Viroid synthesis: the question of inhibition by actinomycin D

Abstract

Viroids, pathogenic, low molecular weight (105) single-stranded circular RNA molecules, represent a unique class of minimal infectious agents. Physical characterisation has culminated in the primary sequence of the 359 nucleotides comprising the potato spindle tuber viroid (PSTV)1. However, other than the dramatic expression of disease symptoms in plants2, little is understood of the synthesis and biological activity of the viroid RNA. Molecular hybridisation techniques resulted in the detection of both viroid complementary RNA3 and DNA4 sequences. The putative template role of the DNA sequences was reinforced by the reported inhibition of viroid synthesis by actinomycin D5–7. However, viroid-specified proteins do not seem to function in replication or pathogenesis, for in vitro translation has not been accomplished8,9. Furthermore, no AUG initiation triplets have been found in the primary sequence of PSTV or in the hypothetical complementary sequence. Viroid synthesis, therefore, may be characterised as a markedly host-dependent process and consequently potentially very sensitive to inhibition of DNA-directed functions by agents such as actinomycin D. The accumulation of viroid progeny in nuclei5,10and nuclear-rich subcellular fractions11 has provided clues to the mechanism of viroid synthesis. What was reported as a viroid complementary DNA sequence from DNA-rich extracts of Citrus exocortis viroid (CEV)-infected tissue6 has subsequently been positively identified as complementary RNA3 that may be associated with DNA-rich preparations. With further purification and treatment, no significant levels of viroid-complementary DNA can be detected in diseased Gynura aurantiaca or tomato. Therefore, we feel an RNA intermediate must assume a predominant role in any scheme for CEV replication and/or pathogenesis. We now report that synthesis of CEV and PSTV are not inhibited by actinomycin D concentrations which reduce host 5S RNA synthesis by 80%. Thus, the primary role of actinomycin D in viroid synthesis may reflect either inhibition of a host-specified function or a general toxic effect on cell metabolism, and not evidence for the replication through a DNA intermediate.