Caspase-Dependent Apoptosis Induction via Viral Protein ORF4 of Porcine Circovirus 2 Binding to Mitochondrial Adenine Nucleotide Translocase 3

Abstract

Apoptosis is an essential strategy of host defense responses and is used by viruses to maintain their life cycles. However, the apoptotic signals involved in virus replication are poorly known. In the present study, we report the molecular mechanism of apoptotic induction by the viral protein ORF4, a newly identified viral protein of porcine circovirus type 2 (PCV2). Apoptosis detection revealed not only that the activity of caspase-3 and -9 is increased in PCV2-infected and ORF4-transfected cells but also that cytochrome c release from the mitochondria to the cytosol is upregulated. Subsequently, ORF4 protein colocalization with adenine nucleotide translocase 3 (ANT3) was observed using structured illumination microscopy. Moreover, coimmunoprecipitation and pulldown analyses confirmed that the ORF4 protein interacts directly with mitochondrial ANT3 (mtANT3). Binding domain analysis further confirmed that N-terminal residues 1 to 30 of the ORF4 protein, comprising a mitochondrial targeting signal, are essential for the interaction with ANT3. Knockdown of ANT3 markedly inhibited the apoptotic induction of both ORF4 protein and PCV2, indicating that ANT3 plays an important role in ORF4 protein-induced apoptosis during PCV2 infection. Taken together, these data indicate that the ORF4 protein is a mitochondrial targeting protein that induces apoptosis by interacting with ANT3 through the mitochondrial pathway.IMPORTANCE The porcine circovirus type 2 (PCV2) protein ORF4 is a newly identified viral protein; however, little is known about its functions. Apoptosis is an essential strategy of the host defense response and is used by viruses to maintain their life cycles. In the present study, we report the molecular mechanism of the apoptosis induced by the ORF4 protein. The ORF4 protein contains a mitochondrial targeting signal and is an unstable protein that is degraded by the proteasome-dependent pathway. Viral protein ORF4 triggers caspase-3- and -9-dependent cellular apoptosis in mitochondria by directly binding to ANT3. We conclude that the ORF4 protein is a mitochondrial targeting protein and reveal a mechanism whereby circovirus recruits ANT3 to induce apoptosis.

Keywords: ANT3; ORF4; mitochondrial apoptosis pathway; porcine circovirus; viral protein.

FIG 1

Viral protein ORF4 of PCV2 induces cellular apoptosis by mitochondria in a caspase-dependent manner. (A) Cells infected with PCV2 or ORF4-deficient PCV2 at an MOI of 1 for 72 h were labeled both by TUNEL (green) and with a MAb to the Cap protein of PCV2 (red) and were observed under a confocal microscope. Yellow areas represent a merged image of the cells labeled both by TUNEL and with anti-Cap MAb. The graph for TUNEL-positive cells shows the percentages of TUNEL-labeled cells with and without Cap antigen in 100 tested cells. (B) Activities of casp-3 and casp-9 in cells infected with PCV2 or ORF4-deficient PCV2 at an MOI of 1 for 72 h. (C) Activities of casp-3 and casp-9 in cells transfected with Flag-ORF4 for 24 h. Caspase activity was analyzed by immunoblotting using MAbs recognizing casp-3, casp-9, and β-actin. (D) Casp-8 activity was detected by use of colorimetric assay kits for cells infected with PCV2 or ORF4-deficient PCV2 at an MOI of 1 for 72 h and for cells transfected with the Flag-ORF4 vector for 24 h. (E and F) Cytochrome c and AIF were detected in the cytoplasm and mitochondria of cells infected with PCV2 or of ORF4-deficient cells at 72 h, as well as in the cytoplasm and mitochondria of ORF4-transfected cells. Tomm20 served as a mitochondrial marker, and β-actin was used as a cytoplasm marker.

FIG 2

Colocalization of viral protein ORF4 of PCV2 with mitochondria in PK15 cells during transfection and infection. (A) Cells were transfected with Flag-ORF4 for 24 h or were infected separately with PCV2 and ORF4-deficient PCV2 for 72 h. The resultant cells were visualized using confocal microscopy. The ORF4 protein was labeled in green, and mitochondria were stained with MitoTracker Red CMXRos (red). The merged image (yellow) reveals viral protein ORF4 colocalization with mitochondria within a cell. (a) Flag-ORF4-transfected PK15 cells. (b) Untransfected PK15 cells. (c) PCV2-infected PK15 cells. (d) M4PCV2-infected PK15 cells. (e) Flag-ORF4-transfected 293T cells. (f) Flag-ORF4-transfected 3D4/31 cells. (B) MTS within the viral protein ORF4 as predicted by APSSP2 and Jpred 4 software. All the GFP-fused ORF4 truncation variants (a to h) were constructed. (C) A series of ORF4 truncation variants were transfected into PK15 cells and their expression detected. (D) PK15 cells transfected with EGFP-ORF4(1–30 aa) or EGFP-ORF4(1–24 aa) for 24 h. The mitochondria of the transfected cells were stained with MitoTracker Red CMXRos. The merged image (yellow) reveals viral protein ORF4-mitochondrial colocalization for residues 1 to 30 of viral protein ORF4 within the cell. (a) EGFP-ORF4(1–30 aa)-transfected PK15 cells. (b) EGFP-ORF4(1–24 aa)-transfected PK15 cells.

FIG 3

Viral protein ORF4 interacts with ANT3 in the mitochondria. (A) Flag-tagged ANT3, VDAC1, and COX8a were either expressed separately or coexpressed with Myc-tagged ORF4 protein in 293T cells for 36 h. Viral protein ORF4 was immunoprecipitated (IP) by use of an anti-Myc MAb and probed for VDAC1, ANT3, and COX8A by use of an anti-Flag MAb. IB, immunoblot. (B) In reciprocal immunoprecipitation assays, cell lysates were immunoprecipitated with anti-Flag MAb and Myc-ORF4 was detected by use of an anti-Myc MAb. (C) PK15 cells were cotransfected with Myc-ORF4 and Flag-ANT3 for 24 h and then stained with MitoTracker Red CMXRos for 30 min. Subsequently, the cells were fixed with 4% paraformaldehyde and incubated with mouse anti-Flag MAb and rabbit anti-Myc pAb as primary antibodies, followed by Alexa Fluor 647-labeled donkey anti-mouse IgG and FITC-conjugated goat anti-rabbit IgG as secondary antibodies. The stained cells were observed using an N-SIM microscope. (D) PK15 cells were cotransfected with various Myc-ORF4 mutants and Flag-ANT3 for 24 h, followed by incubation with rabbit anti-Myc pAb and mouse anti-Flag MAb as primary antibodies and FITC-conjugated goat anti-rabbit IgG and Alexa Fluor 546-labeled donkey anti-mouse IgG as secondary antibodies. Stained cells were visualized using a confocal microscope. (a) Myc-ORF4- and Flag-ANT3-transfected cells; (b) Myc-ORF4(1–46 aa)- and Flag-ANT3-transfected cells; (c) Myc-ORF4(1–30 aa)- and Flag-ANT3-transfected cells; (d) Myc-ORF4(31–59 aa)- and Flag-ANT3-transfected cells. (E) Various truncated forms of viral protein ORF4 tagged with Myc were cotransfected with the Flag-ANT3 expression plasmid into 293T cells for 36 h, and cell lysates were collected, immunoprecipitated with an anti-Myc MAb, and immunoblotted with anti-Flag or anti-Myc MAb. (F) Flag-labeled ANT3 was expressed in 293T cells and incubated with recombinant GST-ORF4, GST-ORF4(1–30 aa), GST-ORF4(1–46 aa), or GST-ORF4(31–59 aa), and then glutathione-Sepharose beads were used to immobilize the GST fusion proteins. Finally, pulled-down proteins were separated by 12% SDS-PAGE and visualized by immunoblotting with an anti-Flag antibody and an anti-GST antibody.

FIG 4

ANT3 silencing inhibits apoptotic induction by viral protein ORF4. (A) Silenced ANT3 expression in shANT3-transfected PK15 cells as observed by fluorescence microscopy. (B) ANT3 expression in shANT3-transfected PK15 cells as detected by Western blotting. (C and D) ANT3-silenced cells were transfected with Flag-ORF4 for 24 h and infected with PCV2 at an MOI of 1 for 72 h. Cleaved casp-3 and -9 were then measured by Western blotting as indicators of apoptosis. ns, not significant. (E) ANT3-silenced cells were infected with PCV2 at an MOI of 1 for 72 h. Cytochrome c, ANT3, AIF, and Tomm20 were analyzed by Western blotting with their corresponding antibodies. (F) Virus titers in ANT3-silenced PK15 cells.

FIG 5

Viral protein ORF4 is unstable and is degraded through the proteasome-dependent pathway. (A) PK15 cells were transfected with Flag-ORF4 for 24 h and then treated with CHX for 0, 3, 6, 9, 12, and 15 h. (B) PK15 cells were transfected with Flag-ORF4 for 24 h and then exposed to both CHX and MG132 for 9 h. (C) PK15 cells stably expressing Flag-ORF4 via a lentivirus were treated with MG132. In panels A to C, the expressed ORF4 protein was analyzed by Western blotting using an anti-ORF4 MAb. (D) PK15 cells infected with PCV2 for 60 h were treated with or without MG132 for 12 h and then analyzed by IFA.

FIG 6

Caspase activity is induced by exogenous viral protein ORF4 in the cytoplasm. (A) PK15 cells infected with M4PCV2 or with wild-type PCV2 were transfected with ORF4 for 48 h. (B) The stably ORF4-expressing PK15 cell line was inoculated with M4PCV2 or with wild-type PCV2 for 72 h. For panels A and B, casp-3 in the lysates was analyzed by immunoblotting. (C) Viral protein ORF4 in the cytoplasm and nuclei of both stably ORF4-expressing cells and transiently ORF4-transfected cells was detected by immunoblotting with anti-ORF4, anti-β-tubulin (cytoplasmic marker), and anti-histone H3 (nuclear marker) antibodies. W, whole-cell extracts; C, cytoplasmic extracts; N, nuclear extracts.