1.11.2
COMPLETE NUCLEOTIDE SEQUENCE AND GENOME ORGANIZATION OF BROAD BEAN WILT FABAVIRUS

S NAKAMURA, T IWAI and R HONKURA

Miyagi Pref Agric Res Ctr, Natori, Japan

Background and objectives
Broad bean wilt virus (BBWV) is type member of the fabavirus group. It has a wide host range among dicotyledons and some families of monocotyledons, and is economically one of the most important viruses in the world [1]. BBWV has a bipartite genome, consisting of two positive-strand RNA molecules denoted RNA1 (6 kb) and RNA2 (3.5 kb). Fabaviruses have been included in the family Comoviridae together with the Comoviruses and the Nepoviruses. Both the Comoviruses and the Nepoviruses have been well investigated, while fabaviruses have not been characterized at the molecular level [2]. We present the complete nucleotide sequences of both BBWV RNA1 and RNA2, and compare the deduced translation products with those of related viruses.

Materials and methods
The MB7 isolate of BBWV was isolated from broad bean (Vicia faba) showing mosaic symptom in Miyagi, Japan, and was propagated in broad bean. Purified viral RNAs were reverse-transcribed by priming with oligo(dT) or random hexanucleotides, and then the cDNAs were cloned and sequenced. The 5'-ends of both RNA1 and RNA2 were cloned by RACE.

Results and conclusions
BBWV RNA1 is 5957 nucleotides in length, excluding the 3' poly(A) tail, and contains a single long open reading frame (ORF) of 5613 nucleotides extending from nucleotide 235 to 5847. The putative polyprotein encoded by this ORF (polyprotein-1) is 1870 amino acids in length with a Mr of 210 kDa. The primary structure of polyprotein-1 was compared with those of other viruses, revealing a similar genetic organization to that of Comoviruses. Four conserved motifs, the protease cofactor, the NTP-binding, the protease and the RNA-dependent RNA polymerase, were identified within polyprotein-1. Of these domains, the RNA polymerase of BBWV shows the greatest similarity to those of Comoviruses, with identities of 38-41%.

BBWV RNA2 is 3587 nucleotides in length, excluding the 3' poly(A) tail, and contains a single long ORF of 3156 nucleotides extending from nucleotide 222 to 3377. The putative polyprotein encoded by this ORF (polyprotein-2) is 1051 amino acids in length with an Mr of 118 kDa. The proteolytic processing sites within the BBWV polyprotein-2 have been deduced by comparison with those of fully-sequenced Comoviruses, indicating that the BBWV polyprotein-2 is divided into three mature proteins. The overall amino acid identity of the BBWV polyprotein-2 compared with those of Comoviruses is low (16-18.7%).

References
1. Taylor RH, Stubbs LL, 1972. CMI/AAB Descriptions No. 81.
2. Goldbach R, Martelli GP, Milne RG, 1995. In Virus Taxonomy, Springer-Verlag, pp. 341-347.