1.11.24
IDENTIFICATION OF COCKSFOOT MOTTLE VIRUS INDIVIDUAL GENE PRODUCTS

T TAMM1, K MÄKINEN2 and E TRUVE1

1Institute of Chemical Physics and Biophysics, Tallinn, Estonia; 2Institute of Biotechnology, University of Helsinki, Finland

Background and objectives
Cocksfoot mottle virus (CfMV), a member of the plant sobemovirus group, has a positive-sense single-stranded RNA genome. The coding region of the virus contains four open reading frames (ORFs) [1]. In contrast to the other sequenced sobemoviruses, CfMV lacks the continuous large ORF for the polyprotein. However, CfMV genomic RNA directs the in vitro synthesis of at least four major proteins of 100, 71, 34 and 16 kDa, in wheat germ extract (WGE) [2], whereas no ORF is able to individually encode a 100-kDa protein. We have shown that CfMV is the first sobemovirus reported to translate its polyprotein by using a -1 ribosomal frameshift [2]. This is achieved by translation from two overlapping ORFs, called 2a and 2b. The purpose of our studies is to identify the virus-encoded proteins and their functions, respective regulatory sequences in viral genomic RNA, and to characterize the possible mechanism(s) regulating the efficiency of the -1 ribosomal frameshifting.

Results and conclusions
We have expressed proteins encoded by all four CfMV ORFs in E. coli and raised rabbit polyclonal antisera recognizing specifically these recombinant proteins. N-terminal sequencing of CfMV coat protein proved that it is encoded by ORF3. Coupled in vitro transcription/translation of overlapping ORF2a and ORF2b revealed that P100 and P71 are the products of these ORFs. The detailed map of genes encoding CfMV proteins was constructed after immunoprecipitation of proteins translated from CfMV genomic RNA and from individually cloned ORFs.

CfMV ORF2a-2b overlap region, comprising 531 nt, contains all cis-acting signals required for -1 ribosomal frameshifting: a shifty heptamer with the sequence U UUA AAC and, 7 nt downstream, a potential stem-loop structure. Chimaeric GUS-CfMV frameshift constructs which differ from each other in one amino-acid codon just downstream from the slippery sequence have been constructed. The in vitro translation of these constructs in WGE showed that the first amino-acid codon after the shifty sequence has a strong influence on the -1 ribosomal frameshifting efficiency.

A full-length cDNA clone of CfMV under the control of the bacteriophage T7 RNA promoter has been constructed. Infectivity tests using barley and oat plants indicated that this clone is infectious but has lower infection frequency compared to the wild-type virus.

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.