Turnip crinkle virus (TCV) is a single-stranded positive-sense RNA virus of the Carmovirus genus. Two of its five open reading frames (ORFs), encoding proteins of 8 and 9 kDa, are required for cell-to-cell movement of the virus. These movement proteins (MPs) were fused to green fluorescent protein (GFP) to determine their cellular localization. In protoplasts, p9-GFP, like GFP itself, is found throughout the cytoplasm, as well as in cell nuclei. In contrast, p8-GFP was confined to the cell nucleus. Similar localization patterns were observed when specific small peptide epitopes were fused to p and p9 proteins instead of GFP. The cytoplasmic localization of p9-GFP and nuclear localization of p8-GFP were also detected in leaves after particle bombardment of DNA encoding these fusion proteins or after overexpression of p8-GFP in transgenic Arabidopsis seedlings. The expression of the GFP fusion proteins by recombinant TCV viruses in infected protoplasts or on inoculated Arabidopsis leaves produced similar patterns. Unlike TMV-MP and other MPs studied to date, no obvious punctuate expression in the cell wall or association with the cytoskeleton was detected. The sequence analysis of p8 revealed two unique nuclear localization signals (NLSs), which were not conserved within p8 homologues of other viruses in the genus Carmovirus. Mutation in either of these NLSs did not disrupt the nuclear localization of p8-GFR However, when both NLSs were mutated, p8-GFP expression was no longer restricted to cell nuclei. The NLSs are not required for cell-to-cell movement; TCV recombinant viruses mutated in one or both NLSs could still facilitate cell-to-cell movement of the virus. The nuclear localization of p8 suggests a novel function for this protein in the cell nucleus. (C) 2000 Academic Press.
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