Nuclear Transport of Tobacco Etch Potyviral RNA-Dependent RNA Polymerase Is Highly Sensitive to Sequence Alterations

Abstract

The putative RNA-dependent RNA polymerase (Nlb protein) of tobacco etch potyvirus accumulates primarily in the nucleus of infected cells, although viral RNA replication is suggested to occur in the cytoplasm. To understand the possible relationship between Nlb nuclear localization and its function, we have studied translocation of Nlb using gene fusion and plant transformation techniques. When expressed as a fusion with a cytoplasmic reporter protein,β-glucuronidase (GUS), Nlb efficiently directed transport to the nucleus in transgenic tobacco plants, confirming that Nlb contains an independent nuclear translocation signal. The effects of site-directed substitutions and deletions in Nlb were analyzed. Substitutions were targeted to three small clusters of basic amino acids that bear some resemblance to well-characterized nuclear localization signals (NLSs) of other karyophilic proteins. Amino acid changes affecting two clusters, between residues 3-5 and residues 303-306, abolished transport activity. However, the assignment of NLS function to these regions was complicated by the fact that substitutions at four additional sites throughout the Nlb sequence also rendered the fusion protein primarily cytoplasmic. Each of six deletions in Nlb debilitated nuclear localization, regardless of whether the basic clusters were deleted. Insertions of Pro-Pro dipeptides, which were predicted to induce protein folding aberrations, at three out of four positions in Nlb reduced translocation. Taken together, these results suggest that nuclear localization activity of Nlb may require a stringent tertiary structure in addition to one or more NLSs.