Orf Virus Encoded Protein ORFV119 Induces Cell Apoptosis Through the Extrinsic and Intrinsic Pathways

Abstract

Apoptosis, a significant form of cell death, has a leading role in the host cell defense against virus infection. Viruses have evolved a series of strategies that block apoptosis during the early stage of viral infection to enhance viral replication, and induce apoptosis in the late stages to facilitate viral particle release from the cells. Here we show that orf virus (ORFV), the causative agent of orf, encodes an apoptosis-inducing protein ORFV119. ORFV119 targets the mitochondria in host cells, inhibits cell proliferation, and induces cell apoptosis. Protein array data indicated that ORFV119 could induce apoptosis via up-regulation of Smac, Bak, and Bax and down-regulation of anti-apoptotic proteins Bcl-2 and cIAP-2. Activation of caspase-9 and caspase-3, and consequent PARP cleavage, ultimately lead to apoptosis. ORFV119 could also directly activate caspase-8 and induce Bid, involved in the extrinsic pathway, to achieve cell death. Furthermore, sequence analysis and experiments with mutants of ORFV119 introduced revealed that ORFV119 contains a key N-terminal domain that is necessary and sufficient to direct the protein to the mitochondria. Together, we report, for the first time, the identification of the novel apoptosis-inducing protein ORFV119 encoded by a parapoxvirus. This provides an important reference for the study of pathogenesis, identification of immunomodulation mechanisms of ORFV, and may lead to new strategies for orf disease control.

Keywords: ORFV119; apoptosis; orf virus; parapoxvirus; protein array.

FIGURE 1

Analysis of ORFV119 amino acid sequence. (A) ORFV119 amino acid sequence. The N-terminal region contains the transmembrane segments on both sides of the basic amino acid (black body); C-terminal region contains the pRb binding motif (LxCxE: red). The deleted fragment in recombinant protein 119Nmut1 is marked with a line above and named as Nmut1. The mutant fragment in 119Nmut2 is underlined with a dotted line and named as Nmut2. (B) Homology comparison of N-terminal sequences of ORFV119, MCVMC007L, and human Tom20. The mitochondrial signal targeting peptide is underlined.

FIGURE 2

Cellular localization of ORFV119. (A) ORFV119 localizes in the mitochondria. OFTu cells were transfected with pEGFP-N1/p119GFP + pMitoDsRed or pERDsRed, fixed, stained and observed under laser confocal fluorescence microscopy after 24 h. GFP and 119GFP are shown in green; Mitochondria (Mito) are red; Endoplasmic reticula (ER) are shown in red. Nuclei were DAPI stained and are shown in blue. ORFV119 co-localized in the mitochondria and appeared yellow when merged with mitochondria marker; arrows denote the mitochondria. (B) The intracellular location of 119Nmut1/119Nmut2. OFTu cells were co-transfected with p119GNmut1/p119GNmut2 and pMitoDsRed. Colocalization was visualized by confocal microscopy.

FIGURE 3

Inhibition of cell proliferation by ORFV119. (A) 293T cells were transfected with pEGFP-N1, p119GFP, p119GNmut1or p119GNmut2. (B) 293T cells were transfected with pCMV-tag4, p119Flag, p119FNmut1 or p119FNmut2. The OD450 values were determined using Cell Counting Kit 8 at 0 h, 24 h, 48 h, and 72 h. Cell growth curves were plotted. Data are presented as the average of three independent experiments performed in duplicate. ^∗P < 0.05.

FIGURE 4

ORFV119 induced apoptosis as evidenced by morphologic changes and TUNEL assay. Cells were transected with pEGFP-N1 or p119GFP. (A) 24 h after transfection, the morphological changes of cells were observed by fluorescent microscopy (400×). For nuclear morphology analysis, cells were fixed, stained with DAPI for 30 min, and analyzed. Arrowheads showing apoptotic cells. (B) 24 h after transfection, ORFV119-induced apoptotic cells were determined qualitatively by labeling (TUNEL) assay.

FIGURE 5

Flow cytometry analysis of ORFV119-induced apoptosis. 293T cells were transfected with pEGFP-N1 or p119GFP for 24 h. Cells were collected, washed and dyed with Annexin V-APC and 7-AAD. (A) Apoptotic cells were detected using a flow cytometer (Q3: the normal cells; Q2: late apoptotic cells; Q2 + Q4: overall apoptotic cells). (B) Data are presented as mean ± standard deviation. ^∗∗P < 0.01 as compared with control.

FIGURE 6

Caspase-3, caspase-8, and caspase-9 were activated by ORFV119. (A–C) 293T cells were transected with vectors able to express ORFV119 or 119Nmut2, as well as pEGFP-N1 or pCMV-tag4 as mock controls. The cells were washed, lysed and caspase substrate was added. Caspase-3, caspase-8, and caspase-9 activities were measured using a micro-plate reader (Bio-Rad). (D) Cells were pre-treated with 50 μM caspase-8 inhibitor (Z-IETD-FMK) or caspase-9 inhibitor (Z-LEHD-FMK) for 1 h. The cells were transfected with p119GFP or pEGFP-N1 for 24 h, and the caspase-3, caspase-8, and caspase-9 activites were measured as above. The data shown in the panels are averages of three independent experiments with standard deviations indicated. ^∗P < 0.05, ^∗∗P < 0.01.

FIGURE 7

Human apoptotic protein array analysis of multiple proteins regulated by ORFV119. 293T cells were transfected with pEGFP-N1 or p119GFP, and apoptosis-related proteins were detected and analyzed using Human Apoptosis Antibody Microarray slides. (A) Characteristic proteins detected in antibody array. (B) Fold changes (ratio of medians among groups) of apoptotic signaling molecules, in comparison to controls, with a cutoff limit of 1.25 fold.

FIGURE 8

ORFV119 upregulated apoptotic promoters and downregulated apoptosis inhibitors. 293T cells were transfected with p119GFP or pEGFP-N1 for 24 h and 48 h. The protein expression levels of cleaved-caspase-3, cleaved-caspase-8, cleaved-caspase-9, cleaved-PARP, Bax, Bak, Bcl-2, Smac, cIAP-2, and BID were assessed by western blot using relevant primary antibodies. GAPDH/Tubulin served as the loading controls. Data are shown as mean ± SD of three independent experiments. ^∗P < 0.05, ^∗∗P < 0.01.

FIGURE 9

ORFV119 promoted the release of Cyto C and TNF-α. 293T cells were transfected with p119GFP or pEGFP-N1 for 24 h and 48 h. Cell lysates were collected and Cyto C and TNF-α were detected using a Human Cytochrome C/Tumor Necrosis Factor ELISA kit. The experiments were repeated three times with similar results. ^∗∗P < 0.01.

FIGURE 10

Pro-apoptotic network map of ORFV119. Caspase-3, caspase-8, and caspase-9 were activated in the current study. Expression of BID, Bax, Bak, Smac, and cleave-PARP were upregulated, while expression of BCL-2 and cIAP-2 were inhibited. Apoptosis was ultimately induced.