Abstract:
A mutational analysis was conducted to investigate the functions of the tobacco etch potyvirus VPgproteinase
(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 activesite
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
