# Recognition and recruitment of RNA viruses into RNA silencing pathways

> **NIH NIH R01** · UNIVERSITY OF NEBRASKA LINCOLN · 2020 · $263,598

## Abstract

ABSTRACT
In eukaryotes, RNA silencing is a conserved mechanism of gene regulation and has essential antiviral roles in
in plants, nematodes, and insects. Antiviral RNA silencing is triggered by viral dsRNA and is characterized by
the accumulation of small interfering RNAs (siRNAs) derived from viral RNA (primary siRNAs) formed by Dicer-
like proteins (DCL). Features of viral RNA that trigger silencing have not been determined. Potential triggers
include viral dsRNA in replication intermediates and self-complementary sequences in viral genomic RNA.
Additionally, during an amplification phase, cellular RNA-dependent-RNA polymerases (RDR) synthesize
dsRNA that is processed into secondary virus-derived siRNA by DCL. In plants, RDR1 and RDR6 are the
genetic determinants of silencing amplification. However, the mechanisms that bring plant cellular RDRs in
contact with their viral RNA substrates are not known. This project is focused in two poorly characterized
phases of the plant antiviral RNA silencing: Initiation and amplification. The goals are to 1) identify the viral
triggers of RNA silencing and 2) to determine the mechanisms that route viral RNA into cellular RDR-
dependent amplification. Our hypothesis is that primary virus-derived siRNAs in association with ARGONAUTE
(AGO) proteins flag viral RNA as a substrate for cellular RDRs. The approach consists on genetically blocking
cellular RDR-dependent amplification to identify viral RNA triggers based on the profiles of primary virus-
derived, and by co-precipitation of DCL and AGO proteins with their viral RNA targets. To determine the
mechanisms of RNA silencing amplification, RDR1 and RDR6 interaction partners will be determined by co-
precipitation and mass spectrometry, and a synthetic system will be developed to induce and measure antiviral
RNA silencing amplification. This system is designed to determine the functionality of structurally different
primary virus-derived siRNAs, in association with AGO proteins, in flagging viral RNA as a substrate for cellular
RDR1 and RDR6. This work will help define how plant viral RNAs are recognized as distinct from cellular (non-
targeted) RNA, and determine the mechanisms of antiviral RNA silencing initiation and amplification. Expected
findings will contribute to our understanding and manipulation of an antiviral immunity system that determines
the outcome, disease or no disease, in plant-virus interactions.

## Key facts

- **NIH application ID:** 9994322
- **Project number:** 5R01GM120108-05
- **Recipient organization:** UNIVERSITY OF NEBRASKA LINCOLN
- **Principal Investigator:** Hernan Garcia-Ruiz
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $263,598
- **Award type:** 5
- **Project period:** 2016-09-01 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9994322

## Citation

> US National Institutes of Health, RePORTER application 9994322, Recognition and recruitment of RNA viruses into RNA silencing pathways (5R01GM120108-05). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9994322. Licensed CC0.

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