Viral Nonstructural Protein 1 Induces Mitochondrion-Mediated Apoptosis in Mink Enteritis Virus Infection

Abstract

Mink enteritis virus (MEV), an autonomous parvovirus, causes acute hemorrhagic enteritis in minks. The molecular pathogenesis of MEV infection has not been fully understood. In this study, we observed significantly increased apoptosis in the esophagus, small intestine, mesenteric lymph nodes, and kidney in minks experimentally infected with strain MEVB. In vitro infection of feline F81 cells with MEVB decreased cell viability and induced cell cycle arrest at G₁ phase and apoptosis. By screening MEV nonstructural proteins (NS1 and NS2) and structural proteins (VP1 and VP2), we demonstrated that the MEV NS1 induced apoptosis in both F81 and human embryonic kidney 293T (HEK293T) cells, similar to that induced during MEV infection in minks. We found that the NS1 protein-induced apoptosis in HEK293T cells was mediated not by the death receptor but by the mitochondrial pathway, as demonstrated by mitochondrial depolarization, opening of mitochondrial transition pore, release of cytochrome c, and activation of caspase-9 and -3. Moreover, in NS1-transfected cells, we observed an increase of Bax expression and its translocation to the mitochondria, as well as an increased ratio of the Bax/Bcl-2, reactive oxygen species (ROS) production, and activated p38 mitogen-activated protein kinase (MAPK) and p53. Taken together, our results demonstrated that MEV induces apoptosis through activation of p38 MAPK and the p53-mediated mitochondrial apoptotic pathway induced by NS1 protein, which sheds light on the molecular pathogenesis of MEV infection.IMPORTANCE MEV causes fatal hemorrhagic enteritis in minks. Apoptosis is a cellular mechanism that effectively sacrifices virus-infected cells to maintain homeostasis between the virus and host. In this study, we demonstrated that MEV induces apoptosis both in vivo and in vitro Mechanistically, the viral large nonstructural protein NS1 activates p38 MAPK, which leads p53 phosphorylation to mediate the mitochondrial apoptotic pathway but not the death receptor-mediated apoptotic pathway. This is the first report to uncover the mechanism underlying MEV-induced apoptosis.

Keywords: NS1 protein; apoptosis; caspase; mink enteritis virus; mitochondrial apoptotic pathways; parvovirus.

FIG 1

In situ TUNEL assay of tissues of minks infected with MEVB. (A) In situ TUNEL staining of a single or serially cut tissue sections from the esophagus, small intestine, mesenteric lymph nodes, and kidneys of infected minks, showing an increase of TUNEL-positive cells compared to that in the uninfected group. Images show the macroscopic appearance of the different tissues with TUNEL assay after MEVB infection of the different groups as indicated. (B) Statistical analysis. The histogram summarizes the average percentage of apoptotic cells in the different tissues of infected minks. Data are means ± SEMs from three independent experiments. *, P < 0.05; **, P < 0.01; these values are statistically significantly different from those of the control group.

FIG 2

MEV infection induced apoptosis in F81 cells. (A) The cell viability of MEV infected cells was compared to control at different time points postinfection. F81 cells were infected with MEVB at 0.5 MOI and harvested at 12, 24, 36, 48, 60, and 72 h. Cell viability was measured by MTT assay. (B) MEV infection led F81 cells to be arrested at G₁ phase. The DNA contents of F81 cells mock infected or infected with MEVB at an MOI of 0.5 were collected at 24, 48, and 72 h postinfection (hpi), and the cell cycle was analyzed by PI staining and flow cytometry. (C) Apoptosis determination. Cells infected with MEVB at an MOI of 0.5 were stained with annexin V-FITC and PI at different time points postinfection and were analyzed by flow cytometry. Annexin V-positive cells were regarded as apoptotic. Data are means ± SEMs from three independent experiments. *, P < 0.05, and **, P < 0.01, compared with control groups.

FIG 3

MEV NS1-induced cell death. (A and B) Effects of MEV NS1, VP1, and VP2 on viability of F81 cells (A) and HEK293T cells (B). NS1-, VP1-, VP2- or vector-transfected cells were harvested at the indicated times postinfection for determination of cell viability using the MTT assay. (C and D) The release of LDH was used to assess the cell lysis of transfected F81 cells (C) and HEK293T cells (D) at different time points posttransfection. Z-DEVD-FMK and cisplatin were used as the negative and positive controls for apoptosis, respectively. DMSO was used as a vehicle control. For all assays, data are means ± SEMs, and results from a representative experiment of three independent experiments are shown. *, P < 0.05, and **, P < 0.01, compared with control groups. hpt, hour posttransfection.

FIG 4

MEV NS1-induced apoptosis. (A and B) F81 (A) and HEK293T cells (B) were transfected with NS1-, VP1-, and VP2-expressing plasmids, as indicated, and subjected to cell cycle analysis by cytometry at 24, 48, and 72 h posttransfection. Empty-vector- and mock-transfected cells were used as negative controls. The histograms show representative cell cycle analyses of NS1-, VP1-, VP2-, empty-vector-, and mock-transfected cells, and the bar charts show percentages of averages and standard error of the mean of the cells in each phase of the cell cycle. (C and D) Apoptosis analysis using annexin V/PI staining. Cells were transfected with the NS1, VP1, VP2, or empty vectors or mock transfected. F81 (C) and HEK293T cells (D) at 24, 48, and 72 h posttransfection were analyzed. The annexin V-positive cells were regarded as apoptotic cells. For all assays, data are representative of results from three independent experiments. *, P < 0.05, and **, P < 0.01, compared with control groups.

FIG 5

MEV NS2 did not induce apoptosis. HEK293T cells were transfected with NS2-expressing plasmid or empty vector. At 24, 48, and 72 h posttransfection, the cell cycle (A) and apoptotic cells (B) were analyzed by flow cytometry. The histograms show results from representative experiments. The bar charts show the percentages of the cells in G₁ and G₂ phases (A) and the rates of apoptotic cells (B), respectively.

FIG 6

NS1-induced mitochondrial damage with an accumulation of ROS. (A) NS1-induced mitochondrial transmembrane potential depolarization. HEK293T cells were transfected with plasmids as indicated, incubated with MitoCapture reagent, and analyzed by flow cytometry at different time points posttransfection. Vector- and mock-transfected cells were used as negative controls. (B) Loss of mitochondrial membrane integrity in NS1-transfected HEK293T cells. Transfected and stained HEK293T cells were captured as overlay histograms. The bar chart presents the significant increase in apoptotic cells with reduced mitochondrial membrane potential after NS1 transfection. (C) Accumulation of ROS in NS1-transfected HEK293T cells. NS1-transfected HEK293T cells were analyzed at different time points posttransfection, and the production of intracellular ROS was determined fluorometrically. The linear graph presents the increased production of ROS in the NS1-transfected cells compared to the mock and vector-transfected cells. Data are expressed as the means ± SEMs and are representative of results from three independent experiments. *, P < 0.05, and **, P < 0.01, versus the mock control.

FIG 7

Expression of the full-length MEV NS1 protein was necessary to induce apoptosis. (A and B) Schematic diagram of NS1 mutants. (A) Five NS1 truncated proteins and (B) three NS1 mutants. Positions of each domain are indicated, and the mutated amino acids in the origin-binding (OBD) and helicase domains and the deleted region (C-terminal 67 aa) in the transactivation (TAD) domain are indicated as well. (C) Apoptosis analysis. HEK293T cells were transfected with the five different truncated NS1 proteins and the three NS1 mutants. At 24, 48, and 72 h posttransfection, the cells were analyzed for apoptosis using annexin V/PI staining, followed by flow cytometry.

FIG 8

NS1 expression activated caspase-9 and -3. (A and B) Caspase activity in NS1-transfected cells. HEK293T cells were transfected with plasmids as indicated. At the indicated time points posttransfection, the caspase-9 and -3 activities of the transfected cells were measured using Caspase-Glo 9 (A) and Caspase-Glo 3/7 (B) luminescent assay kits, respectively. The bar chart presents the means ± SEMs from three independent experiments. *, P < 0.05, and **, P < 0.01, versus the control group. (C) Caspase-9 and -3 are activated by NS1 expression. HEK293T cells were transfected with pEGFP-NS1 or pEGFP vector. Cells expressing pEGFP-NS1 demonstrated active caspase-9 and -3 with fluorescence in red, while the active caspases were not observed in cells expressing enhanced green fluorescent protein (EGFP) only. (D) Western blotting of pro- and cleaved caspases and PARP. NS1- transfected cells were collected at various time points posttransfection and analyzed by Western blotting for expression of caspase-9 and -3, PARP, and their cleaved proteins. β-Actin was probed as a loading control.

FIG 9

NS1-induced apoptosis was mediated by the activation of mitochondrial pathway. (A and B) Bax and Bcl-2 expression. HEK293T cells were transfected with pCMV-MYC-NS1. At various time points posttransfection as indicated, the cells were analyzed for expression of Bax, Bcl-2, and phosphorylation of Bcl-2 using Western blotting (A). The ratio of Bax/Bcl-2 bands was quantified by densitometry using ImageJ software and plotted (B). (C) Western blot analysis of Bax translocation. HEK293T cells were transfected with pCMV-MYC-NS1. At various time points posttransfection as indicated, the cells were extracted for mitochondria and cytoplasm portions. Bax and Cyt c in various compartments were probed by Western blotting. β-Actin and COX-IV were used as endogenous controls for proteins in the cytosolic and mitochondrial fractions, respectively. Representative images from three independent experiments are shown.

FIG 10

NS1 activated the p38 MAPK and p53 signaling pathways. (A) Analysis of p38 MAPK and p53 expression. HEK293T cells were transfected with pCMV-MYC-NS1 (left) or pCMV-MYC (right). At various time points posttransfection as indicated, the cells were analyzed for levels of total p38 MAPK and p53 and their phosphorylated forms by Western blotting. (B) Inhibition of p38 MAPK and p53 activation altered the expression of Bcl-2 and Bax. HEK293T cells were pretreated with a 20 μM concentration of the p38 MAPK inhibitor SB203580 or p53 inhibitor pifithrin-α for 18 h, followed by transfection with NS1. At 24 and 48 h posttransfection, the cells were subjected to Western blotting using antibodies against Bcl-2, Bax, and p53. β-Actin was probed as a loading control.

FIG 11

A proposed model for MEV NS1-induced mitochondrion-mediated apoptosis. MEV infection induces apoptosis both in vivo and in vitro. The full-length MEV NS1 protein induces apoptosis through activating the mitochondrial pathway through indirectly activating p38 MAPK by phosphorylation at threonine 180 and tyrosine 182. The activated p38 MAPK then directly activates p53 via phosphorylation at serines 15 and 20, thereby decreasing Bcl-2 expression and increaing Bax expression, which motivates mitochondrial damage and progression of apoptosis.