Different models have been proposed for the nature of the potexvirus transport form that moves from cell to cell over the infected plant: (i) genomic RNA moves as native virions; or (ii) in vitro-assembled non-virion ribonucleoprotein (RNP) complexes consisting of viral RNA, coat protein (CP) and movement protein (MP), termed TGBp1, serve as the transport form in vivo. As the structure of these RNPs has not been elucidated, the products assembled in vitro from potato virus X (PVX) RNA, CP and TGBp1 were characterized. The complexes appeared as single-tailed particles (STPs) with a helical, head-like structure composed of CP subunits located at the 59-proximal region of PVX RNA; the TGBp1 was bound to the terminal CP molecules of the head. Remarkably, no particular non-virion RNP complexes were observed. These data suggest that the CP-RNA interactions resulting in head formation prevailed over TGBp1-RNA binding upon STP assembly from RNA, CP and TGBp1. STPs could be assembled from the 59 end of PVX RNA and CP in the absence of TGBp1. The translational ability of STPs was characterized in a cell-free translation system. STPs lacking TGBp1 were entirely non-translatable; however, they were rendered translatable by binding of TGBp1 to the end of the head. It is suggested that the RNA-mediated assembly of STPs proceeds via two steps. Firstly, non-translatable CP-RNA STPs are produced, due to encapsidation of the 59-terminal region. Secondly, the TGBp1 molecules bind to the end of a polar head, resulting in conversion of the STPs into a translatable form.

No abstract

The primary structures of N-terminal 19-mer peptides, released by limited trypsin treatment of coat protein (CP) subunits in intact virions of three potato virus X (PVX) isolates, were analyzed. Two wild-type PVX strains, Russian (Ru) and British (UK3), were used and also the ST mutant of UK3 in which all 12 serine and threonine residues in the CP N-terminal segment were replaced by glycine or alanine. With the help of direct carbohydrate analysis and MS, it was found that the acetylated N-terminal peptides of both wildtype strains are glycosylated by a single monosaccharide residue (galactose or fucose) at NAcSer in the first position of the CP sequence, whereas the acetylated N-terminal segment of the ST mutant CP is unglycosylated. Fourier transform infrared spectra in the 1000-4000 cm )1 region were measured for films of the intact and in situ trypsindegraded PVX preparations at low and high humidity. These spectra revealed the presence of a broad-band in the region of valent vibrations of OH bonds (3100-3700 cm )1 ), which can be represented by superposition of three bands corresponding to tightly bound, weakly bound, and free OH groups. On calculating difference (ÔwetÕ minus ÔdryÕ) spectra, it was found that the intact wild-type PVX virions are characterized by high water-absorbing capacity and the ability to order a large number of water molecules on the virus particle. This effect was much weaker for the ST mutant and completely absent in the trypsin-treated PVX. It is proposed that the surface-located and glycosylated N-terminal CP segments of intact PVX virions induce the formation of a columnar-type shell from bound water molecules around the virions, which probably play a major role in maintaining the virion surface structure.

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.

No abstract

We found that the fusion of hexahistidine (H)6 tag to the potato virus X (PVX) coat protein (CP) abolished its ability to be assembled with viral RNA into helical nucleoprotein virus-like particles (VLPs). Instead, irregular agglomerates were produced upon incubation of PVX RNA with (H)6-tagged PVX CP. A factor Xa recognition site, IEGR, was inserted upstream of the CP coding sequence. Removal of the (H)6 tag from PVX CP by Xa protease restored its ability to bind RNA and to assemble VLPs. In addition to the canonical IEGR site, the factor Xa protease was found to cleave PVX CP at a second (non-consensus) site, AVTRGR, located close to the C-terminus of PVX CP. The latter cleavage did not affect reassembly of the PVX RNA and CP into VLPs.