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Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Viikinkaari 9, Helsinki, FIN-00014, Finland1
National Institute of Chemical Physics and Biophysics, Akadeemia tee 23, EE12611 Tallinn, Estonia2
Department of Plant Biology, Genetics Centre, SLU, PO Box 7080, S-750 07 Uppsala, Sweden3
Author for correspondence: Andres Merits at University of Helsinki. Fax +358 9 19159560. e-mail merits{at}operoni.helsinki.fi
Processing of the polyprotein encoded by Potato virus A (PVA; genus Potyvirus) was studied using expression of the complete PVA polyprotein or its mutants from recombinant baculoviruses in insect cells. The time-course of polyprotein processing by the main viral proteinase (NIaPro) was examined with the pulse–chase method. The sites at the P3/6K1, CI-6K2 and VPg/NIaPro junctions were processed slowly, in contrast to other proteolytic cleavage sites which were processed at a high rate. The CI-6K2 polyprotein was observed in the baculovirus system and in infected plant cells. In both cell types the majority of CI-6K2 was found in the membrane fraction, in contrast to fully processed CI. Deletion of the genomic region encoding the 6K1 protein prevented proper proteolytic separation of P3 from CI, but did not affect processing of VPg, NIaPro, NIb or CP from the polyprotein. The 6K2-encoding sequence could be removed without any detectable effect on polyprotein processing. However, deletion of either the 6K1 or 6K2 protein-encoding regions rendered PVA non-infectious. Mutations at the 6K2/VPg cleavage site reduced virus infectivity in plants, but had a less pronounced, albeit detectable, effect on proteolytic processing in the baculovirus system. The results of this study indicate that NIaPro catalyses proteolytic cleavages preferentially in cis, and that the 6K1/CI and NIb/CP sites can also be processed in trans. Both 6K peptides are indispensable for virus replication, and proteolytic separation of the 6K2 protein from the adjacent proteins by NIaPro is important for the rate of virus replication and movement.
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