Dissecting Molecular Interactions Between the Hepatitis C Virus (HCV) RNA Genome and the MicroRNA, miR-122

July 18, 2013 

DURHAM, NC – In a study published online in RNA on June 21, 2013, Shelton Bradrick, PhD, an assistant research professor in the Department of Molecular Genetics and Microbiology, in collaboration with two undergraduate researchers, Hilary Novatt and Simardeep Nagyal, analyzed molecular interactions between the hepatitis C virus (HCV) RNA genome and a liver-specific microRNA known as miR-122.

HCV is a significant human pathogen that chronically infects between 130-200 million people worldwide and is associated with cirrhosis (scarring of the liver), liver failure, and in some cases, hepatocellular carcinoma.

“Studying the molecular underpinnings of a clinically-relevant host-virus interaction makes this work exciting,” said Bradrick.  “Hilary and Simar cooperated to perform RNA affinity chromatography experiments that identified the IGF2BP RNA-binding proteins as factors that may regulate HCV replication.”  They will be continuing their research this fall and have been a pleasure to mentor,” he added.

“My undergraduate research experience has been incredibly rewarding, as it has allowed me to explore indepth topics I only briefly learned about in class, but found extremely interesting, said Novatt. “This experience has also afforded me the opportunity to work alongside amazing mentors and peers who constantly challenge me to ask new, interesting questions about my research.”

The overarching goal of this study was to biochemically purify and characterize RNA-protein complexes that form on HCV RNA in a manner that depends on miR-122. To this end, the researchers initially developed a cell-free assay that recapitulated a positive effect of miR-122 on HCV gene expression.

Using this “test tube” system, they subsequently conducted RNA affinity chromatography using HCV RNA as “bait” to isolate protein complexes for analysis by mass spectrometry and Western blotting. This approach specifically pulled down argonaute 2 (Ago2), a protein know to directly bind miRNAs.

The researchers also identified several related RNA-binding proteins, known as insulin-like growth factor 2 mRNA binding proteins (IGF2BPs), that appear to bind HCV RNA in close proximity to the miR-122 binding sites. They are now testing the hypothesis that IGF2BPs may serve as factors that modulate virus replication through regulating miR-122-HCV RNA interactions. In order to test this hypothesis, they are conducting live virus infection experiments in liver cancer cell lines.

“Conducting scientific research as a Duke undergrad has been the ultimate test for all of the biology, chemistry and other lab courses I have had,” said Nagyal. “It has compelled me to integrate and apply relevant information from the various scientific disciplines and then approach the problem. Although an intimidating examination, thankfully, it has been open-book,” she added. “Mentors and other members of the lab family have supported and encouraged me by sharing their wealth of knowledge and experience to guide my learning.”

The study was funded by a Mentored Research Scientist Development Award (K01) from National Institute of Health (NIDDK) to study host-pathogen interactions in the context of HCV infection.