Viroids are single-stranded, circular RNAs of 250 to 400 bases, that replicate 
autonomously in their host plants but do not code for a protein. Viroids of the 
family Pospiviroidae, of which potato spindle tuber viroid (PSTVd) is the type 
strain, are replicated by the host's DNA-dependent RNA polymerase II in the 
nucleus. To analyze the initiation site of transcription from the (+)-stranded 
circles into (-)-stranded replication intermediates, we used a nuclear extract 
from a non-infected cell culture of the host plant S. tuberosum. The 
(-)-strands, which were de novo-synthesized in the extract upon addition of 
circular (+)-PSTVd, were purified by affinity chromatography. This purification 
avoided contamination by host nucleic acids that had resulted in a misassignment 
of the start site in an earlier study. Primer-extension analysis of the de 
novo-synthesized (-)-strands revealed a single start site located in the hairpin 
loop of the left terminal region in circular PSTVd's secondary structure. This 
start site is supported further by analysis of the infectivity and replication 
behavior of site-directed mutants in planta.