XAF1 prevents hyperproduction of type I interferon upon viral infection by targeting IRF7

Abstract

Interferon regulatory factor (IRF) 3 and IRF7 are master regulators of type I interferon (IFN-I)-dependent antiviral innate immunity. Upon viral infection, a positive feedback loop is formed, wherein IRF7 promotes further induction of IFN-I in the later stage. Thus, it is critical to maintain a suitably low level of IRF7 to avoid the hyperproduction of IFN-I. In this study, we find that early expression of IFN-I-dependent STAT1 promotes the expression of XAF1 and that XAF1 is associated specifically with IRF7 and inhibits the activity of XIAP. XAF1-knockout and XIAP-transgenic mice display resistance to viral infection, and this resistance is accompanied by increases in IFN-I production and IRF7 stability. Mechanistically, we find that the XAF1-XIAP axis controls the activity of KLHL22, an adaptor of the BTB-CUL3-RBX1 E3 ligase complex through a ubiquitin-dependent pathway. CUL3-KLHL22 directly targets IRF7 and catalyzes its K48-linked ubiquitination and proteasomal degradation. These findings reveal unexpected functions of the XAF1-XIAP axis and KLHL22 in the regulation of IRF7 stability and highlight an important target for antiviral innate immunity.

Keywords: IFN-I; IRF7; KLHL22; XAF1-XIAP axis; ubiquitination.

Figure 1. IFN‐I‐induced XAF1 expression is negatively correlated with the Irf7 mRNA level

1. A, B

   mRNA levels of IFN‐I (A) and IRF7 (B) in virus‐stimulated WT BMDMs were measured by qRT–PCR at the indicated time points.

2. C

   WT BMDMs were stimulated with SeV or VSV‐WT (MOI = 1) for 9 h and treated with MG132 2 h before harvest. The indicated proteins in whole‐cell lysates were detected by IB.

3. D

   WT BMDMs were stimulated with SeV for the indicated time course, and whole‐cell lysates were subjected to IP using an anti‐IRF7 antibody. Cell lysates were also subjected to direct IB, and actin was used as a loading control.

4. E

   Schematic of the experimental procedure for RNA‐seq and MS.

5. F

   Left panel, KEGG analysis of IRF7‐interacting proteins as determined by MS; middle panel, Venn diagrams illustrating the overlap between DEGs identified through analysis of the RNA‐seq and MS results; right panel, heatmap showing overlapping DEGs.

6. G

   Abundant XAF1 mRNA expression was induced by different influenza viruses at an MOI of 1 for 6 h in PBMCs obtained from healthy donors (n = 64) as measured by qRT–PCR.

7. H, I

   The mRNA (H) and protein (I) levels of XAF1 in virus‐induced WT BMDMs were measured throughout the indicated time course by qRT–PCR and IB, respectively.

8. J, K

   qRT–PCR analysis of XAF1 mRNA in BMDMs derived from IFNAR‐KO mice (J) and STAT1‐KO mice (K).

Data information: All data are representative of at least three biologically independent experiments. Data from the qPCR assay are presented as the fold change relative to the Actin mRNA level. The data are presented as the means ± SDs. The significance of differences was determined by t‐tests. *P < 0.05, **P < 0.01, and ***P < 0.005. Please see Appendix Fig S1 for information regarding replicates, quantification, and statistical evaluation for biochemical data in this figure. Source data are available online for this figure.

Figure 2. XAF1 negatively regulates IFN‐I induction

1. HEK293T cells were transfected with IFN‐β luciferase reporters and XAF1‐expressing plasmids and stimulated with SeV or VSV‐WT (MOI = 1). The readouts were normalized to Renilla luciferase activity and are presented as the fold change relative to activity in untransfected cells.

2. qRT–PCR analysis of IFN‐I induction in WT and XAF1‐deficient BMDMs stimulated with different viruses.

3. WT or XAF1‐deficient BMDMs were infected with GFP‐expressing VSV (VSV‐GFP) at an MOI of 0.1 for 8 h. The data are presented as representative images and FACS data, showing the infected (GFP⁺) and total (bright field) cells. Scale bar, 1,000 μm.

4. qRT–PCR analysis of the mRNA levels of IFN‐I in WT and XAF1‐deficient BMDMs stimulated with distinct TLR agonists.

5. Some of the LPS‐ or polyI:C‐induced ISGs in WT and XAF1‐deficient BMDMs were measured by qPCR.

6. mRNA levels of the indicated cytokines in WT and XAF1‐deficient BMDMs activated by distinct TLR agonists.

7. IFN‐I expression in WT and XAF1‐KO MEFs stimulated with SeV or HSV, evaluated by qPCR.

Data information: All data are representative of at least three biologically independent experiments. Data from the qPCR assay are presented as the fold change relative to the Actin mRNA level. The data are presented as the means ± SDs. The significance of differences was determined by t‐tests. *P < 0.05, **P < 0.01, and ***P < 0.005.

Figure 3. XAF1^MKO mice exhibit resistance to viral infection

WT and XAF1^MKO mice (6–8 weeks) were i.n. infected with a sublethal dose (0.1 hyaluronic acid [HA]) of PR8.

1. A, B

   Survival rate (A) and body weight loss (B) were monitored for 8 days (n = 10).

2. C

   Viral titers in the lung and trachea were quantified by TCID₅₀ assay on day 2 (n = 5).

3. D

   ELISA of IFN‐I levels in the sera of WT and XAF1^MKO mice infected with H1N1 strain PR8 on day 2 (n = 3).

4. E

   H&E‐stained lung tissue sections 2 days after infection. Inflammation scores are presented in a bar graph (n = 4). Scale bar, 200 μm.

5. F–H

   Survival rate (F, n = 12) and viral titer (G, n = 4) in the spleen and IFN‐β in the serum (H, n = 4) of WT and XAF1^MKO mice intravenously injected with HSV‐1 (3 × 10⁶ PFU per mouse).

6. I–K

   WT and XAF1^MKO mice crossed with IFNAR^−/− mice were i.n. infected with a sublethal dose (0.1 HA) of PR8. The survival rate (I, n = 10), viral titer in the lung (J, n = 4), and serum IFN‐β level (K, n = 4) were monitored for the indicated times.

Data information: All data are representative of at least three biologically independent experiments. The data are presented as the means ± SDs. The significance of differences was determined by t‐tests. *P < 0.05, **P < 0.01, and ***P < 0.005.

Figure EV1. XAF1 deficiency in myeloid cells had no effect on the development of peripheral immune cells

1. A

   Genotyping PCR of WT (XAF1^fl/flLyz2^+/+) and myeloid‐conditional XAF1‐KO (XAF1^MKO, XAF1^fl/flLyz2^+/Cre) mice.

2. B

   qRT–PCR analysis showing clear ablation of XAF1 in the BMDMs of XAF1^MKO mice.

3. C, D

   Flow cytometry analysis of macrophages (CD11b⁺F4/80⁺) and neutrophils (CD11b⁺Ly6G⁺) in bone marrow (BM) from 6‐ to 8‐week‐old WT and XAF1^MKO mice (n = 3).

4. E, F

   Flow cytometry analysis of the frequencies of distinct immune cells in the spleen (left), naïve and memory T cells (middle), and Treg cells (right) from WT or XAF1^MKO mice (n = 3).

5. G

   Volcano plot illustrating the upregulated and downregulated DEGs in XAF1‐deficient BMDMs compared with WT littermates after 6 h of LPS stimulation. The KEGG analysis showed that the upregulated DEGs were enriched in biological processes.

6. H

   qRT–PCR analysis of ARG1, MRC1, and YM1 in IL‐4‐treated M2 macrophages in WT and XAF1‐deficient BMDMs.

Data information: All data are representative of at least three biologically independent experiments. Data from the qPCR assay are presented as the fold change relative to the Actin mRNA level. Data are represented as the means ± SDs. The significance of differences was determined by a t‐test. ns, not significant.

Figure 4. XAF1 regulates IRF7 stability

1. A, B

   Virus‐induced phosphorylation of TBK1 and IRF3 in cytoplasmic (CE) and nuclear (NE) extracts of WT and XAF1‐deficient BMDMs was measured by IB.

2. C

   Monomeric and dimeric IRF3 expression (top blot) in whole‐cell lysates of WT and XAF1‐deficient BMDMs stimulated with VSV‐WT (MOI = 1) was measured by IB.

3. D

   ChIP assays were performed, and the results were quantified by qRT–PCR to detect IRF3 binding to an ISRE in WT and XAF1‐deficient BMDMs activated by VSV‐WT (MOI = 1) for 9 h.

4. E

   IB was performed to determine the VSV‐induced phosphorylation level of STAT1.

5. F

   HEK293T cells were transfected with IFN‐β luciferase reporters in the presence (+) or absence (−) of the indicated XAF1 expression plasmids. Luciferase assays were used to determine the fold change in expression with respect to the empty vector group 36 h after transfection.

6. G

   qRT–PCR analysis of Irf7 mRNA in WT and XAF1‐deficient BMDMs stimulated with distinct virus infections.

7. H, I

   IB analysis of VSV‐ or HSV‐1‐induced IRF7 expression as measured in whole‐cell lysates of WT and XAF1‐deficient BMDMs.

8. J

   ChIP assays were performed and quantified by qRT–PCR to detect IRF7 binding to an ISRE in WT and XAF1‐deficient BMDMs stimulated with VSV‐WT (MOI = 1) or HSV‐1.

9. K–N

   WT and XAF1^MKO mice crossed with mice with an IRF7^−/− background were i.n. infected with a sublethal dose (0.1 hyaluronic acid [HA]) of the H1N1 strain PR8. The survival rate (K, n = 15), viral titer in the lung (L, n = 5), serum IFN‐β level (M, n = 5), and intensity of H&E‐stained lung tissue sections (N, n = 5) were determined at the indicated time points. Scale bar, 200 μm.

Data information: All data are representative of at least three biologically independent experiments. Data from the qPCR assay are presented as the fold change relative to the Actin mRNA level. The data are presented as the means ± SDs. The significance of differences was determined by t‐tests. *P < 0.05, **P < 0.01, and ***P < 0.005. Please see Appendix Fig S2 for information regarding replicates, quantification, and statistical evaluation for biochemical data in this figure. Source data are available online for this figure.

Figure EV2. No difference in the colitis model or activation of the MAPK and NF‐κB pathways was observed between WT and XAF1^MKO mice

1. A, B

   Body weight loss (A) and stool diarrhea and hematochezia scores (B) in WT and XAF1^MKO mice of the DSS‐induced colitis model.

2. C, D

   Colon length (C) and proinflammatory cytokine production (D) of DSS‐treated WT and XAF1^MKO mice on day 8.

3. E, F

   The indicated proteins in cytoplasmic (CE) and nuclear extracts (NE) of WT and XAF1‐deficient BMDMs were detected by IB.

4. G

   The activation of MAPKs in total cell lysates was measured by IB.

Data information: The data are representative of at least three biologically independent experiments. Data from the qPCR assay are presented as the fold change relative to the Actin mRNA level. Data are represented as the means ± SDs. The significance of differences was determined by a t‐test. Source data are available online for this figure.

Figure 5. XAF1 promotes the ubiquitination of IRF7

1. A

   After treatment with CQ or MG132, IRF7 in nuclear extracts of WT and XAF1‐deficient BMDMs was detected by IB.

2. B, C

   WT and XAF1‐deficient BMDMs were infected with VSV‐WT (MOI = 1) and treated with MG132 for 2 h before harvest. Whole‐cell lysates (WLs) were subjected to IP using an anti‐IRF7 antibody, and the indicated proteins were detected by IB.

3. D

   HEK293T cells were cotransfected with XAF1 and distinct IRF‐expressing plasmids. IB HA was performed followed by IP with an anti‐FLAG antibody.

4. E

   The interaction between XAF1 and IRF7 was assessed in WT BMDMs stimulated by VSV‐WT (MOI = 1). WLs were subjected to IP using an anti‐IRF7 or anti‐IgG antibody and then to IB and detected with anti‐XAF1 antibody.

5. F

   The associations between XAF1 and various IRF7 truncation mutants were detected through IP and IB of the transfected HEK293T cells.

6. G, H

   The associations between IRF7 and various XAF1 truncation mutants were detected through IP and IB.

7. I

   qRT–PCR analysis of the indicated genes in XAF1‐deficient MEFs expressing XAF1^WT and XAF1^ΔZF6 and subjected to the indicated virus stimulation.

Data information: Data from the qPCR assay are presented as the fold change relative to the Actin mRNA level. The data are presented as the means ± SDs and are representative of at least three biologically independent experiments. The statistical analyses revealed variations among the experimental replicates. Two‐tailed unpaired t‐tests were performed. *P < 0.05. Please see Appendix Fig S3 for information regarding replicates, quantification, and statistical evaluation for biochemical data in this figure. Source data are available online for this figure.

Figure 6. XIAP enhances IRF7 stability and promotes IFN‐I induction

1. A

   Model diagram showing the potential regulatory mechanisms involving XAF1, XIAP, and IRF7.

2. B

   qRT–PCR analysis of XIAP expression in WT and XAF1‐deficient BMDMs stimulated with IFN‐β.

3. C

   HEK293T cells were transfected with IFN‐β luciferase reporters in the presence (+) or absence (−) of the indicated XIAP expression plasmids. Luciferase assays were performed, and the results are reported as fold changes with respect to the empty vector group 36 h after transfection.

4. D, E

   qRT–PCR analysis of IFN‐I expression in WT or XIAP^Tg BMDMs stimulated with distinct viruses (D) or TLR agonists (E).

5. F, G

   The virus‐induced expression of IRF7 in WT and XIAP^Tg BMDMs was measured by IB.

6. H

   WT and XIAP^Tg BMDMs were infected with VSV‐WT (MOI = 1) and treated with MG132 2 h before harvest. Whole‐cell lysates were subjected to IP using an anti‐IRF7 antibody.

7. I

   HEK293T cells were transfected with FLAG‐tagged ubiquitin and the indicated expression plasmids. The ubiquitination level of IRF7 was determined by IB, and total cell lysates were also subjected to direct IB.

8. J

   HEK293T cells were cotransfected with XAF1 and distinct IRF‐expressing plasmids. IBHA was performed, followed by IP with an anti‐FLAG antibody.

9. K

   The interaction between XIAP and IRF7 was assessed in XIAP‐overexpressing MEFs activated by SeV. Whole‐cell lysates (WLs) were subjected to IP using an anti‐FLAG‐XIAP or anti‐IgG antibody and then subjected to IB and detected with the anti‐IRF7 antibody.

10. L

    The associations between IRF7 and various XIAP truncation mutants were detected through IP and IB.

Data information: The data are representative of at least three biologically independent experiments. Data from the qPCR assay are presented as the fold change relative to the Actin mRNA level. The data are presented as the means ± SDs. The significance of a difference was determined by t‐tests. *P < 0.05, **P < 0.01 and ***P < 0.005. Please see Appendix Fig S4 for information regarding replicates, quantification, and statistical evaluation for biochemical data in this figure. Source data are available online for this figure.

Figure EV3. XIAP is not required for the development of peripheral immune cells

1. A–F

   Flow cytometry analysis of the absolute numbers of different immune cells in the spleen (A, B, upper), inguinal lymph nodes (C, D, middle), and thymus (E, F, lower) from WT and XAF1^MKO mice (n = 3).

2. G, H

   Flow cytometry analysis of macrophages (CD11b⁺F4/80⁺) and neutrophils (CD11b⁺Ly6G⁺) in bone marrow (BM) from 6‐ to 8‐week‐old WT and XAF1^MKO mice (n = 3).

Data information: The data are representative of at least three biologically independent experiments. Data are represented as the means ± SDs. The significance of differences was determined by t‐tests. **P < 0.01.

Figure 7. CUL3‐KLHL22 directly promotes the ubiquitination and degradation of IRF7

1. A

   The list of ubiquitination (Ub)‐related proteins that interact with IRF7 according to the MS results.

2. B

   HEK293T cells were transfected with IRF7 and the indicated ubiquitin‐related expression plasmid. IB was performed to detect FLAG, followed by IP with an anti‐hyaluronic acid (HA) antibody.

3. C, D

   HEK293T cells were transfected with IFN‐β luciferase reporters and the indicated expression plasmids. Luciferase assays were performed to determine the fold changes with respect to the empty vector group.

4. E

   qRT–PCR analysis of IFN‐I in WT and KLHL22‐KO MEFs stimulated with polyI:C or SeV. The data are presented as the fold change relative to the Actb mRNA level.

5. F

   Immunoblot analysis of SeV‐induced IRF7 in whole‐cell lysates (WLs) of WT or KLHL22‐KO MEFs.

6. G

   After treatment with MG132, IRF7 was isolated by IP from WLs of KLHL22‐KO MEFs and subjected to IB using anti‐K48‐ubiquitin. Total cell lysates were also subjected to direct IB.

7. H

   HEK293T cells were transfected with IRF7 and FLAG‐K48R‐ubiquitin or FLAG‐ubiquitin in the presence (+) or absence (−) of KLHL22 expression plasmids. HA‐tagged IRF7 was isolated by IP, and the ubiquitination level was then measured by IB.

8. I

   HEK293T cells were transfected with IRF7 and distinct KLHL22 truncation mutants. IB of FLAG was performed, followed by IP with an anti‐HA antibody.

9. J

   HEK293T cells were transfected with KLHL22, FLAG‐K48‐ubiquitin, and various IRF7 point mutants. After treatment with MG132, IB of K48‐Ub was performed, followed by IP with an anti‐HA antibody.

10. K

    HEK293T cells were transfected with IFN‐α4 luciferase reporters and the indicated expression plasmids. Luciferase assays were performed to determine fold changes with respect to the empty vector group.

Data information: The data are representative of at least three biologically independent experiments. Data from the qPCR assay are presented as the fold change relative to the Actin mRNA level. The data are presented as the means ± SDs. The significance of differences was determined by t‐test. *P < 0.05, **P < 0.01, and ***P < 0.005. Please see Appendix Fig S5 for information regarding replicates, quantification, and statistical evaluation for biochemical data in this figure. Source data are available online for this figure.

Figure EV4. KLHL22 mediates K48‐linked ubiquitination of IRF7 and promotes its degradation

1. A

   HEK293T cells were transfected with IRF7 and the indicated expression plasmids. After treatment with MG132, the indicated proteins were detected by IB.

2. B, C

   IB and qRT–PCR analysis showing specific ablation of KLHL22 in KLHL22‐KO MEFs generated by CRISPR.

3. D

   After treatment with MG132, IRF7 was isolated by IP from whole‐cell lysates (WLs) of KLHL22‐KO MEFs and subjected to IB using anti‐ubiquitin. Total cell lysates were also subjected to direct IB.

4. E

   HEK293T cells were transfected with IRF7 and FLAG‐K48‐ubiquitin in the presence (+) or absence (−) of KLHL22 expression plasmids. After pretreatment with MG132 or CQ, HA‐tagged IRF7 was isolated by IP, and the ubiquitination level was then measured by IB.

5. F

   HEK293T cells were transfected with multiple ubiquitin mutants (mutations at K6, K11, K27, K29, K33, K48, and K63) and the indicated expression plasmids. HA‐tagged IRF7 was isolated by IP, and the ubiquitination level was then measured by IB.

6. G, H

   HEK293T cells were transfected with HA‐tagged IRF7 and the inactive mutant variants (S477 and 479A) whose phosphorylation sites at serine 477 and 479 were mutated from serine to alanine. HA‐tagged IRF7 was isolated by IP, and the ubiquitination level was then measured by IB.

Data information: Data from the qPCR assay are presented as the fold change relative to the Actin mRNA level. Data are represented as the means ± SDs. The data are representative of at least three biologically independent experiments. Please see Appendix Fig S7 for information regarding replicates, quantification, and statistical evaluation for biochemical data in this figure. Source data are available online for this figure.

Figure EV5. KLHL22 interacts with IRF7 and promotes its ubiquitination

1. Purification of recombinant proteins was performed by Coomassie blue staining and IB.

2. CUL3‐KLHL22 E3 ligase catalyzed IRF7 ubiquitination in a cell‐free system. Ubiquitin and recombinant FLAG‐IRF7 were incubated with recombinant CUL3–RBX1 and KLHL22 or KLHL22‐Δ6xKelch.

3. The associations between KLHL22 and various truncations of IRF7 were detected through the indicated IP and IB analyses.

4. HEK293T cells were transfected with KLHL22 and IRF7 variants. IB of FLAG was performed followed by IP with an anti‐HA antibody.

5. HEK293T cells were transfected with KLHL22, FLAG‐ubiquitin, and various IRF7 point mutants. IB of FLAG was performed, followed by IP with an anti‐HA antibody. The data are representative of at least three biologically independent experiments.

Data information: Please see Appendix Fig S8 for information regarding replicates, quantification, and statistical evaluation for biochemical data in this figure. Source data are available online for this figure.

Figure 8. XIAP induces the ubiquitination of KLHL22

1. HEK293T cells were transfected with FLAG‐tagged ubiquitin and the indicated expression plasmids. The ubiquitination levels of IRF7 were measured by IB. Cell lysates were subjected to direct IB.

2. HEK293T cells were transfected with KLHL22 in the presence (+) or absence (−) of XIAP. IB of FLAG was performed followed by IP with an anti‐hyaluronic acid (HA) antibody.

3. IB of VSV‐induced KLHL22 expression in whole‐cell lysates (WLs) of WT and Xiap ^Tg mice.

4. After treatment with MG132, KLHL22 was isolated by IP from WLs of WT and Xiap ^Tg mice and subjected to IB using an anti‐ubiquitin or anti‐K48‐ubiquitin antibody. Protein lysates were also subjected to direct IB.

5. IB showed the ablation of KLHL22 and the overexpression of FLAG‐XIAP in KLHL22‐KO MEFs expressing XIAP (KLHL22^−/−XIAP^OE).

6. qRT–PCR analysis of IFN‐I in WT and KLHL22‐KO MEFs expressing XIAP and subjected to SeV stimulation.

Data information: The data are representative of at least three biologically independent experiments. Data from the qPCR assay are presented as the fold change relative to the Actin mRNA level. The data are presented as the means ± SDs. The significance of differences was determined by t‐tests. *P < 0.05. Please see Appendix Fig S6 for information regarding replicates, quantification, and statistical evaluation for biochemical data in this figure. Source data are available online for this figure.