„Potencjalna strategia przeciw wirusowi SARS-CoV-2 oparta na zahamowaniu programowanej zmiany ramki odczytu podczas translacji genomu wirusa”
"Potential SARS-Cov-2 antiviral strategy based on the inhibition of programmed ribosomal frameshifting"
Projekt finansowany z szybkiej ścieżki dostępu do funduszy na badania COVID-19 Narodowego Centrum Nauki, realizowany w grupie badawczej prof. Joanny Kufel.
Project supported by National Science Center via express call to fund research on COVID-19, PI: prof. Joanna Kufel.
Keywords
Ribosomal frameshifting, molecular modelling, genetic screens, viral gene expression, antiviral therapy
Abstract
COVID-19 pandemic calls for rapid reevaluation and reimplementation of research and potential antiviral strategies developed previously for SARS-CoV-1, a coronavirus closely related to SARS-CoV-2 and originator of SARS epidemic. A key mechanism required for translation of coronavirus genome and thus its replication and propagation is -1 programmed ribosomal frameshifting (-1PRF). This mechanism gained some attention as a potential target of antiviral therapies due to its high evolutionary conservation and tight regulation. Based on work performed for SARS-CoV-1 we propose in this research to test and develop promising strategies to inhibit SARS-CoV-2 replication by targeting ribosomal frameshifting, which is necessary for the synthesis of essential viral proteins. We also plan to assess the prospect of applying these approaches as a mean to design a novel therapy. In addition, we will also address the regulatory aspects of the frameshifting process to unravel the impact of cis-acting elements. First, we will model the dynamical behavior of the mRNA fragment responsible for ribosomal frameshifting in SARS-CoV-2 (CoV2prf) and perform in silico design of effective molecular blockers of this process. Next, we will design a library of antisense oligonucleotides that, via enhancing the action of naturally existing frameshifting attenuator hairpin, will supress frameshifting. Such inhibitors may become the basis for a new therapeutic modality against COVID-19 and will be tested in vivo with an optimized dual-reporter system established for frameshifting studies. Selected potential inhibitors will be characterized for their efficiency and tested in toxicity assays in different human epithelial cell lines. Finally, conservation of the CoV2prf will be surveyed by applying two mutagenesis-based approaches. The yeast model will be used for a quick, targeted site-directed mutagenesis of known essential features of this element. In turn, evolutionary constraints on all residues will be probed by massive random mutagenesis. The created "evolutionary sensor" may help to monitor natural variability of the frameshift element that may occur upon selective pressure exerted by anti-SARS-CoV2 treatments available in the future. The outline and scope of this research strongly comply with the objective of the current call as it deals with the key molecular mechanisms of SARS-CoV-2 and may yield a potential therapeutic approach to tackle COVID-19 before a vaccine becomes available.