Fitness of a Turnip Crinkle Virus Satellite RNA Correlates with a Sequence-Nonspecific Hairpin and Flanking Sequences That Enhance Replication and Repress the Accumulation of Virions

Abstract

Most plant viruses possess plus-sense single-stranded RNA genomes. Replication of these genomes is mediated by a replicase complex that in general comprises a virus-encoded RNA-dependent RNA polymerase (RdRp), other virally encoded proteins, and possibly host factors (17). The replicase must recognize its cognate RNA through direct or indirect interaction with specific cis-acting elements in the RNA template (4, 9), which for Brome mosaic virus are functional at various locations relative to the minus-strand initiation site (36). Replicases can accurately identify the correct start site for transcription of the genomic RNA, even when the site is artificially placed distal to the 3Ј end of the template (24,40,43). In addition, many viral RdRps synthesize 3Ј-coterminal subgenomic RNAs by using internal promoters located on minusstrand replication intermediates (18, 25, 48) that do not necessarily contain sequence or structural similarity to promoters that mediate full-length complementary-strand synthesis. Some plant viruses also provide the replication machinery for associated satellite RNAs (satRNAs), most of which share little sequence similarity with the viral genomic RNA (38). Thus, a single viral replicase must recognize a variety of seemingly different promoter elements.In addition to core promoter elements, many RNA virus genomes contain additional cis-acting elements that enhance but are not required for basal level transcription (29, 37). These RNA replication enhancers, which can be found in both plus and minus strands of viral RNAs, are located at variable positions in relation to the initiation site for RNA transcription and are thought to facilitate the recruitment of RdRp to the template (17). Replication enhancers have been characterized for a number of RNA viruses such as bacteriophage Q␤ (2), Alfalfa mosaic virus (46), Tomato bushy stunt virus (37), Brome mosaic virus (10,35,36), and Sindbis virus (11).Since most satRNAs associated with plant viruses are fewer than 500 bases in size and do not encode proteins, they are ideal templates for examining the structure and function of cis replication elements. The Turnip crinkle virus (TCV) (family Tombusviridae, genus Carmovirus) isolate TCV-M, a singlestranded RNA virus of 4,054 bases, was originally found to be associated with three related satRNAs, including satD (194 bases) and satC (356 bases) (1,22,39). satD appears to have originated from numerous short noncontiguous stretches of TCV genomic RNA sequence (5), while satC is a chimeric molecule containing nearly full-length satD at its 5Ј end and two discontinuous segments from TCV genomic RNA at its 3Ј end (Fig. 1A) (39). satC is also an unusually virulent satRNA, strongly intensifying the symptoms of TCV on hosts that display discernible symptoms when infected with TCV alone (20).Most satRNAs are considered to be parasitic agents, selfishly coopting the replication machinery of their helper virus without conferring any obvious advantage to the virus. satC, by interfering with the replication of ...

Structural Plasticity and Rapid Evolution in a Viral RNA Revealed by In Vivo Genetic Selection

“…Asterisks (V ab through Y ab ) indicate the seven of eight clones that feature a second-site mutation at nt 312, just 3Ј of H5, allowing for possible formation of 2 vivo SELEX to isolate a novel sequence for the evolutionarily conserved tetraloop D that interacts with poliovirus 3C protease and regulates viral RNA replication. Additionally, in vivo SELEX has been used extensively to characterize cis-acting sequences and structures in satC (1,6,7,27,29,34,(37)(38)(39).…”

The roles of ascorbic acid and glutathione in symptom alleviation to SA-deficient plants infected with RNA viruses

“…However, when Tobacco mosaic virus (TMV) vectors induced a high expression level of alternative oxidase (AOX), the virus spread was faster, and the symptom became more severe, in spite of the virus replication level (Murphy et al 2004). Another example is the TCV-satC (a satellite RNA associated with TCV) system, where symptoms are substantially enhanced and virus levels are signiWcantly reduced (Sun and Simon 2003). However, for single virus infection (not accompanying with satellite RNAs or other genes), we Wrst report that SA-deWcient plants adapt to RNA virus infections better (lighter symptoms) in spite of the virus replication level.…”

Repression and Derepression of Minus-Strand Synthesis in a Plus-Strand RNA Virus Replicon

“…M1H, replication enhancer on satC minus strands. M1H is also required as a hairpin on satC plus strands to bring together flanking single-stranded sequences that enhance helper virus movement by repressing virion formation (57,71,72). H4a, H4b, and H5 have not been reported previously.…”