Academic journal article Genetics

From Hypo- to Hypersuppression: Effect of Amino Acid Substitutions on the RNA-Silencing Suppressor Activity of the Tobacco Etch Potyvirus HC-Pro

Academic journal article Genetics

From Hypo- to Hypersuppression: Effect of Amino Acid Substitutions on the RNA-Silencing Suppressor Activity of the Tobacco Etch Potyvirus HC-Pro

Article excerpt

ABSTRACT

RNA silencing participates in several important functions: from the regulation of cell metabolism and organism development to sequence-specific antiviral defense. Most plant viruses have evolved proteins that suppress RNA silencing and that in many cases are multifunctional. Tobacco etch potyvirus (TEV) HC-Pro protein suppresses RNA silencing and participates in aphid-mediated transmission, polyprotein processing, and genome amplification. In this study, we have generated 28 HC-Pro amino acid substitution mutants and quantified their capacity as suppressors of RNA silencing in a transient expression assay. Most mutations either had no quantitative effect or completely abolished silencing suppression (10 in each class), 3 caused a significant decrease in the activity, and 5 significantly increased it, revealing an unexpected high frequency of mutations conferring hypersuppressor activity. A representative set of the mutant alleles, containing both hypo- and hypersuppressors, was further analyzed for their effect on TEV accumulation and the strength of induced symptoms. Whereas TEV variants with hyposuppressor mutants were far less virulent than wild-type TEV, those with hypersuppressor alleles induced symptoms that were not more severe than those characteristic of the wild-type virus, suggesting that there is not a perfect match between suppression and virulence.

SMALL RNAs, including micro RNAs (miRNAs) and short interfering RNAs (siRNAs), are key components of an evolutionarily conserved RNA-based gene regulation system documented in fungi, plants, and animals that is implicated in various biological processes from development to antiviral defenses (Ratcliff et al. 1997; Waterhouse et al. 2001; Baulcombe 2002; Voinnet 2002; Ding et al. 2004; Chen et al. 2005; Wilkins et al. 2005; Kim and Nam 2006; Ding and Voinnet 2007). The silencing pathway is triggered by the presence of double-stranded RNAs (dsRNA) or single-stranded RNAs with stem-loop structures that are processed by several Dicer proteins into ~21- to 24-nt short RNAs, including siRNAs, miRNAs, and others, which are incorporated into an RNA-induced silencing complex (RISC) to promote a sequence-specific cleavage or translation arrest of transcripts of complementary sequence (Hammond et al. 2001; Waterhouse et al. 2001; Voinnet 2002; Carrington and Ambros 2003; Bartel 2004; Baulcombe 2004; Pfeffer et al. 2004; Brodersen and Voinnet 2006; Buchon and Vaury 2006; Chapman and Carrington 2007).

RNA silencing is responsible for important endogenous functions, including regulation of cellular transcripts, guiding heterochromatin formation and transcriptional repression of transposon, processing of noncoding RNA precursors that control developmental timing and leaf polarity, and regulation of stress (Hammond et al. 2001; Carrington and Ambros 2003; Bartel 2004; Xie et al. 2004; Voinnet 2005; Brodersen and Voinnet 2006). RNA silencing also represents a natural defense system against viruses because it is activated by the structured RNAs or the dsRNAs produced during the replication cycles of different classes of viruses and subviral pathogens (Ratcliff et al. 1997, 1999; Voinnet 2001;Waterhouse et al. 2001;Ding et al. 2004; Lecellier and Voinnet 2004; Chen et al. 2005; Wilkins et al. 2005; Buchon and Vaury 2006; Ding and Voinnet 2007).

Viruses are inducers and targets of RNA silencing, but they have also evolved strategies to counteract this defense mechanism (Kasschau and Carrington 1998; Voinnet et al. 1999; Roth et al. 2004; Li and Ding 2006). Silencing suppression is a common property of plant viruses and suppressor proteins are considered as pathogenicity determinants, needed for efficient accumulation. Found in most viruses, silencing suppressor proteins show a tremendous structural and sequence diversity that has been explained as an evolutionary convergence toward a common functional necessity (Li and Ding 2006). The sine qua non condition for the operation of natural selection is the existence of genetic variation affecting fitness. …

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