Processing and subcellular localization of the leader papain-like proteinase of Beet yellows closterovirus.
ORF 1a of Beet yellows closterovirus (BYV) encodes the domains of the papain-like proteinase (PCP), methyltransferase (MT) and RNA helicase. BYV cDNA inserts encoding the PCP-MT region were cloned in pGEX vectors next to the glutathione S-transferase gene (GST). In a 'double tag' construct, the GST-PCP-MT cDNA was flanked by the 3'-terminal six histidine triplets. Following expression in E. coli, the fusion proteins were specifically self-cleaved into the GST-PCP and MT fragments. MT-His(6) was purified on Ni-NTA agarose and its N-terminal sequence determined by Edman degradation as GVEEEA, thus providing direct evidence for the Gly(588)/Gly(589) bond cleavage. The GST-PCP fragment purified on glutathione S-agarose was used as an immunogen to produce anti-PCP monoclonal antibodies (mAbs). On Western blots of proteins from virus-infected Tetragonia expansa, the mAbs recognized the 66 kDa protein. Immunogold labelling of BYV-infected tissue clearly indicated association of the PCP with the BYV-induced membranous vesicle aggregates, structures related to closterovirus replication.
Zinovkin
Roman A
RA
Department of Virology and Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia.
Erokhina
Tatyana N
TN
Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, 117871 Moscow, Russia.
Lesemann
Dietrich E
DE
Department of Plant Virology, Microbiology and Biosafety, BBA, Messeweg 11-12, D-38104 Braunschweig, Germany.
Jelkmann
Wilhelm
W
Institute for Plant Protection in Fruit Crops, BBA, Schwabenheimer Str. 101, D-69221 Dossenheim, Germany.
Agranovsky
Alexey A
AA
Department of Virology and Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia.
D035722:Aizoaceae / Q000821:virology; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D027461:Beta vulgaris / Q000821:virology*; D002478:Cells, Cultured; D017790:Closterovirus / Q000201:enzymology* / Q000235:genetics; D008565:Membrane Proteins; D008854:Microscopy, Electron; D010206:Papain / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D013347:Subcellular Fractions / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; C026515:type I signal peptidase; D010206:Papain