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.

Cloning of the Arabidopsis RTM1 gene, which controls restriction of long-distance movement of tobacco etch virus

“…First, RTM1, in cooperation with other factors, could block long-distance movement of TEV by preventing entry into, passage through, or exit from the phloem. This could occur if RTM1, or a RTM1-containing complex, physically interacts with one or more of the TEV factors required for long-distance movement (HC-Pro, CI, NIa, and capsid) (34)(35)(36)(37)(38)(39). The RTM1 protein or complex also could inhibit the function of cellular factors or structures required for long-distance movement.…”

Cell-to-cell movement of three genera (+) ss RNA plant viruses

“…The TEV HC-Pro mutant appeared to move from cell-to-cell less efficiently than the native/natural virus (Kasschau et al 1997), whereas TVMV HC-Pro mutant was unable to spread in inoculated leaves (Klein et al 1994). HC-Pro proteins together with CP were shown to increase plasmodesmal SEL and to promote viral RNA movement from cell to cell, whereas CI and NIa proteins did not induce these effects.…”

“…Some genetic analyses indicate the involvement of the HC-Pro protein in cell-to-cell transport, but its direct role in this process is not clear (Cronin et al 1995;Kasschau et al 1997). The TEV HC-Pro mutant appeared to move from cell-to-cell less efficiently than the native/natural virus (Kasschau et al 1997), whereas TVMV HC-Pro mutant was unable to spread in inoculated leaves (Klein et al 1994).…”

Silencing suppressors: viral weapons for countering host cell defenses

“…The first silencing suppressor discovered is potyvirusencoded helper component-proteinase (HC-Pro) which enhances the replication of many unrelated viruses (Kasschau et al, 1997;Pruss et al, 1997). Tobacco etch virus silencing suppressor is comprised of the 5′-proximal region of the tobacco etch potyviral genomic RNA encoding P1, HC-Pro and a small part of P3, and is termed P1/HC-Pro.…”