title: Genome amplification and long-distance movement functions associated with the central domain of tobacco etch potyvirus helper component-proteinase.
abstract: The tobacco etch potyvirus (TEV) helper component-proteinase (HC-Pro, 460 amino acid residues) is a multifunctional protein involved in aphid-mediated transmission, genome amplification, polyprotein processing, and long-distance movement. To investigate the interrelationships between three of these functions, 25 alanine-scanning mutations affecting clusters of charged residues were introduced into the HC-Pro coding sequence. The resulting mutants were analyzed with respect to HC-Pro proteolytic activity in vitro, genome amplification in protoplasts, and long-distance movement in tobacco plants. Three classes of mutants were identified. Class I mutants (total of 17) were capable of genome amplification, long-distance movement, and HC-Pro proteolysis with efficiencies similar to parental virus. The class III mutant (total of 1) encoded a proteolytically debilitated HC-Pro and was replication-defective. Class II mutants (total of 7) encoded proteolytically active HC-Pro, but each exhibited a suppressed amplification phenotype that was characterized by a progressive shutoff during the course of infection in protoplasts. The class II mutants also exhibited defects in long-distance movement, accumulating to relative levels of 0 to 7.5% in noninoculated tissue. Wild-type HC-Pro supplied in trans was able to partially rescue the class II mutant amplification defects in protoplasts and long-distance movement defects in plants, although the extent of complementation of movement function varied for each mutant. Six of the seven class II mutations affected the central region of HC-Pro between residues 126 and 300, whereas only one affected the C-terminal proteolytic domain. These results indicate that the central region of HC-Pro is necessary for efficient genome amplification and long-distance movement, and that the one or more HC-Pro functions involved in these processes is at least partially trans-active. Additionally, the long-distance movement properties of a previously characterized HC-Pro-defective mutant (TEV-GUS/CCCE) were characterized further using grafted nontransgenic and HC-Pro-expressing transgenic plants. The results indicated that HC-Pro is required in both inoculated and noninoculated tissues to complement the TEV-GUS/CCCE movement defects.
authors.affiliation: Department of Biology, Texas A&M University, College Station 77843, USA.
authors.forename: K D
authors.initials: KD
authors.lastname: Kasschau
authors.forename: S
authors.initials: S
authors.lastname: Cronin
authors.forename: J C
authors.initials: JC
authors.lastname: Carrington
mesh_terms: D000409:Alanine; D001665:Binding Sites; D003546:Cysteine Endopeptidases / Q000235:genetics / Q000502:physiology*; D005784:Gene Amplification; D005816:Genetic Complementation Test; D016679:Genome, Viral; D009068:Movement; D016296:Mutagenesis; D030821:Plants, Genetically Modified; D010947:Plants, Toxic; D017800:Potyvirus / Q000201:enzymology* / Q000235:genetics; D011499:Protein Processing, Post-Translational; D011523:Protoplasts; D014026:Nicotiana / Q000821:virology; D014764:Viral Proteins / Q000235:genetics / Q000502:physiology*
chemical_list: D014764:Viral Proteins; D003546:Cysteine Endopeptidases; C072775:HC-Pro protein, potyvirus; D000409:Alanine