Detection of 6K1 as a mature protein of 6 kDa in plum pox virus-infected Nicotiana benthamiana

The RNA genome of Plum pox virus (PPV) encodes one large polyprotein that is subsequently cleaved into mature viral proteins. One of the products of proteolytic processing, the 6K1 protein, has not yet been identified in vivo for any member of the genus Potyvirus. In this study, 6K1-specific polyclonal antiserum was raised against PPV 6K1 expressed in Escherichia coli as a translational fusion with the N terminus of avian troponin C and an unusual metal-binding cluster of troponin T-1. For detection of 6K1 in vivo, a pPPV-H6K1-NAT infectious clone was constructed, enabling concentration of histidine-tagged 6K1 by affinity chromatography. Affinity-purified 6K1 was detected in locally infected Nicotiana benthamiana leaves at 4, 7 and 14 days post-inoculation (d.p.i.) and, in addition, in systemically infected leaves at 14 d.p.i., 6K1 was detected exclusively as a protein of 6 kDa and no polyprotein precursors were identified with the raised anti-6K1 antiserum.