Changes in populations of cauliflower mosaic virus DNA and RNA forms during turnip callus proliferation

Cauliflower mosaic virus (CaMV) nucleic acids accumulate in the cell in different structural conformations related to their roles in gene expression, replication and virion assembly. We have characterized changes in the population CaMV DNA and RNA replication products which occur following culture of infected turnip leaves under conditions where callus proliferates. After only 5 days in culture, a significant increase in the level of genome-length and subgenomic supercoiled (SC) DNA forms was observed by two-dimensional (2D) gel electrophoresis. Open circular (OC) molecules, corresponding to these SC DNAs, with mobilities consistent with the presence of a single break in each strand, were also detected after 5 days culture. By 10 days culture, the proportion of OC molecules with only one break per double-stranded molecule had increased. After 34 days culture, SC DNA with a range of sizes predominated in the unencapsidated DNA fraction. The change in pattern of OC and SC DNA forms during callus proliferation suggests a possible precursor/product relationship involving generation of deleted molecules from gap-containing virion DNA-like molecules followed by sequential repair of the gaps to produce SC DNA. Moreover, heterogeneity in the mobility of OC DNAs in the neutral dimension of 2D electrophoresis, a feature exhibited by twisted CaMV virion DNA, changed during the time-course suggesting that untwisting occurs during gap repair. Although the relative abundance of SC DNA increased during callus proliferation, CaMV polyadenylated 35S and 19S transcripts declined together with immediate reverse transcription products. We suggest that cellular changes during callus growth lead to a decline in authentic CaMV transcripts in the cytoplasm resulting in cessation of synthesis of viral products and progeny DNA genomes. In consequence, pre-existing virion DNAs return to the nucleus, possibly as a result of a relaxation in a cytoplasmic control mechanism, where they are assembled into various forms of SC DNA. The presence of CaMV SC DNAs in replicating cells might also enhance illegitimate integration into host chromosomes, as hybridization of CaMV DNA to high M(r) DNA was observed.