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 activityin 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 suppliedin transwas 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 partiallytrans-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.