate virus particles in vitro and in protoplasts but fail to do so in intact tissue (Fedorkin et al, 2000;Zayakina et al, 2008;J. Tilsner and K.J.…”

Potato virus X displaying the E7 peptide derived from human papillomavirus type 16: a novel position for epitope presentation

“…The insertion at position 224 was located 13 aa from the C-terminus of PVX CP. Virus particles from PVX CP lacking C-terminal 10 or 18 aa have been reported to be unable to bind TGBp1 and be translationally activated (Zayakina et al 2008). This could explain the lack of expression of our constructs with inserts in these positions.…”