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Processing and subcellular localization of the leader papain-like proteinase of Beet yellows closterovirus

¹ Department of Virology and Belozersky Institute of Physico-Chemical Biology, Moscow State University, 119899 Moscow, Russia
² Shemyakin & Ovchinnikov Institute of Bioorganic Chemistry, 117871 Moscow, Russia
³ Department of Plant Virology, Microbiology and Biosafety, BBA, Messeweg 11-12, D-38104 Braunschweig, Germany
⁴ Institute for Plant Protection in Fruit Crops, BBA, Schwabenheimer Str. 101, D-69221 Dossenheim, Germany

Correspondence
Alexey Agranovsky
aaa{at}genebee.msu.su

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-His₆ was purified on Ni–NTA agarose and its N-terminal sequence determined by Edman degradation as GVEEEA, thus providing direct evidence for the Gly₅₈₈/Gly₅₈₉ 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.

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