The IGD(E)R(K) Motif Of HC-Pro Regulates The Virulence Of Potyviruses And RNA Silencing Suppression

Potyvirus includes more than 200 virus species and many viruses from this genus such as Tobacco vein banding mosaic virus(TVBMV) and Potato virus Y(PVY) can reduce yield production and cause economic loss significantly. Understanding the mechanisms of Potyviruses causing disease will help us to find a way to control it. In this study, we determined the essential amino acid controlling the TVBMV virulence based on a natural attenuated TVBMV mutant. Mutation analysis on the conserved motif IGD(E)R(K) suggested that it is involved in regulating virus virulence, RNA silencing suppression and synergism with Potato virus X. The following is the main results obtained in this study:1) Amino acid at postion 126 in HC-Pro-encoding sequence is essential for TVBMV virulence. A natural attenuated mutant HN39-12 was discovered during TVBMV HN39 propagation. Sequencing the complete genome revealed two amino acids differences between HN39-12 and HN39. The one is located at HC-Pro-encoding region and the other one is in P3-encoding region. It showed that the amino acid Arg(R) at position 126(R126) in HC-Pro is responsible for reducing TVBMV virulenc according to mutation analysis on pCamTVBMV-GFP infectious clone by substituted corresponding amino acid. Futhermore, the mutation R126 also caused the accumulation of both RNA and coat protein decreasing significantly. Mutated R126 to L in HC-Pro resulted in losing its ability to suppress RNA silencing.2) The IGD(E)R(K) motif in HC-Pro is involved regulating potyvirus virulence and suppressing RNA silencing. Comparison of Potyvirus HC-Pro revealed that R126 is located in conserved IGD(E)R(K) motif. Mutated the corresponding amino acid K124 to L in PVYN605 HC-Pro lost the ability to induce necrosis on N. tabacum. Mutation effect on the accumulation of RNA and HC-Pro was similar to that of TVBMV R126 L. Mutation analysis on the conserved motif based on pCamTVBMV-GFP infectious clone suggested that the properties of amino acid within this motif play a important role. These results indicated that the conserved motif IGD(E)R(K) is involved in regulating potyvirus virulence and suppressing RNA silencing.3) R126 in TVBMV HC-Pro affects its synergism with Potato virus X(PVX). Co-infection of PVX and pCamTVBMV-GFP caused server symptoms including death on inoculated N. benthamiana plants, in which the accumulation of PVX was significantly enhanced but that of TVBMV was increased slightly. However, no synergism was observed on N. benthamiana plants co-infected with PVX and TVBMV mutant containing mutation R126 to L in HC-Pro.