Enterovirus 2A^pro Cleavage of the YTHDF m⁶A Readers Implicates YTHDF3 as a Mediator of Type I Interferon-Driven JAK/STAT Signaling

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A High Resolution Method to Monitor Phosphorylation-dependent Activation of IRF3

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Published on: January 24, 2016

Jonathan P Kastan^1,2, Martine W Tremblay², Michael C Brown¹

¹Department of Neurosurgery, Duke University Medical Center, Durham, North Carolina, USA.

|April 14, 2021

Abstract:

Enteroviruses (EV) deploy two proteases that mediate viral polyprotein cleavage and host cell manipulation. Here, we report that EV 2A proteases cleave all three members of the YTHDF protein family, cytosolic N⁶-methyladenosine (m⁶A) "readers" that regulate target mRNA fate. YTHDF protein cleavage occurs very early during infection, before viral translation is detected or cytopathogenic effects are observed. Preemptive YTHDF protein depletion enhanced viral translation and replication but only in cells with restrained viral translation, signs of inefficient 2A protease activity, and protective innate host immune responses. This effect corresponded with repression of interferon (IFN)-stimulated gene (ISG) induction, while type I/III IFN production was not significantly altered. Moreover, YTHDF3 depletion impaired JAK/STAT signaling in cells treated with type I, but not type II, IFN. YTHDF3 depletion's stimulatory effect on viral dynamics was dampened by JAK/STAT blockade and enhanced by type I IFN pretreatment of cells. We propose that EV 2A proteases cleave YTHDF proteins to antagonize ISG induction in infected cells.IMPORTANCE It is believed that ∼7,000 messenger RNAs (mRNAs) are subject to N⁶-methyladenosine modification. The biological significance of this remains mysterious. The YTHDF m⁶A readers are three related proteins with high affinity for m⁶A-modified mRNA, yet their biological functions remain obscure. We discovered that polio/enteroviruses elicit very early proteolysis of YTHDF1 to 3 in infected cells. Our research demonstrates that YTHDF3 acts as a positive regulator of antiviral JAK/STAT signaling in response to positive single-strand RNA virus infection, enabling type I interferon (IFN)-mediated gene regulatory programs to unfurl in infected cells. Our observation of viral degradation of the YTHDF proteins demonstrates that they are key response modifiers in the innate antiviral immune response.

Insights

Enteroviruses cleave YTHDF proteins early in infection, enhancing viral replication by suppressing interferon-stimulated gene induction and antiviral signaling. This reveals a viral strategy to evade host defenses.

Area of Science:

Virology
Molecular Biology
Immunology

Background:

Enteroviruses (EV) utilize proteases for viral replication and host cell manipulation.
Cytosolic N6-methyladenosine (m6A) readers, including the YTHDF protein family, regulate mRNA fate.
The precise roles of YTHDF proteins in antiviral responses remain largely uncharacterized.

Purpose of the Study:

To investigate the interaction between enterovirus 2A proteases and YTHDF proteins.
To elucidate the functional consequences of YTHDF protein cleavage during enterovirus infection.
To understand the role of YTHDF proteins in the host innate immune response to viral infection.

Main Methods:

Western blotting to detect YTHDF protein cleavage.
Viral replication assays in cells with depleted YTHDF proteins.
Interferon-stimulated gene (ISG) induction analysis.
JAK/STAT signaling pathway assessment.
Type I and III interferon production measurements.

Main Results:

Enterovirus 2A proteases cleave all three YTHDF proteins (YTHDF1-3) early in infection.
YTHDF protein depletion enhances viral translation and replication, particularly under conditions of limited viral protease activity.
Cleavage of YTHDF proteins leads to repression of ISG induction.
YTHDF3 depletion impairs JAK/STAT signaling in response to type I interferon.

Conclusions:

Enteroviruses antagonize host antiviral defenses by cleaving YTHDF proteins.
YTHDF proteins, particularly YTHDF3, are crucial positive regulators of antiviral JAK/STAT signaling and ISG induction.
Viral degradation of YTHDF proteins represents a key mechanism for modulating the innate antiviral immune response.

Read full analysis

Keywords:

2A protease Jak/Stat signaling YTHDF proteins enterovirus innate immunity interferon stimulated gene m6A modification poliovirus type I interferon type III interferon

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