# Mechanisms of viral RNA maturation by co-opting cellular exonucleases

> **NIH NIH R01** · UNIVERSITY OF COLORADO DENVER · 2022 · $453,074

## Abstract

PROJECT SUMMARY
Flaviviruses are single-stranded positive-sense RNA viruses that include dangerous human pathogens like
dengue, West Nile, Yellow Fever, Zika, and many others. During infection, these viruses produce a set of non-
coding RNAs called ‘subgenomic flavivirus RNAs’ (sfRNAs) that interact with cellular proteins to manipulate the
cellular environment, to include inhibiting the antiviral response. sfRNAs have been directly linked to cytopathic
and pathogenic outcomes, and viruses that cannot produce sfRNAs are attenuated, motivating efforts to
understand the mechanism of xrRNA production. sfRNAs are made when cellular 5’à3’ exoribonucleases (in
particular, Xrn1) processively degrade the viral genomic RNA but then halt at specifically structured RNA
elements in the viral 3’ UTR called exoribonuclease resistant RNAs (xrRNAs). By solving the structures of
multiple xrRNAs by x-ray crystallography and combining this with biochemistry, biophysics, and virology, we
showed that xrRNAs fold into a unique ring-like topology that creates a mechanical block the exoribonuclease
cannot pass through. Furthermore, we used our discoveries to classify xrRNAs and to find new examples
associated with both non-coding and coding RNAs. These successes now define several new questions. First,
xrRNAs are often found in multiple copies ‘in tandem’ where their function is coupled in some way, but the
structural basis of this coupling, and the effects of breaking the coupling on both sfRNA formation and viral
infection kinetics, are unknown. Second, although we have a good understanding of several classes of xrRNAs,
we have yet to solve the structure of an xrRNA from a tick-borne flavivirus, which appear to have interesting and
unique properties. Third, although we have found many new examples of xrRNAs, it appears there are many
more that are undiscovered, and we also do not understand how the various classes of xrRNA relate
evolutionarily. How do these structures diversify and evolve in 3-D given the tight constraints on their folding?
Here, we propose to answer these questions in three aims, employing a strategy that combines biochemistry, x-
ray crystallography, cryo-EM, virology, and in vitro selections coupled with computational tools. The research
described here will contribute significant basic knowledge regarding an important molecular process of broad
applicability to viral disease, a necessary step between the discovery of a mechanism and the targeting of it for
therapeutic intervention.

## Key facts

- **NIH application ID:** 10463469
- **Project number:** 2R01AI133348-06
- **Recipient organization:** UNIVERSITY OF COLORADO DENVER
- **Principal Investigator:** Jeffrey S Kieft
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $453,074
- **Award type:** 2
- **Project period:** 2022-06-01 → 2023-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10463469

## Citation

> US National Institutes of Health, RePORTER application 10463469, Mechanisms of viral RNA maturation by co-opting cellular exonucleases (2R01AI133348-06). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10463469. Licensed CC0.

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