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 non-structural protein 1, the expression of which enhances neuro-invasiveness 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. 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 non-structural protein 1, the expression of which enhances neuro-invasiveness 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.