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

Abstract

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 CF1l 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 unitlength 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 Tl-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.Viroids and related RNAs are the smallest well-defined microorganisms. Interest in the mechanism of viroid replication has broadened recently with the discovery that the 8 agent, the etiologic agent of an often fatal form of hepatitis in man, has a small circular RNA genome (1-3) and may have additional similarities to the small infectious RNAs of plants. A rolling-circle replication cycle was first proposed for viroids in 1981 (4). Although it is now generally accepted that rolling-circle copying occurs during the replication of viroidlike RNAs (5-7), there have been no direct studies to determine which of the two prevailing models more accurately defines the cycle for any particular pathogen. In the original asymmetric model there is a single rolling circle: a circular plus strand* is copied to give a multimeric linear minus strand which directly serves as the plus-strand template (Fig. 1A). In the alternative, symmetric model presented in 1984 (8), the long minus strand is first cleaved to unit length and circularized; it then initiates a second round of rolling-circle copying (Fig. 1B). We identify methods to distinguish these two pathways and, in particular, to ascertain which of these two models accounts for the replication of potato spindle tuber viroid (PSTV).Several lines of evidence suggest that chromatography on cellulose CF11 could be used to prepare the RNAs of the viroid replication complex. For example, this method (9) separates single-stranded RNA (ssRNA) from doublestranded RNA (dsRNA) and has been widely used to purify and analyze the replication intermediates of pathogens which, like viroids (10), have a minus strand composed of RNA. In addition, Northern analysis of RNA gel blots containing control and nuclease-treated samples have shown that ex...

Rolling-circle replication of viroids, viroid-like satellite RNAs and hepatitis delta virus: Variations on a theme

“…Because only the oligomeric (-) strands, and not their monomeric circular derivative, have been identified in PSTVd-infected tomato, this and other members of the family Pospiviroidae presumably replicate via an asymmetric pathway with a single rolling-circle 55,56 (Fig. 1).…”

A Viroid RNA with a Specific Structural Motif Inhibits Chloroplast Development

“…Viroids are grouped into two taxonomic families on the basis of their biological, structural, and biochemical properties (Flores et al, 2005b). Members of the family Pospiviroidae, type species Potato spindle tuber viroid (PSTVd) (Diener, 1972;Gross et al, 1978), adopt a rod-like or quasi-rod-like conformation with characteristic motifs, prominent among them a central conserved region, and replicate in the nucleus by an asymmetric rolling-circle mechanism (Branch et al, 1988;Harders et al, 1989;Bonfiglioli et al, 1996;Qi and Ding, 2003b). By contrast, members belonging to the family Avsunviroidae Fadda et al, 2003), type species Avocado sunblotch viroid (ASBVd) (Symons, 1981;Hutchins et al, 1986), may adopt branched conformations, do not contain a central conserved region, and replicate in the chloroplast by a symmetric rollingcircle mechanism in which the oligomeric strands of both polarities self-cleave through hammerhead ribozymes (Bonfiglioli et al, 1994;Darò s et al, 1994;Lima et al, 1994;Bussiè re et al, 1999;Navarro et al, 1999).…”

Viroid RNA redirects host DNA ligase 1 to act as an RNA ligase

“…1A), the incoming most-abundant circular RNA, arbitrarily assigned (+) polarity, is reiteratively transcribed into oligomeric RNAs of complementary (−) polarity that do not undergo processing and serve directly as templates for transcription of (+) oligomers. These oligomers of (+) polarity are finally processed to circular monomers (8). In the Avsunviroidae, which replicate in the chloroplasts, the oligomeric (−) RNAs self-cleave through hammerhead ribozymes, and the resulting monomers, once circularized, serve as templates for the synthesis of the (+) oligomers in a second rolling-circle symmetric to the first (9).…”