TY - JOUR
T1 - Analysis of the VPg-proteinase (NIa) encoded by tobacco etch potyvirus
T2 - Effects of mutations on subcellular transport, proteolytic processing, and genome amplification
AU - Schaad, Mary C.
AU - Haldeman-Cahill, Ruth
AU - Cronin, Stephen
AU - Carrington, James C.
PY - 1996/10
Y1 - 1996/10
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=0029819754&partnerID=8YFLogxK
U2 - 10.1128/jvi.70.10.7039-7048.1996
DO - 10.1128/jvi.70.10.7039-7048.1996
M3 - Article
C2 - 8794348
AN - SCOPUS:0029819754
SN - 0022-538X
VL - 70
SP - 7039
EP - 7048
JO - Journal of virology
JF - Journal of virology
IS - 10
ER -