Partial characterisation of different classes of viral DNA, and kinetics of DNA synthesis in turnip protoplasts infected with cauliflower mosaic virus

Summary

Turnip protoplasts infected with cauliflower mosaic virus (CaMV) have been used to examine the kinetics of CaMV DNA synthesis, and the different classes of CaMV DNA found in vivo partially characterised. Differential extraction techniques for DNA from infected protoplasts has identified several distinct classes of viral DNA. The same approach applied to virus preparations revealed that while the majority of virion DNA was stably encapsidated, some small DNAs and a heterogeneous population 3.8-ca. 5.0 Kb were not. The structural relationship of sa-DNA (3) with the particle is such that only its 5′ RNA moeity is susceptible to nuclease attack. Two-dimensional gel electrophoresis of total CaMV DNA from infected protoplasts revealed all the DNA species found in virion DNA, those species representing the ‘free’ DNA class and a further class of molecules, rich in DNA of (−) polarity (24), to which the role of reverse transcription intermediates has been ascribed. ‘Free’ DNA contains 8 Kb supercoiled DNA (Form I DNA), an 8 Kb open circle (Form II), an 8 Kb linear (Form III) and a truncated molecule with an extension of the (−) strand previously observed from infected plants (10). Kinetic experiments show that the accumulation of total CaMV-DNA parallels the accumulation of progeny virions to reach a maximum around 72 h post-inoculation and that there is not a separation of CaMV-DNA synthesis into clearly defined early and late stages.