1.11.6
STUDIES ON POLYPROTEIN OF COCKSFOOT MOTTLE SOBEMOVIRUS

K MÄKINEN1, J LUCCHESI1, T TAMM2, S ZAVRIEV3 and M SAARMA1

1Institute of Biotechnology, Univeristy of Helsinki, Viikki Biocenter 1, PO Box 56, FIN-00014, Finland; 2Institute of Chemical Physics and Biophysics, Akadeemia tee 23, EE00026 Tallinn, Estonia; 3Institute of Agricultural Biotechnology, Timiryazevskaya st. 42, Moscow 127550, Russia

Background and objectives
Cocksfoot mottle sobemovirus (CfMV) has a positive-stranded RNA genome of 4082 nucleotides [1]. Its polyprotein is 103 kDa in size and encodes the putative VPg, serine proteinase and replicase. It is translated from two open reading frames (ORF2a and 2b). ORF2b is translated as part of the polyprotein by a -1 ribosomal frameshifting mechanism [2]. The aim of our study is to analyse further the ribosomal -1 frameshifting mechanism, to find out how the polyprotein is processed and to characterize the functions and interactions of CfMV proteins.

Results and conclusions
We iodinated CfMV RNA, isolated from viral particles, to study the genome-linked protein VPg. SDS-PAGE revealed an iodinated 15-kDa protein. Antiserum raised against the ORF2a protein recognized a protein of 12 kDa in size in a sample derived from isolated CfMV RNA in a Western blot. We conclude that CfMV RNA has a VPg at its 5' end, and currently we are analysing the cleavage sites of VPg in the ORF2a-encoded polyprotein. It is not known what are the target sequences for sobemoviral proteinases. We have expressed the replicase of CfMV in E. coli as a fusion with the maltose-binding protein and in a baculovirus system in order to analyse the functions of this protein.

The frameshifting signal directing the ribosomes from 0-frame to -1-frame is composed of UUUAAAC, a slippery heptamer and a hairpin loop starting seven nucleotides downstream. Deletion mutants were created to identify sequences necessary for frameshifting. Deletion-destabilizing the hairpin loop prevents the frameshifting. However, deletion of 442 nucleotides starting 10 nucleotides after the stem-loop structure does not prevent frameshifting. Also, a construct containing only 12 nucleotides of CfMV sequences upstream from the slippery heptanucleotide and having the rest of the 5' region of the polyprotein coding region deleted, is translated by -1 ribosomal frameshift.

In conclusion, our preliminary data suggest that 70 nucleotides of CfMV sequence, including the frameshifting signal, is enough to direct the ribosomes to translational shift. We are analysing the effect of these deletions on the frameshifting efficiency, when the polyprotein coding ORFs are translated in wheat germ extract at the level of 12-16%. This efficiency is high if compared to the frameshifting efficiencies of related luteoviruses.

References:
1. Mäkinen K, Tamm T, Næss V et al., 1995. Journal of General Virology 76, 2817-2825.
2. Mäkinen K, Næss V, Tamm T et al., 1995. Virology 207, 566-571.