MicroRNA-122 and Poly(C)-Binding Protein 2 Bind to the Hepatitis C Virus Genome, Regulating Viral Replication and Genome Structure

Hepatitis C virus (HCV) has a positive-sense RNA genome which is used as the template for both viral translation and negative-strand RNA synthesis. As both processes work in opposite directions on the genome the virus requires a mechanism to coordinate what process is acting on a given copy of genome. It has been suggested that the host factors, microRNA-122 (miR-122) and poly-(C) binding protein 2 (PCBP2), may regulate these two processes. These host factors promote HCV replication and compete for overlapping binding sites on the 5’ untranslated region (UTR) of the HCV genome. This work investigates how miR-122 and PCBP2 bind the 5’ UTR of the HCV genome, and how the competition between the two factors regulates viral translation and replication. The binding competition of miR-122 and PCBP2 was characterized and the impacts on HCV replication identified. Near the 5’ terminus of the HCV genome there are two miR-122 binding sites, S1 and S2. PCBP2 binds a sequence that overlaps the second miR-122 binding site (S2) causing direct competition with miR-122 for binding. Steady-state binding assays determined that PCBP2 and miR-122 have similar affinities for the overlapping binding sites. Electron microscopy shows the competition of these two factors modulates the PCBP2-mediated circularization of the HCV genome. Additionally, a novel interaction between the viral polymerase, NS5B, and the 5’ UTR was shown. The affinity of this interaction is increased by miR-122 binding to HCV, potentially promoting RNA synthesis. The modulation of genome structure and polymerase affinity may form a mechanism by which the competition of miR-122 and PCBP2 regulates genome templating for translation versus RNA synthesis. The structure of the miR-122 and HCV genome was investigated. The first miR-122 binding site (S1) is the higher affinity of the two sites and is not affected by PCBP2 binding. To determine the structure of the RNA complex via cryo-EM, a proheadRNA-Assisted RNA Imaging Scheme (pARIS) was developed. This novel method can be applied to other small RNA structures that otherwise cannot be reconstructed via cryo-EM. We found that the HCV:miR-122 complex is flexible and accessible in the absence of the argonaut-2 protein.

Name thesis:degree_name

Biochemistry and Molecular Biology (Doctoral)

Discipline thesis:degree_discipline

Biomedical Sciences

Grantor

The University of Texas Medical Branch at Galveston

Year dc:date.issued

2023

Author dc:creator

• Scott, Seth Dylan May 5th 1993-

Advisors dc:contributor.advisor

• Smith, Thomas
• Choi, Kyung

Committee members dc:contributor.committeemember

• Morais, Marc
• Kuyumcu-Martinez, Muge N.
• Lemon, Stanley M.

Language dc:language.iso

English

Handle dc:identifier.uri

https://hdl.handle.net/2152.3/12487

OAI identifier oai:identifier

oai:utmb-ir.tdl.org:2152.3/12487