Fig 1.
IBV nsp15 inhibits de novo protein synthesis.
(A) DF-1 cells were co-transfected with plasmids encoding Flag-tagged IBV nsp7, nsp8, nsp9, nsp12, or nsp15 along with reporter plasmids encoding V5-chMDA5(N), HA-chMAVS, V5-chIRF7, or EGFP. The PXJ40 vector was included as a control. After 24 h, cells were collected for western blot analysis. Protein signals were detected using the indicated antibodies, with β-actin serving as a loading control. The density of the corresponding protein bands was analysed using ImageJ, normalized to β-actin, and the ratio was presented relative to PXJ40 transfected group. (B) DF-1 cells were transfected with a plasmid encoding Flag-nsp15 or PXJ40 for 23 h and treated with puromycin (5 µg/mL) for 1 h to label de novo synthesized peptides. Indirect immunofluorescence staining was performed to detect nsp15 (magenta) and puromycin-labelled de novo synthesized peptides (green). The nuclei were labelled with DAPI (blue). The fluorescence intensity of nsp15 and puromycin in individual cells along the white line (from a to b) is shown in the right panel (histogram plot). Representative images are shown.
Fig 2.
The catalytic activity and oligomeric structure of IBV nsp15 are indispensable for the inhibition of de novo protein synthesis in chicken cells and mammalian cells.
(A) DF-1 cells were co-transfected with plasmids encoding Flag-tagged nsp15, catalytic-deficient nsp15 mutants (H223A, H238A), oligomerization-deficient nsp15 mutants (D285A, D315A), or the vector PXJ40, along with plasmids encoding V5-chMDA5(N), HA-chMAVS, V5-IRF7, or EGFP. After 24 h, Western blot analysis was performed using specific antibodies. β-actin served as a loading control. The density of the protein bands was analyzed using Image J, normalized to the signal of β-actin, and presented relative to the density detected in the corresponding PXJ40-transfected cells. (B-D) DF-1, Vero, and H1299 cells were transfected with plasmids encoding wild-type or mutated nsp15 and treated with puromycin (5 µg/mL) for 1 h at 23 h post-transfection (h.p.t). Indirect immunofluorescence staining was performed to detect nsp15 (magenta) and puromycin-labelled de novo synthesized peptides (green). Nuclei were stained with DAPI (blue). The fluorescence intensity of nsp15 and puromycin signal along the white line (from point a to b) is indicated by histogram plot. Representative images are shown.
Fig 3.
Inhibition of exogenous protein expression by nsp15 from different coronaviruses.
Plasmids encoding wild-type or catalytic-deficient nsp15 from various coronaviruses were co-transfected with plasmids encoding EGFP or IBV N into Vero cells. After 24 h, Western blot analysis was conducted using specific antibodies. β-actin served as a loading control. Band densities of EGFP or IBV N were quantified using ImageJ, normalized to the signal of β-actin, and presented relative to the PXJ40 group.
Fig 4.
Nsp15 from various coronaviruses inhibits de novo protein synthesis in PK15 cells and HeLa cells.
(A) PK15 cells were transfected with wild-type nsp15 or the corresponding catalytic-deficient nsp15 from porcine coronaviruses PEDV, TGEV, or PDCoV. (B) HeLa cells were transfected with wild-type nsp15 or the corresponding catalytic-deficient nsp15 from human coronaviruses SARS-CoV-1, MERS-CoV, or SARS-CoV-2. At 23 h.p.t, cells were treated with puromycin (5 µg/mL) for 1 h. (A-B) Indirect immunofluorescence analysis was performed to detect nsp15 (magenta), puromycin-labelled de novo synthesized peptides (green), and nuclei (DAPI, blue). Fluorescence intensity of nsp15 and puromycin signal along the white line (from a to b) is indicated by histogram plot. Representative images are shown.
Fig 5.
IBV nsp15 alters PABPC1 localization without affecting cellular mRNA distribution.
(A) DF-1 cells were transfected with plasmids encoding wild-type IBV nsp15 or catalytic/oligomerization-deficient nsp15 mutants (H223A, H238A, D285A, D315A). After 24 h, indirect immunofluorescence staining was performed to visualize nsp15 (magenta) and PABPC1 (green). Nuclei were labelled with DAPI (blue). Cells expressing nsp15 with nuclear accumulation of PABPC1 are indicated by yellow arrows. (B-D) DF-1, Vero, or H1299 cells were transfected with plasmids encoding IBV nsp15 or catalytic-deficient H223A, or the vector PXJ40. After 24 h, mRNA was visualized using oligo dT probes (green) via in situ hybridization, followed by indirect immunofluorescence to detect IBV nsp15 (magenta) and PABPC1 (red). Nuclei were labelled with DAPI (blue). Representative images are shown. White arrows indicate the cells expressing IBV nsp15, red arrows indicate the distribution of mRNA, and yellow arrows indicate the cells with PABPC1 nuclear retention. Treatment of DF-1 cells with 1 mM ARS for 30 minutes was used as a positive control for stimulating PABPC1 and mRNA nuclear localization. Representative images from are shown.
Fig 6.
Nsp15 of PEDV, TGEV, PDCoV, and SARS-CoV-1 alters the localization of PABPC1, while nsp15 of MERS-CoV and SARS-CoV-2 does so in most, but not all, cells.
Wild-type nsp15 or catalytic-deficient nsp15 of the indicated coronaviruses was transfected into the PK15 or Vero cell lines. After 24 h, in situ hybridization followed by indirect immunofluorescence was performed to visualize the location of nsp15 (magenta), PABPC1 (red), and mRNA (green). Nuclei were labelled with DAPI (blue). White arrows indicate cells expressing nsp15, yellow arrows indicate cells expressing nsp15 with nuclear localization of PABPC1, blue arrows indicate nsp15 expressing cells without nuclear retention of PABPC1, magenta arrows indicate nsp15-expressing cells with weaker mRNA signal, and red arrows indicate cells with overlapping nsp15 and mRNA distribution. Treatment with 1 mM ARS for 30 min was used as a positive control for visualizing of PABPC1 and mRNA nuclear localization. Representative images are shown.
Fig 7.
The nsp15-mediated relocation of PABPC1 is accompanied by inhibition of de novo protein synthesis.
(A-C) The PK15 (A), Vero cells and DF-1 cells (B and C) were transfected with PXJ40 or with a plasmid encoding wild type or catalytic-deficient Flag-tagged nsp15 of the indicated coronaviruses. After 23 h, puromycin labelling (5 µg/mL) was performed for 1 h. Indirect immunofluorescence was performed to detect nsp15 (magenta), PABPC1 (red), and puromycin-labelled de novo synthesized peptides (green). The nuclei were stained with DAPI (blue). White arrows indicate nsp15-expressing cells, yellow arrows indicate nsp15-expressing cells with PABPC1 nuclear retention, blue arrows indicate nsp15-expressing cells without PABPC1 nuclear retention, red arrows indicate cells with PABPC1 nuclear retention and reduced puromycin labelling signals, and magenta arrows indicate cells expressing nsp15 in which puromycin labelling signals remained stable. Representative images are shown.
Fig 8.
IBV nsp15 targets cytoplasmic factors to inhibit protein synthesis.
(A) Plasmids encoding wild-type or catalytic-deficient IBV nsp15 and reporter plasmids encoding IBV N or IBV M, or luciferase DNA, were co-incubated with Rabbit Reticulocyte Lysate for 1 h, followed by Western blot analysis or luciferase assay. Density of the bands corresponding to the reporter proteins was normalized to the signal of β-actin and presented relative to the sample transfected with the empty vector PXJ40. ****:p<0.0001; *:p<0.05. (B) Plasmids encoding wild-type or catalytic-deficient IBV nsp15 and reporter plasmids encoding IBV N or IBV M, or EGFP, were co-transfected into HEK 293T cells for 24 h, followed by Western blot analysis. Band densities of IBV N, IBV M, or EGFP were quantified using ImageJ, normalized to the signal of β-actin, and presented relative to the PXJ40 group.
Fig 9.
IBV nsp15 suppresses the transcriptional expression of antiviral genes IFN
β, IFITM3, and IL-8. (A) DF-1 cells were transfected with PXJ40 or plasmids encoding IBV proteins. At 24 h post-transfection, cells were either transfected with poly(I:C) or treated with IFNβ or TNFα, followed by real time qRT-PCR analysis to measure the transcription of IFNβ, IFITM3, and IL-8. (B) Vero cells were transfected with PXJ40 or plasmids encoding IBV nsp15, H223A, or H238A. At 24 h post-transfection, cells were either transfected with poly(I:C) or treated with IFNβ or TNFα. Immunofluorescence was performed to detect the nuclear translocation of IRF3, STAT1, and p65. Representative images are shown. Real time qRT-PCR was also conducted to measure the transcription of IFNβ, IFITM3, and IL-8. Statistical significance levels are denoted as follows: ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig 10.
Coronavirus nsp15 suppresses the transcriptional expression of antiviral genes IFN
β, IFITM3, and IL-8. (A-C) PK15 or HeLa cells were transfected with PXJ40 or plasmids encoding nsp15 or catalytic-deficient mutants from porcine coronavirus (PEDV, TGEV, PDCoV) and human coronavirus (SARS-CoV-1, MERS-CoV, SARS-CoV-2). At 24 h post-transfection, cells were either transfected with poly(I:C) or treated with IFNβ or TNFα. Immunofluorescence staining was performed to detect the nuclear translocation of transcription factors IRF3, STAT1, and p65. Representative images are shown. Real time qRT-PCR was conducted to measure the transcription of IFNβ, IFITM3, and IL-8. Statistical significance levels are denoted as follows: ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
Fig 11.
Nsp15 plays a dual role in regulating host protein synthesis efficiency during IBV infection.
(A) DF-1 cells or (B) H1299 cells were infected with wild-type IBV or rIBV-nsp15-H1238A at an MOI of 1. At 6, 12, 24 h.p.i., cells were treated with puromycin (5 µg/mL) for 1 h, followed by western blot analysis to detect puromycin-labelled de novo peptides, IBV-N, p-PKR, PKR, p-eIF2α, eIF2α, and β-actin. Dot blot analysis was performed to detects dsRNA. (C) H1299 cells were infected with wild-type IBV or rIBV-nsp15-H1238A for 18 h, followed by Northern blot analysis to detects viral RNA. (D) H1299 cells were transfected with siRNA targeting PKR or control siRNA for 36 h, followed by infection with IBV or rIBV-nsp15-H1238A for 24 h and incubation with puromycin for 1 h. Subsequently, cells were subjected to western blot analysis to detect p-PKR, PKR, p-eIF2α, eIF2α, puromycin-labelled de novo peptides, IBV-N, and β-actin.
Fig 12.
Infection with rIBV-nsp15-H238A, but not with wild-type IBV, induces nuclear retention of both PABPC1 and mRNA, and this is associated with reduced viral protein synthesis.
(A) Vero cells were infected with wild-type IBV or treated with ARS (left panel), or infected with rIBV-nsp15-H1238A (right panel). At 8, 12, 16, 20 and 24 h.p.i, indirect immunofluorescence was performed to detect IBV-N protein (red) and PABPC1 (green). Nuclei were stained with DAPI (blue). White circles indicate the rIBV-nsp15-H238A-infected cells with nuclear localization of PABPC1 and weaker IBV-N signals. (B) Vero and DF-1 cells were infected with wild-type IBV or rIBV-nsp15-H1238A. At 16 h.p.i., FISH and indirect immunofluorescence were performed to detected PABPC1(red), IBV N (magenta), and mRNA (green). Nuclei were stained with DAPI (blue). The white circle and white arrows indicate wild-type IBV-infected cells in which no nuclear accumulation of PABPC1 and re-distribution of mRNA were observed, while the yellow arrows indicate rIBV-nsp15-H238A-infected cells that exhibit nuclear localization of both PABPC1 and mRNA. Representative images are shown.
Fig 13.
IBV nsp15 is associated with the RTC major component nsp12.
(A) DF-1 cells were transfected with a plasmid encoding Flag-tagged nsp15 or infected with either IBV or rIBV-nsp15-H238A. At 24 h.p.t. or 18 h.p.i., indirect immunofluorescence was performed with anti-Flag antibody or with mouse anti-IBV-nsp15 monoclonal antibody (red). Nuclei were stained with DAPI (blue). (B) Vero cells were infected with IBV or rIBV-nsp15-H238A. At 18 h.p.i, indirect immunofluorescence was performed with mouse anti-IBV-nsp15 monoclonal antibody and rabbit anti-IBV-nsp12 polyclonal antibody. The nuclei were stained with DAPI (blue). (C) H1299 cells were infected with wild-type IBV for 24 h. Whole-cell lysates were subjected to immunoprecipitation using an anti-nsp15 monoclonal antibody or a Mouse IgG antibody as a control, followed by RNA extraction. Western blot was performed to confirm the expression and successful immunoprecipitation using anti-nsp15 monoclonal antibody. RNA yields were quantified using a NanoDrop spectrophotometer, and the eluted RNA was analyzed via high-throughput sequencing. (D) The left panel presents the enrichment of GO annotations for proteins encoded by nsp15-associated RNAs, while the right panel highlights the enrichment of KEGG pathways for these proteins.
Fig 14.
H1299 cells were infected with wild-type IBV for 24 hours.
Whole-cell lysates were subjected to immunoprecipitation using an anti-nsp15 antibody, with a Mouse IgG antibody serving as the control. Immunoprecipitated proteins were subsequently analyzed by mass spectrometry. The results display the enrichment of Gene Ontology (GO) annotations and KEGG pathways associated with nsp15-interacting proteins.
Fig 15.
Working model of the mode of action on host gene expression of nsp15 in the context of IBV infection.
Upon IBV infection, nsp15 plays a crucial role in regulating both viral and host protein synthesis simultaneously. It localizes to the RTC, where it regulates the abundance of viral dsRNA intermediates. This function is essential for the virus to evade the activation of the PKR-eIF2α pathway, which can lead to global protein synthesis inhibition for both viral and host mRNAs. This mechanism allows the virus to evade the host’s defense mechanisms and maintain translation of viral mRNAs. Meanwhile, nsp15 specifically targets host RNAs including anti-viral genes, suppressing host mRNA translation while ensuring the translation of viral mRNA, ultimately promoting viral protein synthesis. Upon catalytic mutant rIBV-nsp15-H238A infection, catalytic-deficient nsp15 is no longer able to control the levels of viral dsRNA intermediates, leading to a dsRNA-PKR-eIF2α-mediated global translation shutoff that limits both, host and viral protein synthesis, thereby leading to nuclear accumulation of PABPC1. This figure was created using Biorender (BioRender, Toronto, ON, Canada).