Institute of Biotechnology, University of Helsinki, PL56, Viikinkaari 9, Helsinki, FIN-00014, Finland

Background and objectives
Potyviruses are the largest group of plant viruses with single-stranded positive RNA genomes. Approximately 10 kb long, the genomic RNA has a VPg at the 5' end and a poly(A) tail at the 3' end, and is translated into single polyprotein which is subsequently cleaved up to 10 functional proteins: P1, HC, P3, 6K1, CI, 6K2, VPg, NIA-Pro, NIb and CP. These proteins function in such virus-specific activities as replication, cell-to-cell and long-distance movement, particle formation and interactions with different plant factors such as host proteins and RNAs. In those processes interaction(s) between RNAs and viral protein(s) might play a crucial role, and therefore the RNA-binding properties of several potyvirus-encoded proteins have been studied for several years. So far RNA-binding properties were reported only for P1, HC, CI and NIa-Pro from different potyviruses. However, the complete picture of RNA-binding properties of proteins for a single potyvirus is still missing. In 1994, the complete nucleotide sequence of potato A potyvirus (PVA) genomic RNA was determined by our group [1] and now we analyse the RNA-binding activities of all PVA encoded proteins, except the 6K1 and 6K2 peptides.

Materials and methods
All PVA protein 'genes' were cloned into pQE vectors (Qiagen) and recombinant proteins expressed in Escherichia coli and purified by Ni-NTA chromatography. RNA-binding activities were detected by Northwest blotting and membrane-binding assays in solution.

Results and conclusions
Purity of E. coli-expressed proteins was verified on SDS-PAGE, and for CI protein the enzymatic activity (NTPase) was also proven. In Northwest assays, the following labelled RNAs were used as probes: 5' NTR (165 b) and 3' NTR with poly(A) (260 b), both in sense and antisense orientations, and full-length infectious transcript from icDNA clone. Four labelled RNAs were used as controls: full-length transcript from icDNA of potato virus X, two non-viral transcripts for genes of plant origin, and a double-stranded RNA probe. The results of Northwest assays, carried out under various conditions, clearly identified P1, HC, CI, VPg, NIa-Pro, NIb and CP as RNA-binding proteins. Using nine different RNA probes in Northwest blotting and membrane-binding assays we found that all seven PVA-encoded RNA-binding proteins show no sequence preference in RNA binding. We were unable to detect any RNA binding for the P3 protein in both assays used in this study. Thus our results indicate that at least seven PVA-encoded proteins bind RNA in a sequence-independent manner, while one protein lacks RNA-binding activity. Sequence-independent RNA binding by potyvirus-encoded proteins might be an important mechanism both in the virus replication cycle and in pathogenesis, since it allows virus to interfere in host plant metabolism via possible binding to different host RNAs at different stages of potyvirus infection.

1. Puurand Ü, Mäkinen K, Paulin L, Saarma M, 1994. Journal of General Virology 75, 461-465.