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.

Bovine herpesvirus 1 (BoHV-1) is an important pathogen of cattle that causes lesions in mucosal surfaces, genital tracts and nervous systems. As a novel oncolytic virus, BoHV-1 infects and kills numerous human tumor cells. However, the mechanisms underlying the virus-induced cell damages are not fully understood. In this study, we demonstrated that virus infection of MDBK cells induced high levels of DNA damage, because the percentage of comet tail DNA (tailDNA%) determined by comet assay, a direct indicator of DNA damage, and the levels of 8-hydroxyguanine (8-oxoG) production, an oxidative DNA damage marker, consistently increased following the virus infection. The expression of 8-oxoguanine DNA glycosylase (OGG-1), an enzyme responsible for the excision of 8-oxoG, was significantly decreased due to the virus infection, which corroborated with the finding that BoHV-1 infection stimulated 8-oxoG production. Furthermore, the virus replication in human tumor cells such as in A549 cells and U2OS cells also induced DNA damage. Chemical inhibition of reactive oxidative species (ROS) production by either ROS scavenger N-Acetyl-l-cysteine or NOX inhibitor diphenylene iodonium (DPI) significantly decreased the levels of tailDNA%, suggesting the involvement of ROS in the virus induced DNA lesions. Collectively, these results indicated that BoHV-1 infection of these cells elicits oxidative DNA damages, providing a perspective in understanding the mechanisms by which the virus induces cell death in both native host cells and human tumor cells.

During bovine herpesvirus 1 (BoHV-1) productive infection in cell cultures, partial of intranuclear viral DNA is present in nucleosomes, and viral protein VP22 associates with histones and decreases histone H4 acetylation, indicating the involvement of histone H4 acetylation in virus replication. In this study, we demonstrated that BoHV-1 infection at the late stage (at 24 h after infection) dramatically decreased histone H3 acetylation [at residues K9 (H3K9ac) and K18 (H3K18ac)], which was supported by the pronounced depletion of histone acetyltransferases (HATs) including CBP/P300 (CREB binding protein and p300), GCN5L2 (general control of amino acid synthesis yeast homolog like 2) and PCAF (P300/CBP-associated factor). The depletion of GCN5L2 promoted by virus infection was partially mediated by ubiquitin-proteasome pathway. Interestingly, the viral replication was enhanced by HAT (histone acetyltransferase) activator CTPB [N-(4-Chloro-3-trifluoromethylphenyl)-2-ethoxy-6-pentadecylbenzamide], and vice versa, inhibited by HAT inhibitor Anacardic acid (AA), suggesting that BoHV-1 may take advantage of histone acetylation for efficient replication. Taken together, we proposed that the HAT-dependent histone H3 acetylation plays an important role in BoHV-1 replication in MDBK (Madin-Darby bovine kidney) cells.

Bovine herpesvirus type 1 (BoHV-1) is a significant cofactor for bovine respiratory disease complex (BRDC), the most important inflammatory disease in cattle. BoHV-1 infection in cell cultures induces overproduction of pathogenic reactive oxygen species (ROS) and the depletion of nuclear factor erythroid 2 p45-related factor 2 (Nrf2), a master transcriptional factor regulating a panel of antioxidant and cellular defense genes in response to oxidative stress. In this study, we reported that the virus productive infection in MDBK cells at the later stage significantly decreased the expression levels of heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase-1 (NQO1) proteins, the canonical downstream targets regulated by Nrf2, inhibited Nrf2 acetylation, reduced the accumulation of Nrf2 proteins in the nucleus, and relocalized nuclear Nrf2 proteins to form dot-like staining patterns in confocal microscope assay. The differential expression of Kelch-like ECH associated protein 1 (KEAP1) and DJ-1 proteins as well as the decreased association between KEAP1 and DJ-1 promoted Nrf2 degradation through the ubiquitin proteasome pathway. These data indicated that the BoHV-1 infection may significantly suppress the Nrf2 signaling pathway. Moreover, we found that there was an association between Nrf2 and LaminA/C, H3K9ac, and H3K18ac, and the binding ratios were altered following the virus infection. Taken together, for the first time, we provided evidence showing that BoHV-1 infection inhibited the Nrf2 signaling pathway by complicated mechanisms including promoting Nrf2 degradation, relocalization of nuclear Nrf2, and inhibition of Nrf2 acetylation.