Structural characterization of HC-Pro, a plant virus multifunctional protein.
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 post-transcriptional 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 (DeltaHC-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 Ni2+-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 DeltaHC-Pro allowed us to calculate a projection map at 9-A resolution. Our data from electron microscopy, biochemical analysis, and secondary structure predictions lead us to suggest a model for structure/function relationships in the HC-Pro protein.
Plisson
Célia
C
Université Rennes I, Unité Mixte de Recherche 6026 CNRS, Campus de Beaulieu, Rennes 35042, Cedex, France.
Drucker
Martin
M
Blanc
Stéphane
S
German-Retana
Sylvie
S
Le Gall
Olivier
O
Thomas
Daniel
D
Bron
Patrick
P
D020285:Cryoelectron Microscopy; D003460:Crystallization; D003546:Cysteine Endopeptidases / Q000737:chemistry*; D019281:Dimerization; D009154:Mutation; D010449:Peptide Mapping; D017800:Potyvirus / Q000737:chemistry*; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D014764:Viral Proteins / Q000737:chemistry*
D014764:Viral Proteins; D003546:Cysteine Endopeptidases; C072775:HC-Pro protein, potyvirus