Proteomics informed by transcriptomics reveals Hendra virus sensitizes bat cells to TRAIL-mediated apoptosis

Abstract

Background: Bats are a major reservoir of emerging infectious viruses. Many of these viruses are highly pathogenic to humans however bats remain asymptomatic. The mechanism by which bats control viral replication is unknown. Here we utilize an integrated approach of proteomics informed by transcriptomics to compare the response of immortalized bat and human cells following infection with the highly pathogenic bat-borne Hendra virus (HeV).

Results: The host response between the cell lines was significantly different at both the mRNA and protein levels. Human cells demonstrated minimal response eight hours post infection, followed by a global suppression of mRNA and protein abundance. Bat cells demonstrated a robust immune response eight hours post infection, which led to the up-regulation of apoptosis pathways, mediated through the tumor necrosis factor-related apoptosis inducing ligand (TRAIL). HeV sensitized bat cells to TRAIL-mediated apoptosis, by up-regulating death receptor transcripts. At 48 and 72 hours post infection, bat cells demonstrated a significant increase in apoptotic cell death.

Conclusions: This is the first study to comprehensively compare the response of bat and human cells to a highly pathogenic zoonotic virus. An early induction of innate immune processes followed by apoptosis of virally infected bat cells highlights the possible involvement of programmed cell death in the host response. Our study shows for the first time a side-by-side high-throughput analysis of a dangerous zoonotic virus in cell lines derived from humans and the natural bat host. This enables a way to search for divergent mechanisms at a molecular level that may influence host pathogenesis.

Figure 1

HeV infection and transcription in bat and human cells. Confocal microscopy was used to visualize HeV-infected cells. Nuclei are stained with 4′,6-diamidino-2-phenylindole dihydrochloride, and HeV-N protein was immunodetected with an anti-N polyclonal antibody. PaKiT03 cells at (A) 0 hpi, (B) 8 hpi and (C) 24 hpi are shown. HEK293T cells infected with HeV for (D) 0 hpi, (E) 8 hpi and (F) 24 hpi are shown. Scale bar is 30 μm. (G) Transcription profile of HeV in PaKiT03 and HEK293T at 24 hpi. (H) Genome structure of HeV. bp, base pairs; F, fusion protein; G, glycoprotein (attachment protein); L, large protein (polymerase); M, matrix protein; N, nucleocapsid; P, phosphoroprotein (includes proteins V, W and C).

Figure 2

Statistics of differential expression. (A) Differential expression of Trinity transcripts at 8 and 24 hpi was assessed as pairwise comparisons to 0 hpi. The number and overlap of significantly differentially expressed transcripts (adjusted P <0.05) for 8 and 24 hpi are illustrated as two-way proportional Venn diagrams. Venn diagrams are organized in a 2 × 2 matrix where up- and down-regulated statistics are given in the columns and the cell type is given in the rows. (B) Differential expression of Trinity proteins were defined as proteinGroups with SILAC ratios ≥2 fold relative to 0 hpi. Two-way proportional Venn diagrams for proteins differentially expressed at 8 and/or 24 hpi are illustrated as for (A).

Figure 3

Relationship between mRNA and protein expression of orthologous transcripts. (A) Scatterplot illustrating the relationship in baseline (0 hpi) mRNA expression as log10 normalized fragments per kilobase of transcript per million mapped reads (FPKM) for all ortholog transcripts (identity >70%). Pearson’s correlation coefficient is indicated in the top right hand corner. (B) Scatterplot matrix of the correlation between transcript expression (log2-fold change) at 8 and 24 hpi in PaKiT03 and HEK293T cells. Pearson’s correlation coefficients are given in upper boxes. Simple linear regression models were fitted and are illustrated as red lines through each scatterplot. (C) Scatterplot matrix of the correlation between protein expression (log10 SILAC ratio) at 8 and 24 hpi in PaKiT03 and HEK293T cells. Pearson’s correlation coefficients are given in the upper boxes. Simple linear regression models were fitted and are illustrated as red lines through each scatterplot. (D) Heatmap of orthologous transcripts that were significantly differentially expressed at 8 and/or 24 hpi in either cell line. Expression values for transcripts are given as log2-fold change.

Figure 4

Activation of apoptosis pathways in HeV-infected PaKiT03 cells. (A) GO terms enriched in the up-regulated genes/proteins in PaKiT03 cells were analyzed and visualized using REVIGO, where redundant GO terms are removed. The size of the circle represents the number of enriched child GO terms contributing to the parent term. The circle color represents the uncorrected log10 P-value for each parent GO term. Semantic space is the result of multi-dimensional scaling where similar GO terms cluster together. A full list of enriched GO terms, including false discover rate-corrected P-values, are presented in Additional file 5. (B) Top 10 most over-represented KEGG pathways, and the number of significantly differentially expressed genes/proteins contributing to each pathway. The full list of over-represented KEGG pathways is presented in Additional file 6. (C) Expression profile of significantly up-regulated genes/proteins in the PaKiT03 cells which were assigned to the GO term regulation of apoptotic process [GO:0042981] or apoptotic process [GO:0006915]. Gray cells represent proteins where no peptides were observed. Expression values for mRNA are given as log2-fold change, while expression values for proteins are normalized SILAC ratios. Validation of RNAseq differential expression with real-time quantitative PCR (qPCR) for (D) CASP13, (E) TNFRSF10A, (F) TNFRSF11B, (G) CD40 and (H) TNFAIP3/A20 in PaKiT03 cells. Real-time PCR differential expression was calculated based on relative expression to 0 hpi and normalized to GAPDH. Log2-fold change is presented; *P <0.05, **P <0.01. (I) Relative protein expression profile of CD40 in PaKiT03 cells measured by SILAC. (J) Protein expression profile of CD40 and TNFAIP3/A20 in PaKiT03 cells measured by Western blot. β2-tubulin served as the load control.

Figure 5

Interferon production and signaling in HeV-infected HEK293T cells. (A) GO terms enriched in the up-regulated genes/proteins in HEK293T cells were analyzed and visualized using REVIGO, where redundant GO terms are removed. The size of the circle represents the number of enriched child GO terms contributing to the parent term. The circle color represents the uncorrected log10 P-value for each parent GO term. Semantic space is the result of multi-dimensional scaling where similar GO terms cluster together. A full list of enriched GO terms, including false discover rate-corrected P-values are presented in Additional file 5. (B) Top 10 most over-represented KEGG pathways, and the number of significantly differentially expressed genes/proteins contributing to each pathway. The full list of over-represented KEGG pathways is presented in Additional file 6. Validation of RNAseq differential expression with real-time qPCR for (C) IFNB1, (D) IFIT2 and (E) IFIT3. Real-time PCR differential expression was calculated based on relative expression to 0 hpi and normalized to GAPDH. Log2-fold change is presented; *P <0.05, **P <0.01.

Figure 6

Hendra virus induces apoptosis in PaKiT03, but not HEK293T cells. (A) TRAIL-mediated apoptosis pathway. (B) TRAIL relative protein expression in PaKiT03 cells measured by SILAC. (C) mRNA fold change relative to 0 hpi and normalized to GAPDH of TRAIL, TNFRSF10A, TNFRSF11B and CASP13 in PaKiT03 cells at 24, 48 and 72 hpi. (D) Cell viability relative to 0 hpi of PaKiT03 and HEK293T at 24, 48 and 72 hpi with HeV at a multiplicity of infection (MOI) of 5. One-way analysis of variance (ANOVA) was used to compare cell viability between time points. (E) Relative activity of caspase 3/7 normalized to cell viability for PaKiT03 and HEK293T at 0, 24, 48 and 72 hpi with HeV at an MOI of 5. One-way ANOVA was used compared the relative activity of caspase 3/7 between the PaKiT03 and HEK293T cells at each time point. (F) Western blot of PARP1 cleavage at 0, 8 and 24 hpi in HEK293T. The human PARP1 antibody was not cross-reactive with the PaKiT03 cells. *P <0.05, **P <0.01.

Figure 7

TUNEL staining of HeV-infected (A) PaKiT03 and (B) HEK293T cells. Cells were infected with HeV for either 8, 24 or 48 h using an MOI of 5. TUNEL staining was achieved using the Click-iT® TUNEL Alexa Fluor® 488 kit (green, fluorescence) and HeV-N was immunodetected as described above (red, fluorescence). DNase I treatment was used as a positive control on HEK293T cells that demonstrated clear nuclear staining. Owing to the decrease in cell viability at 24 hpi and beyond in the HEK293T cells, reliable TUNEL staining could not be performed after 8 hpi. Scale bar is 30 μm in all panels.

Figure 8

Apoptosis in other (A) bat and (B) human cells. Relative fold change of caspase 3/7 activity of bat cells from brain (PaBrT03), fetus (PaFeb5) and lung (PaLuT02), and common human cells from lung (A549), embryonic fibroblasts (HEF) and HeLa cells. All cells were infected with HeV for 24, 48 and 72 hpi at an MOI of 5. Uninfected cells served as the control for each time point. Two sample t-tests were used to compare the relative activity of caspase 3/7 between the control and infected cells at each time point. *P <0.05, **P <0.01.

Figure 9

HeV sensitizes PaKiT03 cells to TRAIL-mediated apoptosis. Cells were either treated with 500 ng/ml human recombinant TRAIL and/or infected with virus (MOI = 5) for 24, 48 and 72 hpi. The activities of caspase 3/7 within the experimental samples were compared to the control (no TRAIL or virus) using one-way analysis of variance for each time point. *P <0.05, **P <0.01. All assays were performed in triplicate. (A) Increase of caspase 3/7 activity over control in PaKiT03 cells following stimulation with human recombinant TRAIL and/or HeV. (B) Increase of caspase 3/7 activity over control in HEK293T cells following stimulation with human recombinant TRAIL and/or Nelson Bay virus (NBV).

Figure 10

TRAIL expression and apoptosis in bats in vivo . Tissue samples were obtained from a previous unpublished HeV infection of P. alecto bats (Australian black flying fox). The expression of CD40 and TRAIL mRNA was compared in (A) kidney and (B) lung and from bats infected with HeV for 36 and 60 hpi compared to uninfected bats. TUNEL staining was also performed on spleen and kidney sections from the experimentally infected and control bats. TUNEL staining of the (C) spleen and (D) kidney of a representative control bat. Arrows highlight TUNEL-positive cells in the spleen. TUNEL staining from a representative HeV-infected bat (60 hpi) showed no increase in the number of TUNEL-positive cells in the (E) spleen and/or (F) kidney compared to the control. Scale bar is 100 μm in all panels.