A Replication Cycle for Viroids and Other Small Infectious RNA's

Abstract

Experimental data concerning viroid-specific nucleic acids accumulating in tomato plants establish, together with earlier studies, the major features of a replication cycle for viroid RNA in plant cells. Many features of this pathway, which involves multimeric strands of both polarities, may be shared by other small infectious RNA's including certain satellite RNA's and "virusoid" RNA's which replicate in conjunction with conventional plant viruses. The presence, in host plans, of an elaborate machinery for replicating these disease agents suggests a role for endogenous small RNA's in cellular development.

How RNA acts as a nuclease: some mechanistic comparisons in the nucleolytic ribozymes

“…The hammerhead ribozyme was the first nucleolytic ribozyme to be discovered (52)(53)(54), and yet its catalytic mechanism is still incompletely understood. Early forms of the ribozyme lacked a key tertiary contact element (55) required to facilitate folding (56) and permit the active site to adopt the correct conformation (16,57).…”

HDV Family of Self-Cleaving Ribozymes

“…4.1). 19 In the first pathway, the RNA undergoes capping and polyadenylation to yield a subgenomic sequence that serves as the mRNA for the sole protein produced from the HDV genome, the delta antigen. [20][21][22] Two variants of the protein are present in vivo, the small and large delta antigens.…”

Specific HDV RNA-templated transcription by pol II in vitro

“…The rolling circle mechanism for HDV replication during which multimeric intermediate transcripts are synthesized requires intact circular RNA template for transcription (Branch & Robertson, 1984;Chen et al+, 1986)+ Transcription reaction detected in our in vitro system depends on a specific cleavage of the RNA template, implying that it cannot directly represent the initiation step of HDV RNA replication+ However, initiation of the rolling circle replication involving a similar mechanism is possible by considering a hypothesis of the RNA template switching+ According to this model, pol II would cleave one molecule of HDV RNA and proceed into the elongation phase only after switching to another circular template, using the cleaved template as a primer for initiation of replication+ In fact, the use of primers to initiate replication is a common theme among the viruses+ For example, tRNAs (Gilboa et al+, 1979), capped oligonucleotides cleaved from host mRNAs (Plotch et al+, 1981), or a polypeptide primer (Andino et al+, 1993) are used by reverse transcriptases, influenza, and poliovirus polymerase, respectively+ Utilization of the 39 end of a cleaved HDV RNA molecule as a primer for initiation of HDV replication may represent a similar situation in which RNA structural elements control recognition, specificity of cleavage (initiation) as well as the specificity and efficiency of template switching+ HDV and viroids may share a similar pol II-mediated replication mechanism that requires common sequence/structure elements in the RNA (Branch & Robertson, 1984)+ The template used in this study is derived from the highly conserved, viroid-like region of HDV RNA (Lai, 1995)+ Identification of the Hepatitis Delta Interacting Protein A (DIPA), a human homolog of HDAg, led to the hypothesis that HDV evolved from a viroid-like RNA that acquired HDAg ORF as a result of pol II switching templates from a viroid ancestor to the cellular mRNA (Brazas & Ganem, 1996)+ Furthermore, sequence comparison of a number of viroid variants suggested that RNA template switching may frequently occur during viroid replication (Hammond et al+, 1989;Koltunow & Rezaian, 1989;Rezaian, 1990;Hernandez et al+, 1992;Kofalvi et al+, 1997)+ Notably, stem-loops present at both ends of viroid RNAs are directly implicated in template switching during viroid replication (Semancik et al+, 1994;Diener, 1995)+ Further studies are necessary to determine if a similar template switching may also occur during HDV RNA replication+ Alternatively, it is possible that in contrast to the initiation of HDV transcription observed in vitro, initiation of HDV replication in vivo proceeds by a mechanism that does not require RNA cleavage, but still depends on a specific recognition of the same AG HDV RNA element for de novo initiation by pol II+ In that case, the observed template cleavage could reflect suboptimal conditions of the in vitro reac...…”

“…RNA synthesis in eukaryotes is carried out by RNA polymerases I, II, and III (Roeder, 1976)+ Each polymerase transcribes functionally distinct DNA sequences controlled by specific promoter elements+ In particular, RNA polymerase II (pol II) responsible for transcription of pre-mRNAs has been extensively studied both in terms of DNA template requirements and protein factors that regulate its activity+ Pol II has also been implicated in replication of two classes of RNA pathogens: plant viroids and human Hepatitis Delta Virus (HDV) (Robertson & Branch, 1987;Taylor, 1992;Lai, 1995)+ Viroids are small (;250-600 nt), single-stranded, circular RNAs that can be folded into unbranched rod-like structures (Keese & Symons, 1987)+ They replicate in plant cells by a rolling circle mechanism, in which a circular template of one polarity is used to generate multimeric intermediates of the opposite polarity+ These multimeric RNAs are self-cleaved by ribozyme elements encoded in the viroid sequence, yielding monomeric RNAs that are subsequently ligated, possibly by a host RNA ligase activity, to generate circular products (Branch & Robertson, 1984;Robertson & Branch, 1987)+ Similarly, HDV RNA, a satellite of Hepatitis B Virus (HBV), is a 1+7-kb single-stranded circular RNA that, like viroids, can be folded into an unbranched rod-like structure and is thought to replicate by a rolling circle mechanism (Taylor, 1992, and references therein; Lai, 1995)+ Both polarities of HDV RNA [genomic (G) and antigenomic (AG)] contain HDV ribozyme domains (Fig+ 1A) that are responsible for processing of multimeric intermediates to monomeric products (Taylor, 1992, and references therein; Lai, 1995)+ One significant difference between viroids and HDV concerns their coding potential+ Although viroids do not code for any polypeptides, HDV RNA encodes a single protein, Hepatitis Delta Antigen (HDAg)+ Although HDAg is essential for replication of HDV, it is not a replicase itself (Kuo et al+, 1989;MacNaughton et al+, 1991)+ Furthermore, HBV helper virus does not provide replicase function either, as HDV cDNA-or RNA-transfected cell lines efficiently support HDV RNA replication in the absence of any HBV proteins (Kuo et al+, 1989;Glenn et al+, 1990)+ Thus, while viroids and HDV must fully depend on the host cell machinery for their replication, the identity of the polymerase activity involved has not been demonstrated+ Synthesis of viroid RNAs has been studied in vitro using intact protoplasts or cell free nuclear homogenates of different plant cells in the presence of inhibitors of specific RNA polymerases+ Inhibition of viroid synthesis by a-amanitin at concentrations known to inhibit pol II suggested the involvement of this activity in viroid replication …”