Functional and structural characterization of the chikungunya virus translational recoding signals

Abstract

Climate change and human globalization have spurred the rapid spread of mosquito-borne diseases to naïve populations. One such emerging virus of public health concern is chikungunya virus (CHIKV), a member of the Togaviridae family, genus Alphavirus. CHIKV pathogenesis is predominately characterized by acute febrile symptoms and severe arthralgia, which can persist in the host long after viral clearance. CHIKV has also been implicated in cases of acute encephalomyelitis, and its vertical transmission has been reported. Currently, no FDA-approved treatments exist for this virus. Recoding elements help expand the coding capacity in many viruses and therefore represent potential therapeutic targets in antiviral treatments. Here, we report the molecular and structural characterization of two CHIKV translational recoding signals: a termination codon read-through (TCR) element located between the nonstructural protein 3 and 4 genes and a programmed ؊1 ribosomal frameshift (؊1 PRF) signal located toward the 3 end of the CHIKV 6K gene. Using Dual-Luciferase and immunoblot assays in HEK293T and U87MG mammalian cell lines, we validated and genetically characterized efficient TCR and ؊1 PRF. Analyses of RNA chemical modification data with selective 2-hydroxyl acylation and primer extension (SHAPE) assays revealed that CHIKV ؊1 PRF is stimulated by a tightly structured, triple-stem hairpin element, consistent with previous observations in alphaviruses, and that the TCR signal is composed of a single large multibulged hairpin element. These findings illuminate the roles of RNA structure in translational recoding and provide critical information relevant for design of live-attenuated vaccines against CHIKV and related viruses.

Cotranslational folding stimulates programmed ribosomal frameshifting in the alphavirus structural polyprotein

“…The portion of the transcript containing the EE and LL mutations is over 100 nucleotides upstream from the ribosomal slip site, and should therefore not perturb the stimulatory RNA structures that are currently believed to modulate -1PRF. 2,22,23 These mutations instead alter the portion of the nascent chain that falls just outside of the ribosomal exit tunnel during -1PRF, which suggests the nascent chain itself may stimulate frameshifting. Though it has yet to be implicated in ribosomal frameshifting, the cotranslational membrane integration and/ or folding of the nascent chain is known to generate tension on the ribosome.…”

“…Current evidence suggests -1PRF is stimulated by a canonical poly-U slip site and a downstream RNA hairpin. 23 However, an effort to map the stimulatory RNA structures within alphavirus polyproteins revealed that deletions within the predicted hairpin region are capable of reducing the efficiency of -1PRF, but appear to be insufficient to knock out frameshifting completely. 22 This observation suggests there may be multiple regulatory mechanisms that mediate -1PRF within the alphavirus structural polyprotein.…”

Cotranslational Folding Stimulates Programmed Ribosomal Frameshifting in the Alphavirus Structural Polyprotein

“…The portion of the transcript containing the EE and LL mutations is over 100 nucleotides upstream from the ribosomal slip site, and should therefore not perturb the stimulatory RNA structures that are currently believed to modulate -1PRF. 1,16,17 These mutations instead alter the portion of the nascent chain that falls just outside of the ribosomal exit tunnel during -1PRF, which suggests the nascent chain itself may stimulate frameshifting. Though it has yet to be implicated in ribosomal frameshifting, the cotranslational membrane integration and/ or folding of the nascent chain is known to generate tension on the ribosome.…”

“…The frameshift reporter (mKate) was fused 100 nucleotides downstream from the slip site in order to avoid disrupting the stimulatory RNA hairpin downstream from the slip sites. 16,17 This design avoids recently described artefacts associated with dual-luciferase PRF reporters in two ways. 33 First, the transcripts of our fluorescent expression reporter (eGFP) does not contain any cryptic splice sites.…”

“…Current evidence suggests -1PRF is stimulated by a canonical poly-U slip site and a downstream RNA hairpin. 17 However, an effort to map the stimulatory RNA structures within alphavirus polyproteins revealed that deletions within the predicted hairpin region are capable of reducing the efficiency of -1PRF, but appear to be insufficient to knock out frameshifting completely. 16 This observation suggests there may be multiple regulatory mechanisms that mediate -1PRF within the alphavirus structural polyprotein.…”