MITD1 is a brain-specific interferon-inducible factor that inhibits flavivirus replication

Abstract

West Nile virus (WNV) and Usutu virus (USUV) are closely related mosquito-borne neurotropic flaviviruses that share common transmission cycle and can infect humans. However, while human infections by WNV are widespread, infections by USUV are comparatively less frequent, less severe, and currently limited to Africa and Europe. To identify human host factors that contribute to the pathogenic signatures of these two flaviviruses, we carried out an arrayed expression screen of over 1,300 interferon-stimulated genes (ISGs). Several ISGs known to target flaviviruses, including IFI6, SHFL, and RTP4 were among the strongest hits. Interestingly, we also found MITD1, an ISG with no previously reported antiviral activity, among the strongest hits. We demonstrated that the antiviral activity of MITD1 was not limited to USUV and WNV, since it also inhibited Zika and dengue virus replication. We found MITD1 to interfere with viral RNA replication by sequestering specific endosomal sorting complexes required for transport-III (ESCRT-III) proteins involved in the formation of viral replication factories. MITD1 expression was not increased by type I interferon (IFN-I) in most human cells and mouse tissues that we examined, although WNV and USUV replication was strongly inhibited by IFN-I. Strikingly, MITD1 was induced in the brain of USUV-infected mice and importantly, in human monocyte-derived microglia. Using human microglial-like cells, we confirmed that MITD1 is an essential mediator of the anti-flavivirus activity of IFN-I in these cells. We conclude that MITD1 plays a key role in the cellular defenses against neurotropic flaviviruses.

Keywords: flavivirus; innate immune response; interferon-stimulated genes; microglial cells.

Fig. 1.

ISG arrayed expression screen identifies MITD1 as a restriction factor for flaviviruses. (A–C) A549 cells were infected with USUV or WNV (MOI of 0.1 or 1) for 24 h. (A) IFN-β expression was assessed by RT-qPCR. (B) Secretion of IFN-I was quantified using STING-37 reporter cells. (C) MX1 expression was assessed by RT-qPCR. (D) A549 cells were treated with increasing concentration of IFN-I for 16 h, and infected with USUV or WNV (MOI of 0.1) for 24 h. Viral replication was assessed by RT-qPCR. (E) Venn diagram of the lSG libraries used in the screen, schematic of the SCRPSY lentiviral vector used for ISG delivery, and schematic of the flow cytometry screening strategy. (F) Normalized USUV or WNV infection (mean centered) of HEK 293T cells expressing ISGs. Each dot represents infection with a single ISG. The screen was performed once. (G) Confirmation screen of ISGs of interest, each dot representing the mean of two experiments. Gray areas indicate the 95% confidence (dark) and prediction (light) bands. (H and I) Generation of stable empty vector or bovine MITD1-expressing Vero E6 cells. (H) TagRFP expression was assessed by flow cytometry. (I) MITD1 expression was assessed by western blot. (J) Stable Vero E6 cells were infected with the indicated flaviviruses (MOI of 1) for 48 h; normalized infection (control centered) was assessed by flow cytometry. (K) Validation of transient expression of bovine and human MITD1 orthologs in HEK 293T cells at 24 h posttransduction, assessed by western blot. (L and M) E2 crimson, bovine or human MITD1-expressing HEK 293T cells were infected with USUV or WNV (MOI of 1) for 48 h. (L) Normalized infection (control centered), assessed by flow cytometry. (M) Viral production, assessed by TCID₅₀ titration. Data are presented as biological replicates and mean ± SD. ***P ≤ 0.001; **P ≤ 0.01; ns, P > 0.05. Statistical analyses: one-way ANOVA with Tukey’s multiple comparisons test (A–D, L, and M) and two-tailed unpaired t tests (J). Data were log-transformed prior to analysis (A–D). Abbreviations: NI, not infected; NT, not treated; EV, empty vector; WT, wild type; E2C, E2 Crimson; PAC, puromycin N-acetyl transferase.

Fig. 2.

MITD1 blocks flavivirus replication by an indirect mechanism. (A–C) Vero E6 cells stably expressing human MITD1 were infected with WNV (MOI of 20) for attachment and internalization assays. (A) Schematic of the assay. (B) WNV binding to MITD1-expressing cells, assessed by RT-qPCR; heparin at 200 µg/mL was used as control. (C) WNV entry into MITD1-expressing cells, assessed by RT-qPCR; PitStop2 at 50 µM was used as control. (D) Schematic of WNVII Rep genome replicon used to generate RVPs. (E) Stable MITD1-expressing Vero E6 cells were infected with WNV RVPs (MOI of 1) for 48 h. Normalized replication (eGFP fluorescence, control centered) was assessed by flow cytometry; stable IFI6, SHFL, and RTP4-expressing cells were used as controls. (F) A549 cells were transduced with 1 × TCID₅₀ of lentivectors expressing Myc-tagged MITD1 for 24 h, then infected with WNV (MOI of 5) for 24 h. The viral replication factories (dsRNA) in transduced cells or not were assessed by confocal microscopy and quantified. (Scale bar: 10 µm). (G) A549 cells were infected with WNV (MOI of 1) for 24 h. The colocalization of endogenous MITD1 with viral replication factories (dsRNA) was assessed by confocal microscopy and estimated by calculating a Pearson’s correlation score. (Scale bar: 5 µm). Data are presented as biological replicates and mean ± SD. ***P ≤ 0.001; ns, P > 0.05. Statistical analyses: one-way ANOVA followed by Tukey’s multiple comparisons test (B, C, and E), two-tailed unpaired t test (F), and Pearson correlation score (r) calculated on linear (15 µm each) measurements of gray values (G). Abbreviations: NT, not transduced; EV, empty vector; vRNA, viral RNA; RVP, reporter viral particle.

Fig. 3.

MITD1 binds to and sequesters CHMP1B and CHMP4B within lipid-rich regions. (A–E) A549 cells were transfected with Myc-tagged MITD1 for 24 h. (A) Differential localization of ESCRT-III component proteins ALIX, CHMP1B, CHMP2A, and CHMP4B in transfected cells or not; assessed by confocal microscopy. MITD1 expression is outlined in black on the single channel fields. (Scale bar: 10 µm). (B) ESCRT-III component protein foci size alteration in MITD1-expressing cells was measured. (C) Schematic of the functional domains of MITD1. (D) Fate of CHMP1B aggregates in MITD1-expressing cells, compared to Nile Red phospholipid staining (561 nm excitation; 640/20 nm emission); assessed by confocal microscopy. Arrowheads indicate CHMP1B aggregates on the single channel fields. Cross-section gray value analyses corresponds to the yellow line. (Scale bar: 5 µm). (E) Fate of CHMP4B aggregates in MITD1-expressing cells, compared to Nile Red lipid droplet staining (514 nm excitation; 583/20 nm emission); assessed by confocal microscopy. Zooms show representative images of lipid-filled “f” and empty “e” aggregates. Cross-section analysis corresponds to the yellow line. (Scale bar: 5 µm). (F) Soluble ESCRT-III component proteins availability after MITD1 expression in HEK 293T cells, assessed by western blot. (G) Schematic of the Myc-tagged MITD1 individual domain constructs. (H) HEK 293T cells were cotransfected with Myc-tagged MIT domain of MITD1 and either GFP or FLAG-tagged CHMP1B, CHMP2A, and CHMP4B for 24 h. The interaction of ESCRT-III component protein with MIT domain of MITD1 was tested by immunoprecipitation with Myc, FLAG, GFP, or HA-Trap beads, and assessed by western blot. (I) HEK 293T cells were transfected with full-length or truncated Myc-tagged MITD1 constructs for 24 h. CHMP1B, CHMP2A, and CHMP4B foci size alteration in transfected cells was assessed by confocal microscopy and measured. (J) HEK 293T cells were transduced with lentivectors expressing full-length or truncated Myc-tagged MITD1 constructs for 24 h, then infected with WNV (MOI of 1) for 48 h. Normalized infection (control centered) was assessed by flow cytometry. Data are presented as biological replicates and mean ± SD. ***P ≤ 0.001; **P ≤ 0.01; ns, P > 0.05. Statistical analyses: two-tailed unpaired t tests (B) and one-way ANOVA followed by Tukey’s multiple comparisons test (H and I). Abbreviations: NT, not transfected; EV, empty vector; NR, Nile Red; C1B, CHMP1B; C2A, CHMP2A; C4B, CHMP4B.

Fig. 4.

Dysregulation of CHMP1B and CHMP4B blocks flavivirus replication. (A and B) A549 cells were infected with WNV (MOI of 5) for 24 h. (A) The interaction of dsRNA with CHMP1B, CHMP2A, CHMP4B, and MITD1 was tested with a proximity ligation assay (PLA) and assessed by confocal microscopy. (Scale bar: 10 µm). (B) PLA foci were quantified. (C–G) A549 cells were transfected with siRNAs targeting PPIB (as a control), CHMP1B, CHMP2A, or CHMP4B (alone or two together) for 24 h, then infected with WNV (MOI of 2) for 24 h. (C) Validation of single CHMP knockdowns, assessed by RT-qPCR and western blot. (D) Normalized viral RNA level (control centered) in single CHMP knockdown A549 cells infected with WNV, assessed by RT-qPCR. (E) Normalized viral RNA level (control centered) in double CHMP knockdown A549 cells infected with WNV, assessed by RT-qPCR. (F) Viral replication factories (dsRNA), assessed by confocal microscopy. (Scale bar: 10 µm). (G) The number of viral replication factories (dsRNA) per cell was quantified. (H) A549 cells were transfected with siRNAs targeting PPIB (as a control), CHMP1B, CHMP2A, and CHMP4B alone, or coupled CHMP1B/CHMP4B for 24 h, then transduced with lentivectors expressing MITD1 for 24 h, then infected with WNV (MOI of 1) for 24 h. Normalized viral RNA level (control centered) was assessed by RT-qPCR. Data are presented as biological replicates and mean ± SD. ***P ≤ 0.001; **P ≤ 0.01; ns, P > 0.05. Statistical analyses: two-tailed unpaired t tests (C), one-way ANOVA followed by Tukey’s multiple comparisons test (D, E, and G), and two-way ANOVA followed by Šidák’s multiple comparisons test (H). Abbreviations: NI, not infected; NT, not transduced; KD, knockdown; vRNA, viral RNA; C1B, CHMP1B; C2A, CHMP2A; C4B, CHMP4B.

Fig. 5.

MITD1 is a brain-specific ISG specifically induced in microglial cells. (A) A549, HT1080, Huh7, primary monocytes, MDDCs, and MDMs were treated with 100 UI/mL IFN-I for 16 h. Expression of the indicated genes was assessed by RT-qPCR. Genes are ordered from lower to higher mean induction. (B) Schematic of the mouse experiment pipeline with harvested organs. (C) Viral replication and expression of IFN-β and MITD1 transcripts in organs from control or USUV-infected mice, assessed by RT-qPCR. (D) Schematic representation of the simplified cell composition of the brain. (E) hNGCs, astrocytes, MDMis, and peripheral MDMs were treated with 100 UI/mL IFN-I for 8 h. MX1, ISG15, and MITD1 expression was assessed by RT-qPCR. Data are presented as biological replicates and mean ± SD. ***P ≤ 0.001; *P ≤ 0.05; ns, P > 0.05. Statistical analyses: two-way ANOVA followed by Šidák’s multiple comparisons test (E and C). Abbreviations: NI, not infected; NT, not treated.

Fig. 6.

MITD1 blocks flavivirus replication by sequestering CHMP1B and CHMP4B away from replication factories. (A) HMC3 cells were transfected with Myc-tagged MITD1 for 24 h. Differential localization of CHMP1B, CHMP2A, and CHMP4B in transfected cells or not was assessed by confocal microscopy. MITD1 expression is outlined in black on the single channel fields. Arrowheads indicate colocalization of MITD1 and CHMP1B. Fate of CHMP4B was further compared to Nile Red lipid droplet staining (514 nm excitation; 583/20 nm emission). (Scale bar: 10 µm). (B) MDMis were infected with WNV (MOI of 2) for 24 h. The interaction of CHMP1B, CHMP2A, and CHMP4B with MITD1 was tested with a proximity ligation assay (PLA) and assessed by confocal microscopy. Arrowheads indicate foci of MITD1 and CHMP1B condensation. (Scale bar: 10 µm). (C) PLA foci were quantified. (D) MDMis were transduced with lentivectors expressing MITD1 for 24 h, then infected with WNV (MOI of 2) for 24 h. The interaction of CHMP1B, CHMP2A, and CHMP4B with viral replication factories (dsRNA) was tested by PLA, assessed by confocal microscopy and quantified. (E–G) MDMis were transfected with siRNAs targeting MITD1 or SHFL for 24 h, and treated with 100 UI/mL IFN-I for 16 h. (E) Validation of MITD1 and SHFL knockdowns, assessed by RT-qPCR. (F) Control or IFN-I-treated MITD1^KD and SHFL^KD MDMis were infected with WNV (MOI of 1) for 24 h. Normalized viral replication (control centered) was assessed by RT-qPCR. (G) Viral production of IFN-I treated MITD1^KD and SHFL^KD MDMis, assessed by TCID₅₀ titration. Data are presented as biological replicates and mean ± SD. ***P ≤ 0.001; **P ≤ 0.01; *P ≤ 0.05; ns, P > 0.05. Statistical analyses: two-tailed unpaired t tests (C and E), one-way ANOVA followed by Tukey’s multiple comparisons test (D and G), and two-way ANOVA followed by Šidák’s multiple comparisons test (F). Data were log-transformed prior to analysis (F). Abbreviations: NI, not infected; NT, not treated; KD, knockdown; vRNA, viral RNA; C1B, CHMP1B; C2A, CHMP2A; C4B, CHMP4B.