Use of pentapeptide-insertion scanning mutagenesis for functional mapping of the plum pox virus helper component proteinase suppressor of gene silencing

Abstract

Helper component proteinase (HC-Pro) of Plum pox virus is a multifunctional potyvirus protein that has been examined intensively. In addition to its involvement in aphid transmission, genome amplification and long-distance movement, it is also one of the better-studied plant virus suppressors of RNA silencing. The first systematic analysis using pentapeptide-insertion scanning mutagenesis of the silencing suppression function of a potyvirus HC-Pro is presented here. Sixty-three in-frame insertion mutants, each containing five extra amino acids inserted randomly within the HC-Pro protein, were analysed for their ability to suppress transgene-induced RNA silencing using Agrobacterium infiltration in transgenic Nicotiana benthamiana plants expressing green fluorescent protein. A functional map was obtained, consisting of clearly defined regions with different classes of silencing-suppression activity (wild-type, restricted and disabled). This map confirmed that the N-terminal part of the protein, which is indispensable for aphid transmission, is dispensable for silencing suppression and supports the involvement of the central region in silencing suppression, in addition to its role in maintenance of genome amplification and synergism with other viruses. Moreover, evidence is provided that the C-terminal part of the protein, previously known to be necessary mainly for proteolytic activity, also participates in silencing suppression. Pentapeptide-insertion scanning mutagenesis has been shown to be a fast and powerful tool to functionally characterize plant virus proteins.

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

“…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).…”

Structure of the Autocatalytic Cysteine Protease Domain of Potyvirus Helper-component Proteinase

“…Although the exact role of CPD in RSS activity remains to be defined, an intact CPD structure seems to be important. Many mutations that abolish RSS activity appear to disrupt the CPD structure (23,24). We identified a surface on the CPD that is conserved in Potyvirus, but not in other genera of Potyviridae that contain a homologous CPD.…”

“…In addition to proteolysis, the CPD of potyvirus has been associated with RSS activity (23,24). Although the exact role of CPD in RSS activity remains to be defined, an intact CPD structure seems to be important.…”

“…RNA silencing serves as an antiviral defense system in plants, in which viral doublestranded RNAs are processed by Dicer into small interfering RNAs (siRNAs) that guide Argonaute proteins to cleave viral RNAs (22). The RSS activity of HC-Pro was mainly associated with its central region (13,14,21) and was also affected by mutations in the CPD (23,24). The molecular mechanism of HC-Pro RSS activity remains elusive.…”

Soybean mosaic virus Infection and Helper Component-protease Enhance Accumulation of Bean pod mottle virus-Specific siRNAs

“…The amino acid sequences of HC-Pros of SMV G2 and G5 differed at 13 of 457 positions. While some research has suggested the existence of functional domains within the HC-Pro protein with the central domain responsible for suppression of RNA silencing (Varrelmann et al, 2007), other research has not supported that hypothesis (TorresBarceló et al, 2008;Wu et al, 2010). Hence, it was not possible to predict the effects of the individual amino acid sequence differences on suppression of RNA silencing from their positions alone.…”