TY - JOUR
T1 - Internal cleavage and trans-proteolytic activities of the VPg-proteinase (NIa) of tobacco etch potyvirus in vivo
AU - Carrington, J. C.
AU - Haldeman, R.
AU - Dolja, V. V.
AU - Restrepo-Hartwig, M. A.
PY - 1993
Y1 - 1993
N2 - The NIa protein of plant potyviruses is a bifunctional protein containing an N-terminal VPg domain and a C-terminal proteinase region. The majority of tobacco etch potyvirus (TEV) NIa molecules are localized to the nucleus of infected cells, although a proportion of NIa is attached covalently as VPg to viral RNA in the cytoplasm. A suboptimal cleavage site that is recognized by the NIa proteinase is located between the two domains. This site was found to be utilized in the VPg-associated, but not the nuclear, pool of NIa. A mutation converting Glu-189 to Leu at the P1 position of the processing site inhibited internal cleavage. Introduction of this mutation into TEV-GUS, an engineered variant of TEV that expresses a reporter protein (β-glucuronidase [GUS]) fused to the N terminus of the helper component-proteinase (HC-Pro), rendered the virus replication defective in tobacco protoplasts. Site- specific reversion of the mutant internal processing site to the wild-type sequence restored virus viability. In addition, the trans-processing activity of NIa proteinase was tested in vivo after introduction of an artificial cleavage site between the GUS and HC-Pro sequences in the cytoplasmic GUS/HC- Pro polyprotein encoded by TEV-GUS. The novel site was recognized and processed in plants infected by the engineered virus, indicating the presence of excess NIa processing capacity in the cytoplasm. The potential roles of internal NIa processing in TEV-infected cells are discussed.
AB - The NIa protein of plant potyviruses is a bifunctional protein containing an N-terminal VPg domain and a C-terminal proteinase region. The majority of tobacco etch potyvirus (TEV) NIa molecules are localized to the nucleus of infected cells, although a proportion of NIa is attached covalently as VPg to viral RNA in the cytoplasm. A suboptimal cleavage site that is recognized by the NIa proteinase is located between the two domains. This site was found to be utilized in the VPg-associated, but not the nuclear, pool of NIa. A mutation converting Glu-189 to Leu at the P1 position of the processing site inhibited internal cleavage. Introduction of this mutation into TEV-GUS, an engineered variant of TEV that expresses a reporter protein (β-glucuronidase [GUS]) fused to the N terminus of the helper component-proteinase (HC-Pro), rendered the virus replication defective in tobacco protoplasts. Site- specific reversion of the mutant internal processing site to the wild-type sequence restored virus viability. In addition, the trans-processing activity of NIa proteinase was tested in vivo after introduction of an artificial cleavage site between the GUS and HC-Pro sequences in the cytoplasmic GUS/HC- Pro polyprotein encoded by TEV-GUS. The novel site was recognized and processed in plants infected by the engineered virus, indicating the presence of excess NIa processing capacity in the cytoplasm. The potential roles of internal NIa processing in TEV-infected cells are discussed.
UR - http://www.scopus.com/inward/record.url?scp=0027437292&partnerID=8YFLogxK
U2 - 10.1128/jvi.67.12.6995-7000.1993
DO - 10.1128/jvi.67.12.6995-7000.1993
M3 - Article
C2 - 8230423
AN - SCOPUS:0027437292
SN - 0022-538X
VL - 67
SP - 6995
EP - 7000
JO - Journal of Virology
JF - Journal of Virology
IS - 12
ER -