Viral RNA secondary structures : novel biology and therapeutic implications

RNA viruses cause a variety of acute and chronic diseases in humans. In addition to encoding for viral proteins, the genomes of RNA viruses often harbor secondary and tertiary RNA structures that have been associated with functions essential for successful virus replication and propagation. While antiviral research has focused almost exclusively on targeting viral proteins, the importance of RNA structural elements in virus lifecycles suggest these motifs could be promising therapeutic targets. In this thesis, I will dissect the contributions and druggability of RNA structures and complexes in two distinct phases of the viral lifecycle: first, in the virus packaging and production of influenza A virus (IAV), a negative-strand segmented RNA virus responsible for severe, sometimes fatal respiratory disease with pandemic potential; and second, in the replication complex of hepatitis C virus (HCV), a positive-strand RNA virus associated with chronic liver disease and cancer. To characterize the role of RNA secondary structures in IAV virus packaging, I employed the chemical mapping technique, Selective 2′ Hydroxyl Acylation analyzed by Primer Extension (SHAPE), which uses experimental probing data to guide computational modeling of high resolution RNA structure. This method enabled me to determine the RNA secondary structure of the IAV 5′ packaging signal region of virus genome segment PB2, the structures of known mutations in PB2 that disrupt virus packaging, as well as the discovery and characterization of novel mutants. Cutting-edge multidimensional mapping methods, Mutate-and-Map and Mutate-Map-Rescue, built upon this technical base and provided structural validation through discovery of compensatory mutant partners, which later informed both the in vivo exploration of packaging-defective viruses, as well as the design of locked nucleic acids (LNAs) targeting this structure. In vitro and in vivo drug targeting by LNAs of this IAV packaging signal structure exhibited powerful prophylactic and therapeutic efficacy. Combined, this work points to the promise of RNA secondary structures as valid antiviral targets, and sheds new light on IAV genome packaging mechanisms. The second section of my thesis built upon previous research in the lab. SHAPE was again employed to map both the RNA secondary structure of the HCV 5′ internal ribosomal entry site (IRES) and the structural interaction between the virus's 5′ untranslated region (UTR) and the miR-122 complex found to mediate viral replication. As an extension of this investigation, we sought to better parse the interaction of the viral replication complex with the host membrane in real-time. To do so, we employed a supported lipid-bilayer (SLB) platform and coupled it with quartz crystal microbalance with dissipation (QCM-D) to reconstitute and explore how components of the HCV replicase complex affect viral RNA replication and in vitro RNA transcription. The core enzyme of this complex, the NS5B RNA-dependent RNA polymerase, is required for in vivo virus replication and is a prime drug target for current and future HCV antiviral therapies. The reconstitution of full length NS5B in a membranous environment capable of active polymerase function, however, had not been demonstrated. Here, the SLB/QCM-D platform made it possible to reconstruct an in silico biological mimic of the host membrane to quantitatively probe and observe interactions between the membrane, the HCV viral RNA template and its associated viral proteins in real time. Together, these provide strong proof-of-concept for use of this technique in further understanding of the molecular details of HCV genome replication and future drug screening applications.

Keywords: Microbiology and Immunology.
Advisor: Glenn Jeffrey S.
Das Rhiju
Greenberg Harry B. 
Sarnow P. (Peter)