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 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.
Highlights
The helper component proteinase (HC-Pro) is a key protein encoded by plant viruses of the genus Potyvirus
Construction of Recombinant Viruses—Recombinant Lettuce mosaic virus (LMV)-E mutants were obtained by insertion in the plasmid p70SLMVE of a His6 tag fused to the N terminus of either wild-type HC-Pro or of a deleted form (99 AA) of HC-Pro. p70SLMVE contains an infectious, full-length cDNA copy of LMV-E RNA, under the control of the enhanced cauliflower mosaic virus (CaMV) 35S promoter and the NOS terminator [24]
Expression of Functional Recombinant HC-Pro—Two LMV recombinants were constructed (Fig. 1), where a histidine tag (His tag) was inserted upstream of the wild-type HC-Pro sequence or of a mutant deleted of AA 4 to 102
Summary
The helper component proteinase (HC-Pro) is a key protein encoded by plant viruses of the genus Potyvirus. Mutagenesis studies and sequence alignments suggest that HC-Pro can be schematically divided into three regions: an N-terminal region essential for the transmission process, a C-terminal region harboring the proteinase activity, and a central region implicated in all other functions (see Fig. 1). The proteinase domain has been mapped to the C-terminal 155 AA and characterized as a cysteine protease-like activity with Cys344 and His417 residues in the active site [4] This region might overlap with a cell-to-cell movement domain, as in Bean common mosaic necrosis virus HC-Pro, a C-terminal deletion of 87 and 293 AA, respectively, partially or totally abolished cell-to-cell movement of heterogeneously expressed protein in microinjection studies [12]
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