Reverse transcription products generated by defective plus-strand synthesis during cauliflower mosaic virus replication

Abstract

Cauliflower mosaic virus (CaMV) replicates by reverse transcription of an RNA template producing DNA (−)-strands on which DNA (+)-strands are initiated at two specific priming sites. We have analysed unencapsidated DNA forms of the CaMV genome isolated from infected leaf tissue to understand detailed aspects of the replicative process. This complex population of molecules contains linear single-stranded (ss), double-stranded (ds) and hairpin (hp) DNAs, and open-circular (oc) forms generated by reverse transcription. Purification by hydroxylapatite chromatography of ss-DNA (−)-strand molecules, revealed heterogeneous DNAs of up to genome length on which priming at both of the recognised (+)-strand priming sites had apparently failed to occur. Analysis by 2-dimensional gel electrophoresis suggested that the ss-DNA molecules had also lost the tRNA primer of reverse transcription necessary for the second strand-switch involving the (+)-strand. Loss of this bridging sequence might be responsible for many of the putative defective linear DNA forms observed in the unencapsidated fraction. Amongst these were DNA molecules in which (+)-strand priming had not occurred at either one or other of the recognised sites and were resolved as linear hairpin DNAs. Defective (+)-strand priming could also produced potentially infectious oc- forms with only one (+)-strand discontinuity. These latter DNAs were similar in structure to virion DNA of a CaMV isolate (CM4-184) with only one authentic (+)-strand priming site and were found preferentially in the unencapsidated dated DNA fraction. The relationship between the oc-forms and CaMV suppercoiled DNA suggest the possibility that defective (+)-strand synthesis might play a regulatory role in the CaMV multiplication cycle.