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 D^5–7. However, viroid-specified proteins do not seem to function in replication or pathogenesis, for in vitro translation has not been accomplished^8,9. 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 nuclei^5,10and 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.