Mitochondria are organelles critical for cell survival because they produce ATP and modulate programmed cell death (PCD) pathways. PCD pathways are important in many clinical disorders, such as ischemia/reperfusion injuries, trauma, and toxic/metabolic syndromes, as well as in chronic neurodegenerative conditions, such as amyotrophic lateral sclerosis, Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease. Moreover, many viruses and other pathogens target the mitochondria. Viruses induce the production of various proteins in their hosts that have proapoptotic and anti-apoptotic activities, depending on the cellular environment. More specifically, many viruses that target mitochondria regulate the balance between the anti- and proapoptotic Bcl-2 family proteins and thereby increase their own survival within the host cell. Recent studies indicated that mitochondria centralize several critical innate immune responses based on the presence of several important signaling proteins within the mitochondria: mitochondrial antiviral signaling (MAVS), stimulation of interferon genes (STING), and NLR family member X1. Therefore, mitochondria are not only vital because they regulate cell survival and death but also they have broad roles in the control of cell functions following pathogen invasion. This chapter highlights the tight interplay between viral infection and mitochondria.
- mitochondrial membrane potential (MMP)
- Bcl-2 family
- viral death genes
1.1. Viruses and hosts
Viral diseases are becoming increasingly common worldwide, so it is important to identify the causative species and examine the underlying pathogenesis to prevent future epidemics and reduce the spread of new diseases. Although many host responses can contribute to the pathogenesis of viral diseases, little is known about the role of mitochondria in viral pathogenesis. Mitochondria are suitable targets for infectious microorganisms, such as viruses, because they act as powerhouses of the cell and have various other important functions. Therefore, “hijacking” the mitochondria disrupts overall cell function and makes it easy for a virus to control the cell and propagate. The host, in turn, has several responses that it uses to protect itself from viral invasions. One defense mechanism is based on the immune system, and another is based on cell autonomy, in which cells undergo certain physiological changes upon the onset of infection, such as unscheduled activation of the cell cycle following induction by certain viral proteins. When an infected cell undergoes programmed cell death (PCD), which includes apoptosis, autophagy, and necroptosis, this can prevent the spread of the virus infection to neighboring cells. A further complication is that viruses often promote apoptosis, but they can also block apoptosis by interacting with different signaling molecules of the host cell. Many viruses, especially human viruses, can perform either function, depending on the underlying conditions. Recent studies have shown that most viruses force cells to undergo apoptosis. However, from the perspective of the virus, apoptosis of the host seems to provide no benefit, so this is a topic of the current research. Therefore, additional studies that elucidate the mechanisms underlying the induction of the virus-induced PCD and cell lysis may help to identify new drug targets for the treatment of viral infections.
1.2. Intrinsic and extrinsic pathways of apoptosis in viral infections
Programmed cell death has a key role in the pathogenesis of many conditions including viral diseases, cancer, inflammation, and neurodegenerative diseases. Apoptosis is a highly complex process that is controlled by numerous cell signaling pathways . The common event at the end point is activation of a set of cysteine-aspartic proteases (caspases) [2, 3]. Apoptosis may benefit host cells by limiting the production and dissemination of viruses . However, apoptosis may benefit viruses if it allows them to increase production and dissemination of progeny [5, 6]. The PCD induced by a virus infection is often described as “typical apoptosis” . However, recent studies reported that nonapoptotic forms of PCD are important for the pathogenesis of certain RNA viruses, including the JC virus, hepatitis C virus (HCV), coxsackievirus B3, enterovirus, and dengue virus . The mechanism of DNA virus-induced nonapoptotic cell death is not well understood. Although not all signaling pathways that induce apoptosis are fully understood, the fate of a cell undergoing apoptosis mainly depends on the balance between the Bcl-2 family sensor proteins, which can promote and inhibit apoptosis (Figure 1) [9, 10, 11]. Recent studies have shown viral pathogenesis that involves oxidative damage and apoptosis.
1.3. Role of ROS in viral diseases
Recent studies have shown that mitochondria are the targets of the reactive oxygen species (ROS) that are produced inside a cell during viral infections, and that mtDNA is a major target of these ROS . Mitochondrial ATP generation requires proteins from the nuclear and mitochondrial genomes. ROS disrupt the oxidative production of ATP, which is required for normal cellular function, because damage of mtDNA disrupts the normal synthesis of proteins needed for mitochondria function and making them suitable targets for attack by ROS produced during infections by viruses and other microorganisms, although ROS also have other cellular targets. In HIV and hepatitis C virus infections, oxidative stress (OS) always plays a dominant pathogenic role. Peterhen and other researchers showed that almost all viruses (DNA/RNA viruses) cause cell death by generating oxidative stress in infected cells [12, 13, 14]. The OS generated during chronic hepatitis is associated with hepatic damage, a decrease in reduced glutathione (GSH) and decrease in plasma and hepatic zinc concentration [15, 16]. In case of influenza virus infection, the activated phagocytes release not only produces ROS but also cytokine and TNF. The pro-antioxidant effect of TNF may be relevant to influenza virus as shown by children with Rey’s syndrome . OS ultimately results in decrease in the functioning of the immune system.
2. Mitochondrial functions
Mitochondria are multifunctional organelles that are covered by an outer membrane (OM), within which lie the intermembrane space (IMS) and the inner membrane (IM). The IM is folded into special structures called cristae, so its surface area is much greater than that of the OM. The cristae function as matrices for protein complexes required for the electron transport chain (ETC), and they contain many integral membrane proteins, including adenine nucleotide translocator (ANT) and ATP synthase. The IM remains almost entirely impermeable under normal physiological conditions, thereby allowing the respiratory chain to create an electrochemical gradient. This electric potential is important for maintaining the mitochondrial membrane potential (MMP, Δψm) of the IM. The pumping of protons by the ETC out of the IM activates ATP synthase, which phosphorylates ADP to ATP. The ATP generated on the matrix side of the IM is then exported by ANT in exchange for ADP. The OM has many voltage-dependent anion channels (VDACs), which maintains permeability of solutes up to 5000 Da in size under normal physiological conditions. The IMS is chemically equivalent to the cytosol in terms of low molecular weight solutes and has its own special set of proteins. The human mitochondrial genome (about 16,500 bp), which is in the matrix (within the IM), only codes for 13 subunits of the respiratory chain. More than 99% of mitochondrial proteins are encoded by the nuclear genome and then selectively imported into one of the mitochondrial compartments. Thus, the mitochondrial OM, IM, IMS, and matrix have highly unique protein compositions.
Mitochondria act “powerhouse” of the cell and have several other important functions. The numerous functions of mitochondria make them indispensable to the cell, so when a virus “hijacks” mitochondrial function, it allows it to control the whole cell. Mitochondria have important roles in several signal transduction pathways [18, 19], the process of aging , regulation of different biochemical pathways related to cell metabolism [21, 22], PCD [23, 24], development [25, 26], the pathogenesis of numerous diseases immune responses , and cell cycle control [19, 27]. The circular mitochondrial genome encodes 13 polypeptides, 2 rRNAs, and 22 tRNAs. All of these mtDNA products are essential for function of the ETC and the generation of ATP by oxidative phosphorylation [9, 27], although many proteins from the nuclear genome are also required. Thus, any injury to mtDNA can affect the whole cell. The mtDNA is more susceptible to damage from ROS because it lacks protective histones, the mitochondrion has more limited DNA repair enzymes, and it is close to ETC, the main center of ATP and ROS production. The matrix of IMS, which contains cytochrome c oxidase (Cyt C, encoded by mtDNA), SMAC/DIABLO (encoded by nuclear DNA), and endonuclease G (encoded by nuclear DNA), acts as a buffer zone between the IM and OM. This matrix region contains many of the enzymes needed for aerobic respiration, dissolved oxygen, water, carbon dioxide, and recyclable intermediates that serve as energy shuttles and have other functions.
2.1. Loss of mitochondrial membrane potential
A loss of the MMP (Δψm) leads to imbalances in the membrane potentials of the IM and OM, and then to arrest of normal cellular biosynthetic function and bioenergetics, and finally to a “crisis” within the cell. A loss of the MMP (Δψm) also leads to release of several proapoptotic proteins from the IMS, such as Cyt C and Smac/DIABLO, as well as caspase independent death effectors, such as apoptosis-inducing factor (AIF) and endonuclease G (EndoG) , which have important roles in caspase-independent and caspase-dependent cell death . The MMP (Δψm) transition occurs during the pathogenesis of exogenous factors (e.g., viral proteins, toxins, and prooxidants [9, 10]). A prolonged loss of the MMP (Δψm) leads to serious cell damage, from which the cell cannot recover. Therefore, in the intrinsic pathway of apoptosis, any viral factor that influences the MMP (Δψm) has a major impact on cell fate, either by inducing or by blocking cell death .
In recent years, there has been an increasing focus on the role of the MMP (Δψm) in disease and health. Thus, several recent models based on in vivo and in vitro studies explain the mechanisms underlying the maintenance and loss of the MMP. A loss of the MMP by any mechanism leads to functional and structural collapse of the mitochondria and cell death . A recent study for first time has shown that dengue virus (DV) infection of human hepatoma cell line (HepG2) leads alteration in the bioenergetic function of mitochondrial morphology leading to MMP loss . The alteration in respiratory properties of HepG2 cells in DV infection results due to decrease in respiratory control ratio (PCR) and ADP/O ratio, which suggest significant alteration in mitochondrial morphology. Another additional feature observed by an increase in proton leak termed mitochondrial uncoupling which occurs by leaking of protons through FoF1 ATP synthase from inner membrane into matrix resulting in decrease in MMP loss. Thus, creating an imbalance in ATP synthesis ultimately affects the bioenergetic functions of cell. The biochemical mitochondrial damage induced in cell infected with HCV showed that E1 Protein together with core and NS3 are responsible for ROS production. Core and NS3 induce NO production which causes MMP loss by opening of transition pore . NO could also interact with another free radical superoxide (O2−) to form strong peroxynitrite anion (ONOO–), which irreversibly inhibits multiple respiratory complexes (complexes I, II and IV) and aconitase, and activate proton leak and permeability transition pore [30, 31]. Therefore, interfering with energy metabolism by disrupting the ATP synthesis of cell results in modulation of mitochondrial function.
2.2. Effects of viral infections on mitochondrial processes
Mitochondria undergo a number of processes, such as fusion and fission, in normally functioning healthy cells. However, when mitochondria develop abnormalities, the cell can destroy it by the process of mitophagy . Cells typically eliminate unhealthy mitochondria by mitochondrial fission; they typically use mitochondrial fusion to recycle matrix metabolites, including mtDNA and mitochondrial membranes, for the assembly of new and healthy mitochondria. Therefore, these three processes—mitophagy, fission, and fusion—are interlinked, and they all play prominent roles in maintaining healthy cells . Mitophagy plays an important role in maintaining mitochondrial homeostasis, but can also eliminate healthy mitochondria in cases such as cell starvation, viral invasion, and erythroid cell differentiation [34, 35]. The mitochondrial fusion and fission are highly dynamic. Viruses interfere with these processes to distort mitochondrial dynamic to facilitate their proliferation. Thus, interfering with these processes promotes the interference of different cellular signaling pathways [36, 37, 38]. New castal virus (NDV) uses strategy that interferes with P62-mediated mitophagy to promote viral propagation . The severe acute respiratory syndrome coronovirus (SARS-CoV) escapes the innate immune response by translocating its ORF-9b to mitochondria and promotes proteosomal degradation of dynamin-like protein (Drp1) leading to mitochondrial fission . However, still more studies are needed to explain the exact role of mitophagy in the viral disease pathogenesis, which regulates the cell death process .
2.3. Role of mitochondria in host immune response
During the coevolution of viruses and hosts, some viruses have evolved proteins that mimic the activity of host proteins, thereby allowing the virus to complete its life cycle without inducing an immune response in the host. For example, Mimivirus, a genus with a single species in the newly created Mimiviridae, has genes for many proteins, including a viral mitochondrial carrier protein (VMC-I) , which mimics the host cell’s mitochondrial carrier protein, allowing it to control mitochondrial transport in infected cells. Therefore, this virus takes control of the host cell’s transportation of ADP, dADP, TTP, dTTP, and UTP in exchange for dATP, which the virus uses as an energy source for genome replication and production of progeny .
2.4. Viruses target mitochondrial DNA and disable host cells
Numerous viruses appear to have adopted a “strategy” of damaging the host cell mitochondrial DNA to control the whole cell. For example, the herpes simplex virus (HSV) causes productive and latent infections in human hosts by disruption of mitochondrial function. The HSV-1UL12 gene encodes two distinct yet similar proteins, UL12 and UL12.5. UL12 is an alkaline nuclease, and UL12.5 is an N terminally truncated 500-aa polypeptide that lacks the first 126 residues of UL12. UL12 plays a crucial role in viral genome replication and processing; UL12.5 also has nuclease and strand-exchange activities but does not accumulate in the host cell nucleus. Instead, UL12.5 localizes predominantly to the mitochondria, where it triggers massive degradation of mitochondrial DNA during early HSV replication. In particular, UL12.5 occurs directly within the mitochondrial matrix, and its nuclease activity degrades mtDNA . HIV and hepatitis C virus infections cause metabolic stress due to mtDNA depletion in coinfected patients . The Zta protein encoded by Ebola virus (EBV) genome translocates into mitochondria and interacts with mitochondrial single-strand protein, which ultimately affects mtDNA replication . The OS generated during HCV infection also interferes with mtDNA .
3. Human viruses in mitochondria-mediated diseases
3.1. Epstein: Barr virus
The Epstein–Barr virus (EBV) is an oncovirus that is associated with breast cancer, gastric cancer, and numerous other cancers. Recent studies showed that EBV-encoded latent membrane protein 2A (LMP2A) leads to increased mitochondrial fission . Further, molecular studies showed that LMP2A activates the NOTCH pathway, which alters the expression of Drp1 and then increases mitochondrial fission. Although increased Drp expression does not directly increase mitochondrial fission, the recruitment of Drp by mitochondria and activation of GTPase leads to mitochondrial fission. Therefore, the EBV-induced changes in mitochondrial function, due to the LMP2A protein, may play a major role in the pathogenesis of several cancers .
3.2. Herpes simplex virus (alphaherpesvirus)
Infections by the herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) lead to imbalances in the calcium homeostasis of host cells. A study on rodents indicated that this calcium imbalance leads to disruption of mitochondrial function in the cervical ganglion neurons . An imbalance in the cellular calcium pool affects the Miro protein (a calcium-binding protein), altering its interaction with kinesin-1. Therefore, HSV-1 and PRV “hijack” host cell proteins, and this disrupts mitochondrial dynamics, thereby allowing the viruses to replicate and spread to neighboring cells.
3.3. Influenza A virus
3.3.1. Influenza A virus induces oxidative stress in mitochondria
Reactive oxygen species have important roles in the overall function of normal cells and play a vital role in the host adaptive immune response [49, 50]. For example, production of O2.− is an important defense against microbial infections. However, a large increase in the production of O2.−during influenza A virus infection leads to damage of lung parenchyma cells . Further studies that OS increased lung injuries caused by the influenza virus and viral replication, irrespective of the viral strain [52, 53]. Excessive and nonspecific knockdown of stress-related enzymes, such as superoxide dismutase 2 (SOD2), led to T-cell apoptosis and many developmental defects, resulting in overall weakening of the adaptive immune system and an increased susceptibility to influenza A virus subtype H1N1 [54, 55].
3.3.2. Influenza A virus-encoded protein PB1-F2 targets mitochondria
Influenza A viruses encode the proapoptotic protein PB1-F2, which localizes to the mitochondria due to a target sequence on its C terminal domain. PB1-F2 is conserved within the influenza family . After PB1-F2 binds to mitochondria, it interacts with two important mitochondrial proteins, VDAC1 on the OM and ANT3 on the IM [57, 58]. This leads to alterations in mitochondria morphology, release of proapoptotic proteins, loss of MMP, and then cell death.
3.4. HCV virus
3.4.1. HCV induces oxidative stress that damages mitochondria
HCV infection typically leads to generation of ROS, which interferes with the calcium signaling pathways of the cell . This disruption of calcium homeostasis alters the structure of the endoplasmic reticulum, and increased calcium is taken up by the mitochondria, leading to disruption of the MMP. Recent molecular studies have shown that many other HCV proteins, such as E2 , and NS4B , are important in generating oxidative stress. In addition, the nonstructural HCV protein NS5A is an integral membrane protein that is important for viral replication, apoptosis, immune responses (such as interferon resistance), and changes in cellular calcium . The proteins NS5Aand NS3 have roles in increasing calcium uptake and in the oxidation of glutathione (GSH) to glutathione disulfide (GSSG) in mitochondria, which ultimately leads to oxidative stress [61, 62, 63]. The imbalance of ROS created in the mitochondria leads to activation and translocation of NF-𝜅B and STAT3 to the nucleus, as part of disease progression. Antioxidants block the NS5A-mediated activation of NF-𝜅B and STAT3 . NS4B also promotes translocation of NF-𝜅B to the nucleus in a PTK-mediated pathway. The resulting production of ROS and nitric oxide (NO) causes oxidative damage and inhibits DNA repair  and leads to apoptosis. ROS-mediated disruption of mitochondria is believed to be the sole cause of liver inflammation in HCV infections .
3.4.2. HCV-encoded proteins target mitochondria
HCV remains persistent in its host because it lowers the host cell immune response. The HCV protein NS3/4A is a serine protease that inhibits interferon beta production by the retinoic acid-inducible gene I (RIG-I) pathway. Studies of the NS3/4A protein show that this protease cleaves MAVS at Cys-508, a few residues before its mitochondrial targeting domain. Cleavage of MAVS inactivates this protein because its soluble form is not functional. NS3/4A has a mitochondrial localizing signal, so it can directly cleave MAVS in the mitochondria [65, 66]. Substitution of Cys-508 with arginine prevents cleavage of MAVS. Cleavage of MAVS is thus an important mechanism by which HCV reduces host cell defenses .
3.5. Hepatitis B virus targets mitochondria
Hepatitis B virus x protein (HBx) is potentially essential for viral replication, and it has oncogenic properties in animal models . HBx sensitizes hepatocytes to apoptosis induced by stimuli such TNF-α . Studies of the overexpression of HBx showed that this protein causes apoptosis by causing a perinuclear clustering of mitochondria and a loss of the MMP . Studies of HBx mutants identified that certain hydrophobic residues (a mitochondrial targeting sequence, MTS) are important for its induction of mitochondrial localization, loss of MMP, and cell death . The HBx protein usually interacts with at least two mitochondrial proteins, heat shock protein 60 (HSP60)  and HVDAC3 . The interaction of HBx with these two proteins (which are important in maintaining mitochondrial integrity) ultimately disrupts mitochondrial function in infected cells. These two mitochondrial proteins play major roles in chronic liver disease and carcinogenesis. Therefore, HBx plays a major role in the pathogenesis of HBV infection due to its alteration of host cell mitochondria.
4. Control strategies
4.1. RNA interference treatment of viral diseases that target mitochondria
Despite the many advances in molecular biology and in treatment of viral diseases, the prevention and control of viral infections remains a challenge. Alteration of the interaction of the virus and host is one general strategy. Therefore, a complete understanding of the interactions of the host and virus at the molecular level is needed to develop new antiviral drugs and vaccines. There is an urgent need to find more effective therapeutic agents for the treatment of viral infections. Researchers have recently started testing treatments based on RNA interference (RNAi), using either microRNA (miRNA) or small interfering RNA (siRNA). Although this approach is still in its infancy, there has been some success in silencing the viral genes responsible for virulence [73, 74].
RNAi is an endogenous defense that cells use as a defense against harmful nucleic acids, either generated by the cell itself or from external environment (such as a viral invasions) . RNAi is successful against many virus infections, but the delivery and stability of RNAi molecules within the cell are major concerns. The stability of RNAi is affected by its charge and biochemical activity within a cell, so these two parameters must be considered when designing RNAi-based therapies. In addition, the effectiveness of RNAi-based therapies depends on the delivery route , target gene [77, 78], target pathogen [75, 78], and target tissue . The adverse effects of using RNAi-based treatment on the environment and treatment costs must also be considered, and we must have a deeper understanding of RNAi at the molecular level. The growing interest of molecular virologists in the use of RNAi suggests that this is one of the most exciting new therapeutic approaches for treatment of viral diseases .
4.2. Host antioxidant defense system fights viral invasion
The increased generation of ROS and reactive nitrogen species (RNS) is a key part of the pathogenesis of many virus infections. OS induces loss of the MMP, so mitochondria are become more susceptible to ROS damage. However, cells also have defenses against ROS, such as reduced glutathione (GSH),which acts as an antioxidant during the oxidative production of ATP in healthy cells [9, 79, 80, 81]. An imbalance between the generation of ROS and ROS quenching by the cell’s endogenous antioxidant defense system usually leads to a disease and is common during viral invasion. In recent years, due to the unavailability of antiviral drugs, researchers have proposed a number of new strategies to protect against free radical-induced OS. These strategies may be characterized as repair and protection. Protection is achieved by enzymes and by nonenzymatic compounds, such as carotenoids, vitamin C, vitamin E, GSH, and flavonoids . Recent studies have shown the importance of both classes of these molecules in defense against oxidative stress [9, 83, 84, 85, 86].
5. Concluding remarks
Identifying the main cause of a new epidemic is the most important factor in controlling disease outbreak. Many host responses appear to contribute to the pathogenesis of viral infections, and recent cellular and molecular studies have shown that many viruses specifically target mitochondria. Several different host responses and viral proteins directly or indirectly act on the mitochondria and lead to loss of the MMP. Mitochondria play important roles in cell survival and cell death, so a better understanding how different viruses use mitochondrial responses to control cells may provide a foundation for the development of new treatments for different viral diseases. More specifically, clarification of the roles of viruses and viral proteins in host mitochondria may help to develop methods that protect against pathogenic viruses. Therefore, molecular examination of the exact roles of viruses and viral proteins on mitochondria may help to guide the discovery of novel therapeutic strategies and provide important insights into different mitochondrial viral diseases. However, there are major unanswered questions regarding the mechanism of virus- and protein-induced loss of the MMP. Answering these questions may lead to the discovery of key molecules or pathways involved in loss of MMP, a common feature in the pathogenesis of many viral diseases. The research summarized in this review clearly shows that mitochondria are the main target of invading viruses, and that disruption of mitochondrial function is a major part of the pathogenesis of viral diseases. Although the prevention and treatment of viral diseases is challenging, molecular pathogenesis studies examining virus-host interactions will help in the design of new drugs and therapeutic strategies against different viral diseases.
This work was supported by a grant (NSC 98-2313-B-006-004-MY3 and NSC 101-3011-P-006-006) awarded to Dr. Jainn-Ruey Hong from the National Science Council, Taiwan, Republic of China.
Conflict of interest
The authors declare no conflicts of interest in terms of funding or authorship.
|DISC||death-inducing signaling complex|
|HCV||hepatitis C virus|
|HBV||hepatitis B virus|
|KPSV||Kaposi sarcoma virus|
|Bid||BH3 interacting-domain death agonist|
|WDSV||walleye dermal saecoma virus|
|SARS-CoV||severe acute respiratory syndrome coronovirus|
|VDAC||voltage-dependent anion channel|
|HIV||human immunodeficiency virus|
|IAPs||inhibitor of apoptosis proteins.|