Structural Characterization of HC-Pro, a Plant Virus Multifunctional Protein

Abstract

The helper component proteinase (HC-Pro) is a key protein encoded by plant viruses of the genus Potyvirus. HC-Pro is involved in different steps of the viral cycle, aphid transmission, replication, and virus cell-to-cell and systemic movement and is a suppressor of posttranscriptional gene silencing. Structural knowledge of HC-Pro is required to better understand its multiple functions. To this aim, we purified His-tagged wild-type HC-Pro and a N-terminal deletion mutant (⌬HC-Pro) from plants infected with recombinant potyviruses. Biochemical analysis of the recombinant proteins confirmed that HC-Pro is a dimer in solution, that the N terminus is not essential for self-interaction, and that a large C-terminal domain is highly resistant to proteolysis. Two-dimensional crystals of the recombinant proteins were successfully grown on Ni 2؉ -chelating lipid monolayers. Comparison of projection maps of negatively stained crystals revealed that HC-Pro is composed of two domains separated by a flexible constriction. Cryo-electron crystallography of ⌬HC-Pro allowed us to calculate a projection map at 9-Å resolution. Our data from electron microscopy, biochemical analysis, and secondary structure predictions lead us to suggest a model for structure/function relationships in the HCPro protein.

MicroRNAs and their regulatory roles in animals and plants

“…Some of these miRNAs may get involved in virus-induced gene silencing. Helper component-proteinase (HC-Pro), p19, p21, and p69, are unrelated viral suppressors of gene silencing, and they play important roles in the virus response to plant antiviral silencing response (Plisson et al, 2003;Chapman et al, 2004). Several investigations demonstrated that several miRNAs are related the activity of these viral suppressors (Kasschau et al, 2003;Chapman et al, 2004;Chen et al, 2004;Llave, 2004).…”

A Violaxanthin Deepoxidase Interacts with a Viral Suppressor of RNA Silencing to Inhibit Virus Amplification

“…In order to investigate which domain of HC-Pro is necessary for the interaction with ZmVDE, the coding sequences of each functional domain of HC-Pro reported by Plisson et al (2003;Fig. 4F) were constructed into pGBKT7 and then cotransformed with ZmVDEm into yeast cells.…”

Attenuated plant viruses: preventing virus diseases and understanding the molecular mechanism

“…Dotted square in b represents arginine-rich region in 2b protein (Goto et al 2007). KITC, FRNK, IGN, CC/SC, C344 and H417 in c represent domains (single letter of amino acid and number of amino acids from N-terminal of HC-Pro) in HC-Pro (Plisson et al 2003). Asterisk shows CDNQLD domain, 12 amino acids downstream from FRNK (Desbiez et al 2010).…”

“…In studies with HC-Pro mutants of Tobacco etch virus, hyposuppression of RNA silencing was correlated with milder virulence, but hypersuppression did not correlate with stronger virulence than with the wild type (TorresBarcelo et al 2008). HC-Pro is known as a multifunctional protein: aphid transmissibility, viral cell-to-cell and longdistance movement, viral amplification, symptom expression, synergism with other viruses, interaction with 20S proteasome and cysteine type proteolysis, siRNA binding and silencing suppressor including miR-guided cleavage of target mRNAs (Maia et al 1996;Plisson et al 2003;Shiboleth et al 2007). It is important to determine the structural requirements for RNA silencing suppressor activity, virus attenuation and cross protection.…”