Figure 1.
Generation of a recombinant TGEV virus lacking protein 7 expression (rTGEV-Δ7).
(A) Genus α1 CoV protein 7a sequence alignment, using T-COFFEE [135]. Protein 7a sequences from the canine (CCoV) and porcine respiratory (PRCV) CoVs, transmissible gastroenteritis (TGEV) and feline infectious peritonitis (FIPV) viruses were used. GenBank accession numbers are ADB28914.1, ABG89313.1, CAA80842.1, and CAA62190.1, respectively. In silico prediction of TGEV protein 7 domains is represented. Transmembrane domains (TM) are in green [PredictProtein, [136]], the signal peptide in blue [Signal P3.0 Server, [137]], and a conserved phosphorilable Serine in red (S-Phos) [NetPhos 2.0 Server, [138]]. The predicted topology of TGEV protein 7 is also represented in lower panel [PSORTII [139]]. Signal peptide cleavage is indicated by a red arrowhead. S-Phos is indicated by a red star. (B) Mutations introduced to generate a rTGEV-Δ7 virus, right panel. The scheme of TGEV gRNA is shown in the upper part. The white letters represent the CS. Nucleotide change is indicated with a blue square, and the deletion (Δ) as a white square. Northern blot of subgenomic mRNAs (sgmRNAs) produced during rTGEV infections, right panel. ST cells were infected with rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) viruses, at a moi of 5. Total RNA was extracted at 8 hpi. The sgmRNAs for the spike (S), 3a, envelope (E), membrane (M), nucleocapsid (N) proteins, and protein 7 were detected. (C) In vitro growth kinetics of the rTGEV viruses. ST cells were infected with the rTGEV-wt (wt, blue) and rTGEV-Δ7 (Δ7, red) viruses, at a moi of 5. Culture medium and total intracellular RNA were collected at different hours post infection. Intracellular RNA was only analyzed during those hours post infection in which viable cells were bound to the plate. Viral titers (left panel), and genomic RNA (gRNA) amounts (right panel), determined by RT-qPCR, were analyzed. Error bars represent the standard deviation from three independent experiments.
Figure 2.
Cell death caused by rTGEV-Δ7.
(A) ST cells were infected with rTGEV-wt and rTGEV-Δ7 (Δ7) viruses. The cytophatic effect induced by both viruses was analyzed by optical microscopy, at 12, 16 and 24 hpi. Images were taken with a 40x objective. (B) Quantification of cell death induced by rTGEV viruses. ST cells were infected with rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) viruses. Cells were collected at 4, 8, 10, 12 and 16 hpi, permeabilized, and stained with propidium iodide. Dead cell population was measured by flow cytometry. Error bars indicate the standard deviation from three independent experiments. **, p-value<0.01.
Figure 3.
(A) Apoptosis levels in mock, rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) infected cells were evaluated at 4, 8 and 12 hpi, by flow cytometry. Annexin V-PI double staining was performed to differentiate cells in early apoptosis (Annexin V+, PI−) from those in late apoptosis (Annexin V+, PI+) stages. (B) Detection of active caspase 3 by Western-blot. Total protein was extracted from ST cells infected with rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) viruses, at the indicated times post infection. Active caspase 3 was detected using specific antibodies for the cleaved form. β-actin was detected as a loading control.
Figure 4.
De novo protein synthesis in rTGEV infections.
(A) At the indicated times post infection, ST cells were infected at a moi of 1 with rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) viruses. Cells were labeled with35S Met-Cys for 30 min. Protein extracts were obtained and SDS-PAGE electrophoresis was performed to detect labeled proteins. Viral spike (S), nucleocapsid (N), and membrane (M) proteins are indicated. Densitometric analysis was performed to determine the levels of host protein synthesis. The boxes represent the region of the gel used for densitometry analysis, and the numbers below represent the relative radioactivity compared with mock-infected cells. (B) Viral-to-cell protein synthesis ratio. The amount of radiolabeled N protein, estimated by densitometry, was related to the estimated total amount of protein, at the indicated hpi. Error bars indicate the standard deviation from three independent experiments. r.u., relative units.
Figure 5.
(A) Total RNA extracted from infected ST cells, at indicated times post infection, was analyzed using a Bioanalyzer. The position of 28S and 18S rRNAs are indicated. (B) 28S rRNA integrity. Graph of 28S fluorescence intensity, as measured by Bioanalyzer, in the RNA samples from ST cells infected with rTGEV-wt (blue) or rTGEV-Δ7 (red), collected at different times post infection. f.u., fluorescence units. Error bars indicate the standard deviation from three independent experiments. *, p-value <0.05. (C) ST cells were treated with caspase inhibitor ZVAD, and infected. Total RNA was extracted and analyzed using a Bioanalyzer. (D) ST cells were transfected with Poly(I:C), and total RNA was extracted 16 hours post transfection. ST cells were also infected with a vaccinia virus expressing T7 polymerase (T7), or with the vaccinia expressing T7 polymerase, and two additional vaccinia viruses expressing 2′-5′ OAS and RNase L (RL+OAS). Total RNA was extracted 24 hpi. In all cases, cell RNA integrity was analyzed using a Bioanalyzer.
Figure 6.
Quantification of 2′-5′OAS expression during rTGEV infection.
(A) Scheme of 2′-5′OAS/RNase L activation pathway. (B) Quantification of porcine 2′-5′OAS mRNA accumulation during rTGEV-wt (blue) or rTGEV-Δ7 (red) infections, by RT-qPCR, at indicated time post infection. r.u., relative units. Error bars indicate the standard deviation from three independent experiments.
Figure 7.
(A) Quantification of viral N and M sg mRNAs accumulation during rTGEV-wt (blue) or rTGEV-Δ7 (red) infections by RT-qPCR at indicated hpi. The ratio of sg mRNA to genomic RNA is represented. r.u., relative units. Error bars indicate the standard deviation from three independent experiments. (B) Northern blot analysis of intracellular viral sg mRNAs. ST cells were infected with rTGEV-wt or rTGEV-Δ7 viruses. Total RNA was extracted at indicated hours post infection and analyzed by Northern blot using a probe complementary to the 3′ end of all sg mRNAs. Total RNA amount loaded from rTGEV-Δ7 infected cells was 1.5 to 2 fold higher than that loaded from rTGEV-wt infected ones, in order to detect possible degradation species. Viral mRNAs for the spike (S), 3a, envelope (E), membrane (M), nucleocapsid (N) proteins, and protein 7 are indicated on the left.
Figure 8.
eIF2α phosphorylation during rTGEV infection.
(A) Total protein was extracted, at indicated times post infection, from ST cells infected at a moi of 5 with rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) viruses. Accumulation of total eIF2α and phosphorylated eIF2α (eIF2α-P), was analyzed by Western-blot. (B) eIF2α and eIF2α-P amounts were estimated by densitometric analysis. The graph represented eIF2α/eIF2α-P ratio in mock (green), rTGEV-wt (blue) and rTGEV-Δ7 (red) infected cells at indicated hpi. Error bars indicate the standard deviation from six independent experiments. r.u., relative units. *, p-value <0.05; **, p-value <0.01.
Figure 9.
Complementation of rTGEV-Δ7 produced apoptosis and RNA degradation by protein 7 provided in trans.
Generation of ST cells expressing TGEV protein 7 in trans. (A) Scheme of TGEV protein 7 expressed by the gene transfected into ST cells. Hemaglutinin tag (HA, light blue) was inserted after signal peptide (blue). (B) Protein 7 expression levels for the three ST-HA-7 selected cellular clones (C1, C2 and C3), were analyzed by immunofluorescence (left). Tagged protein 7 was detected with an anti-HA antibody stained in green, and cell nucleus were stained in blue. Percentage of HA-7 expressing cells is indicated. HA-7 protein accumulation was evaluated by Western-blot (right). HA-7 band is indicated, and corresponds to tagged protein cleaved form (7 KDa). (C) ST cells, or ST cells expressing HA-tagged protein 7 (ST-HA-7) were used to analyze apoptosis levels by flow cytometry. Apoptosis levels in mock, rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) infected cells were evaluated at 12 hpi. Annexin V-PI double staining was performed to differentiate cells in early apoptosis (Annexin V+, PI−) from those in late apoptosis (Annexin V+, PI+) stages. (D) ST cells and the three ST-HA-7 cell clones obtained were mock, rTGEV-wt or rTGEV-Δ7 infected. Total RNA was extracted at 18 hpi. Cellular RNA integrity was analyzed using a Bioanalyzer. 28S and 18S rRNAs are indicated on the right.
Figure 10.
PP1c binding motif in genus α1 CoV protein 7.
(A) Fragment from the alignment of genus α1 CoV 7a proteins. Canonical PP1c-binding motif is represented by the red box [ELM server [105], [106]]. (B) Consensus PP1 binding motif, including a short sequence (R/K)VxF (red), surrounded by non-polar residues. This motif is present in other viral and cellular proteins, such as human simplex virus-1 (HSV-1) γ134.5, African swine fever virus (ASFV) DP71L, human papilomavirus (HPV) E6 oncoprotein, and human growth arrest DNA-damage 34 (GADD34). GenBank accession numbers are ADB28914.1, P36313, Q65212, ACR78108 and O75807, respectively. Dark blue, non-polar aa; light blue, basic aa; green, polar aa; and red, PP1 binding motif core sequence. (C) Proposed model for protein 7 function during TGEV infection. (D) Coimmunoprecipitation of TGEV protein 7 and PP1. TGEV protein 7-PP1 interaction was evaluated using ST cells, ST-HA-7 cells (7), or ST cells transiently expressing SARS-CoV E protein (E), or a protein 7 mutant lacking the PP1 binding motif (7-mut). Cell extracts were incubated with anti-HA agarose. Input, flow through (FT), and final elution (CoIP) samples were resolved by SDS-PAGE. The presence of HA-tagged proteins, PP1 and eIF2α was analyzed by Western-blot using specific antibodies.
Figure 11.
Effect of mutated protein 7 provided in trans on RNA degradation and eIF2α phosphorylation.
ST cells, or ST cells expressing native TGEV protein 7 or the mutated protein 7 lacking PP1 binding motif were used. Cells were mock infected or rTGEV-wt (wt) and rTGEV-Δ7 (Δ7) infected. (A) Total RNA was extracted at 18 hpi and cell RNA integrity was analyzed using a Bioanalyzer. 28S and 18S rRNAs are indicated on the right. (B) Total protein was extracted at 10 hpi and eIF2α and eIF2α-P protein levels were analyzed by Western-blot. Protein amounts were estimated by densitometry, and the ratio of eIF2α-P to total eIF2α was represented. Error bars represented the standard deviation of three independent experiments.
Figure 12.
In vivo growth kinetics of rTGEV-Δ7 virus.
(A) Two- to three-day-old piglets were inoculated with 1×107 pfu/pig of rTGEV-wt and rTGEV-Δ7 viruses by two routes (oral and nasal) in combination. At 0.5, 1, 2, 3, 4 and 5 days post inoculation two animals per group were sacrificed, and the lungs were harvested. rTGEV-wt (blue) and rTGEV-Δ7 (red), recovered from lung, were titrated. Triangles indicated sentinel animals. (B) Two- to three-day-old piglets were inoculated with 1×107 pfu/pig of rTGEV-SC11-wt and rTGEV-SC11-Δ7 viruses by three routes (oral, intranasal and intragastric) in combination. At indicated days post inoculation two animals per group were sacrificed, and the lung and the gut were harvested. rTGEV-SC11-wt (blue) and rTGEV-SC11-Δ7 (red) titers in gut are represented. Triangles indicate sentinel animals. Error bars indicate the standard deviation from three independent experiments.
Figure 13.
Lung histopathology caused by rTGEV-Δ7 infection.
Two- to three-day-old piglets were inoculated with 1×107 pfu/pig of rTGEV-wt and rTGEV-Δ7. Lung samples, collected at 1 and 4 days post infection, were stained with hematoxylin-eosin (H&E). Pictures were obtained with a 10x objective. TGEV membrane protein (M) and cleaved caspase 3, were also immunodetected with specific antibodies. Pictures were obtained with a 20x objective.
Figure 14.
dsRNA induced antiviral pathway.
Schematic overview of the dsRNA-induced antiviral pathway analyzed. Differential effects observed during rTGEV-Δ7 infection are in red. PP1, the proposed target of protein 7, is in blue.
Table 1.
Accession numbers of proteins mentioned in the text.