DNA forms of the geminivirus African cassava mosaic virus consistent with a rolling circle mechanism of replication

Abstract

We have analysed DNA from African cassava mosaic virus (ACMV)-infected Nicotiana benthamiana by two-dimensional agarose gel electrophoresis and detected ACMV-specific DNAs by blot-hybridisation. ACMV DNA forms including the previously characterised single-stranded, open-circular, linear and supercoiled DNAs along with five previously uncharacterised heterogeneous DNAs (H1-H5) were resolved. The heterogeneous DNAs were characterised by their chromatographic properties on BND-cellulose and their ability to hybridise to strand-specific and double-stranded probes. The data suggest a rolling circle mechanism of DNA replication, based on the sizes and strand specificity of the heterogeneous single-stranded DNA forms and their electrophoretic properties in relation to genome length single-stranded DNAs. Second-strand synthesis on a single-stranded virus-sense template is evident from the position of heterogeneous subgenomic complementary-sense DNA (H3) associated with genome-length virus-sense template (VT) DNA. The position of heterogeneous virus-sense DNA (H5), ranging in size from one to two genome lengths, is consistent with its association with genome-length complementary-sense template (CT) DNA, reflecting virus-sense strand displacement during replication from a double-stranded intermediate. The absence of subgenomic complementary-sense DNA associated with the displaced virus-sense strand suggests that replication proceeds via an obligate single-stranded intermediate. The other species of heterogeneous DNAs comprised concatemeric single-stranded virus-sense DNA (H4), and double-stranded or partially single-stranded DNA (H1 and H2).

Molecular characterization of banana bunchy top virus isolate from Sri Lanka and its genetic relationship with other isolates

“…The nine nucleotide sequence within the 11 nt conserved sequence were identical to the invariant loop sequence in geminiviruses [28]. A model for implicating the loop sequence in rolling circle replication has been described for Gemini viruses [36]. Thus, it is possible that the loop sequence of the BBTV has a similar function.…”

TGMV replication protein AL1 preferentially binds to single‐stranded DNA from the common region

“…Geminiviruses are a group of plant viruses with small circular single-stranded DNA genomes [l] which replicate in the nucleus of the plant cell by a rolling circle mechanism [2,3]. Tomato golden mosaic virus (TGMV) belongs to a subgroup of the geminiviruses which have genomes with two components, DNA A and DNA B, which encode several proteins involved in virus DNA replication, cell-to-cell movement, transcriptional regulation or particle structure [4].…”

Identification of the nicking tyrosine of geminivirus Rep protein

“…Geminiviruses replicate by a rolling circle mechanism [6][7][8], initiated in the intergenic region (IR) at the nonanucleotide sequence TAATATTAC, universally conserved in all geminiviruses [9,10]. The protein that mediates replication initiation is the Mr 41 kDa Rep protein, the sole viral protein essential for replication [11][12][13].…”