DNA forms of the geminivirus African cassava mosaic virus consistent with a rolling circle mechanism of replication.
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).
Saunders
K
K
Department of Virus Research, John Innes Institute, John Innes Centre for Plant Science Research, Norwich, UK.
Lucy
A
A
Stanley
J
J
D002845:Chromatography; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000235:genetics / Q000302:isolation & purification; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D015180:Electrophoresis, Gel, Two-Dimensional; D009029:Mosaic Viruses / Q000235:genetics*
D004277:DNA, Single-Stranded; D004279:DNA, Viral