Structural studies of 5´ UTR of Hepatitis C viral RNA by NMR-based structural biology

Abstract

RNA is a single-stranded biopolymer that plays a myriad of roles in physiological and pathological processes and is the carrier of genetic information in many human pathogens. Hepatitis C virus (HCV) is one of the most impactful representatives of RNA viruses. Liver-abundant human microRNA-122 (miR-122) binds to two tandem sites within domain I of the 5´ untranslated region (5´ UTR) of HCV, ultimately resulting in upregulation of viral propagation. Despite many studies of the interaction between HCV and miR-122, the exact mechanism by which this recognition event leads to increased viral propagation is unknown. In this thesis, I have studied the 5´ UTR HCV–miR-122 interaction at different levels of structural complexity (domain I, domains I-II and the full 5´ UTR) using an integrative NMR-based structural biology approach. First, I have performed the near-complete assignment of domain I resonances and determined its secondary structure. Isolated domain I binds two copies of miR-122 with different affinities, and the binding kinetics fall into the slow-to-intermediate exchange-regime on the NMR chemical-shift timescale. Magnesium ions promote structural rearrangement of domain I, which in turn changes its interaction pattern with miR-122. Next, I have determined the secondary structures of the isolated domain II and a domain I-II construct, both in their apo (without miR-122) and holo (bound to miR-122) states. The data demonstrates that, in the domain I-II construct, domains I and II maintain independent folds; furthermore, the secondary structure of domain II remains intact upon domain I binding two copies of miR-122. However, the binding of miR-122 to the domain I-II construct does lead to a structural rearrangement that changes the relative orientation of the two domains, resulting in more open and extended conformation. Finally, I have investigated the interaction of miR-122 with the full 5´UTR. Since the differences between the low-resolution scattering data of the 5´ UTR in the apo and holo states were minimal, no major structural changes in the 5´ UTR upon miR-122 binding appear to occur. To study the local structural details of the 5´ UTR, I have explored the use of solid-state NMR. While there were clear changes in chemical shifts of the 5´ UTR upon miR-122 binding, indicating conformational changes in the 5´ UTR, acquisition of solid-state NMR data on segmentally labeled samples and isolated domain I was challenging and could not provide definitive answers at this stage. Overall, using an NMR-based integrative structural biology approach, I could show that miR-122 binding to domain I causes both widespread local rearrangements within domain I and a significant reorientation of domain I relative to domain II, while the effect of miR-122 binding on the overall structure of the full 5’ UTR was found to be minimal.