Structural and Functional Characterization of Programmed Ribosomal Frameshift Signals in West Nile Virus Strains Reveals High Structural Plasticity Among cis-Acting RNA Elements

Abstract

West Nile virus (WNV) is a prototypical emerging virus for which no effective therapeutics currently exist. WNV uses programmed ؊1 ribosomal frameshifting (؊1 PRF) to synthesize the NS1 protein, a C terminally extended version of its nonstructural protein 1, the expression of which enhances neuroinvasiveness and viral RNA abundance. Here, the NS1 frameshift signals derived from four WNV strains were investigated to better understand ؊1 PRF in this quasispecies. Sequences previously predicted to promote ؊1 PRF strongly promote this activity, but frameshifting was significantly more efficient upon inclusion of additional 3 sequence information. The observation of different rates of ؊1 PRF, and by inference differences in the expression of NS1, may account for the greater degrees of pathogenesis associated with specific WNV strains. Chemical modification and mutational analyses of the longer and shorter forms of the ؊1 PRF signals suggests dynamic structural rearrangements between tandem stem-loop and mRNA pseudoknot structures in two of the strains. A model is suggested in which this is employed as a molecular switch to fine tune the relative expression of structural to non-structural proteins during different phases of the viral replication cycle.

Complex dynamics under tension in a high-efficiency frameshift stimulatory structure

“…S1): the pseudoknot proposed by Firth and Atkins (35), denoted by PK A , which unfolded over a broad range of forces ( Fig. 2B, orange); a pseudoknot similar to that proposed by Moomau et al (21), denoted by PK D , which unfolded partially at low force ( Fig. 2B, green); a partially folded version of PK D , denoted by PK D −, which unfolded over a broad range of forces ( Fig.…”

“…This correlation was supported by subsequent work extending SMFS studies of −1 PRF to other pseudoknots (30), different types of stimulatory structures such as hairpins (31), and the effects of antiframeshifting ligands (32). Evidence supporting the importance of conformational plasticity has also been found from single-molecule fluorescence experiments of ribosomes translocating through pseudoknots (33) and ensemble structural studies of stimulatory structures using such methods as selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) (21,34). However, structures stimulating extremely high levels of frameshifting, which should pose the most stringent test of any hypotheses relating stimulatory structure properties and −1 PRF efficiency, have not yet been studied to probe their mechanical properties or dynamics.…”

“…Here we report SMFS measurements of the frameshift signal from West Nile virus (WNV), a mosquito-borne flavivirus causing neurologic disease that uses −1 PRF to control the relative expression levels of structural and nonstructural proteins encoded within a single open reading frame (9). Whereas most viral frameshift signals stimulate −1 PRF with an efficiency in the range of 5% to 30% (16,17), the WNV frameshift signal has been reported to do so at up to 70% efficiency (21), among the highest levels measured to date. The WNV frameshift signal is also notable because the nature of the stimulatory structure is unclear.…”

“…Initial studies of a 75-nt segment of the WNV mRNA suggested that, based on bioinformatic predictions, the stimulatory structure is a 61-nt pseudoknot (35). However, subsequent work extending the RNA by ∼50 nt and applying SHAPE analysis suggested that the frameshift signal forms competing tandem stem loop and 109-nt pseudoknot structures (21), with the tandem stem loops sharing stem 1 in common with the 61-nt pseudoknot proposed previously but the 109-nt pseudoknot being mutually exclusive with the other structures.…”

“…The level of −1 PRF stimulated by different structures can vary enormously, from a few percent up to ∼70% to 80% (16)(17)(18)(19)(20)(21). However, the features that contribute to efficient stimulation remain under debate (2), in part because the mechanisms driving frameshifting remain incompletely understood.…”