Cruciferous vegetable-derived indole-3-carbinol prevents coronavirus cell egression mechanisms in tracheal and intestinal 3D in vitro models.

The potential antiviral effects of indole-3-carbinol (I3C), a phytochemical found in Cruciferous vegetables, were investigated. Fibroblasts and epithelial cells were co-cultured on Alvetex® scaffolds, to obtain ad hoc 3D in vitro platforms able to mimic the trachea and intestinal mucosae, which represent the primary structures involved in the coronavirus pathogenesis. The two barriers generated in vitro were treated with various concentrations of I3C for different incubation periods. A protective effect of I3C on both intestinal and trachea models was demonstrated. A significant reduction in the transcription of the two main genes belonging to the Homologous to E6AP C-terminus (HECT)-E3 ligase family members, namely NEDD4 E3 Ubiquitin Protein Ligase (NEDD4) and WW Domain Containing E3 Ubiquitin Protein Ligase 1 (WWP1), which promote virus matrix protein ubiquitination and inhibit viral egression, were detected. These findings indicate I3C potential effect in preventing coronavirus cell egression processes that inhibit viral production. Although further studies are needed to clarify the molecular mechanisms whereby HECT family members control virus life cycle, this work paves the way to the possible therapeutic use of new natural compounds that may reduce the clinical severity of future pandemics.

HDAC6 Degrades nsp8 of Porcine Deltacoronavirus through Deacetylation and Ubiquitination to Inhibit Viral Replication.

Porcine deltacoronavirus (PDCoV) is an emerging swine enteropathogenic coronavirus that has the potential to infect humans. Histone deacetylase 6 (HDAC6) is a unique type IIb cytoplasmic deacetylase with both deacetylase activity and ubiquitin E3 ligase activity, which mediates a variety of cellular processes by deacetylating histone and nonhistone substrates. In this study, we found that ectopic expression of HDAC6 significantly inhibited PDCoV replication, while the reverse effects could be observed after treatment with an HDAC6-specific inhibitor (tubacin) or knockdown of HDAC6 expression by specific small interfering RNA. Furthermore, we demonstrated that HDAC6 interacted with viral nonstructural protein 8 (nsp8) in the context of PDCoV infection, resulting in its proteasomal degradation, which was dependent on the deacetylation activity of HDAC6. We further identified the key amino acid residues lysine 46 (K46) and K58 of nsp8 as acetylation and ubiquitination sites, respectively, which were required for HDAC6-mediated degradation. Through a PDCoV reverse genetics system, we confirmed that recombinant PDCoV with a mutation at either K46 or K58 exhibited resistance to the antiviral activity of HDAC6, thereby exhibiting higher replication compared with wild-type PDCoV. Collectively, these findings contribute to a better understanding of the function of HDAC6 in regulating PDCoV infection and provide new strategies for the development of anti-PDCoV drugs. IMPORTANCE As an emerging enteropathogenic coronavirus with zoonotic potential, porcine deltacoronavirus (PDCoV) has sparked tremendous attention. Histone deacetylase 6 (HDAC6) is a critical deacetylase with both deacetylase activity and ubiquitin E3 ligase activity and is extensively involved in many important physiological processes. However, little is known about the role of HDAC6 in the infection and pathogenesis of coronaviruses. Our present study demonstrates that HDAC6 targets PDCoV-encoded nonstructural protein 8 (nsp8) for proteasomal degradation through the deacetylation at the lysine 46 (K46) and the ubiquitination at K58, suppressing viral replication. Recombinant PDCoV with a mutation at K46 and/or K58 of nsp8 displayed resistance to the antiviral activity of HDAC6. Our work provides significant insights into the role of HDAC6 in regulating PDCoV infection, opening avenues for the development of novel anti-PDCoV drugs.

Heat shock protein 90 facilitates SARS-CoV-2 structural protein-mediated virion assembly and promotes virus-induced pyroptosis.

Inhibition of heat shock protein 90 (Hsp90), a prominent molecular chaperone, effectively limits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection but little is known about any interaction between Hsp90 and SARS-CoV-2 proteins. Here, we systematically analyzed the effects of the chaperone isoforms Hsp90α and Hsp90ß on individual SARS-CoV-2 viral proteins. Five SARS-CoV-2 proteins, namely nucleocapsid (N), membrane (M), and accessory proteins Orf3, Orf7a, and Orf7b were found to be novel clients of Hsp90ß in particular. Pharmacological inhibition of Hsp90 with 17-DMAG results in N protein proteasome-dependent degradation. Hsp90 depletion-induced N protein degradation is independent of CHIP, a ubiquitin E3 ligase previously identified for Hsp90 client proteins, but alleviated by FBXO10, an E3 ligase identified by subsequent siRNA screening. We also provide evidence that Hsp90 depletion may suppress SARS-CoV-2 assembly partially through induced M or N degradation. Additionally, we found that GSDMD-mediated pyroptotic cell death triggered by SARS-CoV-2 was mitigated by inhibition of Hsp90. These findings collectively highlight a beneficial role for targeting of Hsp90 during SARS-CoV-2 infection, directly inhibiting virion production and reducing inflammatory injury by preventing the pyroptosis that contributes to severe SARS-CoV-2 disease.

The E3 ligase RNF5 restricts SARS-CoV-2 replication by targeting its envelope protein for degradation.

The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a severe global health crisis; its structural protein envelope (E) is critical for viral entry, budding, production, and induction of pathology which makes it a potential target for therapeutics against COVID-19. Here, we find that the E3 ligase RNF5 interacts with and catalyzes ubiquitination of E on the 63rd lysine, leading to its degradation by the ubiquitin-proteasome system (UPS). Importantly, RNF5-induced degradation of E inhibits SARS-CoV-2 replication and the RNF5 pharmacological activator Analog-1 alleviates disease development in a mouse infection model. We also found that RNF5 is distinctively expressed in different age groups and in patients displaying different disease severity, which may be exploited as a prognostic marker for COVID-19. Furthermore, RNF5 recognized the E protein from various SARS-CoV-2 strains and SARS-CoV, suggesting that targeting RNF5 is a broad-spectrum antiviral strategy. Our findings provide novel insights into the role of UPS in antagonizing SARS-CoV-2 replication, which opens new avenues for therapeutic intervention to combat the COVID-19 pandemic.

PRPF19 Limits Porcine Epidemic Diarrhea Virus Replication through Targeting and Degrading Viral Capsid Protein.

Porcine epidemic diarrhea (PED) indicates the disease of the acute and highly contagious intestinal infection due to porcine epidemic diarrhea virus (PEDV), with the characteristics of watery diarrhea, vomiting, and dehydration. One of the reasons for diarrhea and death of piglets is PEDV, which leads to 100% mortality in neonatal piglets. Therefore, it is necessary to explore the interaction between virus and host to prevent and control PEDV. This study indicated that the host protein, pre-mRNA processing factor 19 (PRPF19), could be controlled by the signal transducer as well as activator of transcription 1 (STAT1). Thus, PEDV replication could be hindered through selective autophagy. Moreover, PRPF19 was found to recruit the E3 ubiquitin ligase MARCH8 to the N protein for ubiquitination. For the purpose of degradation, the ubiquitin N protein is acknowledged by the cargo receptor NDP52 and transported to autolysosomes, thus inhibiting virus proliferation. To conclude, a unique antiviral mechanism of PRPF19-mediated virus restriction was shown. Moreover, a view of the innate immune response and protein degradation against PEDV replication was provided in this study. IMPORTANCE The highly virulent porcine epidemic diarrhea virus (PEDV) emerged in 2010, and causes high mortality rates in newborn pigs. There are no effective and safe vaccines against the highly virulent PEDV. This virus has caused devastating economic losses in the pork industry worldwide. Studying the relationship between virus and host antiviral factors is important to develop the new antiviral strategies. This study identified the pre-mRNA processing factor 19 (PRPF19) as a novel antiviral protein in PEDV replication and revealed its viral restriction mechanisms for the first time. PRPF19 recruited the E3 ubiquitin ligase MARCH8 to the PEDV N protein for ubiquitination, and the ubiquitin N protein was acknowledged by the cargo receptor NDP52 and transported to autolysosomes for degradation. Our findings provide new insights in host antiviral factors PRPF19 that regulate the selective autophagy protein degradation pathway to inhibit PEDV replication.

SARS-CoV-2 ORF10 impairs cilia by enhancing CUL2ZYG11B activity.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causal pathogen of the ongoing global pandemic of coronavirus disease 2019 (COVID-19). Loss of smell and taste are symptoms of COVID-19, and may be related to cilia dysfunction. Here, we found that the SARS-CoV-2 ORF10 increases the overall E3 ligase activity of the CUL2ZYG11B complex by interacting with ZYG11B. Enhanced CUL2ZYG11B activity by ORF10 causes increased ubiquitination and subsequent proteasome-mediated degradation of an intraflagellar transport (IFT) complex B protein, IFT46, thereby impairing both cilia biogenesis and maintenance. Further, we show that exposure of the respiratory tract of hACE2 mice to SARS-CoV-2 or SARS-CoV-2 ORF10 alone results in cilia-dysfunction-related phenotypes, and the ORF10 expression in primary human nasal epithelial cells (HNECs) also caused a rapid loss of the ciliary layer. Our study demonstrates how SARS-CoV-2 ORF10 hijacks CUL2ZYG11B to eliminate IFT46 and leads to cilia dysfunction, thereby offering a powerful etiopathological explanation for how SARS-CoV-2 causes multiple cilia-dysfunction-related symptoms specific to COVID-19.

Trim69 is a microtubule regulator that acts as a pantropic viral inhibitor.

Through a screen that combines functional and evolutionary analyses, we identified tripartite motif protein (Trim69), a poorly studied member of the Trim family, as a negative regulator of HIV-1 infection in interferon (IFN)-stimulated myeloid cells. Trim69 inhibits the early phases of infection of HIV-1, but also of HIV-2 and SIVMAC in addition to the negative and positive-strand RNA viruses vesicular stomatitis virus and severe acute respiratory syndrome coronavirus 2, with magnitudes that depend on the combination between cell type and virus. Mechanistically, Trim69 associates directly to microtubules and its antiviral activity is linked to its ability to promote the accumulation of stable microtubules, a program that we uncover to be an integral part of antiviral IFN-I responses in myeloid cells. Overall, our study identifies Trim69 as the antiviral innate defense factor that regulates the properties of microtubules to limit viral spread and highlights the cytoskeleton as an unappreciated battleground in the host-pathogen interactions that underlie viral infections.

The RING finger protein family in health and disease.

Ubiquitination is a highly conserved and fundamental posttranslational modification (PTM) in all eukaryotes regulating thousands of proteins. The RING (really interesting new gene) finger (RNF) protein, containing the RING domain, exerts E3 ubiquitin ligase that mediates the covalent attachment of ubiquitin (Ub) to target proteins. Multiple reviews have summarized the critical roles of the tripartite-motif (TRIM) protein family, a subgroup of RNF proteins, in various diseases, including cancer, inflammatory, infectious, and neuropsychiatric disorders. Except for TRIMs, since numerous studies over the past decades have delineated that other RNF proteins also exert widespread involvement in several diseases, their importance should not be underestimated. This review summarizes the potential contribution of dysregulated RNF proteins, except for TRIMs, to the pathogenesis of some diseases, including cancer, autoimmune diseases, and neurodegenerative disorder. Since viral infection is broadly involved in the induction and development of those diseases, this manuscript also highlights the regulatory roles of RNF proteins, excluding TRIMs, in the antiviral immune responses. In addition, we further discuss the potential intervention strategies targeting other RNF proteins for the prevention and therapeutics of those human diseases.

Suppression of ACE2 SUMOylation protects against SARS-CoV-2 infection through TOLLIP-mediated selective autophagy.

In addition to investigating the virology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), discovering the host-virus dependencies are essential to identify and design effective antiviral therapy strategy. Here, we report that the SARS-CoV-2 entry receptor, ACE2, conjugates with small ubiquitin-like modifier 3 (SUMO3) and provide evidence indicating that prevention of ACE2 SUMOylation can block SARS-CoV-2 infection. E3 SUMO ligase PIAS4 prompts the SUMOylation and stabilization of ACE2, whereas deSUMOylation enzyme SENP3 reverses this process. Conjugation of SUMO3 with ACE2 at lysine (K) 187 hampers the K48-linked ubiquitination of ACE2, thus suppressing its subsequent cargo receptor TOLLIP-dependent autophagic degradation. TOLLIP deficiency results in the stabilization of ACE2 and elevated SARS-CoV-2 infection. In conclusion, our findings suggest selective autophagic degradation of ACE2 orchestrated by SUMOylation and ubiquitination as a potential way to combat SARS-CoV-2 infection.

UBR5 Acts as an Antiviral Host Factor against MERS-CoV via Promoting Ubiquitination and Degradation of ORF4b.

Within the past 2 decades, three highly pathogenic human coronaviruses have emerged, namely, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The health threats and economic burden posed by these tremendously severe coronaviruses have paved the way for research on their etiology, pathogenesis, and treatment. Compared to SARS-CoV and SARS-CoV-2, MERS-CoV genome encoded fewer accessory proteins, among which the ORF4b protein had anti-immunity ability in both the cytoplasm and nucleus. Our work for the first time revealed that ORF4b protein was unstable in the host cells and could be degraded by the ubiquitin proteasome system. After extensive screenings, it was found that UBR5 (ubiquitin protein ligase E3 component N-recognin 5), a member of the HECT E3 ubiquitin ligases, specifically regulated the ubiquitination and degradation of ORF4b. Similar to ORF4b, UBR5 can also translocate into the nucleus through its nuclear localization signal, enabling it to regulate ORF4b stability in both the cytoplasm and nucleus. Through further experiments, lysine 36 was identified as the ubiquitination site on the ORF4b protein, and this residue was highly conserved in various MERS-CoV strains isolated from different regions. When UBR5 was knocked down, the ability of ORF4b to suppress innate immunity was enhanced and MERS-CoV replication was stronger. As an anti-MERS-CoV host protein, UBR5 targets and degrades ORF4b protein through the ubiquitin proteasome system, thereby attenuating the anti-immunity ability of ORF4b and ultimately inhibiting MERS-CoV immune escape, which is a novel antagonistic mechanism of the host against MERS-CoV infection. IMPORTANCE ORF4b was an accessory protein unique to MERS-CoV and was not present in SARS-CoV and SARS-CoV-2 which can also cause severe respiratory disease. Moreover, ORF4b inhibited the production of antiviral cytokines in both the cytoplasm and the nucleus, which was likely to be associated with the high lethality of MERS-CoV. However, whether the host proteins regulate the function of ORF4b is unknown. Our study first determined that UBR5, a host E3 ligase, was a potential host anti-MERS-CoV protein that could reduce the protein level of ORF4b and diminish its anti-immunity ability by inducing ubiquitination and degradation. Based on the discovery of ORF4b-UBR5, a critical molecular target, further increasing the degradation of ORF4b caused by UBR5 could provide a new strategy for the clinical development of drugs for MERS-CoV.

A C-terminal glutamine recognition mechanism revealed by E3 ligase TRIM7 structures.

The E3 ligase TRIM7 has emerged as a critical player in viral infection and pathogenesis. However, the mechanism governing the TRIM7-substrate association remains to be defined. Here we report the crystal structures of TRIM7 in complex with 2C peptides of human enterovirus. Structure-guided studies reveal the C-terminal glutamine residue of 2C as the primary determinant for TRIM7 binding. Leveraged by this finding, we identify norovirus and SARS-CoV-2 proteins, and physiological proteins, as new TRIM7 substrates. Crystal structures of TRIM7 in complex with multiple peptides derived from SARS-CoV-2 proteins display the same glutamine-end recognition mode. Furthermore, TRIM7 could trigger the ubiquitination and degradation of these substrates, possibly representing a new Gln/C-degron pathway. Together, these findings unveil a common recognition mode by TRIM7, providing the foundation for further mechanistic characterization of antiviral and cellular functions of TRIM7.

PRMT5 epigenetically regulates the E3 ubiquitin ligase ITCH to influence lipid accumulation during mycobacterial infection.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), triggers enhanced accumulation of lipids to generate foamy macrophages (FMs). This process has been often attributed to the surge in the expression of lipid influx genes with a concomitant decrease in those involved in lipid efflux. Here, we define an Mtb-orchestrated modulation of the ubiquitination of lipid accumulation markers to enhance lipid accretion during infection. We find that Mtb infection represses the expression of the E3 ubiquitin ligase, ITCH, resulting in the sustenance of key lipid accrual molecules viz. ADRP and CD36, that are otherwise targeted by ITCH for proteasomal degradation. In line, overexpressing ITCH in Mtb-infected cells was found to suppress Mtb-induced lipid accumulation. Molecular analyses including loss-of-function and ChIP assays demonstrated a role for the concerted action of the transcription factor YY1 and the arginine methyl transferase PRMT5 in restricting the expression of Itch gene by conferring repressive symmetrical H4R3me2 marks on its promoter. Consequently, siRNA-mediated depletion of YY1 or PRMT5 rescued ITCH expression, thereby compromising the levels of Mtb-induced ADRP and CD36 and limiting FM formation during infection. Accumulation of lipids within the host has been implicated as a pro-mycobacterial process that aids in pathogen persistence and dormancy. In line, we found that perturbation of PRMT5 enzyme activity resulted in compromised lipid levels and reduced mycobacterial survival in mouse peritoneal macrophages (ex vivo) and in a therapeutic mouse model of TB infection (in vivo). These findings provide new insights into the role of PRMT5 and YY1 in augmenting mycobacterial pathogenesis. Thus, we posit that our observations could help design novel adjunct therapies and combinatorial drug regimen for effective anti-TB strategies.

The Multifaceted Role of the Ubiquitin Proteasome System in Pathogenesis and Diseases.

Ubiquitin is a small protein that is conjugated to target proteins to signal a great number of critical biological processes. Impaired ubiquitin signaling and defects in the ubiquitin proteasome system (UPS) surveillance are implicated in many human diseases, including cancer. Characterization of the physiological roles of UPS components and their regulatory mechanisms is therefore vital for the identification of therapeutic targets and the development of tools and paradigms to better understand and treat human diseases. In this Special Issue, we assembled seven original research and review articles to provide insights on the multifaceted role of the UPS in pathogenesis and disease, covering the areas of molecular and cellular mechanisms of UPS enzymes, biochemical and biophysical characterization strategies, drug development, and targeted protein degradation.

A close shave: How SARS-CoV-2 induces the loss of cilia.

Wang et al. report in this issue (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202108015) that the SARS-CoV-2 protein ORF10 increases the activity of the E3 ligase CUL2ZYG11B, leading to the degradation of multiple ciliary proteins. The resulting loss of cilia may facilitate the spread of SARS-CoV-2 in the respiratory tree.

Structural insights into ORF10 recognition by ZYG11B.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a major threat to human health. As a unique putative protein of SARS-CoV-2, the N-terminus of ORF10 can be recognized by ZYG11B, a substrate receptor of the Cullin 2-RING E3 ubiquitin ligase (CRL2). Here we elucidated recognition mechanism of ORF10 N-terminus by ZYG11B through presenting the crystal structure of ZYG11B bound to ORF10 N-terminal peptide. Our work expands the current understanding of ORF10 interaction with ZYG11B, and may also inspire the development of novel therapies for COVID-19.

The Role of E3 Ligase Pirh2 in Disease.

The p53-dependent ubiquitin ligase Pirh2 regulates a number of proteins involved in different cancer-associated processes. Targeting the p53 family proteins, Chk2, p27Kip1, Twist1 and others, Pirh2 participates in such cellular processes as proliferation, cell cycle regulation, apoptosis and cellular migration. Thus, it is not surprising that Pirh2 takes part in the initiation and progression of different diseases and pathologies including but not limited to cancer. In this review, we aimed to summarize the available data on Pirh2 regulation, its protein targets and its role in various diseases and pathological processes, thus making the Pirh2 protein a promising therapeutic target.

Adenovirus entry: Stability, uncoating, and nuclear import.

Adenoviruses (AdVs) are widespread in vertebrates. They infect the respiratory and gastrointestinal tracts, the eyes, heart, liver, and kidney, and are lethal to immunosuppressed people. Mastadenoviruses infecting mammals comprise several hundred different types, and many specifically infect humans. Human adenoviruses are the most widely used vectors in clinical applications, including cancer treatment and COVID-19 vaccination. AdV vectors are physically and genetically stable and generally safe in humans. The particles have an icosahedral coat and a nucleoprotein core with a DNA genome. We describe the concept of AdV cell entry and highlight recent advances in cytoplasmic transport, uncoating, and nuclear import of the viral DNA. We highlight a recently discovered "linchpin" function of the virion protein V ensuring cytoplasmic particle stability, which is relaxed at the nuclear pore complex by cues from the E3 ubiquitin ligase Mind bomb 1 (MIB1) and the proteasome triggering disruption. Capsid disruption by kinesin motor proteins and microtubules exposes the linchpin and renders protein V a target for MIB1 ubiquitination, which dissociates V from viral DNA and enhances DNA nuclear import. These advances uncover mechanisms controlling capsid stability and premature uncoating and provide insight into nuclear transport of nucleic acids.

Host E3 ligase HUWE1 attenuates the proapoptotic activity of the MERS-CoV accessory protein ORF3 by promoting its ubiquitin-dependent degradation.

With the outbreak of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), coronaviruses have begun to attract great attention across the world. Of the known human coronaviruses, however, Middle East respiratory syndrome coronavirus (MERS-CoV) is the most lethal. Coronavirus proteins can be divided into three groups: nonstructural proteins, structural proteins, and accessory proteins. While the number of each of these proteins varies greatly among different coronaviruses, accessory proteins are most closely related to the pathogenicity of the virus. We found for the first time that the ORF3 accessory protein of MERS-CoV, which closely resembles the ORF3a proteins of severe acute respiratory syndrome coronavirus and SARS-CoV-2, has the ability to induce apoptosis in cells in a dose-dependent manner. Through bioinformatics analysis and validation, we revealed that ORF3 is an unstable protein and has a shorter half-life in cells compared to that of severe acute respiratory syndrome coronavirus and SARS-CoV-2 ORF3a proteins. After screening, we identified a host E3 ligase, HUWE1, that specifically induces MERS-CoV ORF3 protein ubiquitination and degradation through the ubiquitin-proteasome system. This results in the diminished ability of ORF3 to induce apoptosis, which might partially explain the lower spread of MERS-CoV compared to other coronaviruses. In summary, this study reveals a pathological function of MERS-CoV ORF3 protein and identifies a potential host antiviral protein, HUWE1, with an ability to antagonize MERS-CoV pathogenesis by inducing ORF3 degradation, thus enriching our knowledge of the pathogenesis of MERS-CoV and suggesting new targets and strategies for clinical development of drugs for MERS-CoV treatment.

K63 ubiquitination in immune signaling.

Ubc13-catalyzed K63 ubiquitination is a major control point for immune signaling. Recent evidence has shown that the control of multiple immune functions, including chronic inflammation, pathogen responses, lymphocyte activation, and regulatory signaling, is altered by K63 ubiquitination. In this review, we detail the novel cellular sensors that are dependent on K63 ubiquitination for their function in the immune signaling network. Many pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can target K63 ubiquitination to inhibit pathogen immune responses; we describe novel details of the pathways involved and summarize recent clinically relevant SARS-CoV-2-specific responses. We also discuss recent evidence that regulatory T cell (Treg) versus T helper (TH) 1 and TH17 cell subset regulation might involve K63 ubiquitination. Knowledge gaps that merit future investigation and clinically relevant pathways are also addressed.

Epigenetic and transcriptional control of interferon-ß.

The three classes of interferons (IFNs) share the ability to inhibit viral replication, activating cell transcriptional programs that regulate both innate and adaptive responses to viral and intracellular bacterial challenge. Due to their unique potency in regulating viral replication, and their association with numerous autoimmune diseases, the tightly orchestrated transcriptional regulation of IFNs has long been a subject of intense investigation. The protective role of early robust IFN responses in the context of infection with SARS-CoV-2 has further underscored the relevance of these pathways. In this viewpoint, rather than focusing on the downstream effects of IFN signaling (which have been extensively reviewed elsewhere), we will summarize the historical and current understanding of the stepwise assembly and function of factors that regulate IFNß enhancer activity (the "enhanceosome") and highlight opportunities for deeper understanding of the transcriptional control of the ifnb gene.