The E3 ligase subunit FBXO45 binds the interferon-λ receptor and promotes its degradation during influenza virus infection
2022; Elsevier BV; Volume: 298; Issue: 12 Linguagem: Inglês
10.1016/j.jbc.2022.102698
ISSN1083-351X
AutoresMuChun Tsai, Wissam Osman, J. Adair, Rabab ElMergawy, Lexie Chafin, Finny Johns, Daniela Farkas, Ajit Elhance, J.D. Londino, Rama K. Mallampalli,
Tópico(s)interferon and immune responses
ResumoInfluenza remains a major public health challenge, as the viral infection activates multiple biological networks linked to altered host innate immunity. Following infection, IFN-λ, a ligand crucial for the resolution of viral infections, is known to bind to its cognate receptor, IFNLR1, in lung epithelia. However, little is known regarding the molecular expression and regulation of IFNLR1. Here, we show that IFNLR1 is a labile protein in human airway epithelia that is rapidly degraded after influenza infection. Using an unbiased proximal ligation biotin screen, we first identified that the Skp-Cullin-F box E3 ligase subunit, FBXO45, binds to IFNLR1. We demonstrate that FBXO45, induced in response to influenza infection, mediates IFNLR1 protein polyubiquitination and degradation through the ubiquitin-proteasome system by docking with its intracellular receptor domain. Furthermore, we found ectopically expressed FBXO45 and its silencing in cells differentially regulated both IFNLR1 protein stability and interferon-stimulated gene expression. Mutagenesis studies also indicated that expression of a K319R/K320R IFNLR1 variant in cells exhibited reduced polyubiquitination, yet greater stability and proteolytic resistance to FBXO45 and influenza-mediated receptor degradation. These results indicate that the IFN-λ–IFNLR1 receptor axis is tightly regulated by the Skp-Cullin-F box ubiquitin machinery, a pathway that may be exploited by influenza infection as a means to limit antiviral responses. Influenza remains a major public health challenge, as the viral infection activates multiple biological networks linked to altered host innate immunity. Following infection, IFN-λ, a ligand crucial for the resolution of viral infections, is known to bind to its cognate receptor, IFNLR1, in lung epithelia. However, little is known regarding the molecular expression and regulation of IFNLR1. Here, we show that IFNLR1 is a labile protein in human airway epithelia that is rapidly degraded after influenza infection. Using an unbiased proximal ligation biotin screen, we first identified that the Skp-Cullin-F box E3 ligase subunit, FBXO45, binds to IFNLR1. We demonstrate that FBXO45, induced in response to influenza infection, mediates IFNLR1 protein polyubiquitination and degradation through the ubiquitin-proteasome system by docking with its intracellular receptor domain. Furthermore, we found ectopically expressed FBXO45 and its silencing in cells differentially regulated both IFNLR1 protein stability and interferon-stimulated gene expression. Mutagenesis studies also indicated that expression of a K319R/K320R IFNLR1 variant in cells exhibited reduced polyubiquitination, yet greater stability and proteolytic resistance to FBXO45 and influenza-mediated receptor degradation. These results indicate that the IFN-λ–IFNLR1 receptor axis is tightly regulated by the Skp-Cullin-F box ubiquitin machinery, a pathway that may be exploited by influenza infection as a means to limit antiviral responses. The RNA virus influenza robustly activates the immune system through molecular sensing of the viral RNA by host cells via pattern recognition receptors (Toll-like receptor, retinoic acid-inducible gene (RIG-I)), sequential activation of the mitochondrial antiviral-signaling protein signaling cascade, transcriptional activation of the DNA-binding component interferon (IFN) regulatory factors and NF-κB, and then type I (e.g., IFN-α) and type III interferon (IFN-λ) production (1Weis S. Te Velthuis A.J.W. Influenza virus RNA synthesis and the innate immune response.Viruses. 2021; 13: 780Crossref PubMed Scopus (15) Google Scholar). IFN-λ is critical in the resolution of viral, bacterial, and fungal infections and acts through engagement of its cognate receptor complex, comprised of interferon lambda receptor 1 (IFNLR1) and interleukin (IL)-10R2 (2Ye L. Schnepf D. Staeheli P. Interferon-lambda orchestrates innate and adaptive mucosal immune responses.Nat. Rev. Immunol. 2019; 19: 614-625Crossref PubMed Scopus (145) Google Scholar, 3Kotenko S.V. Rivera A. Parker D. Durbin J.E. Type III IFNs: beyond antiviral protection.Semin. Immunol. 2019; 43101303Crossref PubMed Scopus (52) Google Scholar). Four IFN-λ ligands (type III IFN) have been described with IFNλ1–3 sharing high amino acid sequence homologies, whereas IFNλ4 is more divergent with only 40.8% amino acid similarity to IFNλ3 (4Lazear H.M. Nice T.J. Diamond M.S. Interferon-lambda: immune functions at barrier surfaces and beyond.Immunity. 2015; 43: 15-28Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar, 5Donnelly R.P. Kotenko S.V. Interferon-lambda: a new addition to an old family.J. Inter. Cytokine Res. 2010; 30: 555-564Crossref PubMed Scopus (337) Google Scholar). Only IFNλ2 and 3 are conserved in mice. IFNLR1 (also known as IL-28R) is a single pass 520 AA type I membrane receptor. Upon ligand activation, STAT1 and STAT2 are recruited by IFNLR1 and IL-10rb, respectively, which are then phosphorylated and transported to the nucleus to mediate transcriptional activation of interferon-stimulated genes (ISGs) (4Lazear H.M. Nice T.J. Diamond M.S. Interferon-lambda: immune functions at barrier surfaces and beyond.Immunity. 2015; 43: 15-28Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). IFNLR1 expression is largely restricted to mucosal surfaces such as the lung and gut epithelial layer in mice with a notable exception being high IFNLR1 expression in neutrophils (4Lazear H.M. Nice T.J. Diamond M.S. Interferon-lambda: immune functions at barrier surfaces and beyond.Immunity. 2015; 43: 15-28Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar). Several studies have shown that mice treated with IFN-λ after influenza infection exhibited significantly lower mortality, decreased viral burden, with reduced inflammatory cytokines compared to control underscoring the indispensability of the IFN-λ–IFNLR1 axis (6Galani I.E. Triantafyllia V. Eleminiadou E.E. Koltsida O. Stavropoulos A. Manioudaki M. et al.Interferon-lambda mediates non-redundant front-line antiviral protection against influenza virus infection without compromising host fitness.Immunity. 2017; 46: 875-890.e876Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 7Klinkhammer J. Schnepf D. Ye L. Schwaderlapp M. Gad H.H. Hartmann R. et al.IFN-lambda prevents influenza virus spread from the upper airways to the lungs and limits virus transmission.Elife. 2018; 7e33354Crossref PubMed Scopus (157) Google Scholar). Further, the requirement of the IFNλ-IFNLR1 pathway for antiviral protection is underscored given that targeted disruption of IFNLR1 results in widespread viral dissemination and lethality (7Klinkhammer J. Schnepf D. Ye L. Schwaderlapp M. Gad H.H. Hartmann R. et al.IFN-lambda prevents influenza virus spread from the upper airways to the lungs and limits virus transmission.Elife. 2018; 7e33354Crossref PubMed Scopus (157) Google Scholar). In human tissues, IFNLR1 has a more diverse expression profile, but notably, the expression, signaling, and molecular regulation of IFNLR1 in the human lung has not been fully elucidated. In humans, IFN-λ is the earliest and most profuse interferon produced during influenza infection, triggering its engagement with IFNLR1 in providing host innate immunity (6Galani I.E. Triantafyllia V. Eleminiadou E.E. Koltsida O. Stavropoulos A. Manioudaki M. et al.Interferon-lambda mediates non-redundant front-line antiviral protection against influenza virus infection without compromising host fitness.Immunity. 2017; 46: 875-890.e876Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 8Cao Y. Huang Y. Xu K. Liu Y. Li X. Xu Y. et al.Differential responses of innate immunity triggered by different subtypes of influenza a viruses in human and avian hosts.BMC Med. Genomics. 2017; 10: 70Crossref PubMed Scopus (25) Google Scholar). Like rodents, IFN-λ may partake in antiviral defense at barrier surfaces, such as within the human lung and gastrointestinal epithelial lining. Remarkably, although both IFN-λ and IFN-α confer antiviral protection, IFN-α leads to the upregulation of proinflammatory genes (6Galani I.E. Triantafyllia V. Eleminiadou E.E. Koltsida O. Stavropoulos A. Manioudaki M. et al.Interferon-lambda mediates non-redundant front-line antiviral protection against influenza virus infection without compromising host fitness.Immunity. 2017; 46: 875-890.e876Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar). Multiple immune cells, including bone marrow–derived macrophages, dendritic cells, and neutrophils, stimulated with IFN-α, but not IFN-λ, secrete proinflammatory cytokines (6Galani I.E. Triantafyllia V. Eleminiadou E.E. Koltsida O. Stavropoulos A. Manioudaki M. et al.Interferon-lambda mediates non-redundant front-line antiviral protection against influenza virus infection without compromising host fitness.Immunity. 2017; 46: 875-890.e876Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 9Grandvaux N. tenOever B.R. Servant M.J. Hiscott J. The interferon antiviral response: from viral invasion to evasion.Curr. Opin. Infect. Dis. 2002; 15: 259-267Crossref PubMed Scopus (178) Google Scholar). IFN-λ interestingly has been observed to inhibit several inflammatory mechanisms including ROS production, granule mobilization, and the release of neutrophil extracellular traps (10Chrysanthopoulou A. Kambas K. Stakos D. Mitroulis I. Mitsios A. Vidali V. et al.Interferon lambda1/IL-29 and inorganic polyphosphate are novel regulators of neutrophil-driven thromboinflammation.J. Pathol. 2017; 243: 111-122Crossref PubMed Scopus (69) Google Scholar, 11Staitieh B.S. Egea E.E. Fan X. Azih N. Neveu W. Guidot D.M. Activation of alveolar macrophages with interferon-gamma promotes antioxidant defenses via the nrf2-ARE pathway.J. Clin. Cell Immunol. 2015; 6: 365PubMed Google Scholar). The IFNLR1 gene is robustly induced after influenza infection in humans, predicting interferon signaling (12Davenport E.E. Antrobus R.D. Lillie P.J. Gilbert S. Knight J.C. Transcriptomic profiling facilitates classification of response to influenza challenge.J. Mol. Med. (Berl). 2015; 93: 105-114Crossref PubMed Scopus (30) Google Scholar). Viral pathogenesis has been shown to exploit host cellular pathways for numerous steps of the infection cycle including molecular hijacking of the host protein degradation apparatus (13Rudnicka A. Yamauchi Y. Ubiquitin in influenza virus entry and innate immunity.Viruses. 2016; 8: 293Crossref PubMed Scopus (53) Google Scholar). The ubiquitin-proteasome system (UPS), executed via actions of E1-activating, E2-conjugating enzymes, and E3-ubiquitin ligases, are central to intracellular protein degradation. The UPS is fundamental to control of numerous cell functions involving protein turnover, cellular sorting and cell cycle progression, stress responses, transcriptional control, and surface receptor turnover (14Hochstrasser M. Biochemistry. All in the ubiquitin family.Science. 2000; 289: 563-564Crossref PubMed Scopus (102) Google Scholar, 15Hershko A. Ciechanover A. The ubiquitin system.Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (7027) Google Scholar). Of the many E3 ligases, the understanding of Skp-Cullin1-F- box (SCF) superfamily is growing, comprised of a catalytic core consisting of Skp1, Cullin1, and Rbx1 (16Cardozo T. Pagano M. The SCF ubiquitin ligase: insights into a molecular machine.Nat. Rev. Mol. Cell Biol. 2004; 5: 739-751Crossref PubMed Scopus (895) Google Scholar, 17Deshaies R.J. Joazeiro C.A. RING domain E3 ubiquitin ligases.Annu. Rev. Biochem. 2009; 78: 399-434Crossref PubMed Scopus (1944) Google Scholar). The SCF complex also contains an adaptor receptor subunit, termed F-box protein, that engages numerous substrates to the E3 catalytic core (18Skowyra D. Craig K.L. Tyers M. Elledge S.J. Harper J.W. F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex.Cell. 1997; 91: 209-219Abstract Full Text Full Text PDF PubMed Scopus (1036) Google Scholar, 19Tyers M. Willems A.R. One ring to rule a superfamily of E3 ubiquitin ligases.Science. 1999; 284: 603-604Crossref Scopus (142) Google Scholar). F-box proteins are categorized within three families (FBXW, FBXO, FBXL) according to their substrate-binding motifs with the FBXO family containing a variety of yet unknown substrate-binding motifs (20Skaar J.R. Pagan J.K. Pagano M. Mechanisms and function of substrate recruitment by F-box proteins.Nat. Rev. Mol. Cell Biol. 2013; 14: 369-381Crossref PubMed Scopus (471) Google Scholar). In particular, F-box proteins have been shown to partake in host defense in the pathogenesis of influenza infection, as FBXW7 antagonizes viral replication perhaps via RIG-I stabilization (21Yan H.Y. Wang H.Q. Zhong M. Wu S. Yang L. Li K. et al.PML suppresses influenza virus replication by promoting FBXW7 expression.Virol. Sin. 2021; 36: 1154-1164Crossref PubMed Scopus (7) Google Scholar, 22Song Y. Lai L. Chong Z. He J. Zhang Y. Xue Y. et al.E3 ligase FBXW7 is critical for RIG-I stabilization during antiviral responses.Nat. Commun. 2017; 814654Crossref Scopus (48) Google Scholar). The FBXO family F-box protein, FBXO45, has been previously recognized to play a role in neoplasia, nervous system and psychiatric disorders, and inflammatory disorders (23Lin M. Wang Z.W. Zhu X. FBXO45 is a potential therapeutic target for cancer therapy.Cell Death Discov. 2020; 6: 55Crossref PubMed Scopus (19) Google Scholar). However, a biological role of this F-box protein remains largely unknown especially with respect to viral pathogenesis. In this study, we have observed that influenza infection triggers IFNLR1 degradation mediated by FBXO45 that targets IFNLR1 for ubiquitination and degradation in epithelia to impair IFN-λ signaling. The results suggest a potential mechanism whereby the virus subverts host immune responses essential for tissue repair. We examined the short-term (<12 h) effect of influenza infection on IFNLR1 protein levels in THP-1 macrophages and BEAS-2B lung epithelial cells. Influenza infection significantly decreased IFNLR1 protein at 8 h post infection in both cell types (Fig. 1, A and B) without decreased receptor mRNA (Fig. 1C). We next examined the role of IFNLR1 signaling in BEAS-2B cells in transient knockdown studies using various amounts of siRNA to induce a modest reduction in IFNLR1 mRNA and protein. Here, despite modest knockdown of IFNLR1, we observed that levels of several antiviral genes were reduced in a trend toward a dose-dependent manner after PR8 infection (Fig. 1, D–F). Thus, the results demonstrate that epithelial cells are sensitive to availability of the IFNLR1 receptor that impact vital antiviral responses. To understand the upstream effect of influenza infection on IFNLR1 levels, we infected HBEC3KT cells, which are human telomerase reverse transcriptase-immortalized human bronchial lung epithelial cells. With increasing MOI of PR8 influenza infection of these cells over 6 h, we observed a significant decrease in IFNLR1 with PR8 infection via flow cytometry. (Fig. 1G). Next, we applied recombinant IFN-λ (IL-29) to HBEC3KT cells for 6 h at different concentrations. IFNLR1 signals via flow cytometry decreased with inclusion of IFN-λ (Fig. 1H) in the culture medium, suggesting that downregulation of IFNLR1 occurs due to autocrine signaling. To understand the degradation process further, we next ectopically expressed V5- and histine (HIS)-tagged IFNLR1 and HA-tagged–ubiquitin in HEK-293T cells. This was followed by treatment with the proteasomal inhibitor MG-132 or autophagosome/lysosomal inhibitor Bafilomycin A1 (BafA1) to preserve the ubiquitinated proteins. MLN7243, an inhibitor of ubiquitin activating enzyme, was added as a control to confirm that the detected bands were due to ubiquitination. Cell lysates were harvested, and samples were processed for HIS-tagged pull-down assays to compare the differences in polyubiquitination. BafA1 did not significantly increase ubiquitination above basal levels, whereas MG-132 led to a robust increase in IFNLR1 polyubiquitination (Fig. 2A). Next, in cells treated with the protein synthesis inhibitor, cycloheximide (CHX), IFNLR1 protein was largely degraded by 8 h, an effect significantly blocked by inclusion of MG-132 and MLN7243 in the culture medium but not BafA1 (Fig. 2, B and C). To map the region of IFNLR1 protein necessary for degradation, we constructed plasmids expressing IFNLR1 with truncated C-terminal domains and after transfection into BEAS-2B cells, we measured stabilization of each variant by MG-132 (Fig. 2D). When truncations expressed in cells do not accumulate in the presence of MG-132, this suggests the loss of regions necessary for degradation. Although these constructs expressed to variable degrees in cells, our data showed that both the WT full-length protein and a construct harboring the first 460 AA of the IFNLR1 protein showed accumulation after MG-132 unlike other truncations (Fig. 2D). These data suggest the presence of molecular signatures spanning residues AA 360-460 that may be required for receptor degradation. To examine this region more closely, we constructed a series of 30 AA deletions between the AA 370-460 domain. When AA 430-460 was excised and the plasmid expressed in cells, the resulting protein was both less responsive to MG-132 and more stable in the presence of CHX (Fig. 2E). Using protein motif prediction software (http://emboss.bioinformatics.nl/cgi-bin/emboss/epestfind), we identified that this region corresponds to a PEST domain (AA 432–486), a canonical destabilizing element (24Rechsteiner M. Rogers S.W. PEST sequences and regulation by proteolysis.Trends Biochem. Sci. 1996; 21: 267-271Abstract Full Text PDF PubMed Scopus (1419) Google Scholar). Together, the data suggest that IFNLR1 is (i) ubiquitinated and proteasomally degraded and (ii) that there might reside a specific destabilizing motif necessary for degradation within the cytoplasmic domain. We used BioID2 and miniTurbo proximity ligation assays as unbiased screens to determine molecular binding partners that might mediate IFNLR1 degradation. The BioID2 biotin ligase was conjugated to the carboxyl-terminus of IFNLR1. As specificity controls, we overexpressed the IFN-γ receptor IFNGR1-BioID2 and the transmembrane receptor SIRPA-BioID2. HEK-293T cells were transiently transfected with these constructs for 48 h followed by overnight biotin treatment and isolation of biotinylated proteins. We identified 169 unique IFNLR1 proteins, 7 of which were E3 ligases, including FBXO45 (Fig. 3A). In separate experiments, we also conjugated the biotin ligase miniTurbo to the carboxyl-terminus of IFNLR1, transiently transfected into cells, and treated cells with biotin for 1 h prior to harvest. Compared to miniTurbo alone, we identified 100 unique interactions with the IFNLR1-miniTurbo biotin ligase. Six of these proteins were E3 ligases. FBXO45 was again identified as a unique IFNLR1-interacting partner (Fig. S1). Due to our identification of an IFNLR1–FBXO45 interaction in multiple experiments using different biotin ligase constructs, we further examined FBXO45-mediated changes in IFNLR1 signaling. We examined several ubiquitin-related proteins uniquely associated with IFNLR1, including FBXO45, TRIM25, and USP7, as well as the diphosphate DUSP9 as a potential regulator of IFNLR1 function. Cellular depletion of all four genes using two different DsiRNA was executed in BEAS-2B cells that were transfected with these corresponding DsiRNA (Fig. 3B) and treated with IFN-λ. Here, we observed an increase in IFNLR1-mediated STAT1 phosphorylation and downstream IFIT3 gene induction with both DsiRNA's targeting FBXO45 (Fig. 3C). Thus, we focused on FBXO45. To also map the location of FBXO45 and IFNLR1 protein interaction, we utilized a series of truncated IFNLR1 C-terminal domain proteins and performed coimmunoprecipitation experiments using the truncation mutants of V5-tagged IFNLR1 and FLAG-tagged FBXO45 to locate the receptor region that binds FBXO45. We found that the IFNLR1 constructs lacking the region between AA360 and 410 exhibited substantially reduced ability to bind to FBXO45, suggesting that there might exist a motif in this region necessary for FBXO45 docking (Fig. 3, D and E). Additional studies were conducted to assess viral responses of FBXO45 in cells and effects of the F-box protein on interferon signaling. In BEAS-2B cells, increasing MOI's of PR8 influenza increased FBXO45 levels coupled with a reduction in levels of IFNLR1 (Fig. 4A). Interestingly, FBXO45 silencing and subsequent PR8 infection reduced matrix 1/matrix 2 (M1/M2) proteins (Fig. 4B). Further, FBXO45 overexpression in BEAS-2B cells selectively decreased IFN-λ signaling (Fig. 4C). When FBXO45 was depleted in cells and subsequently treated with IFN-λ, IFN-α, or IFN-γ, only IFN-λ induced IFIT3 after FBXO45 knockdown (Fig. 4D). Infection of BEAS-2B cells with various MOI of PR8 and FBXO45 silencing also led to a substantial increase in ISG expression, an effect not observed after IFNLR1 cellular depletion (Fig. 4E). Specifically, the proteins STAT1 and IFIT3 were notably increased in a dose-dependent manner compared to the control with FBXO45 silenced. Last, the antiviral genes ISG15 and IFIT3 mRNAs were also increased with knockdown of FBXO45 but reduced with IFNLR1 silencing. Together, these data suggest that FBXO45 plays a key role in attenuating antiviral host defense to facilitate influenza virulence. Given the molecular interaction of FBXO45 with IFNLR1 and its ability to antagonize interferon signaling, we next assessed the F-box protein on its ability to modulate receptor protein degradation. We transfected BEAS-2B cells with FLAG-tagged FBXO45 plasmid or empty vector at 0.5 or 1.0 μg for 48 h and observed a reduction of endogenous IFNLR1 by ∼35% versus empty vector (Fig. 5A). We also transfected BEAS-2B cells with V5-tagged IFNLR1 plasmid and either V5-tagged FBXO45 or an empty vector at 0.5, 1, or 2 μg for 24 h and observed a dose-dependent reduction in steady-state IFNLR1 mass (Fig. 5B). When BEAS-2B cells were transfected as above with an empty vector or with a FBXO45-mutant plasmid (lacking E3 ligase catalytic activity) followed by a CHX chase, we found that WT FBXO45 overexpression accelerated IFNLR1 protein decay and tended to reduce its half-life (Fig. 5C), whereas there was no change in half-life with expression of a FBXO45-mutant versus an empty construct (Fig. S2). Conversely, when we silenced FBXO45 in BEAS-2B cells, we found increased endogenous IFNLR1 lifespan (Fig. 5D). Similar findings were observed when we transfected BEAS-2B cells with V5-tagged IFNLR1 and two separate DsiRNA's targeting FBXO45 (Figs. 5E and S3). These results strongly implicate FBXO45 as an E3 ligase component that specifically regulates IFNLR1 protein turnover. To further assess the molecular signatures involved in IFNLR1 protein destabilization, we constructed IFNLR1 point mutants at candidate lysines within the cytoplasmic region: K319/320 (double site) and K410. After cellular expression, we analyzed IFNLR1-WT, IFNLR1-K319R/K320R, and IFNLR1-K410R t1/2 using CHX over 6 h (Fig. 6A). Notably, IFNLR1-K319R/K320R remained stable with a longer t1/2, whereas IFNLR1-WT and IFNLR1-K410R degraded rapidly over time. We then evaluated levels of polyubiquitination of these mutants by cotransfecting V5-tagged constructs and HA-tagged ubiquitin in HEK-293T cells followed by treatment with MG-132. Cell lysates were harvested with DUB inhibitors, 1,10-phenanthroline, PR-619, and N-ethylmaleimide (NEM). Polyubiquitination was evaluated by immunoprecipitation, which revealed greater signal intensity in IFNLR1-WT and IFNLR1-K410R in comparison to the IFNLR1-K319R/K320R variant (Fig. 6B). To inspect whether the IFNLR1 double mutant could resist degradation with the presence of FBXO45, HEK-293T cells were cotransfected with either V5-tagged IFNLR1-WT or IFNLR1-K319R/K320R and FLAG-tagged FBXO45 or GFP-tagged empty vector. Here, the IFNLR1-K319R/K320R was stable despite ectopic expression of FBXO45 (Fig. 6C). Finally, because influenza infection increased IFNLR1 degradation, we evaluated the stability of the IFNLR1-K319R/K320R variant after PR8 infection for 3 h. The data show that IFNLR1-K319R/K320R was more resistant to viral-induced degradation than IFNLR1-WT (Fig. 6D). Thus, the results suggest that degradation of IFNLR1 occurs via polyubiquitination via the SCFFBXO45 E3 ligase, possibly at the K319/K320 molecular site(s) during influenza infection.Figure 6Ectopically expressed IFNLR1 variant displays proteolytic resistance to influenza infection and FBXO45. A, HEK-293T cells transfected with V5-tagged IFNLR1 plasmid, IFNLR1-K319R/K320R, or IFNLR1-K410R for 48 h followed by a CHX (40 μg/ml) chase and cells processed for V5 immunoblotting. Shown on the right graph is decay of the V5-IFNLR1 protein over time after densitometric analysis of immunoblots. ∗p < 0.5 by one-way ANOVA. B, HEK-293T cells were transfected with V5-tagged IFNLR1, IFNLR1-K319R/K320R, or IFNLR1-K410R and HA-tagged ubiquitin. Forty eight hours later, cells were treated with MG-132 (20 μM) for 4 h. Cell lysates were harvested and a V5 pull-down was performed. Whole cell lysate (below) and pull-down (above) were immunoblotted to determine ubiquitinated products, (n = 2). C, HEK-293T cells were cotransfected with V5-tagged IFNLR1 or IFNLR1-K319R/K320R and FLAG-tagged FBXO45 versus GFP-tagged empty vector for 48 h and processed for V5 and FLAG immunoblotting. ∗p < 0.5 by students t test. D, HEK-293T cells were transfected with V5-tagged IFNLR1 or IFNLR1-K319R/K320R. After 48 h, the cells were infected with PR8 (MOI = 0.01) for 3 h and cells harvested for V5 immunoblotting. In (C and D), the graphs on the right show the relative levels of expressed proteins after densitometric quantitation of immunoblots. Results were shown as mean ± SD. Data represent n = 3 experiments unless specified otherwise. CHX, cycloheximide; IFNLR1, interferon lambda receptor 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In this study, we demonstrate for the first time that IFNLR1 is potentially a new substrate for the E3 ligase component FBXO45 that may modulate viral pathogenesis. IFN-λ, acting via IFNLR1, has been shown to be important in the resolution of viral, bacterial, and fungal infections (2Ye L. Schnepf D. Staeheli P. Interferon-lambda orchestrates innate and adaptive mucosal immune responses.Nat. Rev. Immunol. 2019; 19: 614-625Crossref PubMed Scopus (145) Google Scholar, 3Kotenko S.V. Rivera A. Parker D. Durbin J.E. Type III IFNs: beyond antiviral protection.Semin. Immunol. 2019; 43101303Crossref PubMed Scopus (52) Google Scholar). IFNLR1 is predominately found on epithelial cells, which can provide localized antiviral protection along barrier surfaces. Studies have shown that early IFN-λ treatment in mice during influenza virus infection offers significant antiviral protection without the proinflammatory responses associated with Type 1 interferons (6Galani I.E. Triantafyllia V. Eleminiadou E.E. Koltsida O. Stavropoulos A. Manioudaki M. et al.Interferon-lambda mediates non-redundant front-line antiviral protection against influenza virus infection without compromising host fitness.Immunity. 2017; 46: 875-890.e876Abstract Full Text Full Text PDF PubMed Scopus (300) Google Scholar, 25Davidson S. McCabe T.M. Crotta S. Gad H.H. Hessel E.M. Beinke S. et al.IFNlambda is a potent anti-influenza therapeutic without the inflammatory side effects of IFNalpha treatment.EMBO Mol. Med. 2016; 8: 1099-1112Crossref PubMed Scopus (167) Google Scholar). Although we have previously demonstrated the presence of the IFN-λ–IFNLR1 signaling axis in human lung macrophages and its role in combating influenza infection, the expression, signaling, and molecular regulation of IFNLR1 in human bronchial epithelial cells has not been fully elucidated (26Mallampalli R.K. Adair J. Elhance A. Farkas D. Chafin L. Long M.E. et al.Interferon lambda signaling in macrophages is necessary for the antiviral response to influenza.Front. Immunol. 2021; 12735576Crossref PubMed Scopus (7) Google Scholar). The new contributions in this study provide associations between influenza infection of epithelia, FBXO45, and its interaction with IFNLR1 triggering its degradation. The F-box protein is induced after viral infection and in an unbiased screen, FBXO45 binds IFNLR1 that appears to be localized to a region spanning 50 AA within the cytoplasmic domain. This region (AA 360–410) appears downstream of previously identified binding sites for human Janus kinase 1 (JAK1) that also binds the intracellular domain of IFNLR1 (27Zhang D. Wlodawer A. Lubkowski J. Crystal structure of a complex of the intracellular domain of interferon λ receptor 1 (IFNLR1) and the FERM/SH2 domains of human JAK1.J. Mol. Biol. 2016; 428: 4651-4668Crossref PubMed Scopus (26) Google Scholar) and excludes IFN-λ1–induced STAT2 tyrosine phosphorylation sites within the receptor (28Dumoutier L. Tounsi A. Michiels T. Sommereyns C. Kotenko S.V. Renauld J.C. Role of the interleukin (IL)-28 receptor tyrosine residues for antiviral and antiproliferative activity of IL-29/interferon-lambda 1: similarities with type I interferon signaling.J. Biol. Chem. 2004; 279: 32269-32274Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). The identification of phosphorylation sites within the cytoplasmic or soluble region of IFNLR1 may be important given that F-box proteins are recruited to substrates through phosphosite recogn
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