Comparison of multimeric plus and minus forms of viroids and virusoids

Abstract

In order to investigate the mechanism of replication of viroids and virusoids, we have compared the replication intermediates of three members of each group in nucleic acid extracts of infected plants. Viroids were avocado sunblotch viroid (ASBV), citrus exocortis viroid (CEV) and coconut cadang cadang viroid (CCCV). Virusoids were from velvet tobacco mottle virus (VTMoV), solanum nodiflorum mottle virus (SNMV) and lucerne transient streak virus (LTSV). Analysis of intermediates was by the Northern hybridization technique with single-strand DNA and RNA probes prepared from recombinant DNA clones. The results obtained are discussed in terms of current models of viroid and virusoid replication.The plus RNA species consisted of an oligomeric series up to decamers based on the unit of full-length viroid or virusoid, which was always the major component, except for CEV where only monomer and dimer species were found. In the case of ASBV and the virusoids of VTMoV and SNMV, a minor, multimeric series of components (X-bands) was superimposed on the main oligomeric series.The complementary minus species proved more difficult to detect and characterise, with each viroid and virusoid exhibiting a unique pattern on Northern hybridization. However, they all had greater than unit-length minus species. In addition, minus species analogous to the plus X-bands were found in ASBV and CEV. The experimental difficulties encountered in this work are discussed in terms of the problem of detecting minus species by Northern analysis in the presence of excess complementary plus species.

Arabidopsis thaliana has the enzymatic machinery for replicating representative viroid species of the family Pospiviroidae

“…This asymmetric version of the rolling-circle mechanism is followed by Potato spindle tuber viroid (PSTVd) (21,22) and other members of the family Pospiviroidae, which replicate in the nucleus (23)(24)(25)(26)(27)(28). In contrast, Avocado sunblotch viroid (ASBVd) and other members of the family Avsunviroidae, which replicate in the chloroplast, follow a symmetric version in which the (Ϫ) multimers are processed to the monomeric (Ϫ) circular forms, the template for the second half of the replication cycle that is symmetric to the first (29)(30)(31)(32). Remarkably, cleavage of (ϩ) and (Ϫ) multimers is autocatalytic in the family Avsunviroidae and mediated by hammerhead ribozymes (refs.…”

The predominant circular form of avocado sunblotch viroid accumulates in planta as a free RNA adopting a rod-shaped secondary structure unprotected by tightly bound host proteins

“…Moreover, in infected avocado, the mc ASBVd (+) form is by far the most abundant viroid RNA [12][13][14]21], thus facilitating its analysis in vivo and its isolation and purification. The mc ASBVd (À) strand, although considerably less prevalent than its (+) counterpart, still reaches in planta a titre high enough to obtain the amount needed for dissection in vitro with several approaches.…”

“…ASBVd, the type member of the family Avsunviroidae, replicates in plastids (mostly chloroplasts) through a symmetric rolling-circle mechanism [12][13][14][15]. Reiterative transcription of the infecting (+) monomeric circular (mc) RNA by a nuclear-encoded polymerase (NEP) translocated into chloroplasts [16] results in oligomeric head-to-tail (À) RNA intermediates, which after self-cleaving into monomeric linear (ml) forms via cis-acting hammerhead ribozymes [17] most likely acting co-transcriptionally [18], are circularized subsequently by a chloroplastic tRNA ligase [19].…”

Resistencia a viroides inducida por ribozimas de cabeza de martillo y RNAs interferentes

“…La estructura circular de los viroides junto con la presencia en tejidos infectados por el PSTVd de otros RNAs viroidales oligoméricos de polaridad negativa (Branch et al, 1981;Rohder y Sänger, 1981;Owens y Diener, 1982), así como de RNAs viroidales oligoméricos de ambas polaridades en tejidos infectados por el ASBVd (Bruening et al, 1982;Hutchins et al, 1985), condujeron a proponer un mecanismo de replicación de círculo rodante con sólo intermediarios de RNA (Branch y Robertson, 1984;Ishikawa et al, 1984;Hutchins et al, 1985).…”

“…Por lo (Branch et al, 1988). Por el contrario, la detección en tejidos infectados por los miembros de la familia Avsunviroidae de los monómeros circulares de polaridad negativa (Hutchins et al, 1985;Bussière et al, 1999;Delgado et al, 2005), así como la observación de que los RNAs de ambas polaridades son capaces de autocortarse in vitro Hernández y Flores, 1992;Navarro y Flores, 1997;Fadda et al, 2003a), indican que estos viroides se replican siguiendo la variante simétrica del modelo.…”