Non-canonical modification of viral RNA

Introduction

Viruses cause significant disease, exemplified by the COVID-19 pandemic. Most studies of virus-host interactions focused on proteins; however, RNA holds great promise for basic and therapeutic exploration. Viruses evolved elaborate strategies for RNA protection, including 5’ capping and internal modification.

Project Goal

The goal of this proposal is to discover and characterize viral RNA modifications installed by viral enzymes, including their role in innate immune evasion. This could uncover novel RNA-based mechanisms of viral replication and host modulation and lead to therapeutic targets.

Background on Hepatitis C Virus

Many viruses encode methyltransferases (MTases) for canonical RNA 5’ capping. Curiously, no cap was identified for hepatitis C virus (HCV), an important human pathogen. We recently found that the cellular metabolite, flavin adenine dinucleotide (FAD), is used as a noncanonical initiating nucleotide by the HCV polymerase at high frequency, resulting in a 5’FAD cap on HCV RNA.

This is the first description of a virus using this cap type for protecting its RNA and, remarkably, the first robust description of FAD capping across any kingdom of life.

Aim 1: Investigating HCV 5’FAD Cap

In Aim 1, we will investigate the functional role of the HCV 5’FAD cap, including:

1. Viral evasion of innate immune sensing
2. RNA stability

We will also explore the evolutionary conservation of metabolite capping across RNA viruses and its potential as an antiviral target.

Aim 2: Mapping 2’-O-Methylation

Viral MTases further perform 2’-O-methylation (2’OMe) of internal RNA residues, a modification that may also protect from innate recognition. In Aim 2, the extent of 2’OMe on viral RNA will be mapped, and the individual contribution of 5’ and internal modification to innate immune evasion will be dissected.

Conclusion

In aggregate, these aims will uncover how viral enzymes modify the termini and internal viral RNA residues and their associated evasion of innate immunity. The outcome could reshape understanding of viral RNA biology, open novel research directions, and lead to antiviral targets.