The Conserved FRNK Box in HC-Pro, a Plant Viral Suppressor of Gene Silencing, Is Required for Small RNA Binding and Mediates Symptom Development

Plants infected with viruses exhibit diverse symptoms, including pleiotropic developmental defects unique to virus infection (27, 71). To combat the damage caused by these intercellular parasites, plants possess an antiviral defense based on degradation of foreign RNA, known as RNA silencing or RNA interference (33, 65). In order to counteract RNA silencing within their hosts, plant viruses have acquired genes that encode suppressors that prevent degradation of their genomes and mRNAs (50, 65). RNA-silencing suppressors (RSS) in various plant virus families have been identified (67), and interestingly, they share no obvious similarities at either the nucleic acid level or the protein level, suggesting that they function through a variety of mechanisms. The RSS of the Potyviridae family is a multifunctional protein named the helper component-proteinase (HC-Pro) (57). HC-Pro is essential for systemic infection in the genus Potyvirus, which is in contrast to the case for Wheat-streak mosaic virus (a tritimovirus), from which the entire HC-Pro can be removed without abolishing infectivity (55). Several different HC-Pro functions have been identified and mapped, including protease cleavage of the viral polyprotein (64), insect vector transmission (47), capsid binding (7), two single-stranded RNA-binding areas (58), viral movement and replication (11, 26), virus synergism (53, 68), and RNA-silencing suppression (3, 9, 25). Biochemical studies have shown that HC-Pro binds rgs-CaM (an endogenous suppressor of gene silencing) (2), inhibits the 20S proteosome endonuclease activity (6), and limits methylation of virus-derived small RNAs (smRNAs) (13, 74). HC-Pro forms dimers/multimers and is active in these forms (48, 51, 59). Schematically, the HC-Pro protein can be divided into three regions: an N-terminal region associated with aphid transmission, a C-terminal region associated with proteinase and RSS activity (62), and a central region implicated in many functions, including RSS activity (48). The central region contains several motifs that are highly conserved in all potyviruses, including the FRNK box at amino acid positions 179 to 182 in mature HC-Pro, which is associated with Zucchini yellow mosaic virus (ZYMV) symptom severity (18). The mutation of the FRNK box to FINK (R180I) causes a drastic reduction in symptom severity of the leaves of various cucurbit species without noticeably affecting virus accumulation or infectivity, and this mutation has been exploited for use in cross-protection (19). Other mutations in the central region of different potyviruses which are not located in the FRNK box also cause attenuation of symptoms without affecting virus accumulation (52). Recently, a smRNA duplex-binding function has been discovered for HC-Pro (29, 40). This ability implies an effect on plant defense mechanisms and plant development, because short interfering RNAs (siRNAs) and microRNAs (miRNAs) are sequestered in an inactive duplex form. This smRNA binding strategy is similar to that of other RSS, such as the tombusvirus P19 protein and the P21 of closteroviruses (30, 54, 73). RNA interference is based on smRNAs of 20 to 25 nucleotides (nts) which confer sequence specificity to regulatory or defensive pathways (22). Regulatory smRNAs include endogenous miRNAs (49) and trans-acting siRNAs (46) that originate from distinct loci and processing mechanisms. Other siRNAs are formed from double-stranded RNA (dsRNA) produced by RNA-dependent RNA polymerase (RDR) activity and derived from transposable elements and viral RNA. siRNAs can also originate from regions of viral RNA with intrinsic secondary structure (42). All smRNAs are processed by cleavage of dsRNA by one of several members of the DICER-like (DCL) enzyme family (23, 45). The cleavage products are 20- to 25-nt duplex dsRNA molecules that are bound by the RNA-induced silencing complex (RISC). From this duplex, the “passenger” or “star” strand (miRNA*) is released and rapidly degraded, while the remaining guide RNA molecule is incorporated into active RISC. This strand acts as a guide to bring the activated RISC complex into contact with complementary target RNAs, thereby mediating their cleavage and degradation (63). Some symptoms of potyvirus infection can be phenocopied by the expression of HC-Pro in the absence of virus. Transgenic Arabidopsis thaliana and soybeans (Glycine max) with high and low HC-Pro expression levels exhibit severe and mild developmental abnormalities, respectively (35, 41). It has been hypothesized that these symptoms are caused by deregulation of the endogenous smRNA pathways (27). Because genes with mutations conferring defects in miRNA accumulation, such as dcl1, hen1, hyl1, ago1, and se1, have pleiotropic developmental phenotypes in arabidopsis (37), it was deduced that the similarity between virus symptoms and these phenotypes is caused by the disruption of shared components in these pathways. Indeed, miRNA and, strikingly, miRNA* levels rise upon viral infection (10, 12, 27, 39, 76). As a consequence of sequestration of miRNA duplexes by the viral suppressor, their mRNA targets initially accumulate to higher levels in virus-infected plants due to reduced cleavage by RISC (27, 76). Following the discovery that HC-Pro binds duplex smRNA (29, 40), we analyzed the effects of the expression of this protein in attenuated-symptom-causing forms of ZYMV. We present data indicating that the FRNK box of HC-Pro is required for smRNA binding and that this activity is correlated with differential miRNA sequestration and symptom severity.