Mitochondria in the biology, pathogenesis, and treatment of hepatitis virus infections

Summary Hepatitis virus infections affect a large proportion of the global population. The host responds rapidly to viral infection by orchestrating a variety of cellular machineries, in particular, the mitochondrial compartment. Mitochondria actively regulate viral infections through modulation of the cellular innate immunity and reprogramming of metabolism. In turn, hepatitis viruses are able to modulate the morphodynamics and functions of mitochondria, but the mode of actions are distinct with respect to different types of hepatitis viruses. The resulting mutual interactions between viruses and mitochondria partially explain the clinical presentation of viral hepatitis, influence the response to antiviral treatment, and offer rational avenues for novel therapy. In this review, we aim to consider in depth the multifaceted interactions of mitochondria with hepatitis virus infections and emphasize the implications for understanding pathogenesis and advancing therapeutic development.


DNA (mtDNA) is able to elicit innate immune response through
Toll-like receptor 9 (TLR9) and stimulator of interferon genes (STING) signaling. 6 Finally, the release of citric acid cycle intermediates from the mitochondrial matrix into the cytosol following viral infection also regulates host innate immunity. 7 Together, these mechanisms likely impact on the infection course, pathogenesis, and the clinical outcome of IFN-α treatment in hepatitis virus infections.
The liver is a metabolic powerhouse, and accordingly hepatocytes contain abundant numbers of mitochondria to support the energy requirement associated with high metabolic activity. 8 Viruses require energy and macromolecule building blocks from the host to complete their life cycle but on the other hand can modulate the host metabolic machineries. 9 Hepatitis viruses are known to regulate the number, quality, and dynamics of mitochondria, resulting in altered mitochondrial morphology and function. 10 Accordingly, morphological and functional alterations of mitochondria are commonly observed in liver tissues obtained from viral hepatitis patients. [11][12][13] Intriguingly, accumulating evidences have suggest that mitochondrial products serve as mediators of many cellular signaling pathways, including inflammatory responses that are prominent features of viral hepatitis. Adenosine 5'-triphosphate (ATP), the primary carrier of energy, plays pleiotropic roles in inflammation by acting as an extracellular signaling molecule. 14,15 HCV replication actively consumes intracellular ATP. 16 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), an activator of ATP production, counteracts both HCV and HEV infection. 17,18 HBV infection decrease ATP levels in hepatocytes. 19 Several other metabolites from mitochondria, in particular, citrate and succinate, are implicated in the pathological processes of viral hepatitis and cirrhosis. 20,21 Given the complexity, whether it is a sequential or causal relationship between mitochondrial alteration and hepatitis remains unclear.

| MITOCHONDRIAL DYSFUNCTION IN VIRAL HEPATITIS PATIENTS
Mitochondrial dysfunction is associated with many common disorders. 22 It is a prominent feature of liver cell injury and is often seen in patients with viral hepatitis. HBV and HCV infections are frequently accompanied by mitochondrial dysfunction. In patients, HCV infection results in morphological alteration of mitochondria, reduction in the copy number, and oxidative-damage-triggered mutations in the genome of mtDNA. [11][12][13]23 Interestingly, mitochondrial abnormalities in HCV patients vary in a genotype-dependent manner. Their frequency is higher in genotype 1b than genotype 2a/c or 3a infection, suggesting a greater intrinsic cytopathic effect of genotype 1b HCV. 11,24 The current direct-acting antivirals are highly effective in inhibiting HCV infection. However, whether mitochondrial dysfunction persists in patients after HCV eradication remains an interesting question to be investigated. In HBV patients, a lower level of serum mtDNA content is related to an increased risk of HCC development, indicating that circulating mtDNA may be a potential noninvasive marker of HCC risk. 25 Extensive mitochondrial gene dysregulation and global downregulation of mitochondrial function have been observed in HBV-specific CD8 T cells from patients with chronic infection. Treatment with mitochondria-targeted antioxidants restores antiviral activity of these exhausted HBV-specific CD8 T cells. 26 Data regarding the mitochondrial status in hepatitis A and E patients remain limited, identifying a need for future research.

| Apoptosis in the pathogenesis of viral hepatitis
Accumulating evidence supports the role of liver cell apoptosis in the pathogenesis of viral hepatitis. 27 Although there are multiple modes of programmed cell death, pyroptosis and apoptosis cascades through the extrinsic and intrinsic pathways are the predominant forms for viral hepatitis. 28 The extrinsic signaling is activated via the cell surface death receptors including TNFR1, TRAIL-R1, and Fas. The intrinsic pathway is mainly triggered by nonreceptor stimuli but characterized by the permeabilization of the outer mitochondrial membrane. This leads to the release of proapoptotic factors from the mitochondrial intermembrane space into the cytosol. 29 A recent study demonstrates that the extrinsic and intrinsic apoptotic pathways activate pannexin-1 to drive NLRP3 inflammasome assembly, which is involved in the pathogenesis of viral hepatitis. 30,31 The numbers of apoptotic hepatocytes in chronic hepatitis B and C patients are small but higher than those in healthy individuals. 32 It is now generally accepted that cytotoxic T lymphocytes mediate the immune clearance of hepatitis virus-infected hepatocytes. Immunemediated apoptosis plays an important role in liver damage and pathogenesis. 33 However, hepatitis viruses may also have direct effects on apoptosis. The role of the HBV X gene product (HBx) in hepatocyte apoptosis is multifaceted. Proapoptotic function of HBx has been reported in hepatocytes of transgenic mice, 34 whereas it also blocks Fas-induced apoptosis in liver cells. 35 Similarly, HCV infection enhances susceptibility to Fas-mediated apoptosis, 36 whereas several HCV proteins (core, E1, E2, and NS proteins) inhibit TNF-α-mediated apoptosis. 37 Recently, HEV has been reported to induce hepatocyte apoptosis via mitochondrial pathway in Mongolian gerbils. 38 However, the underlining interaction between apoptosis and HEV infection remains largely obscure.
Cytochrome c, an essential component of the electron transport chain (ETC) transferring electrons from complex III to complex IV, plays a key role in the early events of mitochondria-mediated apoptosis. Serum cytochrome c has been suggested as a potential new marker for fulminant hepatitis in patients. 39 During apoptosis, cytochrome c is released from the mitochondrial intermembrane space to induce caspase activation. HCV can induce, 40 whereas HEV can block the release of cytochrome c from mitochondria to cytosol ( Figure 1). 41 The possible correlation between the amount of serum cytochrome c and the severity of hepatitis should be further explored for potential diagnostic relevance. Besides cytochrome c, mutual interactions between caspase activation and viral infection have also been observed. 42 Several viruses express proteins that could be cleaved by the caspase protease, resulting in inhibition of apoptosis. 43,44 For example, the HCV viral nonstructural protein 5A can be cleaved by activated caspase, which subsequently translocates to nucleus to enhance the transcription of several NF-κB target genes to inhibit apoptosis. 45 The protein from HEV ORF2 has different forms and could translocate to the cell nucleus. 46 However, whether ORF2 protein is cleaved by the host protease and whether it regulates apoptotic pathway remain to be further studied. Taken together, apoptosis is likely an important mechanism in pathogenesis of viral hepatitis. Hepatitis viruses can modulate apoptotic pathways at various levels. Thus, detection and quantification of particular apoptosis-related molecules may be explored as potential biomarkers for disease diagnosis in viral hepatitis patients.

| MAVS and mtDNA-mediated Innate Immune Response
The early and non-specific detection of hepatitis viruses is generally through the recognition by pathogen-associated molecular patterns Because mtDNA contains remnants of bacterial nucleic acid sequences and is methylated in a different way from nuclear DNA, it resembles non-self DNA and is thus easily to be degraded after transferring to the cytosol, leading to the activation of innate immune system. 54 mtDNA-mediated immune activation involves TLR9 and cGAS-STING signaling pathways, which contribute to the clearance of invading pathogens and provoke inflammasome activation, interleukin-1 production, and pyroptosis. 55

| Mitochondrial morphodynamics in response to hepatitis virus infection
The mitochondrial life cycle entails frequent fusion (in which two mitochondria form a single organelle) and fission (the division of one mitochondrion into two daughter organelles) events. 61 66 This correlates with oxidative stress, presenting as excessive lipid peroxidation and deficiency of tissue hepatocellular antioxidant stores, which in turn contributes to steatosis that is highly prevalent in HCV infection. 67,68 In contrast, HEV is able to trigger mitochondrial fusion to promote viral replication ( Figure 2B). 69 Because mitochondrial fission is the initial step of mitophagy, the differential regulation of mitochondrial morphodynamics by HEV compared with HCV may suggest a negative regulation of mitophagy during its propagation.
The fission and fusion processes in hepatocytes are responsible for the exchange and reallocation of mitochondrial contents including  70 Importantly, the equilibrium between fission and fusion is crucial for stabilizing mtDNA copy number and maintaining healthy liver function. 71 Hence, modulation of mitochondrial morphodynamics could potentially affect virus-induced liver dysfunction.
In addition, morphodynamics also regulates innate immunity by affecting the distribution of MAVS on the mitochondrial outer membrane. As reorganization of MAVS spatial distribution is a key event in IFN production in response to viral infection, such spatial reorganization has important consequences. Mitochondrial fusion promotes, whereas fission inhibits, RIG-I-like receptor (RLR) signaling.
Fibroblasts lacking mitofusin proteins produce less IFN and proinflammatory cytokines upon viral infection. 72,73 Small molecules, such as mitochondrial division inhibitor 1 (Mdivi1) that inhibits Drp1 activity, have been developed. 74 Hence, the effects of these agents on different hepatitis viruses are interesting be investigated. In contrast, increased ROS production counteracts HCV replication. 81 Thus, the ETC emerges as a primary target for viral infection, although hepatitis viruses likely target its functionality indirectly, for instance, by modifying mitochondrial morphodynamics.

| Mitochondrial permeability transition pore and hepatitis viruses
Mitochondria actively communicate with the cytosol and nuclear compartments. The signals involved are mediated through proteins located on the mitochondrial membrane, including the mitochondrial permeability transition pore (MPTP). Mitochondrial contents can escape from the mitochondrial matrix during MPTP opening. 82,83 The products related to the action of ETC, such as ATP and cytochrome c, are transferred through MPTP to cytosol to exert biological functions. MPTP is composed of voltage-dependent anion channel (VDAC) in the outer mitochondrial membrane, the adenine nucleotide translocator (ANT) in the inner mitochondrial membrane, and cyclophilin D (CypD) as its regulator in the matrix.
Hepatitis viruses have various interactions with MPTP. HBx protein has been shown to colocalize with VDAC, leading to alteration of mitochondrial transmembrane potential. The 68-117 region of HBx interacts with mitochondria and is necessary for membrane permeabilization. 84 HEV ORF3 protein sustains high levels of oligomeric VDAC to preserve mitochondrial potential and membrane integrity, thereby protecting infected cells from mitochondrial depolarization and death. 41 HBV and HCV core proteins provoke MPTP opening, whereas HEV prevents such an event. In line with this, the MPTP inhibitor cyclosporine A (CsA) inhibits HBV and HCV 85-87 but promotes HEV replication. 18,88 As highlighted, the importance of mtDNA in innate immunity, mtDNA fragments in fact are also released through MPTP. Thus, targeting MPTP opening represents a potential antiviral strategy.

| THE IMPACT OF MITOCHONDRIAL METABOLITES
Metabolites produced from the mitochondrial tricarboxylic acid cycle, including citrate, succinate, fumarate, and acetyl-CoA, are important regulators of signaling transduction when released from the mitochondria. 56,89 Citrate synthase and succinate dehydrogenase are upregulated in HBV-infected cells, leading to elevation of the corresponding metabolites such as fumarate and succinate. 90 Succinate has been recognized as an emerging signal transducer to activate inflammatory pathways. 7 An example is the increase in antigenpresenting capacity of dendritic cells if cytosolic succinate levels increase. 91 Thus, it is rational to suggest that such molecules may modulate innate immunity in hepatocytes as well. 92 HCV infection has been related to elevated level of acetyl-CoA, a metabolite that participates in many biochemical reactions in protein, carbohydrate, and lipid metabolism. 93 It has been widely recognized that acetyl-CoA contributes to lysine acetylation by donating its acetyl group. 94 Lysine modification controls many aspects of protein function and provides an obvious mechanism as to how acetyl-CoA can influence cellular function. HBV replication is regulated by the acetylation status of the cccDNA-bound H3/H4 histones. 95,96 Acetylation of retinoic acid-inducible gene I (RIG-I) regulates its antiviral functions, 97 and RIG-I is essential for sensing HAV, 98 HBV, 99 HCV, 100 and HEV infections. 101 Importantly, adequate cytosolic acetyl-CoA level is required for interferon-γ (IFNγ) production. 102 Other metabolites can inhibit inflammatory responses. For example, lactate acts through the lactate receptor to reduce hepatitis in mouse models. 103 There is an increase in lactate production in HCV-infected cells, probably because the corruption of mitochondrial function provokes increased dependency in the hepatocyte on glycolysis to support its energy needs. 104 In apparent agreement, targeting mitochondrial metabolism has been proposed to prevent chronic neuroinflammation. 105 112 The toxicity is primary due to damaging mitochondria, particularly in nerves, liver, skeletal, and cardiac muscle, as these tissues contain many mitochondria. 113 The degree of these side-effects limits development of this class of drugs, even though the antiviral effect may be very promising.
Despite the launch of many antiviral drugs, new therapeutics are still required for eliminating viral hepatitis. Unlike HCV, the persistence of cccDNA prevents cure but only inhibits viral replication in HBV patients. 114 115 It has also been shown to attenuate liver fibrosis in mice. 117 The mitochondrial ETC complexes have long been recognized as an antiviral target. 118 The complex I inhibitor, metformin, inhibits HBV and HCV infections in experimental models, 119,120 although the effects in patients remain unclear. Complex III sustains HEV replication and can be targeted by pharmacological inhibitors to inhibit viral replication in experimental models but requires further clinical validation. 18 Lastly, mitochondria-mediated apoptosis is essential in the pathogenesis of viral hepatitis; however, no optimal drug has been identified to prevent or treat liver injury. In this respect, mitochondria-targeted antioxidants or caspase inhibitors, look promising, but require further investigation.

CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.