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.Banana bunchy top virus (BBTV) is the type member of the genus Babuvirus in the family Nanoviridae and has a genome comprising at least six circular, single-stranded DNA components (referred to as DNA-R, -S, -C, -M, -N and -U3; Vetten et al., 2005), each of approximately 1 kb (Burns et al., 1995). Based on similarities among nanovirus DNAs and those of the geminiviruses, it has generally been accepted that BBTV replication occurs by a rolling-circle-type mechanism (Stenger et al., 1991). In geminiviruses, iterated DNA sequences (iterons) play an important role in the replication process by acting as recognition sites for sequencespecific binding of their cognate replication initiation proteins (Reps). Mutation of these sites can affect Rep binding negatively in vitro and replication in vivo (Chatterji et al., 2000;Choi & Stenger, 1996; Fontes et al., 1994a, b;Orozco et al., 1998). Although putative iteron sequences have been identified in the non-coding regions of some nanovirus DNAs, including Faba bean necrotic yellows virus (FBNYV), Milk vetch dwarf virus (MDV) and Subterranean clover stunt virus (SCSV) , their exact role in replication has not been demonstrated experimentally.Analysis of the intergenic regions of BBTV DNA-R, -S, -C, -M, -N and -U3 (Horser, 2000) has identified a putative iteron sequence (GGGAC) occurring as a tandem repeat (designated iterons F1 and F2, respectively) on the virion-sense strand, 39 of the stem-loop, and as a single iteron (designated iteron R) on the complementary strand, 59 of the stem-loop. The direct repeat iterons, F1 and F2, were located 2 nt 39 of the stem-loop in DNA-R, -S, -C, -M and -N, whereas in DNA-U3, they commenced 1 nt 39 of the stem-loop. However, the location of iteron R varied, being 10 (DNA-N), 19 (DNA-R, -S, -C and -M) and 90 (DNA-U3) nt upstream of the 59 base of the stem-loop.We investigated the role of the F1, F2 and R iterons in BBTV replication by assessing the ability of the BBTV 'master' Rep (M-Rep) (encoded by DNA-R) to replicate native and iteron mutants of DNA-N in banana embryogenic cells. DNA-N...

Genetic structure and diversity of the Banana bunchy top virus population on Sumatra Island, Indonesia

“…The CR-SL is important for replication and contains a stem-loop structure (TATTATTAC) and an Biterated sequence^(F1, F2, and R) region (Herrera-Valencia et al 2006). The CR-M is known to be a GC-rich region, but its function is not clear (Burns et al 1995).…”

Molecular characterization of an Indian isolate of Banana bunchy top virus based on six genomic DNA components

“…Alignment of this region revealed variations at 11 positions. Three iterated sequences designated as F1, F2, and R with the sequence 'GGGAC' were found to be present in the CR-SL region of each component as shown earlier by Herrera-Valencia et al [22], and these iterated sequences are known to perform important role in the replication of BBTV. However, DNA-M contained an A in place of a G at position one of iteron F1 and iteron R is not found in DNA-U3 (Fig.…”

Molecular characterisation of Banana bunchy top virus (BBTV) from Pakistan

“…Recently Herrera-Valencia et al [15] have identified socalled ''iterated'' sequences in the genomic components of BBTV that are involved in sequence-specific interaction with the Rep encoded by the DNA-R component of BBTV. The three ''iterons'' described by them (designated F1, F2 and R), with the sequence GGGAC, are present in all the components analysed here.…”

“…The first, known as the stem-loop common region (CR-SL), contains a predicted stem-loop structure with the loop sequence TAG/TTATTAC and repeated sequences that are likely the sequence-specific binding motifs for Rep recognition [11,15]. With similarity to the replication origins of geminiviruses (reviewed by Hanley-Bowdoin et al [16]), the CR-SL likely forms the origin of replication of BBTV.…”