An iterated sequence in the genome of Banana bunchy top virus is essential for efficient replication

Abstract

Banana bunchy top virus (BBTV) has a multi-component genome of circular, single-stranded DNA. BBTV replicates via a rolling-circle mechanism, probably involving sequence-specific interaction of the replication initiation protein (Rep) with iterated sequences (iterons) within the viral genome. Three putative iterons (designated F1, F2 and R), with the sequence GGGAC, have been identified in the intergenic region of each BBTV component. To investigate their role in replication, each of the iterons was mutated, singularly and in tandem, in a BBTV DNA-N 1.1mer and the ability of these molecules to be replicated by the BBTV 'master' Rep was evaluated in banana cells using transient biolistic assays. All iteron mutants were replicated less efficiently than the native DNA-N. Mutation of the F1 and R iterons caused a 42 and 62 % reduction in DNA-N replication, respectively, whereas mutation of the F2 and combined F1F2 iteron virtually abolished DNA-N replication.