Analysis of the VPg-proteinase (NIa) encoded by tobacco etch potyvirus: effects of mutations on subcellular transport, proteolytic processing, and genome amplification.

A mutational analysis was conducted to investigate the functions of the tobacco etch potyvirus VPg-proteinase (NIa) protein in vivo. The NIa N-terminal domain contains the VPg attachment site, whereas the C-terminal domain contains a picornavirus 3C-like proteinase. Cleavage at an internal site separating the two domains occurs in a subset of NIa molecules. The majority of NIa molecules in TEV-infected cells accumulate within the nucleus. By using a reporter fusion strategy, the NIa nuclear localization signal was mapped to a sequence within amino acid residues 40 to 49 in the VPg domain. Mutations resulting in debilitation of NIa nuclear translocation also debilitated genome amplification, suggesting that the NLS overlaps a region critical for RNA replication. The internal cleavage site was shown to be a poor substrate for NIa proteolysis because of a suboptimal sequence context around the scissile bond. Mutants that encoded NIa variants with accelerated internal proteolysis exhibited genome amplification defects, supporting the hypothesis that slow internal processing provides a regulatory function. Mutations affecting the VPg attachment site and proteinase active-site residues resulted in amplification-defective viruses. A transgenic complementation assay was used to test whether NIa supplied in trans could rescue amplification-defective viral genomes encoding altered NIa proteins. Neither cells expressing NIa alone nor cells expressing a series of NIa-containing polyproteins supported increased levels of amplification of the mutants. The lack of complementation of NIa-defective mutants is in contrast to previous results obtained with RNA polymerase (NIb)-defective mutants, which were relatively efficiently rescued in the transgenic complementation assay. It is suggested that, unlike NIb polymerase, NIa provides replicative functions that are cis preferential

Long-distance movement factor: a transport function of the potyvirus helper component proteinase.

Transport of viruses from cell to cell in plants typically involves one or more viral proteins that supply dedicated movement functions. Transport from leaf to leaf through phloem, or long-distance transport, is a poorly understood process with requirements differing from those of cell-to-cell movement. Through genetic analysis of tobacco etch virus (TEV; potyvirus group), a novel long-distance movement factor was identified that facilitates vascular-associated movement in tobacco. A mutation in the central region of the helper component proteinase (HC-Pro), a TEV-encoded protein with previously described activities in aphid-mediated transmission and polyprotein processing, inactivated long-distance movement. This mutant virus exhibited only minor defects in genome amplification and cell-to-cell movement functions. In situ histochemical analysis revealed that the mutant was capable of infecting mesophyll, bundle sheath, and phloem cells within inoculated leaves, suggesting that the long-distance movement block was associated with entry into or exit from sieve elements. The long-distance movement defect was specifically complemented by HC-Pro supplied in trans by a transgenic host. The data indicate that HC-Pro functions in one or more steps unique to long-distance transport

NIa and NIb of Peanut Stripe Potyvirus Are Present in the Nucleus of Infected Cells, but Do Not Form Inclusions

AbstractWe investigated, by immunological and gene-fusion methods, whether the failure of peanut stripe potyvirus (PStV)-encoded nuclear inclusion proteins a (NIa) and b (NIb) to form nuclear inclusions is due to the lack of theirin vivoaccumulation or the inability of one or both proteins to be transported into the nucleus. NIa domains (NIa-VPg and NIa-proteinase), full-length NIb, and full-length cylindrical inclusion (CI) protein of PStV were cloned, expressed inEscherichia coli,and used for antisera production. Immunoblot analysis of accumulation of NIa, NIb, and CI in time course experiments revealed that they accumulated to similar levels in PStV-infectedNicotiana benthamiana.In immunocytochemical studies with electron microscopy, antiserum against NIa-VPg, NIa-Pro, and NIb specifically labeled NIa and NIb proteins throughout the nuclei of PStV-infected cells, in the absence of nuclear inclusions. Translational fusions were made between NIa and NIb to either the green fluorescence protein or the β-glucuronidase in vectors for transient gene expression or stable expression in transgenic plants, respectively. Fusion proteins containing NIa accumulated in the nucleus, whereas fusion proteins containing NIb accumulated in a punctate pattern in the cytoplasm. These data indicate that at least NIa possesses a nuclear localization signal