| Popis: |
While infection from communicable diseases has posed a longstanding threat to human health throughout history, the modern realities of population expansion, global travel, and climate change have facilitated the rapid emergence and worldwide distribution of RNA viruses at an unprecedented scale. Of particular concern are the alphaviruses, mosquito borne viruses from the Togaviridae family. These viruses were previously relegated to rare outbreaks in isolated forested regions but have dramatically spread across the globe in the past decade. One of these viruses, Venezuelan equine encephalitis virus (VEEV), is a noted bioterror threat due to its ability for aerosol transmission and successful weaponization during the Cold War. While no FDA approved drugs exist against alphaviruses, their reliance on programmed translational recoding mechanisms to regulate gene expression presents a potential vulnerability for therapeutic exploitation. Two instances of translational recoding have been identified but poorly characterized in the alphavirus genome. The first is a termination codon readthrough (TCR) event required for expression of the alphavirus replicase. The second is a programmed -1 ribosomal frameshift (-1 PRF) that produces a C-terminally extended variant of viroporin 6K. In this work, the cis-acting RNA elements that mitigate alphavirus recoding were functionally and structurally characterized. The predicted TCR and -1 PRF sequences were cloned into dual luciferase reporter vectors and their ability to promote efficient recoding was verified in several mammalian cell lines. Chemical probing assays elucidated the presence of highly structured stemloop elements downstream of the alphavirus recoding sites, which function as a kinetic trap for elongating ribosomes. Notably, mutations that abrogate efficient -1 PRF not only attenuated pathogenesis of VEEV in mice, but also provided protective immunity to subsequent wild-type challenge. These findings suggest a novel approach to the development of a safe and effective live attenuated vaccine strategy against VEEV, closely related alphaviruses, and potentially all viruses that rely on translational recoding mechanisms for optimal gene expression. |
