Genome amplification and long-distance movement functions associated with the central domain of tobacco etch potyvirus helper component-proteinase

Virology. 1997 Feb 17;228(2):251-62. doi: 10.1006/viro.1996.8368.

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alanine
  • Binding Sites
  • Cysteine Endopeptidases / genetics
  • Cysteine Endopeptidases / physiology*
  • Gene Amplification
  • Genetic Complementation Test
  • Genome, Viral
  • Movement
  • Mutagenesis
  • Plants, Genetically Modified
  • Plants, Toxic
  • Potyvirus / enzymology*
  • Potyvirus / genetics
  • Protein Processing, Post-Translational
  • Protoplasts
  • Tobacco / virology
  • Viral Proteins / genetics
  • Viral Proteins / physiology*

Substances

  • Viral Proteins
  • Cysteine Endopeptidases
  • HC-Pro protein, potyvirus
  • Alanine