Biologically-supported structural model for a viral satellite RNA

Abstract

Satellite RNAs (satRNAs) are a class of small parasitic RNA replicon that associate with different viruses, including plus-strand RNA viruses. Because satRNAs do not encode a polymerase or capsid subunit, they rely on a companion virus to provide these proteins for their RNA replication and packaging. SatRNAs recruit these and other required factors via their RNA sequences and structures. Here, through a combination of chemical probing analysis of RNA structure, phylogenetic structural comparisons, and viability assays of satRNA mutants in infected cells, the biological importance of a deduced higher-order structure for a 619 nt long tombusvirus satRNA was assessed. Functionally-relevant secondary and tertiary RNA structures were identified throughout the length of the satRNA. Notably, a 3′-terminal segment was found to adopt two mutually-exclusive RNA secondary structures, both of which were required for efficient satRNA accumulation. Accordingly, these alternative conformations likely function as a type of RNA switch. The RNA switch was also found to engage in a required long-range kissing-loop interaction with an upstream sequence. Collectively, these results establish a high level of conformational complexity within this small parasitic RNA and provide a valuable structural framework for detailed mechanistic studies.

A conserved RNA structure is essential for a satellite RNA-mediated inhibition of helper virus accumulation

“…Compensatory mutation analyses suggest that γBs might be non-functional in the accumulation of sat-T1 (mutant series 4, Figure 8). Such effects are also observed in other plant virus-associated satRNAs, such as sat-Cym (72). γBs might contribute to a biological process that could not be monitored in our assays.…”

“…To our knowledge, Ψ1 is the first tertiary structure identified in CMV satRNA so far. Pseudoknots also have been identified in a few satellite RNAs associated with other plant viruses, such as TCV and cymbidium ringspot virus (72–74). All of these pseudoknots are important for the accumulation of these satRNA species, except for Ψ3 in TCV satC (73).…”

Variation Profile of the Orthotospovirus Genome

“…Structural conformations in viral RNA regulate binding to RNA or protein interaction partners, and structures formed by intergenic regions participate in diverse biological functions [67][68][69]. Sequences that fold into similar structures may support similar functions, and mutation within these regions will be tolerated only if they preserve a functional structure [57,70].…”

Physical and Functional Analysis of Viral RNA Genomes by SHAPE

“…SHAPE has been implemented by diverse laboratories to study viral genomes in a variety of both simplified and complex biologically relevant states. These states include RNA transcribed in vitro and refolded (referred to as in vitro RNA) (62)(63)(64)(65)(66)(67)(68)(69)(70)(71)(72), RNA gently extracted from virus particles (ex virion RNA) (37,58,64,(73)(74)(75)(76)(77)(78)(79)(80)(81) or from infected cells (cell-free RNA) (78), RNA in native virus particles (in virion RNA) (58,64,74,80), RNA in infected cell lysates (81), and RNA in infected cells (72, 74) (Figure 2a). Comparisons of SHAPE reactivity profiles obtained for viral RNA molecules probed in different biological states reveal state-specific RNA conformations (31,58,64,72,74,(80)(81)(82)(83)(84)(85), and sites occupied by RNA-binding proteins (58,64,72,(80)…”