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.
Interactions between the major proteins of Clover yellow vein virus (ClYVV) were investigated using a GAL4 transcription activator-based yeast two-hybrid system (YTHS). Self-interactions manifested by VPg and HCPro and an interaction between NIb and NIaPro were observed in ClYVV. In addition, a strong HCPro-VPg interaction was detected by both YTHS and by in vitro far-Western blot analysis in ClYVV. A potyvirus HCPro-VPg interaction has not been reported previously. Using YTHS, domains in ClYVV for the VPg self-interaction and the HCPro-VPg interaction were mapped. The VPg C-terminal region (38 amino acids) was important for the VPg-VPg interaction and the central 19 amino acids were needed for the HCPro-VPg interaction. INTRODUCTIONClover yellow vein virus (ClYVV) is a member of the genus Potyvirus and has a single-stranded, positive-sense RNA genome of about 10 kb (Takahashi et al., 1997). Mature functional proteins (P1, HCPro, P3, 6K1, CI, 6K2, VPg, NIaPro, NIb and CP) are generated by viral polyprotein processing by three virus-encoded proteases (P1, HCPro and NIa) (Riechmann et al., 1992). Although substantial information has been reported on the similarity of the interactions between potyviral proteins, differences are also observed within members of this genus.Most potyviral proteins are multifunctional. For example, HCPro is involved in aphid transmission (Atreya et al., 1990), genome replication (Klein et al., 1994;Kasschau et al., 1997), long-distance movement (Cronin et al., 1995;Kasschau et al., 1997;Saenz et al., 2002) and autoproteolysis (Carrington et al., 1989). In addition, it has been demonstrated that HCPro has plasmodesmatal gating (Rojas et al., 1997) and nucleic acid binding properties (Maia & Bernardi, 1996). Recently HCPro has been identified as a suppressor of post-transcriptional gene silencing (reviewed by Marathe et al., 2000;Anandalakshmi et al., 1998; Brigneti et al., 1998;Kasschau & Carrington, 1998).VPg is also multifunctional and needed for virus replication (Shahabuddin et al., 1988;Murphy et al., 1990Murphy et al., , 1991Murphy et al., , 1996. It is also needed for virus cell-to-cell and long-distance movement (Nicolas et al., 1997;Schaad et al., 1997;Keller et al., 1998; Rajamäki & Valkonen, 2002). It contains a nuclear localization signal that is important for virus replication (Schaad et al., 1996) and a sequence-non-specific RNA-binding domain (Merits et al., 1998). Furthermore, the plant cap-binding proteins eIF4E and eIF(iso)4E have been found to bind the VPg of potyviruses (Wittmann et al., 1997; Leonard et al., 2000;Schaad et al., 2000). This interaction was found to be important for the replication of the Turnip mosaic virus (TuMV), Lettuce mosaic virus (LMV), Potato virus Y (PVY) and Tobacco etch virus (TEV) in Arabidopsis thaliana and pepper (Duprat et al., 2002;Lellis et al., 2002;Ruffel et al., 2002).Protein-protein interactions play important roles in the virus infection cycle. Several interactions between potyviral proteins have been studied using the yeast two-h...
The two adjacent genes of coat protein 1 and 2 of rice tungro spherical virus (RTSV) were amplified from total RNA extracts of serologically indistinguishable field isolates from the Philippines and Indonesia, using reverse transcriptase polymerase chain reaction (RT-PCR). Digestion with HindIII and BstYI restriction endonucleases differentiated the amplified DNA products into eight distinct coat protein genotypes. These genotypes were then used as indicators of virus diversity in the field. Inter- and intra-site diversities were determined over three cropping seasons. At each of the sites surveyed, one or two main genotypes prevailed together with other related minor or mixed genotypes that did not replace the main genotype over the sampling time. The cluster of genotypes found at the Philippines sites was significantly different from the one at the Indonesia sites, suggesting geographic isolation for virus populations. Phylogenetic studies based on the nucleotide sequences of 38 selected isolates confirm the spatial distribution of RTSV virus populations but show that gene flow may occur between populations. Under the present conditions, rice varieties do not seem to exert selective pressure on the virus populations. Based on the selective constraints in the coat protein amino acid sequences and the virus genetic composition per site, a negative selection model followed by random-sampling events due to vector transmissions is proposed to explain the inter-site diversity observed.
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