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

For RNA virus families except Picornaviridae, viral RNA sensing includes Toll-like receptors and/or RIG-I. Picornavirus RNAs, whose 5' termini are shielded by a genome-linked protein, are predominately recognized by MDA5. This has important ramifications for adaptive immunity, as MDA5-specific patterns of type-I interferon (IFN) release are optimal for CD4+T cell TH1 polarization and CD8+T cell priming. We are exploiting this principle for cancer immunotherapy with recombinant poliovirus (PV), PVSRIPO, the type 1 (Sabin) PV vaccine containing a rhinovirus type 2 internal ribosomal entry site (IRES). Here we show that PVSRIPO-elicited MDA5 signaling is preceded by early sensing of the IRES by the double-stranded (ds)RNA-activated protein kinase (PKR). PKR binding to IRES stem-loop domains 5-6 led to dimerization and autoactivation, displaced host translation initiation factors, and suppressed viral protein synthesis. Early PKR-mediated antiviral responses tempered incipient viral translation and the activity of cytopathogenic viral proteinases, setting up accentuated MDA5 innate inflammation in response to PVSRIPO infection. IMPORTANCE Among the RIG-I-like pattern recognition receptors, MDA5 stands out because it senses long dsRNA duplexes independent of their 5' features (RIG-I recognizes viral [v]RNA 5'-ppp blunt ends). Uniquely among RNA viruses, the innate defense against picornaviruses is controlled by MDA5. We show that prior to engaging MDA5, recombinant PV RNA is sensed upon PKR binding to the viral IRES at a site that overlaps with the footprint for host translation factors mediating 40S subunit recruitment. Our study demonstrates that innate antiviral type-I IFN responses orchestrated by MDA5 involve separate innate modules that recognize distinct vRNA features and interfere with viral functions at multiple levels.

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

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.

Viral infection of cells within the tumor microenvironment mediates antitumor immunotherapy via selective TBK1-IRF3 signaling.

Activating intra-tumor innate immunity might enhance tumor immune surveillance. Virotherapy is proposed to achieve tumor cell killing, while indirectly activating innate immunity. Here, we report that recombinant poliovirus therapy primarily mediates antitumor immunotherapy via direct infection of non-malignant tumor microenvironment (TME) cells, independent of malignant cell lysis. Relative to other innate immune agonists, virotherapy provokes selective, TBK1-IRF3 driven innate inflammation that is associated with sustained type-I/III interferon (IFN) release. Despite priming equivalent antitumor T cell quantities, MDA5-orchestrated TBK1-IRF3 signaling, but not NFκB-polarized TLR activation, culminates in polyfunctional and Th1-differentiated antitumor T cell phenotypes. Recombinant type-I IFN increases tumor-localized T cell function, but does not mediate durable antitumor immunotherapy without concomitant pattern recognition receptor (PRR) signaling. Thus, virus-induced MDA5-TBK1-IRF3 signaling in the TME provides PRR-contextualized IFN responses that elicit functional antitumor T cell immunity. TBK1-IRF3 innate signal transduction stimulates eventual function and differentiation of tumor-infiltrating T cells.

CReP mediates selective translation initiation at the endoplasmic reticulum.

Eukaryotic protein synthesis control at multiple levels allows for dynamic, selective responses to diverse conditions, but spatial organization of translation initiation machinery as a regulatory principle has remained largely unexplored. Here we report on a role of constitutive repressor of eIF2α phosphorylation (CReP) in translation of poliovirus and the endoplasmic reticulum (ER)-resident chaperone binding immunoglobulin protein (BiP) at the ER. Functional, proximity-dependent labeling and cell fractionation studies revealed that CReP, through binding eIF2α, anchors translation initiation machinery at the ER and enables local protein synthesis in this compartment. This ER site was protected from the suppression of cytoplasmic protein synthesis by acute stress responses, e.g., phosphorylation of eIF2α(S51) or mTOR blockade. We propose that partitioning of translation initiation machinery at the ER enables cells to maintain active translation during stress conditions associated with global protein synthesis suppression.