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.
Highlights
Climate change and human globalization have spurred the rapid spread of mosquito-borne diseases to naïve populations
The chikungunya virus (CHIKV) termination codon readthrough (TCR) is located at an opal (UGA) termination codon in the genomic RNA beginning at nucleotide 5656, which marks the boundary between the nsP3 and nsP4 genes [11]
In the subgenomic RNA, a Ϫ1 PRF located in the 3Ј region of the 6K viroporin gene enables synthesis of a C-terminally extended trans-frame variant dubbed TF that has been identified in the envelope of mature alphavirus particles [16]
Summary
A study with SINV mutants that disrupted production of TF resulted in reduced virus production in mammalian and insect cell lines that was independent of genome replication, particle infectivity, or envelope protein transport to the cellular membrane. These findings suggest potential roles for TF in virus particle assembly and budding. Ablation of Ϫ1 PRF in Venezuelan equine encephalitis virus (VEEV), a new world alphavirus, had minimal effects on viral replication but abrogated neurovirulence in mouse models [21] These findings suggest that ablation of alphavirus recoding elements could serve as the foundation for live-attenuated vaccines for this virus family. Key mutations that significantly ablated recoding functionality were identified for both signals, laying a foundation for follow-up attenuated live virus experiments in animal models
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