Structural studies on tobacco streak virus coat protein: Insights into the pleomorphic nature of ilarviruses.

Tobacco streak virus (TSV), the type member of Ilarvirus genus, is a major plant pathogen. TSV purified from infected plants consists of a ss-RNA genome encapsidated in spheroidal particles with diameters of 27, 30 and 33nm constructed from multiple copies of a single species of coat protein (CP) subunits. Apart from protecting the viral genome, CPs of ilarviruses play several key roles in the life cycle of these viruses. Unlike the related bromo and cucumoviruses, ilarvirus particles are labile and pleomorphic, which has posed difficulties in their crystallization and structure determination. In the current study, a truncated TSV-CP was crystallized in two distinct forms and their structures were determined at resolutions of 2.4Å and 2.1Å, respectively. The core of TSV CP was found to possess the canonical ß-barrel jelly roll tertiary structure observed in several other viruses. Dimers of CP with swapped C-terminal arms (C-arm) were observed in both the crystal forms. The C-arm was found to be flexible and is likely to be responsible for the polymorphic and pleomorphic nature of TSV capsids. Consistent with this observation, mutations in the hinge region of the C-arm that reduce the flexibility resulted in the formation of more uniform particles. TSV CP was found to be structurally similar to that of Alfalfa mosaic virus (AMV) accounting for similar mechanism of genome activation in alfamo and ilar viruses. This communication represents the first report on the structure of the CP from an ilarvirus.

Sequence analysis of RNA 1 of lilac leaf chlorosis virus supports a close relationship to subgroup 3 ilarviruses.

Analysis of the nucleotide sequence of the RNA 1 of lilac leaf chlorosis virus (LLCV) supports a close relationship with subgroup 3 ilarviruses. LLCV RNA 1 consists of 3404 nucleotides (nt) and encodes a single open reading frame consisting of 3111 nt. The deduced protein (M(r) 117 kDa) contains the putative methyltransferase domain in the N-terminal region, and the NTPase/helicase domain in the C-terminal region. A conserved 41-nt region was identified at the distal end of the 3'UTR of all three genomic fragments of LLCV, with hairpin structures that may constitute putative coat protein binding sites.

In vitro and in vivo mapping of the Prunus necrotic ringspot virus coat protein C-terminal dimerization domain by bimolecular fluorescence complementation.

Interactions between viral proteins are critical for virus viability. Bimolecular fluorescent complementation (BiFC) technique determines protein interactions in real-time under almost normal physiological conditions. The coat protein (CP) of Prunus necrotic ringspot virus is required for multiple functions in its replication cycle. In this study, the region involved in CP dimerization has been mapped by BiFC in both bacteria and plant tissue. Full-length and C-terminal deleted forms of the CP gene were fused in-frame to the N- and C-terminal fragments of the yellow fluorescent protein. The BiFC analysis showed that a domain located between residues 9 and 27 from the C-end plays a critical role in dimerization. The importance of this C-terminal region in dimer formation and the applicability of the BiFC technique to analyse viral protein interactions are discussed.

Remarkable variability of apple mosaic virus capsid protein gene after nucleotide position 141.

Eight new sequences of European isolates from almond, apple, hop, prune and pear of the Apple mosaic ilarvirus (ApMV) capsid protein gene are presented. A consensus sequence was established as having 654 nucleotides (nt) and two American and two European isolates were identified to have insertions 6 to 15 nucleotides after nt position 141. The insertion resulted in the American isolate A inframeshift repaired with two point insertions 17 and 68 nt downstream. The RNA around the insertion point can potentially form a stable secondary structure with three hairpins. The insertions could stabilise this structure or could be neutral. The predicted folding of the translated protein is not influenced by the insertions or frameshift, and we speculate that the region after nt position 141 is without reasonable selection pressure and represents a hot spot for the accumulation of insertion mutations in ApMV.

The use of short and long PCR products for improved detection of prunus necrotic ringspot virus in woody plants.

The reverse transcriptase-polymerase chain reaction (RT-PCR) was used for detection of prunus necrotic ringspot virus (PNRSV) in dormant peach and almond trees by the application of two different pairs of primers yielding a short and a long product, respectively. The relative amount of the short (200 base pair, bp) product was higher than the longer (785 bp) product. PNRSV was detected better in plant tissues with a low virus concentration (e.g. dormant trees) by amplification of the short PCR product, whereas the long product was product was produced at higher virus titers. Simultaneous amplification of both short and long products was demonstrated using a three-primer mixture in a single reaction tube. In this assay, amplification of either PCR product indicated the presence of PNRSV-specific sequences in the plant tissue examined, thus covering a wide range of virus concentrations in a single test. Dilution of the RNA extracted from infected plant material resulted in a steep decline in the amplification of both short and long PCR products. In contrast, serial dilutions of the intermediate cDNA template differentially affected the amplification patterns: the relative amount of the short product increased whereas that of the long product decreased. These results may explain the preferential amplification of the short PCR product observed in samples containing low virus concentrations.

The complete nucleotide sequence of prune dwarf ilarvirus RNA-1.

The sequence of prune dwarf ilarvirus (PDV) RNA-1 has been determined; it consists of 3,374 nucleotides and contains a single open reading frame of 3,168 nucleotides. The putative translation product is 1,055 amino acids in length with a calculated molecular mass of 118.9 kDa. Both the nucleic acid and the translated amino acid sequences show stronger homology to the corresponding RNA-1 and ORF-1 of apple mosaic ilarvirus and alfalfa mosaic alfamovirus than to spinach latent mosaic ilarvirus or citrus leaf rugose ilarvirus. These findings are consistent with the inclusion of alfalfa mosaic virus in the ilarvirus genus. The reported sequence of PDV RNA-1 and its single ORF conform to the genomic organization typical of the Bromoviridae family.