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, 36 (11), 652-665

Type I Interferon-Mediated Induction of Antiviral Genes and Proteins Fails to Protect Cells From the Cytopathic Effects of Sendai Virus Infection

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Type I Interferon-Mediated Induction of Antiviral Genes and Proteins Fails to Protect Cells From the Cytopathic Effects of Sendai Virus Infection

Jacquelyn R Bedsaul et al. J Interferon Cytokine Res.

Abstract

Sendai virus (SeV), a murine paramyxovirus, has been used to study the induction of type I interferon (IFN) subtypes in robust quantities. Few studies have measured whether the IFN that SeV induces actually fulfills its intended purpose of interfering with virus-mediated effects in the cells in which it is produced. We determined the effects of IFN on SeV-mediated cytopathic effects (CPE) and the ability of IFN to protect against virus infection. SeV-induced biologically active IFN resulted in Jak/STAT activation and the production of a number of interferon-stimulated genes (ISGs). However, these responses did not inhibit SeV replication or CPE. This observation was not due to SeV effects on canonical IFN signaling. Furthermore, pretreating cells with type I IFN and establishing an antiviral state before infection did not mediate SeV effects. Therefore, the induction of canonical IFN signaling pathways and ISGs does not always confer protection against the IFN-inducing virus. Because type I IFNs are approved to treat various infections, our findings suggest that typical markers of IFN activity may not be indicative of a protective antiviral response and should not be used alone to determine whether an antiviral state against a particular virus is achieved.

Keywords: Sendai virus; antiviral state; interferon; signaling.

Conflict of interest statement

Author Disclosure Statement No competing financial interests exist.

Figures

<b>FIG. 1.</b>
FIG. 1.
Multiple cell types produce biologically active type I IFNs upon SeV infection. (A) Supernatants from primary monocytes that were either UI or infected with SeV (150 HA U/mL) for 24 h were treated with β-propiolactone to inactivate the virus and then used to pretreat fresh U937 cells for 24 h. The U937 cells were then infected with VSV for 72 h and cell viability was measured via MTT assay. Cell viability is shown as the % of cell control. Cell control: UI, untreated. Virus control: VSV infected, untreated. Supernatants from UI or SeV infected (B) U937, (C) Namalwa, or (D) A549 cells for 16 h were treated with β-propiolactone to inactive the virus and then used to pretreat fresh U937 cells for 24 h. The U937 cells were then infected with EMCV for 72 h and cell viability was measured via MTT assay. Cell viability is shown as the % of cell control. Cell control: UI, untreated. Virus control: EMCV infected, untreated. Data shown are from 3 independent biological replicates. (E) U937 cells were pretreated with IFN-β (100 U/mL) or supernatants from U937 cells treated with β-propiolactone-inactivated SeV (150 HA U/mL) for 24 h. The U937 cells were then infected with EMCV for 72 h and cell viability was measured via MTT assay. Cell control: UI, untreated. Virus control: EMCV infected, untreated, IFNB ptx: pretreated with IFN-β; SeV/Bprop ptxt: treated with inactivated SeV by β-propiolactone. SeV, Sendai virus; IFN, interferon; UI, uninfected; HA, hemagglutination; VSV, vesicular stomatitis virus; EMCV, encephalomyocarditis virus.
<b>FIG. 2.</b>
FIG. 2.
SeV-induced IFN production activates Jak/STAT signaling and ISG induction. (A) U937 cells were infected with SeV (150 HA U/mL) for 4, 8, 16, and 24 h. Nuclear and cytoplasmic protein extracts were prepared and western blots probing for pIRF-3(S386), IRF-3, IRF-7, and IRF-9 were performed. RNA pol II and β-tubulin were the nuclear and cytoplasmic loading controls, respectively. N, nuclear, C, cytoplasmic. (B) U937 cells were infected with SeV (150 HA U/mL) for 4, 8, 16, and 24 h and whole cell protein lysates were prepared. Western blots probing for pSTAT1(Y701), STAT1(total), pSTAT2(Y689), STAT2(total), ISG15, and IFIT3, and tubulin as the loading control were performed. (C) A549 cells were infected with SeV (150 HA U/mL) for 4, 8, 16, and 24 h and whole protein lysates were prepared. Western blots probing for pSTAT1(Y701), STAT1(total), pSTAT2(Y689), STAT2(total), IRF-9, IRF-7, and Mx1, and tubulin as the loading control were performed. (D) ISG15 and IFIT3 mRNA expression in U937 cells infected with SeV (150 HA U/mL) for 4, 8, 16, and 24 h. (E) IRF-7 and Mx1 mRNA expression in A549 cells infected with SeV (150 HA U/mL) for 4, 8, 16, and 24 h. IRF-7 and Mx1 mRNA induction in (F) primary monocytes and (G) Namalwa cells infected with SeV (150 HA U/mL) for 24 h. All gene induction data are expressed as the % of relative HPRT expression. All data are representative of 3 independent biological replicates. UI, uninfected, untreated. IRF, IFN regulatory factor; ISG, interferon-stimulated gene; HPRT, hypoxanthine-guanine phosphoribosyltransferase.
<b>FIG. 3.</b>
FIG. 3.
Type I IFNs induced by SeV do not protect against SeV-induced CPE. (A) Supernatants from primary monocytes that were either UI or infected with SeV (150 HA U/mL) for 24 h were treated with β-propiolactone to inactivate the virus and then used to pretreat fresh U937 cells for 24 h. The U937 cells were then infected with SeV (150 HA U/mL) for 72 h and cell viability was measured via MTT assay. Results are shown as the % of cell control. Supernatants from (B) U937, (C) Namalwa, or (D) A549 cells that were UI or infected with SeV for 16 h were treated with β-propiolactone to inactive the virus and then used to pretreat fresh U937 cells for 24 h. The cells were then infected with SeV (150 HA U/mL) for 72 h and cell viability was measured via MTT assay. Results are shown as the % of cell control and are representative of 3 independent biological replicates. CPE, cytopathic effects.
<b>FIG. 4.</b>
FIG. 4.
Type I IFN pretreatment does not inhibit SeV-induced CPE. (A) U937 and (B) A549 cells were pretreated with serial dilutions of IFN-α10 (ranging from 0.78 to 50 U/mL) for 24 h and then UI or infected with either EMCV (MOI 0.5) or SeV (150 or 1.5 HA U/mL) for 72 h. Cell viability was measured via MTT assay (U397) or crystal violet staining (A549). CC: untreated, UI; VC: untreated virus infected. (C) Namalwa cells were pretreated with 100 U/mL of all 12 IFN-α subtypes for 24 h and then infected with either VSV (MOI 0.5) or SeV (150 HA U/mL) for 72 h. Cell viability was measured via MTT assay. Results show cell viability as % of cell control. Cell Control: UI, untreated; virus control: virus infected, untreated. MOI, multiplicity of infection.
<b>FIG. 5.</b>
FIG. 5.
Inhibiting canonical type I IFN signaling during SeV infection does not affect SeV-mediated CPE. (A) U937 cells were treated with 1000 U/mL of IFN-α2a, IFN-β, or IFN-ω for 1 h in the presence or absence of the IFNAR2 neutralizing antibody. Western blot analysis was performed to detect phosphorylated STAT1(Y701) and tubulin was used as the loading control. UT: untreated; NA: cells treated with IFNAR2 neutralizing antibody alone; α2a: IFN-α2a treated; α2a + NA: cells treated with both antibody and IFN-α2a; β: IFN-β treated; β + NA: cells treated with both antibody and IFN-β; ω: IFN-ω treated; and ω + NA: cells treated with both antibody and IFN-ω. (B) U937 (panel 1) and A549 cells (panel 2) were infected with SeV (150 HA U/mL) in the presence or absence of IFNAR2 neutralizing antibody. Cell viability was measured 72 h pi. Data are representative of 3 independent biological replicates. Supernatants from cells infected with SeV (150 HA U/mL) for 16 h were inactivated (β-propiolactone) and used to pretreat fresh (C) U937 and (D) A549 cells for 24 h in the presence or absence of the IFNAR or IFNLR neutralizing antibodies. The U937 cells were then infected with either EMCV or SeV (150 HA U/mL) for 72 h and cell viability was measured via MTT assay (U937) or crystal violet assay (A549). Results are shown as the % of cell control. SeV150: virus-inactivated supernatant from SeV-infected cells (150 HA U/mL); SeV150 + IFNAR2 NA: treated with virus-inactivated supernatant from SeV-infected cells (150 HA U/mL) in the presence of IFNAR2-neutralizing antibody; SeV150 + IFNLR NA: treated with virus-inactivated supernatant from SeV-infected cells (150 HA U/mL) in the presence of IFNLR-neutralizing antibody. IFNAR, IFN receptor; IFNLR, IFN-lambda receptor.
<b>FIG. 6.</b>
FIG. 6.
The effect of IFNAR signaling on SeV production is cell type dependent. (A) SeV virus levels were measured via hemagglutination assay in U937 cells (panel 1) and A549 cells (panel 2) at 24 and 72 h pi with SeV (150 HA U/mL), respectively. The data are expressed as HA U/mL. Two biological replicates were performed and resulted in the same titer. (B) U937 cells were pretreated with all 12 IFN-α subtypes, IFN-β or IFN-ω (100 U/mL) for 24 h. Cells were then infected with SeV (150 HA U/mL) for 24 h. Hemagglutination assay was performed to measure SeV virus production in the cell supernatants. Data are expressed as HA U/mL. UT: untreated, virus infected cell supernatant. pi, postinfection.
<b>FIG. 7.</b>
FIG. 7.
Decreasing the amount of infecting virus does not result in IFN-mediated protection against SeV-induced CPE. (A) U937 cells were pretreated with 100 U/mL of type I IFNs for 24 h and then infected with serial 2-fold dilutions of SeV, ranging from 0 to 150 HA U/mL of SeV for 72 h. Cell viability was measured via MTT assay. Results are shown for IFN-α1 pretreatment, but all IFN-α and IFN-β subtypes were tested with similar results. (B) Virus preparations were inactivated using β-propiolactone. U937 cells were then infected with SeV or treated with inactivated SeV (β-propiolactone) for 72 h. Cell viability was measured via MTT assay and results are represented as % cell control.

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