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. 2021 Jun 8;118(23):e2024202118.
doi: 10.1073/pnas.2024202118.

The ORF8 protein of SARS-CoV-2 mediates immune evasion through down-regulating MHC-Ι

Yiwen Zhang  1 , Yingshi Chen  1 , Yuzhuang Li  1 , Feng Huang  2 , Baohong Luo  1 , Yaochang Yuan  1 , Baijin Xia  1 , Xiancai Ma  1 , Tao Yang  1 , Fei Yu  1 , Jun Liu  1 , Bingfeng Liu  1 , Zheng Song  1 , Jingliang Chen  1 , Shumei Yan  1 , Liyang Wu  1 , Ting Pan  1 , Xu Zhang  1 , Rong Li  1 , Wenjing Huang  3 , Xin He  1 , Fei Xiao  4 , Junsong Zhang  5 , Hui Zhang  6
Affiliations

The ORF8 protein of SARS-CoV-2 mediates immune evasion through down-regulating MHC-Ι

Yiwen Zhang et al. Proc Natl Acad Sci U S A. .

Abstract

COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic and has claimed over 2 million lives worldwide. Although the genetic sequences of SARS-CoV and SARS-CoV-2 have high homology, the clinical and pathological characteristics of COVID-19 differ significantly from those of SARS. How and whether SARS-CoV-2 evades (cellular) immune surveillance requires further elucidation. In this study, we show that SARS-CoV-2 infection leads to major histocompability complex class Ι (MHC-Ι) down-regulation both in vitro and in vivo. The viral protein encoded by open reading frame 8 (ORF8) of SARS-CoV-2, which shares the least homology with SARS-CoV among all viral proteins, directly interacts with MHC-Ι molecules and mediates their down-regulation. In ORF8-expressing cells, MHC-Ι molecules are selectively targeted for lysosomal degradation via autophagy. Thus, SARS-CoV-2-infected cells are much less sensitive to lysis by cytotoxic T lymphocytes. Because ORF8 protein impairs the antigen presentation system, inhibition of ORF8 could be a strategy to improve immune surveillance.

Keywords: MHC-Ι; ORF8; SARS-CoV-2; immune evasion.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
SARS-CoV-2 infection leads to MHC-Ι down-regulation through ORF8. (A) The ACE2-expressing HEK293T cells (HEK293T/Hace2) were infected with SARS-CoV-2 (hCoV-19/CHN/SYSU-IHV/2020) (MOI = 1). At 48 h after infection, cells were collected for flow cytometry analysis (n = 6). Mean fluorescence intensity (MFI) was normalized to uninfected group. (B) hACE2 mice were intranasally infected with 4 × 103 PFU (recovered), 4 × 104 PFU (replicating) SARS-CoV-2 virus, or uninfected as control. At day 6 after infection, total suspended cells of the lung tissue were collected for flow cytometry analysis. MFI of H2Kb+ cells (gated on EpCAM+ cells) were shown (n = 3). MFI was normalized to uninfected group. (C) Hematoxylin and eosin staining and immunohistochemistry against H2Kb were evaluated in lungs of infected mice in B. (D) Similarity plot based on the genome sequence of SARS-CoV-2_WHU01 (accession no. MN988668) and the genome sequences of SARS-CoV_BJ01 (AY278488) and SARS-CoV_GZ02 (AY390556) were used as reference sequences. The nucleotide position started from the orf3a gene of SARS-CoV-2. (E–G) The effect of different viral proteins on the expression of HLA-A2. The viral protein–expressing plasmids were transfected into HEK293T cell line, and cells were collected at 48 h after transfection for flow cytometry analysis to analyze the MFI of HLA-A2+ cells (n = 5) normalized to empty vector (EV) group. The plasmids expressing SARS-CoV-2 structural proteins and ORFs (E), SARS-CoV ORF8, ORF8b and ORF8a (F), and L and S subtype of SARS-CoV-2 ORF8 or EV (G) were used. The data were shown as mean ± SD (error bars). Student’s t test and one-way ANOVA was used. P < 0.05 indicates a statistically significance difference; *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 2.
Fig. 2.
ORF8-induced MHC-Ι down-regulation. (A and B) 3.1-GFP (negative control), ORF8-GFP, or HIV-Nef-GFP (positive control)–expressing plasmid was transfected into HEK293T cells, respectively. Cells were collected at 48 h after transfection for flow cytometry analysis. Frequency and mean fluorescence intensity (MFI) of HLA-A2+ and β2-microglobulin (β2M)+ cells (gated on GFP+ cells) were shown (n = 5). MFI was normalized to GFP group. (C) Western blot analysis for A (n = 3). (D) HEK293T/Hace2 cells were infected with SARS-CoV-2 (hCoV-19/CHN/SYSU-IHV/2020) (MOI = 0.1). At 48 h after infection, cells were collected for Western blot (n = 3). (E–G) GFP- (negative control) or ORF8-GFP–expressing plasmids were transfected into FHC, HBE, or Huh7 cells, respectively. At 48 h after transfection, cells were harvested for flow cytometry analysis (gated on GFP+ cells) (n = 3) and normalized to GFP group. The data were shown as mean ± SD (error bars). Student’s t test and one-way ANOVA was used. P < 0.05 indicates statistically significant difference; **P < 0.01; ***P < 0.001.
Fig. 3.
Fig. 3.
ORF8 knockdown restores MHC-Ι expression. (A) Schematic of the mechanism for ORF8-scFv-VIF to induce ORF8 degradation. (B) Schematic for construction of ORF8-scFv-VIF plasmid. (C) Cells were transfected with empty vector (EV), pORF8-scFv-VIF-1, or pORF8-scFv-VIF-2 in combination with ORF8-HA–expressing plasmid. At 48 h after transfection, cells were collected for Western blot (n = 3). (D) HEK293T cells transfected with or without ORF8-scFv-VIF were infected with SARS-CoV-2 (hCoV-19/CHN/SYSU-IHV/2020) (MOI = 0.1). At 48 h after infection, cells were collected for Western blot (n = 3). (E) ORF8 was co-IP with the overexpressed ORF8-scFv-VIF-1. Cells were transfected with ORF8-Scfv-1– or GFP (EV)-expressing plasmids together with ORF8-Flag– and Ubiquitin-HA–expressing plasmids and treated with MG132 (10 μM) for 12 h before harvest. Cells were collected at 48 h after transfection for co-IP with the anti–Flag-tag beads and detected with the indicated antibodies (n = 5). (F) The ACE2-expressing HEK293T cells (HEK293T/Hace2) transfected with EV or ORF8-scFv-VIF-1 were infected with SARS-CoV-2 (hCoV-19/CHN/SYSU-IHV/2020) (MOI = 1). At 48 h after infection, cells were collected for flow cytometry analysis (n = 3). Mean fluorescence intensity was normalized to uninfected group. The data were shown as mean ± SD (error bars). Student’s t test and one-way ANOVA was used. P < 0.05 indicates statistically significant difference; **P < 0.01; ***P < 0.001.
Fig. 4.
Fig. 4.
MHC-Ι is targeted for lysosomal degradation by ORF8. (A and B) GFP- (EV) or ORF8-GFP–expressing plasmid was transfected into HEK293T cells. Before harvest, cells were treated with DMSO and DBeQ (15 μM) for 4 h, MG132 (10 μM) for 4 h, and bafilomycin A1 (Baf A1, autophagy inhibitor, 100 nM) for 16 h. Cells were collected at 36 h after transfection and HLA-A2 mean fluorescence intensity was analyzed by flow cytometry (gated on GFP+ cells) and normalized to GFP (EV) group, and the total HLA-A2 protein expression was analyzed by Western blotting (n = 5). (C) Cells transfected with empty vector (EV)- or ORF8-HA–expressing plasmid were treated with Baf A1 (100 nM) for 16 h before harvest for crude lysosomal fraction. Accumulation of HLA-A2 in lysosomes was analyzed by Western blotting (n = 5). (D) Localization of HLA-A2 (red) relative to LAMP1-positive (green) lysosomes (Scale bars, 5 μm). Cells were transfected with EV- or ORF8-HA–expressing plasmid. At 24 h after transfection, colocalization was visualized by confocal microscopy (n = 20 to 40 fields). (E) Localization of HLA-A2 (red) relative to SARS-CoV-2 ORF8-HA (green) (Scale bars, 5 μm). Cells were transfected with EV- or ORF8-HA–expressing plasmid. At 16 h after transfection, colocalization was visualized by confocal microscopy (n = 14 to 20 fields). (F) ORF8 was co-IP with HLA-A2. EV- or ORF8-HA–expressing plasmid was transfected into HEK293T cells, respectively. Cells were treated with Baf A1 (100 nM) for 16 h before being collected. The cells were collected at 48 h after transfection and treated with cross-linker DSP and co-IP with the anti–HA-tag beads (n = 5). (G) ORF8 was co-IP with the overexpressed HLA-A2. Cells were transfected with HLA-A2-FLAG–expressing plasmid together with ORF8-HA–expressing plasmid or vector and treated with Baf A1 (100 nM) for 16 h before harvest. Cells were collected at 48 h after transfection for co-IP with the anti–HA-tag beads (n = 5). The data were shown as mean ± SD (error bars). t test and one-way ANOVA was used. P < 0.05 indicates statistically significant difference; ***P < 0.001.
Fig. 5.
Fig. 5.
ORF8 mediates MHC-Ι degradation through autophagy pathway. (A) Localization of SARS-CoV-2 ORF8-HA (red) relative to CALNEXIN (green, Top) or LAMP1 (green, Bottom). ORF8-HA–expressing plasmid was transfected into HEK293T cells. At 24 h after transfection, colocalization was visualized by confocal microscopy (Scale bars, 5 μm). (B) Localization of SARS-CoV-2 ORF8 (red) relative to LC3-GFP (green). ORF8-HA– and LC3-GFP–expressing plasmids were cotransfected into HEK293T cells. At 24 h after transfection, colocalization was visualized by confocal microscopy (Scale bars, 5 μm) (n = 14 to 20 fields). (C) Localization of HLA-A2 (red) relative to LC3 (green). ORF8-HA–expressing plasmids were transfected into HEK293T cells. At 24 h after transfection, colocalization was visualized by confocal microscopy (Scale bars, 5 μm) (n = 14 to 20 fields). (D) GFP- (empty vector, EV) or ORF8-GFP–expressing plasmid was transfected into HEK293T cells. Before harvest, cells were then treated with chloroquine (CQ) (50 μM) and E64d (10 μg/mL) and pepstatin A (pep) (10 μg/mL) for 6 h. The HLA-A2 mean fluorescence intensity (MFI) (gated on GFP+ cells) was normalized to GFP group (n = 5). (E) EV- or ORF8-HA–expressing plasmid was transfected into HEK293T cells. Cells were treated with Baf A1 (100 nM) for 16 h before harvest for crude lysosomal fraction. Accumulation of LC3B in lysosomes was analyzed by Western blotting. (F and G) GFP (EV)- or ORF8-GFP–expressing plasmids and the indicated siRNAs were transfected into HEK293T cells. MFI of HLA-A2 (gated on GFP+ cells) was normalized to GFP group (n = 5). (H) Localization of SARS-CoV-2 ORF8-HA (red) relative to Beclin 1-GFP (green) (Scale bars, 5 μm). ORF8-HA– and Beclin 1-GFP–expressing plasmids were cotransfected into HEK293T cells. At 16 h after transfection, colocalization was visualized by confocal microscopy (n = 14 to 20 fields). (I) ORF8 was co-IP with Beclin 1. Empty vector (EV)-, or ORF8-HA–expressing plasmid was transfected into HEK293T cells, respectively. Cells were treated with Baf A1 (100 nM) for 16 h before collected. The cells were collected at 48 h after transfection and treated with cross-linker DSP and co-IP with the anti–HA-tag beads (n = 5). (J) GFP (EV)- or ORF8-GFP–expressing plasmids were transfected into HEK293T cells (WT), or Beclin 1 knockout HEK293T cells. Cells were collected at 48 h after transfection, and HLA-A2 MFI was analyzed by flow cytometry (gated on GFP+ cells) and normalized to GFP (EV) group (n = 5). The data were shown as mean ± SD (error bars). Student’s t test and one-way ANOVA was used. P < 0.05 indicates statistically significant difference; *P < 0.05; **P < 0.01; ***P < 0.001.
Fig. 6.
Fig. 6.
ORF8-mediated resistance of SARS-CoV-2 to antiviral CTLs. (A) Frequency of SSp-1–specific CD8+ T cells (gated on CD8+ cells) generated from HLA-A2+ healthy donors (HD). (B) Killing assay using SSp-1–specific CD8+ T cells generated form HD. CTLs were cocultured with SSp-1 peptide–loaded HEK293T cells (antigen), or with HIV-gag peptide (SL9)-loaded HEK293T cells (nontarget) overnight. The ratios of dead target versus nontarget cells (antigen: nontarget) were determined by flow cytometry. (C) IFN-γ ELISpot analysis of COVID-19 recover patients (Pt) to synthetic peptides, compared to HD. (D) Killing assay using CD8+ T cells from HLA-A2+ COVID-19–recovered Pt 3. Activated CTLs were cocultured with SARS-CoV-2 peptide –loaded HEK293T cells (antigen) or with HIV-gag peptide–loaded HEK293T cells (nontarget) at effector: target ratio 8:1. The ratios of dead target versus nontarget cells (antigen: nontarget) were determined by flow cytometry (n = 5). (E) Killing assay for authentic SARS-CoV-2–infected HEK293T/Hace2 cells using SSp-1–specific CD8+ T cells generated form HD. CTLs were cocultured with infected HEK293T/Hace2 cells (antigen) or uninfected cells (nontarget) at effector: target ratio 30:1. The ratios of dead target versus nontarget cells (antigen: nontarget) were determined by flow cytometry. (F) Schematics showing that ORF8 mediates MHC-Ι lysosome degradation through an autophagy-dependent pathway. In SARS-CoV-2–infected cells, ORF8 directly binds to the MHC-Ι molecule, facilitating its trafficking to autophagosome for lysosome degradation. The data were shown as mean ± SD (error bars). Student’s t test was used. P < 0.05 indicates statistically significance difference; *P < 0.05; **P < 0.01; ***P < 0.001.
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References

    1. Huang C., et al. ., Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet 395, 497–506 (2020). - PMC - PubMed
    1. Zhu N.et al. .; China Novel Coronavirus Investigating and Research Team , A novel coronavirus from patients with pneumonia in China, 2019. N. Engl. J. Med. 382, 727–733 (2020). - PMC - PubMed
    1. Li Q., et al. ., Early transmission dynamics in Wuhan, China, of novel coronavirus-infected pneumonia. N. Engl. J. Med. 382, 1199–1207 (2020). - PMC - PubMed
    1. Wu F., et al. ., A new coronavirus associated with human respiratory disease in China. Nature 579, 265–269 (2020). - PMC - PubMed
    1. Bai Y., et al. ., Presumed asymptomatic carrier transmission of COVID-19. JAMA 323, 1406–1407 (2020). - PMC - PubMed

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