Enterovirus 2A ^pro Cleavage of the YTHDF m ⁶ A Readers Implicates YTHDF3 as a Mediator of Type I Interferon-Driven JAK/STAT Signaling

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.

TDRD3 is an antiviral restriction factor that promotes IFN signaling with G3BP1

“…TDRD3 in innate immunity mechanisms including degradation of IFNAR1 [31]. as well as blocking JAK-STAT signaling downstream from the receptors by induction of KPNA1 degradation [32], YTHDF3 cleavage [33], and induction of SOCS1 and SOCS3 expression (negative regulators of JAK-STAT signaling) upon infection [34]. Consistent with enteroviral-mediated JAK-STAT signaling abrogation, we noted no phosphorylated STAT1 in our infected cell lines (S1A Fig) . During viral infection, interferon and ISGs expression levels only modestly changed, however KD/KO cells generally exhibited decreased magnitudes of expression of ISG15, IFNλ1, and ISG56 compared to WT cells.…”

PKR Binds Enterovirus IRESs, Displaces Host Translation Factors, and Impairs Viral Translation to Enable Innate Antiviral Signaling

“…Apart from a central role of MDA5 ( 50 ) innate immunity to EVs is poorly understood. Yet, their peculiar genomes (5′ VPg, the IRES), a drastic tactic to foil host defenses (lethal 2A pro -directed host protein cleavage [ 4 , 7 ]), and eminent clinical importance call for elucidating EV innate immunity. In this work, we deciphered mechanisms of accentuated innate signaling elicited by the highly attenuated PV recombinant PVSRIPO.…”

“…This results in early, lethal host protein synthesis shutoff, while viral IRES-mediated translation proceeds with C-terminal eIF4G cleavage fragments ( 6 ). Lethal 2A pro -directed cleavages abrogate host innate defenses ( 7 , 8 ). The PV IRES strategy is evident as lethal infection of lymphoid and intestinal dendritic cells (DCs)/macrophages in vivo ( 1 ) and primary human DCs/macrophages in vitro ( 9 ).…”

Minding the message: tactics controlling RNA decay, modification, and translation in virus-infected cells

“…Many ISG mRNAs are m 6 A-modified, and this reportedly stimulates translation of a subset ( McFadden et al 2021 ). Indeed, YTHDF1,2,3 are cleaved by EV71 2a protease, and this has been proposed to antagonize ISG expression in infected cells ( Kastan et al 2021 ). Reduced m 6 A on mRNA encoding α-ketoglutarate dehydrogenase promoted RNA decay, limiting metabolite (itaconate) accumulation required for VSV replication ( Liu et al 2019b ).…”