MHC class II transactivator CIITA induces cell resistance to Ebola virus and SARS-like coronaviruses

Abstract

Recent outbreaks of Ebola virus (EBOV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have exposed our limited therapeutic options for such diseases and our poor understanding of the cellular mechanisms that block viral infections. Using a transposon-mediated gene-activation screen in human cells, we identify that the major histocompatibility complex (MHC) class II transactivator (CIITA) has antiviral activity against EBOV. CIITA induces resistance by activating expression of the p41 isoform of invariant chain CD74, which inhibits viral entry by blocking cathepsin-mediated processing of the Ebola glycoprotein. We further show that CD74 p41 can block the endosomal entry pathway of coronaviruses, including SARS-CoV-2. These data therefore implicate CIITA and CD74 in host defense against a range of viruses, and they identify an additional function of these proteins beyond their canonical roles in antigen presentation.

Fig. 1. Transposon-mediated activation tagging generates mutant cells resistant to Ebola.

(A) Modified PB transposon. SV-Puro-pA, puromycin selection cassette; CMV, cytomegalovirus promoter; SD, splice donor. (B and C) Resistance of selected cells to EboGP-VSV (B) and VSVg-VSV (C). Data are means ± SD of n = 3 replicates for one representative pool. Student’s t test; **P < 0.01. (D) Distribution of transposon insertions. Inner rings show insertions per 1 Mb for individual libraries (black histograms) and CISs (P < 10⁻⁷). Outer ring shows combined insertions for all libraries (black histogram) and lowest P value for CISs (red bubble plot). Point size represents the number of libraries with the CIS. freq, frequency. (E and F) Cumulative independent insertions from all eight libraries mapping to NPC1 (E) and CIITA (F).

Fig. 2. Identification of CIITA as an Ebola restriction factor.

(A) Resistance of CIITA-overexpressing and control (Cntrl) U2OS cells EboGP-VSV. MOI, multiplicity of infection. (B and C) Plaque formation assays (B) and effective viral titer (C) for control and CIITA-overexpressing U2OS cells infected with VSVg-VSV (VSV) and EboGP-VSV (Ebo). undil, undiluted; PFU, plaque-forming units. (D) Representative images of CIITA-transfected (CIITA), control-transfected (Cntrl), and unmanipulated U2OS cells (U2OS) infected with mCherry-expressing EboGP-VSV (red) and stained with Hoechst 33342 to resolve cell nuclei (blue). (E to G) Infection of control and CIITA-expressing U2OS cells by recombinant VSV pseudotyped with EboGP, LFVGP (Lassa virus GP), or VSVg (E); single cycle murine leukemia virus (MLV) pseudotyped with VSVg and EboGP (F); or single cycle HIV pseudotyped with VSVg or GP from EBOV, Taï Forest virus (TAFV), Bundibugyo virus (BDBV), Sudan virus (SUDV), Reston virus (RESTV), or Marburg virus (MARV) (G). (H and I) Internalization (H) and fusion (I) of EboGP-VLPs by control and CIITA-overexpressing U2OS cells. No env, nonenveloped control VLPs. (J to M) Infection of control and CIITA-overexpressing U2OS cells by infectious EBOV measured by imaging of GFP reporter (green) and cell nuclei (blue) (J), cell survival (K), infected cells (L), or plaque formation (M). Data are means ± SEM of three independent experiments [(A) to (I)] or experiments with three independent cell clones [(K) to (M)]. Student’s t test [(A), (C), and (K) to (M)] or analysis of variance (ANOVA) with Tukey’s multiple comparison test [(E) to (I)]; *P < 0.05; **P < 0.01; ND, not detected. Scale bars, 100 μm.

Fig. 3. Transcriptional activity of CIITA and enhanceosome components are required for resistance.

(A) Genes regulated by CIITA in U2OS cells, with strongest induced genes identified. Mean of three independent CIITA-expressing clones and controls. (B) EboGP-VSV infection of CIITA-expressing cells treated with small interfering RNA (siRNA) against CIITA transcriptional targets. Data are from two siRNAs per gene, N = 3 independent screens, and bars indicate means with 95% confidence intervals (CIs). One-way ANOVA with Bonferroni’s multiple comparisons; *P < 0.05; **P < 0.01. Dotted lines indicate 99% CIs from no siRNA control. (C) CD74 CRISPR-targeting in CIITA-overexpressing U2OS cells was verified by immunoblot, and infection and survival were measured after EboGP-VSV challenge. Data are means ± SEM of N = 3 experiments using two independent cell clones. (D and E) Ciita and Cd74 expression in wild-type (wt) or Ciita^−/− mouse BMDMs with or without priming by IFN-γ and LPS. ko, knockout; NS, not significant. (F and G) Fusion of EboGP-VLPs in unprimed (F) or primed (G) mouse BMDM from Ciita^−/− and Cd74^−/− mice, measured as geometric mean fluorescence (GMFI) of cleaved CCF2. BLAM, β-lactamase. Data are means ± SEM for independent cultures from three mice per group. Student’s t test; *P < 0.05; **P < 0.01. Similar results were observed in three independent experiments.

Fig. 4. CD74 p41 inhibits cathepsin-mediated cleavage of EboGP.

(A) Human CD74 isoforms with ER retention signal (ER), CLIP, acidic, and p41 thyroglobulin (Thyro) domains. (B and C) EboGP-VSV infection and survival of Cd74^−/− CIITA-expressing (B) or wt (C) U2OS cells expressing CD74 isoforms. (D) EboGP-VLP fusion in THP-1 macrophage-like cells expressing CD74 p33 and p41. (E) EboGP-VSV infection of U2OS cells expressing CD74 mutant constructs. Cyto, cytoplasmic domain; TM, transmembrane domain; Thyro, thyroglobulin domain; CT del, carboxy-terminus deletion; No TM, deletion of the transmembrane sequence. (F) Transmission electron micrographs of control, CIITA-expressing, and CD74-expressing U2OS cells 3 hours after infection with EboGP-VSV. Dotted-line regions are enlarged in adjacent panels (as indicated by white arrows). Intraluminal vesicles (black arrowheads) and internalized EboGP-VSV (black arrows) are marked. Scale bars, 1 μm (left, center left, and right panels) and 200 nm (center right panels). (G) Confocal microscopy of control and p41-expressing U2OS cells showing EBOV-VLP (red), CD63, or Hrs (green), and nuclei (white). Scale bars, 10 μm. (H) VLPs associated with CD63 endosomes in U2OS cells expressing CIITA and CD74 as indicated. Each point represents a single cell, mean ± SD n ≥ 9. Mann-Whitney U test; **P < 0.01. Similar results were seen in three independent experiments. (I) Immunoblot of EboGP in EboGP-VSV–infected U2OS cells. EboGP-VSV preparation ± thermolysin (Therm) is shown for reference (left). Cells were treated with cathepsin inhibitors (Cat. inhib) E64D (E) or FYDMK (F), or expressed CIITA and CD74. EboGP in virus particles (arrow), after proteolysis (closed arrowhead), and after partial cleavage (open arrowhead) are indicated. (J) EboGP-VSV infection of U2OS cells expressing p41 with CTSL binding site mutations. (K) Infection of control, p33-, or p41-expressing U2OS cells by HIV-GFP pseudotyped with GPs from VSV, EBOV, SARS-CoV, or WIV1-CoV, measured as focus-forming units per milliliter of virus (FFU/ml). (L) Infection of control, p33-, or p41-expressing Vero cells by SARS-CoV-2, showing representative crystal violet-stained monolayers and infection measured as plaque-forming units per milliliter of virus (PFU/ml). Except where indicated, data are means ± SEM of data from ≥3 independent experiments. Student’s t test with Benjamini correction; *P < 0.05; **P < 0.01.