Cellular heme is thought to be distributed between a pool of sequestered heme that is tightly bound within hemeproteins and a labile heme pool required for signaling and transfer into proteins. A heme chaperone that can hold and allocate labile heme within cells has long been proposed but never been identified. Here, we show that the glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) fulfills this role by acting as an essential repository and allocator of bioavailable heme to downstream protein targets. We identified a conserved histidine in GAPDH that is needed for its robust heme binding both in vitro and in mammalian cells. Substitution of this histidine, and the consequent decreases in GAPDH heme binding, antagonized heme delivery to both cytosolic and nuclear hemeprotein targets, including inducible nitric-oxide synthase (iNOS) in murine macrophages and the nuclear transcription factor Hap1 in yeast, even though this GAPDH variant caused cellular levels of labile heme to rise dramatically. We conclude that by virtue of its heme-binding property, GAPDH binds and chaperones labile heme to create a heme pool that is bioavailable to downstream proteins. Our finding solves a fundamental question in cell biology and provides a new foundation for exploring heme homeostasis in health and disease.

Interferon (IFN) regulatory factor 3 (IRF3) is a transcription factor activated by phosphorylation in the cytoplasm of a virus-infected cell; by translocating to the nucleus, it induces transcription of IFN-β and other antiviral genes. We have previously reported IRF3 can also be activated, as a proapoptotic factor, by its linear polyubiquitination mediated by the RIG-I pathway. Both transcriptional and apoptotic functions of IRF3 contribute to its antiviral effect. Here, we report a nontranscriptional function of IRF3, namely, the repression of IRF3-mediated NF-κB activity (RIKA), which attenuated viral activation of NF-κB and the resultant inflammatory gene induction. In Irf3 ^−/− mice, consequently, Sendai virus infection caused enhanced inflammation in the lungs. Mechanistically, RIKA was mediated by the direct binding of IRF3 to the p65 subunit of NF-κB in the cytoplasm, which prevented its nuclear import. A mutant IRF3 defective in both the transcriptional and the apoptotic activities was active in RIKA and inhibited virus replication. Our results demonstrated IRF3 deployed a three-pronged attack on virus replication and the accompanying inflammation.

The interferon (IFN) system represents the first line of defense against a wide range of viruses. Virus infection rapidly triggers the transcriptional induction of IFN-β and IFN Stimulated Genes (ISGs), whose protein products act as viral restriction factors by interfering with specific stages of virus life cycle, such as entry, transcription, translation, genome replication, assembly and egress. Here, we report a new mode of action of an ISG, IFN-induced TDRD7 (tudor domain containing 7) inhibited paramyxovirus replication by inhibiting autophagy. TDRD7 was identified as an antiviral gene by a high throughput screen of an ISG shRNA library for blocking IFN’s protective effect against Sendai virus (SeV) replication. The antiviral activity of TDRD7 against SeV, human parainfluenza virus 3 and respiratory syncytial virus was confirmed by its genetic ablation or ectopic expression in several types of mouse and human cells. TDRD7’s antiviral action was mediated by its ability to inhibit autophagy, a cellular catabolic process which was robustly induced by SeV infection and required for its replication. Mechanistic investigation revealed that TDRD7 interfered with the activation of AMP-dependent kinase (AMPK), an enzyme required for initiating autophagy. AMPK activity was required for efficient replication of several paramyxoviruses, as demonstrated by its genetic ablation or inhibition of its activity by TDRD7 or chemical inhibitors. Therefore, our study has identified a new antiviral ISG with a new mode of action. The antiviral functions of interferons (IFNs) are mediated by the IFN-induced proteins, encoded by the IFN Stimulated Genes (ISGs). Because ISGs are virus-specific, we performed a high throughput genetic screen to identify novel antiviral ISGs against Sendai virus (SeV), a respirovirus of the Paramyxoviridae family. Our screen isolated a small subset of anti-SeV ISGs, among which we focused on a novel ISG, Tudor domain containing 7 (TDRD7). The antiviral activity of TDRD7 was confirmed by genetic ablation of the endogenous, and the ectopic expression of the exogenous, TDRD7 in human and mouse cell types. Investigation of the mechanism of antiviral action revealed that TDRD7 inhibited ‘virus-induced autophagy’, which was required for the replication of SeV. Autophagy, a cellular catabolic process, was robustly induced by SeV infection, and was inhibited by TDRD7. TDRD7 interfered with the ‘induction’ step of autophagy by inhibiting the activation of AMP-dependent Kinase (AMPK). AMPK is a multifunctional metabolic kinase, which was activated by SeV infection, and its activity was required for virus replication. Genetic ablation and inhibition of AMPK activity by physiological (TDRD7) or chemical (Compound C) inhibitors strongly attenuated SeV replication. The anti-AMPK activity of TDRD7 was capable of inhibiting other members of Paramyxoviridae family, human parainfluenza virus type 3 and respiratory syncytial virus. Therefore, our study uncovered a new antiviral mechanism of IFN by inhibiting the activation of autophagy-inducing kinase AMPK.

The immune system defends against invading pathogens through the rapid activation of innate immune signaling pathways. Interferon regulatory factor 3 (IRF3) is a key transcription factor activated in response to virus infection and is largely responsible for establishing an antiviral state in the infected host. Studies in Irf3^−/− mice have demonstrated the absence of IRF3 imparts a high degree of susceptibility to a wide range of viral infections. Virus infection causes the activation of IRF3 to transcribe type-I interferon (e.g., IFNβ), which is responsible for inducing the interferon-stimulated genes (ISGs), which act at specific stages to limit virus replication. In addition to its transcriptional function, IRF3 is also activated to trigger apoptosis of virus-infected cells, as a mechanism to restrict virus spread within the host, in a pathway called RIG-I-like receptor-induced IRF3 mediated pathway of apoptosis (RIPA). These dual functions of IRF3 work in concert to mediate protective immunity against virus infection. These two pathways are activated differentially by the posttranslational modifications (PTMs) of IRF3. Moreover, PTMs regulate not only IRF3 activation and function, but also protein stability. Consequently, many viruses utilize viral proteins or hijack cellular enzymes to inhibit IRF3 functions. This review will describe the PTMs that regulate IRF3′s RIPA and transcriptional activities and use coronavirus as a model virus capable of antagonizing IRF3-mediated innate immune responses. A thorough understanding of the cellular control of IRF3 and the mechanisms that viruses use to subvert this system is critical for developing novel therapies for virus-induced pathologies.

Virus-infected cells trigger a robust innate immune response and facilitate virus replication. Here, we review the role of autophagy in virus infection, focusing on both pro-viral and anti-viral host responses using a select group of viruses. Autophagy is a cellular degradation pathway operated at the basal level to maintain homeostasis and is induced by external stimuli for specific functions. The degradative function of autophagy is considered a cellular anti-viral immune response. However, autophagy is a double-edged sword in viral infection; viruses often benefit from it, and the infected cells can also use it to inhibit viral replication. In addition to viral regulation, autophagy pathway proteins also function in autophagy-independent manners to regulate immune responses. Since viruses have co-evolved with hosts, they have developed ways to evade the anti-viral autophagic responses of the cells. Some of these mechanisms are also covered in our review. Lastly, we conclude with the thought that autophagy can be targeted for therapeutic interventions against viral diseases.

Viral inflammation contributes to pathogenesis and mortality during respiratory virus infections. IRF3, a critical component of innate antiviral immune responses, interacts with pro-inflammatory transcription factor NF-κB, and inhibits its activity. This mechanism helps suppress inflammatory gene expression in virus-infected cells and mice. We evaluated the cells responsible for IRF3-mediated suppression of viral inflammation using newly engineered conditional Irf3 ^Δ/Δ mice. Irf3 ^Δ/Δ mice, upon respiratory virus infection, showed increased susceptibility and mortality. Irf3 deficiency caused enhanced inflammatory gene expression, lung inflammation, immunopathology, and damage, accompanied by increased infiltration of pro-inflammatory macrophages. Deletion of Irf3 in macrophages ( Irf3 ^MKO ) displayed, similar to Irf3 ^Δ/Δ mice, increased inflammatory responses, macrophage infiltration, lung damage, and lethality, indicating that IRF3 in these cells suppressed lung inflammation. RNA-seq analyses revealed enhanced NF-κB–dependent gene expression along with activation of inflammatory signaling pathways in infected Irf3 ^MKO lungs. Targeted analyses revealed activated MAPK signaling in Irf3 ^MKO lungs. Therefore, IRF3 inhibited inflammatory signaling pathways in macrophages to prevent viral inflammation and pathogenesis.