Autophagic degradation of IRF3 induced by the small-molecule auranofin inhibits its transcriptional and proapoptotic activities.

The ubiquitously expressed transcription factor interferon (IFN) regulatory factor 3 (IRF3) is critical for the induction of antiviral genes, e.g., type-I IFN. In addition to its transcriptional function, IRF3 also activates a nontranscriptional, proapoptotic signaling pathway. While the proapoptotic function of IRF3 protects against viral infections, it is also involved in harmful immune responses that trigger hepatocyte cell death and promote liver disease. Thus, we hypothesized that a small-molecule inhibitor of the proapoptotic activity of IRF3 could alleviate fatty-acid-induced hepatocyte cell death. We conducted a high-throughput screen, which identified auranofin as a small-molecule inhibitor of the proapoptotic activity of IRF3. In addition to the nontranscriptional apoptotic pathway, auranofin also inhibited the transcriptional activity of IRF3. Using biochemical and genetic tools in human and mouse cells, we uncovered a novel mechanism of action for auranofin, in which it induces cellular autophagy to degrade IRF3 protein, thereby suppressing IRF3 functions. Autophagy-deficient cells were unable to degrade IRF3 upon auranofin treatment, suggesting that the autophagic degradation of IRF3 is a novel approach to regulate IRF3 activities. Using a physiologically relevant in vitro model, we demonstrated that auranofin inhibited fatty-acid-induced apoptotic cell death of hepatocytes. In summary, auranofin is a novel inhibitor of IRF3 functions and may represent a potential therapeutic option in diseases where IRF3 is deleterious.

The interferon-inducible protein TDRD7 inhibits AMP-activated protein kinase and thereby restricts autophagy-independent virus replication.

The interferon system is the first line of defense against virus infection. Recently, using a high-throughput genetic screen of a human interferon-stimulated gene short-hairpin RNA library, we identified a viral restriction factor, TDRD7 (Tudor domain-containing 7). TDRD7 inhibits the paramyxo-/pneumoviruses (e.g. Sendai virus and respiratory syncytial virus) by interfering with the virus-induced cellular autophagy pathway, which these viruses use for their replication. Here, we report that TDRD7 is a viral restriction factor against herpes simplex virus (HSV-1). Using knockdown, knockout, and ectopic expression systems, we demonstrate the anti-HSV-1 activity of TDRD7 in multiple human and mouse cell types. TDRD7 inhibited the virus-activated AMP-activated protein kinase (AMPK), which was essential for HSV-1 replication. Genetic ablation or chemical inhibition of AMPK activity suppressed HSV-1 replication in multiple human and mouse cells. Mechanistically, HSV-1 replication after viral entry depended on AMPK but not on its function in autophagy. The antiviral activity of TDRD7 depended on its ability to inhibit virus-activated AMPK. In summary, our results indicate that the newly identified viral restriction factor TDRD7 inhibits AMPK and thereby blocks HSV-1 replication independently of the autophagy pathway. These findings suggest that AMPK inhibition represents a potential strategy to manage HSV-1 infections.

A new mechanism of interferon's antiviral action: Induction of autophagy, essential for paramyxovirus replication, is inhibited by the interferon stimulated gene, TDRD7.

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-ß 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.

AP3B1 Has Type I Interferon-Independent Antiviral Function against SARS-CoV-2.

The unprecedented research effort associated with the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) included several extensive proteomic studies that identified host proteins that interact with individual viral gene products. However, in most cases, the consequences of those virus-host interactions for virus replication were not experimentally pursued, which is a necessary step in determining whether the interactions represent pro- or anti-viral events. One putative interaction commonly identified in multiple studies was between the host adaptor protein complex 3 (AP-3) subunit B1 (AP3B1) and the SARS-CoV-2 envelope protein (E). AP3B1 is one subunit of AP-3 required for the biogenesis of lysosomal-related organelles (LROs), and its function impacts important disease processes including inflammation and vascular health. Thus, interactions between AP3B1 and SARS-CoV-2 might influence the clinical outcomes of infection. To determine if AP3B1 has a role in the SARS-CoV-2 replication cycle, we first confirmed the interaction in virus-infected cells using immunoprecipitation (IP) and immunofluorescence assays (IFA). AP3B1 is required by multiple viruses to aid in the replication cycle and therefore may be a therapeutic target. However, we found that the overexpression of AP3B1 suppressed SARS-CoV-2 replication, whereas the siRNA-mediated depletion of AP3B1 increased the release of infectious virus, suggesting an antiviral role for AP3B1. Together, our findings suggest that AP3B1 is an intrinsic barrier to SARS-CoV-2 replication through interactions with the viral E protein. Our work justifies further investigations of LRO trafficking in SARS-CoV-2 target cells and their role in viral pathogenesis.

Interferon-stimulated gene TDRD7 interacts with AMPK and inhibits its activation to suppress viral replication and pathogenesis.

IMPORTANCE: Virus infection triggers induction of interferon (IFN)-stimulated genes (ISGs), which ironically inhibit viruses themselves. We identified Tudor domain-containing 7 (TDRD7) as a novel antiviral ISG, which inhibits viral replication by interfering with autophagy pathway. Here, we present a molecular basis for autophagy inhibitory function of TDRD7. TDRD7 interacted with adenosine monophosphate (AMP)-activated protein kinase (AMPK), the kinase that initiates autophagy, to inhibit its activation. We identified domains required for the interaction; deleting AMPK-interacting domain blocked antiAMPK and antiviral activities of TDRD7. We used primary cells and mice to evaluate the TDRD7-AMPK antiviral pathway. TDRD7-deficient primary mouse cells exhibited enhanced AMPK activation and viral replication. Finally, TDRD7 knockout mice showed increased susceptibility to respiratory virus infection. Therefore, our study revealed a new antiviral pathway of IFN and its contribution to host response. Our results have therapeutic potential; a TDRD7-derived peptide may be an effective AMPK inhibitor with application as antiviral agent.

Autophagy in Virus Infection: A Race between Host Immune Response and Viral Antagonism.

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.

Mitophagy antagonism by ZIKV reveals Ajuba as a regulator of PINK1 signaling, PKR-dependent inflammation, and viral invasion of tissues.

Dysregulated inflammation dominated by chemokine expression is a key feature of disease following infection with the globally important human pathogens Zika virus (ZIKV) and dengue virus, but a mechanistic understanding of how pro-inflammatory responses are initiated is lacking. Mitophagy is a quality-control mechanism that regulates innate immune signaling and cytokine production through selective degradation of damaged mitochondria. Here, we demonstrate that ZIKV nonstructural protein 5 (NS5) antagonizes mitophagy by binding to the host protein Ajuba and preventing its translocation to depolarized mitochondria where it is required for PINK1 activation and downstream signaling. Consequent mitophagy suppression amplifies the production of pro-inflammatory chemokines through protein kinase R (PKR) sensing of mitochondrial RNA. In Ajuba-/- mice, ZIKV induces early expression of pro-inflammatory chemokines associated with significantly enhanced dissemination to tissues. This work identifies Ajuba as a critical regulator of mitophagy and demonstrates a role for mitophagy in limiting systemic inflammation following infection by globally important human viruses.

High Throughput Screening of FDA-Approved Drug Library Reveals the Compounds that Promote IRF3-Mediated Pro-Apoptotic Pathway Inhibit Virus Replication.

Interferon (IFN) regulatory factor 3 (IRF3) is the key transcription factor for the induction of IFN and antiviral genes. The absence of antiviral genes in IRF3 deficiency leads to susceptibility to a wide range of viral infections. Previously, we uncovered a function for nontranscriptional IRF3 (nt-IRF3), RLR (RIG-I-like receptor)-induced IRF3-mediated pathway of apoptosis (RIPA), which triggers apoptotic killing of virus-infected cells. Using knock-in mice expressing a transcriptionally inactive, but RIPA-active, IRF3 mutant, we demonstrated the relative contribution of RIPA to host antiviral defense. Given that RIPA is a cellular antiviral pathway, we hypothesized that small molecules that promote RIPA in virus-infected cells would act as antiviral agents. To test this, we conducted a high throughput screen of a library of FDA-approved drugs to identify novel RIPA activators. Our screen identified doxorubicin as a potent RIPA-activating agent. In support of our hypothesis, doxorubicin inhibited the replication of vesicular stomatitis virus, a model rhabdovirus, and its antiviral activity depended on its ability to activate IRF3 in RIPA. Surprisingly, doxorubicin inhibited the transcriptional activity of IRF3. The antiviral activity of doxorubicin was expanded to flavivirus and herpesvirus that also activate IRF3. Mechanistically, doxorubicin promoted RIPA by activating the extracellular signal-regulated kinase (ERK) signaling pathway. Finally, we validated these results using another RIPA-activating compound, pyrvinium pamoate, which showed a similar antiviral effect without affecting the transcriptional activity of IRF3. Therefore, we demonstrate that the RIPA branch of IRF3 can be targeted therapeutically to prevent virus infection.

Gene Therapy Leaves a Vicious Cycle.

The human genetic code encrypted in thousands of genes holds the secret for synthesis of proteins that drive all biological processes necessary for normal life and death. Though the genetic ciphering remains unchanged through generations, some genes get disrupted, deleted and or mutated, manifesting diseases, and or disorders. Current treatment options-chemotherapy, protein therapy, radiotherapy, and surgery available for no more than 500 diseases-neither cure nor prevent genetic errors but often cause many side effects. However, gene therapy, colloquially called "living drug," provides a one-time treatment option by rewriting or fixing errors in the natural genetic ciphering. Since gene therapy is predominantly a viral vector-based medicine, it has met with a fair bit of skepticism from both the science fraternity and patients. Now, thanks to advancements in gene editing and recombinant viral vector development, the interest of clinicians and pharmaceutical industries has been rekindled. With the advent of more than 12 different gene therapy drugs for curing cancer, blindness, immune, and neuronal disorders, this emerging experimental medicine has yet again come in the limelight. The present review article delves into the popular viral vectors used in gene therapy, advances, challenges, and perspectives.

Biochemical Analysis of Caspase-8-dependent Proteolysis of IRF3 in Virus-infected Cells.

Interferon regulatory factor 3 (IRF3) is a transcription factor, which is critical for the antiviral response against a wide range of viruses (Hiscott, 2007; Ikushima et al., 2013). It gets activated in virus- infected cells via Toll like receptors (TLRs), RIG-I (retinoic acid inducible gene 1) like receptors (RLRs), cyclic GMP-AMP synthase (cGAS) ­ stimulator of interferon genes (STING), which are sensors of viral components in the cells (Chattopadhyay and Sen, 2014a; 2014b; Hiscott, 2007). IRF3 is a cytoplasmic protein, upon activation by virally activated sensors it gets phosphorylated, translocated to the nucleus and binds to the interferon-sensitive response element (ISRE) of the gene promoters to induce their transcription (Hiscott, 2007). IRF3 has other functions, including direct stimulation of apoptosis in virus- infected cells. In this pathway, the transcriptional activity of IRF3 is not required (Chattopadhyay et al., 2013b; Chattopadhyay et al., 2016; Chattopadhyay et al., 2010; Chattopadhyay and Sen, 2010; Chattopadhyay et al., 2011). These pathways are negatively regulated by host factors as well as by viruses. Our studies indicate that IRF3 can be proteolytically processed by caspase-8-dependent cleavage (Sears et al., 2011). A specific site in IRF3 is targeted by caspase-8, activated in RNA or DNA virus-infected and dsRNA-stimulated cells (Sears et al., 2011). The direct involvement of caspase-8 was confirmed by in vitro cleavage assay using recombinant proteins and in vivo by virus activated caspase- 8. The proteolytic cleavage of IRF3 can be inhibited by chemical inhibition or genetic ablation of caspase-8. The cleavage of IRF3 removes the activated pool of IRF3 and thus can be used as a pro- viral mechanism (Figure 1). Using a C-terminally epitope-tagged human IRF3, we analyzed the cleavage of IRF3 in virus-infected cells. Moreover, we used recombinant proteins in vitro to conclude that IRF3 is a substrate of caspase-8 (Sears et al., 2011). In the current protocol, we have outlined a simple and detailed procedure to biochemically analyze the proteolysis of IRF3 in virus-infected cells and the specific role of caspase-8 in this process.