Proteolytic processing of potyviral proteins and polyprotein processing intermediates in insect and plant cells

作者：Merits A^*; Rajamaki ML; Lindholm P; Runeberg Roos P; Kekarainen T; Puustinen P; Makelainen K; Valkonen JPT; Saarma M

来源：Journal of General Virology, 2002, 83: 1211-1221.

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 (NlaPro) was examined with the pulse-chase method. The sites at the P3/6K1, Cl-6K2 and VPg/NlaPro junctions were processed slowly, in contrast to other proteolytic cleavage sites which were processed at a high rate. The Cl-6K2 polyprotein was observed in the baculovirus system and in infected plant cells. In both cell types the majority of Cl-6K2 was found in the membrane fraction, in contrast to fully processed Cl. Deletion of the genomic region encoding the 6K1 protein prevented proper proteolytic separation of P3 from Cl, but did not affect processing of VPg, NlaPro, Nlb 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 NlaPro catalyses proteolytic cleavages preferentially in cis, and that the 6K1/Cl and Nlb/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 NlaPro is important for the rate of virus replication and movement.

• 出版日期2002-5