1887

Abstract

ORF 1a of (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 -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 , the fusion proteins were specifically self-cleaved into the GST–PCP and MT fragments. MT-His was purified on Ni–NTA agarose and its N-terminal sequence determined by Edman degradation as GVEEEA, thus providing direct evidence for the Gly/Gly bond cleavage. The GST–PCP fragment purified on glutathione –agarose was used as an immunogen to produce anti-PCP monoclonal antibodies (mAbs). On Western blots of proteins from virus-infected , 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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2003-08-01
2021-01-22
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