SARS-CoV-2 ORF6 disrupts innate immune signalling by inhibiting cellular mRNA export

Abstract

SARS-CoV-2 is a betacoronavirus and the etiological agent of COVID-19, a devastating infectious disease. Due to its far-reaching effect on human health, there is an urgent and growing need to understand the viral molecular biology of SARS-CoV-2 and its interaction with the host cell. SARS-CoV-2 encodes 9 predicted accessory proteins, which are presumed to be dispensable for in vitro replication, most likely having a role in modulating the host cell environment to aid viral replication. Here we show that the ORF6 accessory protein interacts with cellular Rae1 to inhibit cellular protein production by blocking mRNA export. We utilised cell fractionation coupled with mRNAseq to explore which cellular mRNA species are affected by ORF6 expression and show that ORF6 can inhibit the export of many mRNA including those encoding antiviral factors such as IRF1 and RIG-I. We also show that export of these mRNA is blocked in the context of SARS-CoV-2 infection. Together, our studies identify a novel mechanism by which SARS-CoV-2 can manipulate the host cell environment to supress antiviral responses, providing further understanding to the replication strategies of a virus that has caused an unprecedented global health crisis.

Fig 1. SARS-CoV-2 ORF6 inhibits protein expression.

(A) To generate VLPs, HEK293T cells were co-transfected with plasmids encoding HIV-1 Gag-Pol, VSV-G and a pLVX-StrepII-ORF vector encoding either SARS-CoV-2 ORF3a, ORF3d, ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF9c or ORF10 or an empty vector control. VLPs were harvested and titrated in HeLa cells. The infectious units/ml are shown in the bar graph. Points indicated independent biological repeats. Transfected cell lysates were separated by SDS-PAGE and analysed for Gag(HIV-1) and HSP90 by immunoblotting. (B,C) HEK293T cells were transfected with plasmids encoding HIV-1 Gag-Pol, VSV-G and either increasing amounts of pLVX-StrepII-ORF6 or pLVX-StrepII-ORF9b as well as a GFP reporter plasmid, pCSGW. (B) Transfected cell lysates were separated by SDS-PAGE and analysed for Gag(HIV-1), ORF6 or ORF9b and HSP90 by immunoblotting. (C) HeLa cells were infected with increasing amounts of HIV-1 VLPs. Three days post infection, the percentage of GFP positive cells was determined by flow cytometry. Graph shows the mean and range of two biological repeats. (D) HEK293T cells were transfected with the pLVX-EF1α plasmid encoding strep-tagged GFP(Control), ORF3a, ORF3d, ORF6, ORF7a, ORF7b, ORF8, ORF9b, ORF9c or ORF10 and a plasmid encoding mApple. After 48h, cells were fixed and the mApple median fluorescent intensity (MFI) measured by flow cytometry. The fold change (FC) in MFI is plotted relative to the control for independent biological repeats. Error bars show SEM. Transfected cell lysates were analysed by SDS-PAGE and immunoblotting with anti-strep. (E) Amino acid alignment of the C-terminus region of ORF6 from SARS-CoV-2 and SARS-CoV-1 with conserved residues highlighted in red. (F) HEK293T cells were transfected with pLVX-EF1α encoding strep-tagged ORF9b, ORF6(CoV-2), ORF6(CoV-1) or indicated ORF6(CoV-2) mutant and a plasmid encoding HIV-1 Gag-Pol. After 24h, cell lysates were separated by SDS-PAGE and analysed for Gag(HIV-1), Strep and HSP90 by immunoblotting.

Fig 2. ORF6 inhibits protein expression by interfering with Rae1.

(A) HEK293T cells were co-transfected with plasmids encoding HIV-1 Gag-Pol, ORF6 or ORF9b and Rae1 or indicated Rae1 mutant. Mock cells were untransfected. After 48h, cell lysates were separated by SDS-PAGE and analysed for Gag(HIV-1), Rae1, ORF6 or ORF9b and HSP90 by immunoblotting. Gag expression was quantified, normalised to HSP90, and shown in numbers below the Gag panel. (B) HEK293T cells were transfected with plasmids encoding Twin-Strep-tagged GFP or ORF6 and Nup98 and/or HA-Rae1. After 24h, Twin-Strep-tagged proteins were immunoprecipitated with MagStrep beads and proteins eluted with biotin. Input lysates and eluate were separated by SDS-PAGE and analysed by immunoblotting for Strep, Nup98 and HA. (C) HEK293T cells were transfected with plasmids encoding Twin-Strep-tagged GFP or ORF6, Nup98 and either HA-Rae1(WT) or HA-Rae1(R305G). After 24h, Twin-Strep-tagged proteins were immunoprecipitated and analysed as in (C). (D) HEK293T cells were transfected with plasmids encoding Twin-Strep-tagged proteins; ORF6(CoV-2), ORF6(E55A), ORF6(M58A) or GFP and HA-Rae1(WT). After 24h, Twin-Strep-tagged proteins were immunoprecipitated with MagStep beads and input lysates and eluates separated by SDS-PAGE and analysed by immunoblotting for Strep and HA.

Fig 3. ORF6 inhibits the nuclear export of cellular mRNA.

HEK293T cells were transfected with pLVX-EF1α-GFP or pLVX-EF1α-ORF6 or left untransfected (mock). After 24h, cells were either treated with IFN-β (1,000 units/ml) for 16h or left untreated. Cells were then harvested and fractionated into nuclear (Nucl) and cytoplasmic (Cyto) fractions. Cells were either lysed for immunoblot analysis (see S4A Fig) or RNA was extracted for mRNAseq. (A) Schematic of experiment design. (B) The log2-fold change (Log2-FC) in mRNA abundance was calculated between the Nucl and Cyto fractions for both GFP and ORF6 expressing cells, without and with IFN treatment (see S4B Fig). The log2-FC in mRNA abundance was then directly compared between ORF6 and GFP, without (left panel) and with (right panel) IFN, to determine which mRNAs were specifically enriched in the nucleus or cytoplasm by ORF6. The log-2FC was weighted against the adjusted p-value (shrunken Log2-FC) to show significantly enriched mRNAs (yellow points). The number of mRNAs significantly enriched is shown. The mRNA count was normalised and averaged between the three biological repeats. (C) Venn diagram of the number of mRNAs that were significantly enriched in the nucleus by ORF6 with and without IFN. (D) mRNAs significantly enriched by ORF6 without IFN treatment were analysed for enriched GO terms, which were filtered and mapped using the REVIGO tool. Highly similar GO terms are grouped and linked. Colour intensity reflects significance levels.

Fig 4. ORF6 inhibits the nuclear export of IFN-upregulated mRNA.

(A) Interferon upregulated genes (IUGs) were identified as mRNAs that were significantly upregulated after IFN treatment compared to untreated cells (see S5A Fig). These IUGs were then compared to the list of mRNAs that were significantly upregulated or downregulated by ORF6 compared to the GFP control (see S5E Fig), in either the Cyto (upper panel) or Nucl (lower panel) fractions. The heat maps show the Z-score (scaled and centred per-gene expression) in IUG expression in cells expressing GFP (Control) or ORF6 after IFN treatment, for three biological repeats. (B) Venn diagram showing the number of IUG mRNAs that were significantly enriched in the nucleus by ORF6 with and without IFN treatment. (C) The log2-fold change (Log2-FC) in mRNA abundance was calculated between the Nucl and Cyto fractions for both GFP and ORF6 expressing cells, without and with IFN treatment, as in Fig 3B, to determine which mRNA were specifically enriched by ORF6. The log-2FC was weighted against the adjusted p-value (shrunken Log2-FC) to show significantly enriched mRNA (yellow points). IUGs are highlighted in red and labelled.

Fig 5. SARS-CoV-2 inhibits the export of cellular mRNA.

(A, B) Vero cells were inoculated with four SARS-CoV-2 variants (Eng/2, Alpha, Beta, Delta) at a MOI ≥ 1 or left uninfected for 24h. (A) Cells were fixed and analysed by immunofluorescence with anti-ORF6 and DAPI. (B) Cells were fractionated, RNA was extracted, and cDNA was generated. mRNA levels of four non-antiviral (orange) and four antiviral (blue) proteins in both Nucl and Cyto fractions were measured by qPCR. The ratio of Nucl to Cyto mRNA was calculated and plotted. (C, D) A549-ACE2/TMPRSS2 cells were inoculated with two SARS-CoV-2 variants (Alpha, Delta) at a MOI ≥ 1 or left uninfected for 24h. (C) Cells were fixed and analysed by immunofluorescence with anti-ORF6 and DAPI. (D) Cells were fractionated, RNA was extracted, and cDNA was generated. mRNA levels of GAPDH (orange) and four antiviral (blue) proteins in both Nucl and Cyto fractions were measured by qPCR. The ratio of Nucl to Cyto mRNA was calculated and plotted. Points indicate individual biological repeats and error bars show the mean ± SEM.

Fig 6. ORF6 inhibits mRNA export to favour viral translation and supress innate signalling.

(A) Incoming or expressed ORF6 (yellow rectangles) binds to the Rae1-Nup98 complex (grey/brown) blocking the export of cellular mRNA (black lines). This subsequently reduces the cellular mRNA pool, increasing the availability of cellular translational machinery for viral translation as well as decreasing the expression of cellular proteins. (B) Incoming or expressed ORF6 (yellow) binds to the Rae1-Nup98 complex (grey/brown), inhibiting export of cellular mRNA encoding IRF1 (orange). This prevents the translation of IRF1, blocking IRF1 regulation of the transcription of additional steady state antiviral factors, like RIG-I and BST-2. ORF6 also inhibits nuclear export of mRNA encoding RIG-I (green), preventing detection of viral dsRNA produced during coronavirus replication. This helps reduce IRF1/3/7 activity and subsequent transcription of IFNα/β (purple), which prevents IFNα/β inducing an antiviral state in an autocrine and paracrine manner.