Point mutations in helper component protease of clover yellow vein virus are associated with the attenuation of RNA-silencing suppression activity and symptom expression in broad bean.

Helper component protease (HC-Pro) is a potyvirus-encoded multifunctional protein and a major determinant of symptom expression in a susceptible plant. Here, we show the involvement of clover yellow vein virus (ClYVV) HC-Pro in necrotic symptom expression in broad bean (Vicia faba cv. Wase). In this host, lethal necrosis was induced by ClYVV no. 30, from which a spontaneous, mosaic-inducing mutant (MM) was obtained. Mapping with chimeric viruses between ClYVV no. 30 and MM attributed the symptom attenuation to two mutations at the HC-Pro positions 27 (threonine to isoleucine) and 193 (aspartic acid to tyrosine). Although neither mutant with the single amino acid substitution at position 27 or 193 (ClYVV/T27I or D193Y) induced the lethal necrosis, ClYVV/T27I still retained the ability to induce necrotic symptoms, but ClYVV/D193Y scarcely did so. The virus accumulation of ClYVV/D193Y was also lower than that of ClYVV no. 30. The mutations, T27I and D193Y, are located in a putative zinc finger domain and in one (N-terminal) of the two RNA binding domains, respectively, of HC-Pro. RNA-silencing suppression (RSS) activity of P1/HC-Pro in Nicotiana benthamiana was weakened by both mutations. Our results suggest a correlation between viral virulence and RSS function and the importance of the two domains in HC-Pro.

The 3' terminal region is strictly required for clover yellow vein virus genome replication.

To identify the cis-element in the 3' terminal region of infectious cDNA required for replication of clover yellow vein virus (ClYVV), a series of mutants with duplications or deletions of the 3' terminal non-coding region (3'-NCR) of the genome that did not affect the ORFs in the genome was constructed. These were tested for infectivity, and the 3' terminal regions of their progeny RNAs were sequenced. Deletion mutants that lacked portions of the 3'-NCR were not infectious. Various mutants with duplicated 3' terminal sequences were infective only when the authentic 3' terminal sequence was restored, probably by recombination, and none of the constructs retained the original sequence in progeny viral RNA. When a coat protein gene sequence of bean yellow mosaic virus (BYMV) followed by a termination codon was introduced between the nuclear inclusion b and coat protein genes, infective progeny were generated. Sequence analyses of the progeny viruses showed that the coat protein gene was a chimera of the BYMV N-terminal and CIYVV C-terminal portions. These results suggest that the 3'-NCR of ClYVV contains cis-acting elements and is strictly required for genome replication.

Development of clover yellow vein virus as an efficient, stable gene-expression system for legume species.

A highly infectious cDNA clone of clover yellow vein virus (pClYVV) was tested as a viral vector, especially for legume species. The genes for green fluorescent protein (GFP) and soybean glutamine synthetase (GS) were inserted between the genes for P1 and HC-Pro on pClYVV to create three recombinant plasmids: pClYVV-GFP, pClYVV-GFP-GS, and pClYVV-GFP:GS. In the former two constructs all the junctions between the inserted proteins contained the sequences of protease cleavage recognition sites, whereas the third construct expressed a fusion of GFP and GS. Western blot analyses showed that GFP and GS appeared to have been precisely excised from the viral polyprotein with the viral proteases (P1 and NIa). Under UV irradiation, green fluorescence was detected in infected broad bean, kidney bean, and soybean plants. The stability of the constructs in the symptomatic tissues was confirmed by RT-PCR and Western blot analyses. The plants expressing GS together with GFP became tolerant to the herbicide glufosinate, and flowered early. As the GS gene, one of the nodulin genes for nitrogen fixation, is expressed in legume species, this system will be useful for examining the function of genes important to legume plants.

Restoration of the 3' end of potyvirus RNA derived from Poly(A)-deficient infectious cDNA clones.

Poly(A)-deficient full-length cDNA clones of clover yellow vein virus (ClYVV), a member of the genus Potyvirus, were found to be infectious when expressed from the CaMV 35S promoter. The poly(A) tail was replaced with different short sequences and the infectivities of the cDNA constructs were examined. Although the infectivity of the plasmid varied depending on the sequences introduced, all the constructs were infectious. In all cases, progeny viral RNAs from the cDNA clones had an authentic viral sequence at their 3' regions with poly(A) tails and the downstream nonviral sequences were completely lost. However, two minor mutations, a two-nucleotide deletion at the 3' end and a single-nucleotide addition at the second nucleotide position downstream of the poly(A) site, were also observed. The clones of the viral (-) strand RNAs had poly(U) tracts at their 5' ends, suggesting that their synthesis is primed by the poly(U) sequence. It furthermore suggests that the mutations were introduced during or after primary transcription from the cDNA and were maintained during authentic viral replication. Although the mechanism involved is not known, recovery of the poly(A) tail is an essential step for maintaining the infectivity of the viral cDNAs.