# Molecular Mechanism of Action of the Influenza PA-X Host Shutoff Protein

> **NIH NIH R01** · TUFTS UNIVERSITY BOSTON · 2022 · $394,212

## Abstract

Project summary. The innate immune and inflammatory response to influenza A virus is a major contributor to
influenza disease, because it promotes viral pneumonia and secondary infections through lung damage. As a
consequence, it has recently become clear that blocking influenza virus replication is not sufficient to treat
influenza disease. New strategies to block the innate immune and inflammatory responses need to be
developed. These strategies could take advantage of the role of viral proteins in modulating host responses,
including the host shutoff factor PA-X, which targets host RNAs for degradation. PA-X inhibits immune
responses, and viruses that lack PA-X trigger stronger cytokine responses in mouse and chicken infection
models. However, these stronger responses do not promote clearance, but in many cases result in increased
morbidity and mortality. This indicates that PA-X has a different function from other influenza immune
modulators, and that PA-X can be protective for the host. Defining the mechanism of action of PA-X will be
important to understand the unique contribution of PA-X to virus-host interplay and to exploit it to modulate
host responses to influenza. While host shutoff factors are usually considered indiscriminate, we have found
that PA-X is selective, and targets specific types of RNAs. Particularly, we have uncovered a connection
between PA-X and splicing of host RNAs. We have found that PA-X preferentially degrades spliced RNAs in
infected cells and, through proximity-dependent protein labeling, we have identified candidate interaction
partners of PA-X, which are cellular proteins involved in RNA splicing and 3' end processing. These data have
led us to the central hypothesis of this project: PA-X selectively targets RNAs through interactions with cellular
splicing and RNA processing machinery. The connection between splicing and PA-X activity is novel among
host shutoff RNases. It also suggests that through interactions with splicing factors, PA-X may differentially
regulate host RNAs with important functions in immune and inflammatory responses, including spliced vs.
intronless types of interferons, key antiviral cytokines. Our objective is to examine the connection between
PA-X and cellular mRNA splicing and its effects on cytokine regulation. The long-term goal of our research is to
link the molecular function of PA-X to its role in vivo. In Aim 1, we will determine how the number of splice sites
and their position affects PA-X's ability to target RNAs, and how widely applicable this is to PA-X variants from
divergent influenza strains. We will use a combination of reporter assays and high-throughout RNAseq. In Aim
2, we will define the role of candidate cellular co-factors of PA-X in RNA targeting by PA-X, particularly
association with target RNAs and subcellular localization of PA-X using knock-down approaches. We will then
use viral mutants to test the role of these interactions in mouse infections. We expect t...

## Key facts

- **NIH application ID:** 10381706
- **Project number:** 5R01AI137358-05
- **Recipient organization:** TUFTS UNIVERSITY BOSTON
- **Principal Investigator:** Marta Maria Gaglia
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $394,212
- **Award type:** 5
- **Project period:** 2018-05-25 → 2024-04-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10381706

## Citation

> US National Institutes of Health, RePORTER application 10381706, Molecular Mechanism of Action of the Influenza PA-X Host Shutoff Protein (5R01AI137358-05). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10381706. Licensed CC0.

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