SARS-CoV-2 Spike Antagonizes Innate Antiviral Immunity by Targeting Interferon Regulatory Factor 3

Corona virus disease 2019 (COVID-19) pathogenesis is intimately linked to the severe acute respiratory syndrome corona virus 2 (SARS-CoV-2) and disease severity has been associated with compromised induction of type I interferon (IFN-I) cytokines which coordinate the innate immune response to virus infections. Here we identified the SARS-CoV-2 encoded protein, Spike, as an inhibitor of IFN-I that antagonizes viral RNA pattern recognition receptor RIG-I signaling. Ectopic expression of SARS-CoV-2 Spike blocked RIG-I mediated activation of IFNβ and downstream induction of interferon stimulated genes. Consequently, SARS-CoV-2 Spike expressing cells harbored increased RNA viral burden compared to control cells. Co-immunoprecipitation experiments revealed SARS-CoV-2 Spike associated with interferon regulatory factor 3 (IRF3), a key transcription factor that governs IFN-I activation. Co-expression analysis via immunoassays further indicated Spike specifically suppressed IRF3 expression as NF-κB and STAT1 transcription factor levels remained intact. Further biochemical experiments uncovered SARS-CoV-2 Spike potentiated proteasomal degradation of IRF3, implicating a novel mechanism by which SARS-CoV-2 evades the host innate antiviral immune response to facilitate COVID-19 pathogenesis.

Vaccinia Virus Expressing Interferon Regulatory Factor 3 Induces Higher Protective Immune Responses against Lethal Poxvirus Challenge in Atopic Organism

Vaccinia virus (VACV) is an enveloped DNA virus from the Orthopoxvirus family, various strains of which were used in the successful eradication campaign against smallpox. Both original and newer VACV-based replicating vaccines reveal a risk of serious complications in atopic individuals. VACV encodes various factors interfering with host immune responses at multiple levels. In atopic skin, the production of type I interferon is compromised, while VACV specifically inhibits the phosphorylation of the Interferon Regulatory Factor 3 (IRF-3) and expression of interferons. To overcome this block, we generated a recombinant VACV-expressing murine IRF-3 (WR-IRF3) and characterized its effects on virus growth, cytokine expression and apoptosis in tissue cultures and in spontaneously atopic Nc/Nga and control Balb/c mice. Further, we explored the induction of protective immune responses against a lethal dose of wild-type WR, the surrogate of smallpox. We demonstrate that the overexpression of IRF-3 by WR-IRF3 increases the expression of type I interferon, modulates the expression of several cytokines and induces superior protective immune responses against a lethal poxvirus challenge in both Nc/Nga and Balb/c mice. Additionally, the results may be informative for design of other virus-based vaccines or for therapy of different viral infections.

Mutations in SARS-CoV-2 ORF8 Altered the Bonding Network With Interferon Regulatory Factor 3 to Evade Host Immune System

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been continuously mutating since its first emergence in early 2020. These alterations have led this virus to gain significant difference in infectivity, pathogenicity, and host immune evasion. We previously found that the open-reading frame 8 (ORF8) of SARS-CoV-2 can inhibit interferon production by decreasing the nuclear translocation of interferon regulatory factor 3 (IRF3). Since several mutations in ORF8 have been observed, therefore, in the present study, we adapted structural and biophysical analysis approaches to explore the impact of various mutations of ORF8, such as S24L, L84S, V62L, and W45L, the recently circulating mutant in Pakistan, on its ability to bind IRF3 and to evade the host immune system. We found that mutations in ORF8 could affect the binding efficiency with IRF3 based on molecular docking analysis, which was further supported by molecular dynamics simulations. Among all the reported mutations, W45L was found to bind most stringently to IRF3. Our analysis revealed that mutations in ORF8 may help the virus evade the immune system by changing its binding affinity with IRF3.

Regulation of adipogenic differentiation and adipose tissue inflammation by interferon regulatory factor 3

Dysfunction of adipocytes and adipose tissue is a primary defect in obesity and obesity-associated metabolic diseases. Interferon regulatory factor 3 (IRF3) has been implicated in adipogenesis. However, the role of IRF3 in obesity and obesity-associated disorders remains unclear. Here, we show that IRF3 expression in human adipose tissues is positively associated with insulin sensitivity and negatively associated with type 2 diabetes. In mouse pre-adipocytes, deficiency of IRF3 results in increased expression of PPARγ and PPARγ-mediated adipogenic genes, leading to increased adipogenesis and altered adipocyte functionality. The IRF3 knockout (KO) mice develop obesity, insulin resistance, glucose intolerance, and eventually type 2 diabetes with aging, which is associated with the development of white adipose tissue (WAT) inflammation. Increased macrophage accumulation with M1 phenotype which is due to the loss of IFNβ-mediated IL-10 expression is observed in WAT of the KO mice compared to that in wild-type mice. Bone-marrow reconstitution experiments demonstrate that the nonhematopoietic cells are the primary contributors to the development of obesity and both hematopoietic and nonhematopoietic cells contribute to the development of obesity-related complications in IRF3 KO mice. This study demonstrates that IRF3 regulates the biology of multiple cell types including adipocytes and macrophages to prevent the development of obesity and obesity-related complications and hence, could be a potential target for therapeutic interventions for the prevention and treatment of obesity-associated metabolic disorders.

Transcriptional and Non-Transcriptional Activation, Posttranslational Modifications, and Antiviral Functions of Interferon Regulatory Factor 3 and Viral Antagonism by the SARS-Coronavirus

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.

Interferon Regulatory Factor 3 Supports the Establishment of Chronic Gammaherpesvirus Infection in a Route- and Dose-Dependent Manner

ABSTRACT Gammaherpesviruses are ubiquitous pathogens that establish lifelong infections and are associated with several malignancies, including B cell lymphomas. Uniquely, these viruses manipulate B cell differentiation to establish long-term latency in memory B cells. This study focuses on the interaction between gammaherpesviruses and interferon regulatory factor 3 (IRF-3), a ubiquitously expressed transcription factor with multiple direct target genes, including beta interferon (IFN-β), a type I IFN. IRF-3 attenuates acute replication of a plethora of viruses, including gammaherpesvirus. Furthermore, IRF-3-driven IFN-β expression is antagonized by the conserved gammaherpesvirus protein kinase during lytic virus replication in vitro. In this study, we have uncovered an unexpected proviral role of IRF-3 during chronic gammaherpesvirus infection. In contrast to the antiviral activity of IRF-3 during acute infection, IRF-3 facilitated establishment of latent gammaherpesvirus infection in B cells, particularly, germinal center and activated B cells, the cell types critical for both natural infection and viral lymphomagenesis. This proviral role of IRF-3 was further modified by the route of infection and viral dose. Furthermore, using a combination of viral and host genetics, we show that IRF-3 deficiency does not rescue attenuated chronic infection of a protein kinase null gammaherpesvirus mutant, highlighting the multifunctional nature of the conserved gammaherpesvirus protein kinases in vivo. In summary, this study unveils an unexpected proviral nature of the classical innate immune factor, IRF-3, during chronic virus infection. IMPORTANCE Interferon regulatory factor 3 (IRF-3) is a critical component of the innate immune response, in part due to its transactivation of beta interferon (IFN-β) expression. Similar to that observed in all acute virus infections examined to date, IRF-3 suppresses lytic viral replication during acute gammaherpesvirus infection. Because gammaherpesviruses establish lifelong infection, this study aimed to define the antiviral activity of IRF-3 during chronic infection. Surprisingly, we found that, in contrast to acute infection, IRF-3 supported the establishment of gammaherpesvirus latency in splenic B cells, revealing an unexpected proviral nature of this classical innate immune host factor.