SummaryA highly infectious cDNA clone of clover yellow vein virus (pClYVV) was tested as a viral vector, especially for legume species. The genes for green¯uorescent 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¯uorescence was detected in infected broad bean, kidney bean, and soybean plants. The stability of the constructs in the symptomatic tissues was con®rmed by RT±PCR and Western blot analyses. The plants expressing GS together with GFP became tolerant to the herbicide glufosinate, and¯owered early. As the GS gene, one of the nodulin genes for nitrogen ®xation, is expressed in legume species, this system will be useful for examining the function of genes important to legume plants.

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.

Yth1, a subunit of yeast Cleavage Polyadenylation Factor (CPF), contains five CCCH zinc fingers. Yth1 was previously shown to interact with pre-mRNA and with two CPF subunits, Brr5/Ysh1 and the polyadenylation-specific Fip1, and to act in both steps of mRNA 3' end processing. In the present study, we have identified new domains involved in each interaction and have analyzed the consequences of mutating these regions on Yth1 function in vivo and in vitro. We have found that the essential fourth zinc finger (ZF4) of Yth1 is critical for interaction with Fip1 and RNA, but not for cleavage, and a single point mutation in ZF4 impairs only polyadenylation. Deletion of the essential N-terminal region that includes the ZF1 or deletion of ZF4 weakened the interaction with Brr5 in vitro. In vitro assays showed that the N-terminus is necessary for both processing steps. Of particular importance, we find that the binding of Fip1 to Yth1 blocks the RNA-Yth1 interaction, and that this inhibition requires the Yth1-interacting domain on Fip1. Our results suggest a role for Yth1 not only in the execution of cleavage and poly(A) addition, but also in the transition from one step to the other.

The cleavage/polyadenylation factor (CPF) of Saccharomyces cerevisiae is thought to provide the catalytic activities of the mRNA 3¢-end processing machinery, which include endonucleolytic cleavage at the poly(A) site, followed by synthesis of an adenosine polymer onto the new 3¢-end by the CPF subunit Pap1. Because of similarity to other nucleases in the metallo-blactamase family, the Brr5/Ysh1 subunit has been proposed to be the endonuclease. The C-terminal domain of Brr5 lies outside of b-lactamase homology, and its function has not been elucidated. We show here that this region of Brr5 is necessary for cell viability and mRNA 3¢-end processing. It is highly homologous to another CPF subunit, Syc1. Syc1 is not essential, but its removal improves the growth of other processing mutants at restrictive temperatures and restores in vitro processing activity to cleavage/ polyadenylation-defective brr5-1 extract. Our findings suggest that Syc1, by mimicking the essential Brr5 C-terminus, serves as a negative regulator of mRNA 3¢-end formation.

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.