Exploring folding mechanism of the host-cell resistant subgenomic flavivirus RNA by developing a coarse-grained dynamic ion condensation model

Singh, Rishabh Kumar (2022) Exploring folding mechanism of the host-cell resistant subgenomic flavivirus RNA by developing a coarse-grained dynamic ion condensation model. Masters thesis, Indian Institute of Science Education and Research Kolkata.

Text (MS dissertation of Rishabh Kumar Singh (17MS148))) 17MS148_Thesis_file.pdf - Submitted Version Restricted to Repository staff only Download (894kB)

Abstract

Flaviviruses are single-stranded positive-sense RNA enveloped viruses whose genus includes important human pathogens such as Murray Valley Encephalitis Virus (MVEV), Dengue Virus (DENV), Zika Virus (ZIKV) etc. The functional non-coding RNA derived from 3’ untranslated region (3’ UTR), subgenomic flavivirus RNA (sfRNA) stalls the viral RNA degrading processivity of the host cell enzyme, named XRN1. Here, we have developed a coarse-grained structure-based model integrating the theories and concepts of the folding funnel energy landscape and generalized manning counterion condensation that help capture the appropriate ion environment around a highly charged polymer, like RNA to decipher the structural structure basis of sfRNA resistance against XRN1. In this thesis work, we find the formation of RNA pseudoknot by Mg 2+ -ions, serving a crucial role in sequestering 5’ end of xrRNA by which the enzyme chops up viral RNAs through a hydrolysis mechanism. Furthermore, we have also performed free energy analysis using the umbrella sampling method, which gives us insight into the mechanistic pathway of folding. Thus, contributing to the thermodynamic stability, which eventually results in the mechanical stability of the compact RNA against the enzyme. This thesis has four chapters: Chapter-1 (Introduction) covers the description of various RNA constructs observed in nature like the hierarchical constructs of primary, secondary, and tertiary structures, complex folds like quadruplexes, hairpins and pseudoknots etc. We emphasize the significant structural RNA motifs like pseudoknots and 3-D ring-like fold crucial in xrRNA (MVEV) and their functional importance in conserved and variable regions of RNA structure. We briefly describe the biogenesis of sfRNA vis-a-vis stalling of the XRN1 at the site of xr- RNA(i.e XRN1 resistent RNA) and how it overcome es the hydrolysis processivity of the enzyme. Chapter-2 (Theories and Methods) involves the theories of energy landscape of biopolymers and highly charged polymers like RNA that we exploited to develop our coarse-grained structure-based model. Here we also explain the classical manning counterion condensation theory which Professor Gerald S. Manning proposed in 1960s for a regualr polyelectrolye structure like DNA and its drawbacks for its implementation in an RNA system. We also explain further modifications done in the form of a generalized manning counterion condensation model. We have integrated the dynamic counterion condensation phenomenon with a well-known structure-based model in a coarse- grained manner to get a cohesive picture of the conformational dynamics of RNA. Chapter-3 (Conformational Dynamics of RNA Ring in flavivirus RNA) mainly includes various calculations done to show the ring dynamics as the the ring-fold braces against the enzyme to avoid unfolding. The ring-like fold is possible due to a pseudoknot (PK-2) formation with high cooperativity with Mg 2+ ions. We also compare our computational results with experimental results like SAXS and FRET. The final chapter, Chapter-4 (Understanding folding landscape and pathway of viral sfRNA) is towards understanding the folding pathway of the viral RNA by adopting a free energy calculation technique known as Umbrella Sampling. This analysis explores the folding energy landscape of this RNA and its different structural intermediates and their possible role in resisting host-cell XRN1 promoting viral pathogenicity.

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