The RNA regulatory controls that dictate the outcome of RNA virus infection are not fully understood. In particular, the post-transcriptional RNA modification N6-methyladenosine (m6A) is a potent and dynamic modulator of RNA function. However, little is known about the biological significance of this modification or how it regulates systems that perturb cellular homeostasis, such as RNA virus infection. This project aims to determine how m6A regulates the replication of hepatitis C virus (HCV), a positive strand RNA virus of the Flaviviridae family. Our central hypothesis is that m6A acts directly on the HCV RNA genome to regulate the fate of the RNA for the different stages of the viral life cycle. The rationale for the proposed research is that by understanding how m6A regulates RNA virus replication, we can manipulate and target m6A-targeted processes to design new and innovative approaches for the prevention and treatment of RNA virus infection. Guided by our preliminary data, our hypothesis will be tested by pursuing these three specific aims: 1) Identify the sites of m6A within the HCV RNA genome at single nucleotide resolution, 2) define the mechanism by which m6A regulates HCV RNA replication, 3) determine the mechanism by which m6A-binding proteins regulate HCV assembly. In the first aim, we will define the kinetics and sites of adenosine methylation on the HCV RNA genome at single nucleotide resolution by using m6A individual-nucleotide-resolution cross-linking and immunoprecipitation and direct RNA sequencing. For the second aim, we will use viral mutants with inactivated m6A sites and genetic depletion of the m6A methyltransferases and m6A-demethylase to identify the mechanism by which m6A affects HCV RNA replication. In the third aim, we will determine the mechanism by which the m6A-binding proteins differentially regulate HCV assembly by defining where they bind to the HCV RNA genome using photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation and by identifying the functional domains of these proteins. Taken together, this approach will define how direct modification of the HCV RNA genome by m6A regulates the life cycle of HCV, thereby identifying and characterizing a novel RNA regulatory control to viral infection. Ultimately, a detailed understanding of how m6A affects HCV infection will uncover novel strategies to develop antiviral therapies to target this RNA regulatory control that is exploited by RNA viruses for their replication.

Public Health Relevance

RNA virus infection causes significant morbidity and mortality worldwide, and these viruses remain a constant threat to global public health. The proposed study will define the molecular mechanisms of how the RNA modification N6-methyladenosine (m6A) regulates the life cycle of hepatitis C virus (HCV), an RNA virus in the Flaviviridae family. This study will also uncover new host pathways exploited by RNA viruses like HCV that can be harnessed for the development of novel antivirals designed to limit viral infection and viral-mediated disease. Finally, it will help establish a new line of investigation in the field of m6A biology that will lead to greater understanding of regulatory controls of gene expression in human physiology and disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI125416-04
Application #
9698257
Study Section
Virology - A Study Section (VIRA)
Program Officer
Koshy, Rajen
Project Start
2016-06-01
Project End
2021-05-31
Budget Start
2019-06-01
Budget End
2020-05-31
Support Year
4
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Duke University
Department
Genetics
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
Dethoff, Elizabeth A; Boerneke, Mark A; Gokhale, Nandan S et al. (2018) Pervasive tertiary structure in the dengue virus RNA genome. Proc Natl Acad Sci U S A 115:11513-11518
Imam, Hasan; Khan, Mohsin; Gokhale, Nandan S et al. (2018) N6-methyladenosine modification of hepatitis B virus RNA differentially regulates the viral life cycle. Proc Natl Acad Sci U S A 115:8829-8834
Wang, Liuyang; Pittman, Kelly J; Barker, Jeffrey R et al. (2018) An Atlas of Genetic Variation Linking Pathogen-Induced Cellular Traits to Human Disease. Cell Host Microbe 24:308-323.e6
Liu, Bei; Merriman, Dawn K; Choi, Seung H et al. (2018) A potentially abundant junctional RNA motif stabilized by m6A and Mg2. Nat Commun 9:2761
McFadden, Michael J; Mitchell-Dick, Aaron; Vazquez, Christine et al. (2018) A Fluorescent Cell-Based System for Imaging Zika Virus Infection in Real-Time. Viruses 10:
Ahsanuddin, Sofia; Afshinnekoo, Ebrahim; Gandara, Jorge et al. (2017) Assessment of REPLI-g Multiple Displacement Whole Genome Amplification (WGA) Techniques for Metagenomic Applications. J Biomol Tech 28:46-55
Tighe, Scott; Afshinnekoo, Ebrahim; Rock, Tara M et al. (2017) Genomic Methods and Microbiological Technologies for Profiling Novel and Extreme Environments for the Extreme Microbiome Project (XMP). J Biomol Tech 28:31-39
Afshinnekoo, Ebrahim; Chou, Chou; Alexander, Noah et al. (2017) Precision Metagenomics: Rapid Metagenomic Analyses for Infectious Disease Diagnostics and Public Health Surveillance. J Biomol Tech 28:40-45
Vazquez, Christine; Beachboard, Dia C; Horner, Stacy M (2017) Methods to Visualize MAVS Subcellular Localization. Methods Mol Biol 1656:131-142
O'Hara, Niamh B; Reed, Harry J; Afshinnekoo, Ebrahim et al. (2017) Metagenomic characterization of ambulances across the USA. Microbiome 5:125

Showing the most recent 10 out of 16 publications