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. Author manuscript; available in PMC: 2022 Jun 1.
Published in final edited form as: Virology. 2021 Mar 13;558:67–75. doi: 10.1016/j.virol.2021.02.006

Influenza A virus NS1 induces degradation of sphingosine 1-phosphate lyase to obstruct the host innate immune response

Jennifer J Wolf 1,2,*, Chuan Xia 1,2,5,*, Caleb J Studstill 1,2, Hanh Ngo 1,2, Steven L Brody 3, Paul E Anderson 4, Bumsuk Hahm 1,2,#
PMCID: PMC8109848  NIHMSID: NIHMS1685473  PMID: 33730651

Abstract

The type I interferon (IFN)-mediated innate immune response is one of the central obstacles influenza A virus (IAV) must overcome in order to successfully replicate within the host. We have previously shown that sphingosine 1-phosphate (S1P) lyase (SPL) enhances IKKɛ-mediated type I IFN responses. Here, we demonstrate that the nonstructural protein 1 (NS1) of IAV counteracts the SPL-mediated antiviral response by inducing degradation of SPL. SPL was ubiquitinated and downregulated upon IAV infection or NS1 expression, whereas NS1-deficient IAV failed to elicit SPL ubiquitination or downregulation. Transiently overexpressed SPL increased phosphorylation of IKKɛ, resulting in enhanced expression of type I IFNs. However, this induction was markedly inhibited by IAV NS1. Collectively, this study reveals a novel strategy employed by IAV to subvert the type I IFN response, providing new insights into the interplay between IAV and host innate immunity.

Keywords: influenza virus, sphingosine-1-phosphate lyase, nonstructural protein 1, type I interferon, viral immune evasion, ubiquitination, protein degradation

Graphical Abstract

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Introduction

Influenza A virus (IAV) is a negative sense segmented RNA virus in family Orthomyxoviridae. IAV and influenza B virus (IBV) are well known for causing seasonal influenza outbreaks annually (Lam et al., 2019), resulting in substantial morbidity and mortality worldwide despite widespread vaccination and preexposure (Iuliano et al., 2018). IAV exhibits remarkable genetic and genomic diversity that allows IAV to be both a pandemic and zoonotic threat (Brooke, 2017). Recurrent outbreaks of avian influenza add to mounting concerns of the next potential influenza pandemic (Cowling et al., 2013; Fraser et al., 2009; Salvador et al., 2020; Wang and Palese, 2009). Vaccines targeting influenza viruses must be reformulated annually (Krammer et al., 2018). While antiviral drugs that inhibit the function of IAV proteins such as neuraminidase (NA) and the viral polymerase are available, multiple IAV strains have been found to be resistant to these contemporary antivirals (Cheng et al., 2010; Dharan et al., 2009; Hussain et al., 2017; Irwin et al., 2016; Jones et al., 2018; Marjuki et al., 2015; Poland et al., 2009). Therefore, identifying host factors that could be targeted to broadly counteract infection by many different IAV subtypes has great potential as a therapeutic strategy.

One of the host’s first lines of defense against IAV infection is the powerful type I interferon (IFN)-mediated innate immune response (Hoffmann et al., 2015; Seo and Hahm, 2010). Briefly, host RIG-I senses IAV RNA products by recognizing the 5’-ppp moiety that is present on IAV RNA (Goubau et al., 2013; Killip et al., 2015; Loo et al., 2008). The activation of RIG-I leads to its interaction with the downstream adaptor protein MAVS and subsequent recruitment and activation of IKKε and TBK1, both of which activate transcription factors IRF3 and IRF7 (Sharma et al., 2003). This leads to the production and secretion of IFN-α and IFN-β molecules (Sato et al., 2000). IFNs then bind to their cognate receptor, triggering the activation of the JAK-STAT signaling pathway and ultimately inducing the transcription of IFN-stimulated genes (ISGs) (Aaronson and Horvath, 2002). ISG products promote a cellular antiviral state by inhibiting virus replication (Schoggins, 2014).

Sphingosine 1-phosphate (S1P) lyase (SPL) is a host enzyme that mediates the catabolism of the bioactive lipid S1P into hexadecanol and phosphoethanolamine (Aguilar and Saba, 2012; Bourquin et al., 2010). Due to its ability to metabolize S1P, SPL has been implicated in a diverse array of mammalian cellular processes and diseases, such as cell proliferation, cell survival, cell development, host immunity, and cancer (Bandhuvula and Saba, 2007; Chi, 2011; Fyrst and Saba, 2008; Herr et al., 2003; Kumar et al., 2004; Li et al., 2000; Min et al., 2007; Serra and Saba, 2010). Recessive SPL mutations have been found to lead to SPL Insufficiency Syndrome, which can involve symptoms including nephrosis and immunodeficiency among others (Lovric et al., 2017). However, we have previously shown that SPL has antiviral activity during IAV infection (Seo et al., 2010). SPL enhanced the type I IFN response to IAV independently of its ability to metabolize S1P by interacting with IKKε but not TBK1 (Vijayan et al., 2017), ultimately resulting in the production of more IFN molecules and subsequently more ISGs.

In this study, we report that IAV infection leads to the ubiquitination and subsequent downregulation of SPL via IAV nonstructural protein 1 (NS1). IAV NS1 reduced the previously observed SPL-mediated activation of IKKε, which resulted in reduced IFN responses. This study reveals a novel strategy used by IAV to subvert the type I IFN innate immune response.

Methods

Viruses and Cells

Influenza A/Puerto Rico/8/34 (H1N1) virus (gift from Adolfo Garcia Sastre), influenza A/Puerto Rico/8/34 (H1N1) virus deficient in NS1 (ΔNS1) (gift from Dr. Adolfo Garcia Sastre), influenza A/Hong Kong/8/68 (H3N2 VR-1679) virus (ATCC), 2009 pandemic influenza A/CA/04/09 (H1N1) virus (gift from Dr. Wenjun Ma) (Lee et al., 2017; Xia et al., 2018), and influenza B/Lee/40 (IBV) virus (ATCC VR-1535), were used as previously described. In experiments, IAV denotes Influenza A/Puerto Rico/8/34 virus unless stated otherwise. Viruses used in this study were amplified either on Madin Darby Canine Kidney (MDCK) cells or in chicken eggs as described previously (Eisfeld et al., 2014; Neumann et al., 1999; Seo et al., 2010; Varble et al., 2014). Briefly, for amplification of viruses on MDCK cells, cells were incubated with virus for 1 hour (h). The cells were then washed with PBS and incubated with fetal bovine serum (FBS) free medium containing 0.3% bovine serum albumin (BSA) and TPCK-trypsin (1μg/mL) for amplification. For viral amplification in chicken eggs, serum pathogen-free fertilized chicken eggs were candled then inoculated with virus diluted in 1x phosphate buffered saline via the allantoic route. Infected eggs were incubated for 48 hours without turning at 37°C and ~60% humidity. Eggs were then chilled and the allantoic fluid was collected and centrifuged to remove debris. Virus titers were determined by plaque assay. Briefly, supernatants containing viruses were harvested, serially diluted, and then were adsorbed onto 1 × 106 MDCK cells/well in a 6-well plate for at least 1 h. Cells were then overlayed and incubated with 2X EMEM (Gibco) containing 0.6% BSA and 2 ug/mL TPCK-trypsin mixed with an equal portion of 1% agarose (Seakem ME). Cells were fixed using 25% formalin and stained with 1X crystal violet prior to assessing viral titer. Sources of human embryonic kidney (HEK) 293 cells, MDCK cells, and human lung epithelial A549 cells have been previously described (Min et al., 2007; Seo et al., 2012; Vijayan et al., 2014). HEK293 cells and A549 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco), while the MDCK cells were cultured in Minimum Essential Medium Eagle (MEM, Gibco) as previously described (Seo et al., 2013; Vijayan et al., 2017, 2014; Xia et al., 2018, 2016). All cells were cultured in a CO2 incubator at 37°C. All media to culture immortalized cell lines were supplemented with 10% FBS (HyClone) and 1% penicillin (100 U/mL)/streptomycin (100 μg/mL) (Invitrogen) unless stated otherwise. Undifferentiated primary human tracheal epithelial cells (HTEpCs) derived from excess tissue of lungs donated for transplant from human donors were expanded as previously described (Dickinson et al., 2018) with PneumaCult-Ex Plus medium (StemCell) supplemented with 0.1% hydrocortisone stock solution (96 μg/mL) (StemCell), 1% penicillin (100 U/mL)/streptomycin (100 μg/mL) (Invitrogen), 0.1% amphotericin B (250 μg/mL) (Fungizone), and 2% PneumaCult-Ex Plus 50x supplement (StemCell).

Plasmids and Transfection

Mammalian expression plasmids encoding IKKε, Flag-tagged SPL, wild type SPL, HA-tagged ubiquitin, and IAV neuraminidase were used as described previously (Vijayan et al., 2017; Xia et al., 2016). Plasmid encoding IAV NS1 was a gift from Dr. Adolfo Garcia-Sastre (Mt Sinai). For transfection of cultured cells, cells were seeded onto 6-well plates or 24-well plates at densities of 1 × 106 cells/well or 2.5 × 105 cells/well respectively 24 hours prior to transfection. Cells were then transfected with the indicated plasmids using Lipofectamine2000 Transfection Reagent (Thermo Scientific) or LipoD293 transfection reagent (Signagen) at 80%−90% confluency following the protocols recommended by the manufacturers. A concentration of 500 ng/mL DNA was used for the transfection experiments unless specifically indicated. Empty vector plasmids were used as a control in all transfection experiments to ensure that each transfection sample received the same amount of total DNA.

Reagents and Antibodies

Anti-DYKDDDDK (FLAG) G1 Affinity Resin (GenScript), IP lysis buffer (Thermo Scientific), and cycloheximide (CHX, Sigma-Aldrich) were purchased from the indicated manufacturers. Antibodies against IBV NP (B017) and IAV M1 (GA2B) were purchased from Abcam; antibodies against p-IKKε (Ser172, D1B7), IKKε (D20G4), FLAG tag (9A3), HA tag (C29F4), human GAPDH (D1GH11), RIG-I (D14G6), and ubiquitin (P4D1) were purchased from Cell Signaling Technology; antibodies against SPL (H-300), IAV NS1 (NS1-23-1), and IAV M2 (vI-19) were purchased from Santa Cruz Biotechnology; antibody against IAV H5N1 NA (GTX127984) was purchased from Genetex. Fluorophore-labeled IR-Dye secondary antibodies against mouse and rabbit IgG were purchased from LI-COR.

Denatured immunoprecipitation (denatured-IP), and western blotting

293T cells, seeded in 6-well plates, were transiently transfected or co-transfected with the indicated plasmids (1 μg DNA in total). For detection of ubiquitination of SPL during IAV infection, cells were infected with IAV with an MOI of 1. Cells were harvested and lysed 24 hours post-transfection in 1 mL IP lysis buffer (48 h post-transfection for infection experiments). Denatured IP was performed as previously described (Xia et al., 2016). Briefly, cell lysates were boiled at 95°C for 5 minutes, were chilled on ice, and were centrifuged to clear lysates of cell debris. A small amount of lysate was reserved for analysis via western blotting, and the remainder of the samples were incubated with 20 μl of anti-DYKDDDDK (FLAG) G1 affinity resin (GenScript) overnight with rotation at 4°C. The beads were washed three times with 1 mL IP lysis buffer, and precipitates and lysates were analyzed by western blot analysis. Western blotting was performed as previously described. Briefly, cells were lysed by addition of 2x sample buffer containing β-mercaptoethanol and boiled at 95°C for 10 min. The denatured polypeptides from cell lysates or IP were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred to nitrocellulose membranes (Bio-Rad). Membranes were blocked then incubated with the indicated primary antibodies overnight at 4°C. Membrane-bound antibodies were detected using IRDye secondary antibodies (LI-COR). Bound antibodies were visualized and imaged using an Odyssey Fc (LI-COR), and the resulting data were analyzed using Image Studio software V5.2 (LI-COR). Similar results were obtained from at least two independent experiments.

Real-time quantitative PCR

Total cellular RNA was extracted and purified by using Tri Reagent (Sigma-Aldrich) according to the manufacturer’s instructions. Purified RNA was reverse transcribed with random primers (Invitrogen), and the resulting cDNA was used as a template for real-time quantitative PCR (RT qPCR) using gene-specific primers. Primers for human SPL (5’- GGA TGA AGA TTG TGC GGG TC-3’ and 5’-GAA CAG ACG AGC ATG GCA GT-3’), human OAS-1 (5’-GAT CTC AGA AAT ACC CCA GCC A-3’ and 5’-AGC TAC CTC GGA AGC ACC TT-3’), human IFN-β (5’-CGC CGC ATT GAC CAT CTA-3’ and 5’-GAC ATT AGC CAG GAG GTT CTC A-3’), human IFN-α (5’-GTG AGG AAA TAC TTC CAA AGA ATC AC-3’ and 5’-TCT CAT GAT TTC TGC TCT GAC AA-3’), and human GAPDH (5’ -TCA CCA CCA TGG AGA AGG -3’ and 5’ -GAT AAG CAG TTG GTG GTG CA -3’) were used. After cDNA synthesis, qPCR reactions were performed with Power SYBR green PCR Master Mix (Applied Biosystems) using a Step One real-time PCR instrument (Applied Biosystems), and cDNA quantities were normalized to quantities of GAPDH RNA measured from the same samples.

Statistical analysis

Shapiro-Wilk test and Bartlett’s test of homogeneity of variances were used to determine if the assumptions were met to analyze using a one-way ANOVA. Data were then analyzed and compared pairwise using either one-way ANOVA with Tukey’s range test as post hoc or Kruskal-Wallis with Conover-Iman test of multiple comparisons as post hoc. One-way ANOVA, Tukey’s range test, and Kruskal-Wallis were performed using JASP (JASP Team, 2020). Statistical analysis was conducted in R (R Core Team, 2020) with the indicated packages for the following: Conover-Iman test of multiple comparisons (conover.test package (Alexis Dinno, 2017)), Shapiro-Wilk normality test (dplyr package (Wickham et al., 2021)), and Bartlett’s test of homogeneity of variances (stats package (R Core Team, 2020)). All error bars represent mean ± standard error of the mean (SEM). Data are representative of at least two independent experimental repetitions.

Results:

IAV negatively regulates SPL protein levels

We have previously determined that sphingosine 1-phosphate lyase (SPL) has antiviral capabilities against influenza A virus (IAV) infection by enhancing the type I IFN innate immune response. However, it had yet to be determined if SPL levels could change during IAV infection, either as a response by the host to infection or as a result of the viral infection itself. To investigate if SPL levels change during IAV infection, SPL protein levels were assessed by western blotting at 2, 4, 6, 12, and 24 hours post infection (hpi) with pandemic influenza A/CA/04/09 IAV (pH1N1) in A549 cells. Following infection, SPL protein levels decreased progressively over time as viral protein levels, represented by IAV nonstructural protein 1 (NS1), increased (Fig. 1A). To determine if this reduction of SPL protein was specific to IAV H1N1 infection, A549 cells were infected with either IAV pH1N1, influenza A/Hong Kong/8/68 (H3N2), or influenza B/Lee/40 virus (IBV), and SPL protein levels were assessed using western blot analysis. Compared to the uninfected control, SPL protein levels were reduced when cells were infected by any of the tested influenza viruses (Fig. 1B). Furthermore, primary human tracheal epithelial cells (HTEpCs) were infected with IAV and SPL protein levels were assessed by western blotting. Similar to the infection with various influenza viruses in A549 cells, SPL levels decreased in the IAV-infected HTEpCs (Fig. 1C). Next, to assess whether this downregulation was occurring at a transcriptional level, SPL mRNA levels in A549 cells were measured using reverse transcription followed by real-time quantitative PCR (qPCR) at 12 hpi and 24 hpi with IAV. Interestingly, IAV infection did not alter SPL mRNA expression significantly at either timepoint, indicating post-transcriptional downregulation of SPL. Collectively, these data demonstrate that IAV infection negatively regulates SPL levels at the post-transcriptional stage.

Figure 1: IAV negatively regulates SPL protein levels.

Figure 1:

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(A) A549 cells were infected with influenza A virus (IAV), pandemic influenza A/CA/04/09 (pH1N1) virus, at an MOI of 1. Protein levels of SPL, IAV NS1, and GAPDH were detected respectively at the indicated time points post infection. (B) A549 cells were infected for 24 hours with either pH1N1 virus (IAV H1N1) or influenza A/Hong Kong/8/68 virus (IAV H3N2), or influenza B/Lee/40 virus (IBV) (MOI = 1). Levels of SPL, IBV NP, IAV M1, IAV NS1, and GAPDH were detected using western blot analysis. (C) Primary human tracheal epithelial cells (HTEpCs) were infected with IAV, influenza A/Puerto Rico/8/34 (H1N1), and levels of SPL, M1, and GAPDH were detected. (D) A549 cells were infected with pH1N1 IAV at an MOI of 1 and were harvested at the indicated timepoints. The relative mRNA levels of SGPL1 were analyzed using real time qPCR. Data represent mean ± SEM. Pairwise comparison was performed using one-way ANOVA followed by Tukey post hoc (n = 4 per group; ns = not significant).

IAV infection induces ubiquitination of SPL

To further characterize the effects of IAV infection on SPL, denatured immunoprecipitation (IP) assays were performed. Cells were co-transfected with FLAG-SPL and HA-tagged-ubiquitin expressing plasmids and were subsequently infected with IAV. Denatured IP was performed on the IAV infected and uninfected control cells, and relative ubiquitination of FLAG-SPL was subsequently assessed using western blotting. IAV infection strongly increased ubiquitination of FLAG-SPL compared to the uninfected controls (Fig. 2A). Consistent with these findings, we observed enhanced endogenous ubiquitination of FLAG-SPL upon IAV infection in the absence of ubiquitin-overexpressing plasmid (Fig. 2B). These results led us to conclude that SPL ubiquitination is induced during IAV infection, which could lead to the previously observed decrease in SPL protein levels.

Figure 2: IAV infection induces ubiquitination of SPL.

Figure 2:

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(A) HEK293 cells were co-transfected with plasmid DNA encoding HA-tagged ubiquitin (HA-Ub) and FLAG-tagged SPL (FLAG-SPL) and at 24 hours post transfection were subsequently infected with IAV (MOI = 1). Denatured IP experiments were performed at 1 day post infection (dpi) to detect the ubiquitination of SPL. (B) HEK293 cells were transfected with FLAG-SPL and were infected with IAV (MOI = 1) 24 hours post-transfection. Denatured IP was performed, and endogenous ubiquitin of FLAG-SPL was detected.

IAV NS1 protein is responsible for ubiquitination and downregulation of SPL

To determine which component of IAV infection induces the ubiquitination and subsequent downregulation of SPL, we looked at the effects of both host and viral components of infection. First, because SPL is a newly identified pro-IFN factor, we assessed the effect of the type I IFN response on SPL protein level in cells. To this end, western blotting was performed after induction of the type I IFN innate immune response with human IFN-α or IAV viral RNA (vRNA). SPL protein levels remained relatively unchanged after treatment with human IFN-α (Fig. 3A) or IAV vRNA (Fig. 3B) compared to the uninduced controls, indicating that SPL downregulation was not a result of the type I IFN innate immune response to IAV infection. Because IAV nonstructural protein 1 (NS1) is known to regulate host type I IFN responses (Kochs et al., 2007; Krug, 2015; Muñoz-Moreno et al., 2020), we next tested if NS1 could be responsible for the ubiquitination of SPL. Viral proteins were assessed for their abilities to ubiquitinate SPL by transiently overexpressing either NS1 or neuraminidase (NA) as a negative viral protein control and performing denatured IP followed by western blotting to measure relative ubiquitination of FLAG-SPL. Relative to the controls, NS1, but not NA, was determined to induce ubiquitination of SPL (Fig. 3C). Furthermore, NS1 was found to directly instigate SPL downregulation (Fig. 3D). To eliminate the possibility of protein translation affecting total SPL levels, cycloheximide (CHX) treatment was used to stop new protein synthesis in conjunction with NS1 overexpression; with these conditions, we observed a more dramatic reduction of SPL levels in the presence of NS1 (Fig. 3D). Typically, post-transcriptional downregulation of proteins can be attributed to the proteasomal and/or lysosomal degradation pathway. In order to assess whether IAV NS1 was downregulating SPL via these pathways, we used proteasomal inhibitor MG132 or lysosomal inhibitor NH4Cl in conjunction with cycloheximide treatment. Treatment with MG132 or NH4Cl prevented the downregulation of SPL, demonstrating that NS1-mediated SPL downregulation utilizes both the proteasomal and lysosomal degradation pathways (Fig. 3D).

Figure 3: IAV NS1 induces ubiquitination and degradation of SPL.

Figure 3:

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The type I IFN innate immune response was stimulated in HEK293 cells either by treatment with 1000 U/mL recombinant IFN-α (rIFN-α) (A) or by transfection with 2.5 μg/mL IAV RNA (vRNA) or RNA isolated from uninfected cells (−) (B), and levels of SPL, RIG-I, and GAPDH were measured using western blotting. (C) HEK293 cells were co-transfected with plasmids expressing HA-tagged ubiquitin (HA-Ub) and FLAG-tagged SPL (FLAG-SPL), together with plasmids expressing viral NS1 or NA as indicated. Denatured IP experiments were performed to detect the ubiquitination of SPL. (D) A549 cells were transfected with IAV NS1 expressing plasmid for 16 hours and were then subsequently treated for 6 hours with solvent (DMSO), 20 μM MG132, or 20 mM NH4CL. Samples were also treated with 30 μg/mL cycloheximide (CHX) or solvent (DMSO) for the duration of the 6 hour treatment to inhibit protein synthesis. Western blotting was then used to detect levels of SPL, NS1, and GAPDH in order to assess the manner of SPL downregulation.

To confirm that IAV NS1 is a pivotal factor necessary for triggering SPL ubiquitination and downregulation during IAV infection, we next tested the effect of infection with NS1-deficient (ΔNS1) IAV (IAV-ΔNS1) on SPL ubiquitination and downregulation. Denatured IP of transiently overexpressed FLAG-SPL and subsequent western blotting were used to assess SPL ubiquitination during infection with either IAV or IAV-ΔNS1. Infecting with increasing MOIs of IAV likewise increased ubiquitination of SPL. However, infecting with increasing MOIs of IAV-ΔNS1 at even higher MOIs than used in wild type infection did not show discernible ubiquitination of SPL compared to the uninfected control (Fig. 4A). Next, to assess whether NS1 was important for endogenous SPL protein downregulation during infection, cellular SPL protein levels were measured by western blotting with either IAV (MOI = 1) or IAV-ΔNS1 (MOI = 3). While IAV infection led to endogenous SPL downregulation, IAV-ΔNS1 infection did not lead to significant SPL downregulation despite being infected with a higher MOI (Fig. 4B). Collectively, these data demonstrate that IAV NS1 is essential for IAV-mediated ubiquitination and downregulation of SPL.

Figure 4: NS1-deficient IAV fails to induce the ubiquitination and degradation of SPL.

Figure 4:

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(A) HEK293 cells were co-transfected with HA-Ub and FLAG-SPL. At 1 dpi, cells were infected with IAV or NS1-deficient IAV (IAV-ΔNS1) at MOIs of 0.1 or 1. Denatured IP experiments were performed to detect the ubiquitination of SPL. (B) HEK293 cells were infected with IAV or IAV-ΔNS1 at 1 MOI or 3 MOI respectively. The levels of SPL, NS1, M2, HA, and GAPDH were analyzed with western blotting at 1 dpi.

NS1 inhibits SPL-mediated activation of IKKε and production of type I IFNs

Our previous study revealed a new role for SPL in which SPL interacts with IKKε, increasing IKKε activation (phosphorylation of IKKε) as well as subsequent production of IFNs and ISGs. However, it remained to be determined whether IAV could inhibit this SPL-mediated type I IFN innate immune response. To this end, western blotting was performed to assess protein levels of phosphorylated IKKε (pIKKε) 16 hours post transfection with plasmids expressing IKKε, SPL, and NS1. As we have previously observed, transiently overexpressed IKKε increased pIKKε due to autophosphorylation, and transient overexpression of both SPL and IKKε resulted in a larger increase of pIKKε than IKKε overexpression alone. However, transient overexpression of NS1, SPL, and IKKε decreased pIKKε levels compared to the controls (Fig. 5A). To further assess whether the type I IFN response was subsequently impacted by those conditions, relative levels of IFN-α and IFN-β mRNA molecules were measured using reverse transcription followed by qPCR. Similar to our previous findings, IKKε autophosphorylation-induced expression of IFN-α and IFN-β mRNAs was greatly increased by SPL transient overexpression. However, the expression of IFN-α and IFN-β mRNA transcripts was greatly reduced when NS1 was transiently expressed in addition to IKKε and SPL (Fig. 5B and 5C). ISGs are the ultimate antiviral effectors of the type I IFN innate immune response to IAV infection. In order to determine whether the NS1-mediated inhibition of the SPL-enhanced type I IFN innate immune response impacts the production of ISGs, OAS-1 mRNA transcript levels were subsequently measured with reverse transcription and qPCR. Strikingly, OAS-1 transcription was increased by over 300-fold in conditions where both SPL and IKKε were transiently overexpressed, compared to a 20-fold increase during transient overexpression of IKKε alone. However, the enhanced OAS-1 transcription seen in those conditions was mitigated by the addition of transiently overexpressed NS1 (Fig. 5D). Collectively, these data suggest that IAV NS1 obstructs the pro-IFN function of SPL, enabling IAV to evade the SPL-mediated type I IFN innate immune response during infection.

Figure 5: NS1 inhibits SPL’s function in regulating IKKe-mediated type I IFN response elements.

Figure 5:

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293T cells were co-transfected with the indicated plasmids, and cells were harvested 1 day post transfection. The levels of phosphorylated IKKε (pIKKε), IKKε, SPL, NS1, and GAPDH (A) were analyzed by western blotting. The relative mRNA levels of IFN-β (B), IFN-α (C), and OAS-1 (D), were analyzed by real-time qPCR. Data represent mean ± SEM. One-way ANOVA with Tukey’s range test (B and C) or Kruskal-Wallis with Conover-Iman test (D) were used for pairwise comparisons. (n = 3 per group; **, P ≤ 0.01; ***, P ≤ 0.001).

Discussion

Our study demonstrates that IAV NS1 induces the ubiquitination and subsequent downregulation of host SPL, which allows IAV to subvert the SPL-mediated type I IFN innate immune response to IAV infection.

The IAV NS1 protein has several functions during influenza virus replication (Rosário-Ferreira et al., 2020). NS1 is largely responsible for hindering host IFN antiviral responses, ensuring more effective IAV replication (Kochs et al., 2007; Krug, 2015; Muñoz-Moreno et al., 2020). NS1 was reported to directly limit the antiviral state by inhibiting OAS-1 activation, outcompeting OAS for interaction with double-stranded RNA (dsRNA) (Min and Krug, 2006; Wang et al., 1999). NS1 binding to dsRNA also prevents the activation of dsRNA-activated protein kinase (PKR) (Bergmann et al., 2000). Since the experimental conditions of Figure 5 did not use dsRNA or IAV infection, NS1’s impairment of the SPL-mediated pro-IFN function is not due to this previously known mechanism. Additionally, NS1 inhibits RIG-I activation by blocking the TRIM25 ubiquitin E3 ligase-mediated K63-linked ubiquitination of RIG-I (Gack et al., 2009) or by directly binding to RIG-I to prevent downstream activation of IRF3 (Mibayashi et al., 2007). Our findings of IAV NS1 preventing SPL from promoting IKKε phosphorylation is a novel method utilized by IAV NS1 to dampen host innate immunity.

The underlying mechanism of NS1 triggering the ubiquitination and degradation of SPL remains unknown. It is possible that another host protein, such as a yet-to-be-determined host ubiquitin ligase, is somehow activated by NS1 to mediate SPL’s ubiquitination and subsequent destruction. IAV infection has been previously shown to direct host ubiquitin ligase NEDD4 to target interferon-induced transmembrane protein 3 (IFITM3), a protein involved in the immune defense against several RNA viruses, for ubiquitination and subsequent lysosomal degradation (Chesarino et al., 2015). Investigating how IAV NS1 induces the ubiquitination and degradation of SPL is an exciting avenue of research which remains to be explored.

Viruses must counteract host antiviral activities, especially IFN production and the activity of ISGs, in order to replicate efficiently. We have shown that IAV mediates the ubiquitination and subsequent proteasomal and lysosomal degradation of host SPL. We have previously found that IAV infection induces ubiquitination and subsequent degradation of type I IFN receptor 1 (IFNAR1) in order to dampen the type I IFN innate immune response (Xia et al., 2016). Comparable to our findings, IAV infection was found to mediate both proteasomal and lysosomal degradation of IFNAR1. IAV NS1 has also been previously shown to mediate the ubiquitination and lysosomal degradation of eukaryotic translation initiation factor 4B (eIF4B) protein (Wang et al., 2014). While eIF4B is a key component in regulating the initiation of mRNA translation, it could also regulate the IFN-induced expression of IFITM3. Indeed, several viruses are known to hijack the host ubiquitin system in order to subvert or evade IFN responses. For example, human rotavirus nonstructural protein NSP1 stifles IFN expression by inducing ubiquitination-dependent degradation of interferon regulatory factor 3 (IRF3), a key component in the induction of the IFN α/β cascade (Barro and Patton, 2005). It is likely that IAV also utilizes the host ubiquitin system in order to induce degradation of SPL and further evade host IFN responses.

Sphingolipid-metabolizing enzymes have been shown to affect the IAV replication cycle. Sphingosine kinase 1 and 2 (SphK1 and SphK2) enhance IAV replication in cultured cells, and IAV infection increases the expression and phosphorylation of SphK1 and SphK2 (Xia et al., 2018). SphK1 and SphK2 are also known to regulate the replication of other viruses. For example, measles virus (MV) infection elevates SphK1 and pSphK1 levels (Vijayan et al., 2014). Inhibition of SphK impairs MV replication and suppresses NF-kB activation. These results, among others, demonstrate that SphK impacts viral replication and could be a promising therapeutic target in some instances. Our previous studies have also indicated that SPL has a pro-IFN function upon influenza virus infection (Vijayan et al., 2017). However, here we have shown that IAV NS1 protein counteracts this SPL-mediated enhanced type I IFN response to infection. Determining whether these effects are applicable to other viruses is a promising area of research.

This study sheds light on the intricate struggle between influenza virus and the host immune defense and confirms the important role of SPL in the innate immune response to influenza virus infection. Understanding the precise mechanism of how IAV NS1 targets SPL for ubiquitination and degradation could yield potential new therapeutic targets for the treatment of IAV infection.

Highlights.

  • Influenza virus infection induces host SPL downregulation

  • IAV NS1 is crucial for SPL downregulation during IAV infection

  • IAV NS1 mediates SPL ubiquitination and degradation

  • IAV NS1 inhibits the pro-IFN function of SPL

Acknowledgements

This work was supported by the Department of Surgery at University of Missouri and NIH/NIAID grant AI091797 (B.H.). J.J.W. and C.J.S. were supported by Life Sciences Fellowships from the University of Missouri. H.N. was supported by the McNair Scholars program at University of Missouri. We would like to thank Adolfo Garcia-Sastre (Icahn School of Medicine at Mount Sinai), and Wenjun Ma (University of Missouri) for their kind provision of research reagents as described in the Materials and Methods section. We would also like to thank Paige Williams for her work in lab making and maintaining lab reagents, Regina Wamsley for her work in the lab, and Joseph Mangieri for his help with learning to use R.

Footnotes

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Declarations of Interest:

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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