The Differential Expression of Mitochondrial Function-Associated Proteins and Antioxidant Enzymes during Bovine Herpesvirus 1 Infection: A Potential Mechanism for Virus Infection-Induced Oxidative Mitochondrial Dysfunction

Abstract

Reactive oxidative species (ROS) are important inflammatory mediators. Electrons escaping from the mitochondrial electron transport chain (ETC) during oxidative phosphorylation (OXPHOS) in the mitochondrial respiratory chain (RC) complexes contribute to ROS production. The cellular antioxidant enzymes are important for maintaining ROS release at the physiological levels. It has been reported that BoHV-1 infection induces overproduction of ROS and oxidative mitochondrial dysfunction in cell cultures. In this study, we found that chemical interruption of RC complexes by TTFA (an inhibitor of RC complex II), NaN₃ (an inhibitor of RC complex IV), and oligomycin A (an inhibitor of ATP synthase) consistently decreased virus productive infection, suggesting that the integral processes of RC complexes are important for the virus replication. The virus infection significantly increased the expression of subunit SDHB (succinate dehydrogenase) and MTCO1 (cytochrome c oxidase subunit I), critical components of RC complexes II and IV, respectively. The expression of antioxidant enzymes including superoxide dismutase 1 (SOD1), SOD2, catalase (CAT), and glutathione peroxidase 4 (GPX4) was differentially affected following the virus infection. The protein TFAM (transcription factor A, mitochondrial) stimulated by either nuclear respiratory factor 1 (NRF1) or NRF2 is a key regulator of mitochondrial biogenesis. Interestingly, the virus infection at the late stage (at 16 h after infection) stimulated TFAM expression but decreased the levels of both NRF1 and NRF2, indicating that virus infection activated TFAM signaling independent of either NRF1 or NRF2. Overall, this study provided evidence that BoHV-1 infection altered the expression of molecules associated with RC complexes, antioxidant enzymes, and mitochondrial biogenesis-related signaling NRF1/NRF2/TFAM, which correlated with the previous report that virus infection induces ROS overproduction and mitochondrial dysfunction.

Figure 1

The effects of respiratory complex inhibitors on virus productive infection in MDBK cells. MDBK cells were infected with BoHV-1 (MOI = 1) for 24 h. Throughout infection, the cells were treated with TTFA (a), an inhibitor for mitochondrial RC complex II, NaN₃ (b), an inhibitor for mitochondrial RC complex IV, and oligomycin A (c), an inhibitor for ATP synthase, at indicated concentrations, respectively. After infection, the cell cultures were subjected to frozen-thawing twice, and the viral titer was determined in MDBK cells with the results expressed as TCID₅₀/mL. (d) The cytotoxicity of TTFA (200 μM), NaN₃ (1 mM), and oligomycin A (200 μM) in MDBK cells for 24 h was analyzed by the Trypan-blue exclusion test as described elsewhere [47]. Data represent means of three independent experiments. Significance was assessed with the Student t-test. ^∗p < 0.05.

Figure 2

BoHV-1 infection affected the expression of certain components in mitochondrial RC complexes. (a) MDBK cells in 60 mm dishes were mock infected or infected with BoHV-1 at an MOI of 1 for 2, 4, 8, and 16 hours. The cell lysates were then prepared for Western blots to detect NDUFB8 for complex I, SDHB for complex II, MTCO1 for complex IV, UQCRC2 for complex III, and ATP5A for complex V, using OXPHOS antibody cocktail (Abcam; ab110413, 1 : 2000). Data shown are representative of three independent experiments. (b) The relative band intensity was analyzed with software ImageJ, and each analysis was compared with that of uninfected control which was arbitrarily set as 100%. Data are means of three independent experiments. Significance was assessed with the Student t-test (^∗p < 0.05).

Figure 3

The effects of BoHV-1 infection on the gene expression of antioxidant enzymes. (a, c, e, and g) The total RNA was prepared at 8 and 16 h after infection in MDBK cells, and the mRNA levels of SOD1 (a), SOD2 (c), CAT (e), and GPX4 (g) were measured by qRT-PCR. Each analysis was compared with that of uninfected control which was arbitrarily set as 100%. Data represent three independent experiments. Significance was assessed with the Student t-test (^∗p < 0.05). (b, d, f, and h) MDBK cells in 60 mm dishes were mock infected or infected with BoHV-1 at an MOI of 1 for 8 and 16 h. The cell lysates were then prepared for Western blots to detect the expression of SOD1 (b), SOD2 (d), CAT (f), and GPX4 (h) using SOD1 polyclonal antibody (ABclonal, #A0274, 1 : 1000), SOD2 polyclonal antibody (ABclonal, #A1340, 1 : 1000), CAT polyclonal antibody (ABclonal, #A11780, 1 : 1000), and GPX4 polyclonal antibody (ABclonal, #A11309, 1 : 1000). The band intensity was analyzed with software ImageJ. Each analysis was compared with that of uninfected control which was arbitrarily set as 100%. Data represent two independent experiments. Significance was assessed with the Student t-test (^∗p < 0.05), ns: not significant.

Figure 4

The effects of DPI treatment on the expression of NRF1, NRF2, and TFAM following BoHV-1 infection. (a) MDBK cells in 24-well plates pretreated with DPI (5 μM) or DMSO control for 1 h were infected with BoHV-1 (MOI = 1) along with corresponding chemical treatment. After infection for 16 h, cellular ROS levels were determined using H2DCFDA (5 μM, 30 min) (Sigma-Aldrich, St. Louis, MO, USA) and quantitatively analyzed using software Image-pro Plus 6. Data shown are means of three independent experiments. ^∗Significant differences (p < 0.05), as determined by the Student t-test. (b) MDBK cells in 24-well plates were treated with DPI or MDSO control and infected by BoHV-1 (MOI = 1) at the same condition as described in (a). Finally, the cell cultures were subjected to frozen-thawing twice, and viral production was determined using MDBK cells with results expressed as TCID50/mL. (c–e) MDBK cells in 60 mm dishes pretreated with DPI (5 μM) or DMSO control for 1 h were infected with BoHV-1 (MOI = 1) in the presence of DPI or DMSO control for 16 h; the cell lysates were prepared for Western blots to detect the expression of NRF1 (c), NRF2 (d), and TFAM (e). Data shown are representative of three independent experiments. (f) The relative band intensity was analyzed with software ImageJ, and each analysis was compared with that of the uninfected control which was arbitrarily set as 100%. Data are means of three independent experiments. Significance was assessed with the Student t-test (^∗p < 0.05).

Figure 5

The effects of virus infection on the expression of NRF1, NRF2, and TFAM. MDBK cells in 60 mm dishes were mock infected or infected with BoHV-1 at an MOI of 1 for 2, 4, 8, and 16 hours. The cell lysates were then prepared for Western blots to detect the expression of NRF1 (a), NRF2 (b), and TFAM (c) using NRF1 antibody (GeneTex; # PA5-40912, 1 : 1000), NRF2 antibody (Abcam; # ab137550, 1 : 500), and TFAM polyclonal antibody (Thermo Fisher Scientific; # PA5-68789, 1 : 1000). (d) The relative band intensity was analyzed with software ImageJ, and each analysis was compared with that of uninfected control which was arbitrarily set as 100%. Data are means of three independent experiments. Significance was assessed with the Student t-test (^∗p < 0.05).