Evidence for a single rolling circle in the replication of potato spindle tuber viroid

We analyzed in vivo-labeled RNA to determine which of the two proposed rolling-circle models is more likely to depict the replication cycle of potato spindle tuber viroid. A key feature distinguishing the two models is the presence of a circular monomeric minus strand in one and not the other. Chromatography on cellulose CF11 was used to purify a fraction containing the replication intermediates free from single-stranded progeny. Heat denaturation followed by gel electrophoresis was used to seek possible circular templates--species required for rolling-circle replication to take place. Upon heating, a 32P-labeled RNA was released. Limited nuclease digestion (``nicking'') revealed that this was a unit-length circular RNA. Fingerprinting identified it as a plus strand. No circular minus strands were detected in this population or in nuclease-treated samples containing RNase T1-resistant cores of the replication complex. Thus, potato spindle tuber viroid appears to use an asymmetric pathway in which minus strands are synthesized by rolling-circle copying, but plus strands are not. More details of the replication pathways used by various viroid-like RNAs are needed and will help to establish the evolutionary relationships among these infectious agents.