C HORSER, G HAFNER, R WANITCHAKORN, R HARDING and J DALE

Centre for Molecular Biotechnology, Queensland University of Technology, GPO Box 2434, Brisbane, Queensland 4001, Australia

Background and objectives
Banana bunchy top virus (BBTV) is an isometric virus with a circular single-stranded DNA genome consisting of at least six components (BBTV DNA-1 to 6). Each of these components is approximately 1 kb and each contains one gene in the virion sense, except for BBTV DNA-1 which contains two genes. BBTV appears to be a member of a previously undescribed group of viruses which could include subterranean clover stunt virus, faba bean necrotic yellows virus, coconut foliar decay virus and milk vetch dwarf virus. There is little information available regarding the replication of these viruses although a rolling circle mechanism is highly likely. We have been studying the replication strategy of BBTV and current results strongly support such a mechanism.

Results and conclusions
As the BBTV genome is single-stranded DNA, it is not transcriptionally active. We have demonstrated that the ssDNA components of BBTV can be converted to dsDNA by a DNA polymerase and that this is initiated from an endogenous primer bound to the genomic components. The presence of this primer has been confirmed for five of the six components. BBTV DNA-5 appears to be the most efficiently primed component, suggesting that the protein encoded by this component is important early in infection.

We have demonstrated that the major gene of BBTV DNA-1 encodes a replication-associated protein (Rep) based on (i) the presence of both dNTP binding and rolling circle replication motifs; and (ii) the ability of the expressed protein to nick and ligate within the loop sequence that is conserved between all components. Importantly, we have identified multimeric forms of DNA-1 in infected plants. We have also demonstrated that BBTV DNA-3 encodes the virus coat protein based on N-terminal sequencing of the coat protein and the reaction of the expressed protein with antisera generated against BBTV virions.

BBTV DNA-5 encodes a 18.87-kDa protein which contains a LXCXE motif that is associated with retinoblastoma-like binding proteins. We hypothesize that this protein is involved in switching infected cells to S phase early in the infection process to facilitate viral DNA replication.

We have not identified functions for the small gene of DNA-1 or the genes of DNAs 2, 4 and 6, and have not yet identified proteins involved in the inter- and intracellular movement of BBTV DNA.

Based on our results, we hypothesize that BBTV replicates by a rolling circle mechanism and propose that following inoculation into a banana cell and uncoating, BBTV genomic ssDNA is converted into transcriptionally active dsDNA forms by a host DNA polymerase and the various genes expressed. It is probable that the first of the expressed genes is that encoded by DNA-5 which initiates the switching of the infected cell to S phase, facilitating rolling circle DNA replication, directed by the viral Rep protein. The process of cell-to-cell and long-distance movement of BBTV in bananas is yet to be defined.