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BHV-1 induced oxidative stress contributes to mitochondrial dysfunction in MDBK cells
© Zhu et al. 2016
- Received: 22 November 2015
- Accepted: 4 March 2016
- Published: 22 March 2016
The levels of cellular reactive oxygen species (ROS) and ATP as well as the mitochondrial membrane potential (MMP) in response to bovine herpesvirus 1 (BHV-1) infection of MDBK cells were measured, respectively. BHV-1 infection increased ROS production which depended on viral entry, and de novo protein expression and/or DNA replication. Vice versa, excessive ROS was required for efficient viral replication. Levels of both ATP and MMP were significantly decreased after BHV-1 infection. Interestingly, the loss of MMP was ameliorated by ROS depression. Collectively, ROS dependent mitochondrial damage and ultimately disruption of energy metabolism (ATP depletion) are a potential pathogenic mechanism for BHV-1 infection.
- Reactive Oxidative Species
- Reactive Oxidative Species Production
- Mitochondrial Membrane Potential
- Intracellular Reactive Oxidative Species
- Reactive Oxidative Species
Bovine herpesvirus 1 (BHV-1), an enveloped virus belonging to the alphaherpesvirus subfamily, infects cattle of all ages and breeds worldwide. Acute infection of BHV-1 results in clinical diseases in the upper respiratory tract, nasal cavity, or ocular cavity [1, 2]. Generally, BHV-1-induced immune suppression initiates a polymicrobial respiratory tract disease, commonly referred to bovine respiratory disease complex (BRDC), which costs the US cattle industry approximately three billion dollars, annually .
Reactive oxidative species (ROS) such as superoxide, hydrogen peroxide (H2O2), peroxynitrite (OONO−) and hydroxyl radicals (OH.) have been shown to play important roles in both physiological and pathophysiological processes. Under physiological conditions, the production and release of ROS is tightly controlled at specific time and space, which broadly influences cellular processes such as gene expression, cells proliferation, migration and differentiation . Oxidative stress results from the imbalance between production of ROS and the protective effect of the antioxidant system responsible for their neutralization and removal . And excessive production of ROS generally inflicts cell damage through oxidation of macromolecules, such as protein and lipids . Increasing studies suggested that host ROS contributed greatly to the replication of numerous viruses, as well as inflammation-associated tissues damage, such as in the inflammatory diseases caused by influenza virus, Hepatitis C virus and Dengue virus [6–10]. Thus, suppression of ROS appears to be a potential therapeutic approach primarily through reduction of both viral burden and inflammatory response.
It has been demonstrated that BHV-1 infection of MDBK cells induced apoptotic responses through diverse mechanisms including the mitochondria-mediate pathway [11, 12]. And mitochondrial damage is a potential indicator of cell apoptotic response. To our knowledge what happed to the mitochondria in BHV-1 infected MDBK cells is unknown.
In this study, we demonstrated that high level of cellular ROS was dramatically induced due to BHV-1 virus entry, de novo protein expression and/or DNA replication. The increased ROS was a crucial cellular signaling contributed to viral replication and mitochondrial damage, which consequently interfered with cellular ATP production. Our results suggested that the enhanced ROS production by BHV-1 infection is a double-edged sword, which not only promoted the viral infection, but also brought damage to the cells due to the injury to mitochondrial as well as ATP depletion. This could be a potential mechanism for the pathogenicity of BHV-1 infection induced tissue damage.
Acyclovir (ACY) is a known inhibitor to suppress herpesviruses DNA synthesis . The relation between BHV-1-induced ROS production and viral DNA synthesis was investigated through treatment of MDBK cells with ACY. MDBK cells were pretreated with ACY (Sigma-Aldrich, St. Louis, MO, USA) at concentrations ranged from 100 to 5 mM for 1 h, and then infected with BHV-1 at MOI of 10 along with the inhibitor. At 4 h pi the cellular ROS was detected with H2DCFDA. As a result, cellular ROS induced by viral infection was significantly depleted by ACY with a dose-dependent manner (Figure 1B). Phosphonoacetic acid (PAA), is a more specific inhibitor targeting the viral DNA polymerase . Treatment of MDBK cells with chemical PAA (Sigma-Aldrich) at concentrations ranged from 500 to 50 μM with the same manner as that with ACY led to a reduction of viral induced ROS with a dose-dependent manner (Figure 1C). It is correlated with the result of MDBK cells treated with ACY. Thus, de novo viral protein production and/or DNA replication seems to be correlated to the elevated ROS levels, which was further confirmed by the analysis with UV-inactivated viral particles.
In view of that overproduction of cellular ROS may exhibit toxic effect on the mitochondria, MDBK cells were treated with both NAC and Glutathione (GSH, provided by Beyotime Biotechnology, Jiangsu, China), a main intracellular non-enzymatic antioxidant exerting an efficient buffering role against ROS during the virus infection, to characterize the relation between ROS and the loss of MMP. As a result, both NAC (at a concentration of 5 mM) and GSH (at concentrations of 5 μM and 10 μM) significantly ameliorated the drop of MMP induced by BHV-1 infection at 48 h pi (Figure 3B). It implied that the virus triggered overproduction of ROS was partially associated with the loss of MMP.
Overproduced cellular ROS or oxidative stress is a critical factor that influences disease outcome, in the case of multiple viral infection such as by infection of influenza virus and Hepatitis C virus [6, 8, 21]. In this study, we found that BHV-1 infection of MDBK cells dramatically increased the production of cellular ROS, which is essential for virus replication (Figures 1 and 2).
BHV-1 together with Herpes simplex virus (HSV) and HSV-2 all belongs to the Alphaherpesvirinae subfamily, and shares a number of biological properties. The interplays between ROS and HSV-1 or HSV-2 in various cell cultures have been extensively studied [22–24]. Based on both our studies and studies from others [23–25], we inferred that cellular ROS is broadly regulated by alpha herpesviruses, but diverse manners were presented, which are cell type and virus specific, e.g., HSV-2 induced ROS production in RAW246.7 cells could be uniquely detected at 1 h pi , HSV-1 infection of murine microglial cells and neural cells increased ROS levels at time durations of 24–72 and 1–24 h pi, respectively [23, 24]. Increased ROS levels in MDBK cells was detected at 1–12 h pi during BHV-1 infection (Figure 1), and also it appeared at 24 h pi (data not shown). It is highly possible that ROS have diverse effect on viral replication or even on viral pathogenesis.
ROS mainly originated from Nox family of NADPH oxidases and mitochondria. Mitochondria are also susceptible to insult from ROS, since both mitochondrial proteins and mitochondrial DNA are vulnerable to ROS . It is known that mitochondria is a potential target of oxidative stress for environmental toxicants stimulus like the fine particulate matter (PM2.5) . It is reasonable that mitochondria is a potential target by some viruses that are able to induce excessive ROS. It has been documented that HSV infection results in the loss of MMP and decreases the levels of cellular ATP at the late stage of infection , whereas the role of ROS in this adverse effect is not yet elucidated. In this study, we found that BHV-1 infection of MDBK cells rendered mitochondria dysfunction as demonstrated by the mitochondrial depolarization and reduced ATP levels at late stage of infection. The loss of MMP was partially reversed by antioxidants of GSH and ROS scavenger NAC, which indicated that the overexpressed ROS partially accounts for the mitochondrial dysfunction.
Currently, the pathogenic mechanism of ROS in viral disease mainly reside in the ROS—mediated promotion of viral replication and inflammatory response. We suggested that BHV-1 induced ROS/mitochondrial damage and ultimately interfered with energy metabolism (as demonstrated by ATP depletion) is a novel mechanism by which BHV-1 induced cell injury. In addition to ROS mediated inflammatory response, this is also a potential pathogenic mechanism employed by alpha herpesvirus infection, such as in BHV-1 infection.
The authors declare that they have no competing interests.
LZ participated in design of the study, analyzed the data and prepared the manuscript. CY, DZ, YM and XD carried out the experiments, GZ revised the manuscript and coordinated the research. All authors read and approved the final manuscript.
The authors are grateful to Dr Leonard J. Bello, University of Pennsylvania, for providing the MDBK cells and the Colorado 1 strain of BHV-1. This research was supported by Chinese National Science Foundation Grant (No. 31472172), and partially by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and Natural Science Foundation from Chongqing (cstc2014jcyjA1628), Grants No. 14KJA230001 and No. BE2014358 from the Jiangsu province, and 948 program from Ministry of Agriculture of the People’s Republic of China (Grant No. 2011-G24).
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