The interaction between mitochondria and oncoviruses

Abstract

Mitochondria play important roles in multiple aspects of viral tumorigenesis. Mitochondrial genomes contribute to the host’s genetic background. After viruses enter the cell, they modulate mitochondrial function and thus alter bioenergetics and retrograde signaling pathways. At the same time, mitochondria also regulate and mediate viral oncogenesis. In this context, oncogenesis by oncoviruses like Hepatitis B virus (HBV), Hepatitis C virus (HCV), Human papilloma virus (HPV), Human Immunodeficiency virus (HIV) and Epstein-Barr virus (EBV) will be discussed.

Introduction

According to the latest survey, over 10 percent of human tumors are caused by viral infections [1]. Viruses that transform cells into tumors are called oncoviruses, which are one of the three major carcinogenic factors. The pathogenesis of oncoviral oncogenesis includes: entry of the virus into a cell, synthesis and assembly of oncovirus into the host cell, spread of the virus from infected cells to healthy cells and accumulation of oncogenic changes within the infected cells. Recent studies have implicated mitochondria in multiple aspects of oncoviral oncogenesis [2, 3].

Mitochondria are membrane-enclosed organelles that are ubiquitous in eukaryotic cells. They also possess their own genomes, and mitochondrial DNA (mtDNA) contributes to the genetic background of host cells. On the other hand, the biogenesis, distribution and functions of mitochondria are modulated by internal and external stimuli including viruses, and at the same time, mitochondria mediate the viral pathways as well as antiviral immunity [4].

As part of the process to take control of host cells, oncoviruses modulate mitochondrial functions and bioenergetics by altering mitochondrial pathways, including regulating the production of ATP directly through affecting the assembly of respiratory complexes [74], affecting cell death/survival through participating mitochondrial apoptosis pathways, and even causing mitochondrial damage through over-production of reactive oxygen species (ROS) [13, 34, 57, 60, 74, 77, 102].

At the same time, mitochondria have been shown to be a double-edged sword in viral tumorigenesis. On the one hand, mitochondria may promote the invasion of the oncovirus [20, 58, 75, 101] and facilitate the formation and development of tumors by incorporating viral proteins into the mitochondrial system and modulating retrograde tumorigenesis pathways [11, 33, 57, 71, 96]. On the other hand, mitochondria can also meditate the termination of viral infection via activating immune responses [17, 37] and initiating apoptosis [38, 59, 69, 77].

In this review, we will discuss the interactions between mitochondria and oncoviruses in the context of tumorigenesis. In particular, we will summarize the relevant current research with common human carcinogenic viruses, with HBV, HCV, HPV, HIV and EBV as examples.

1. HBV

Hepatitis B virus (HBV) is a DNA virus with a diameter of 42 nm. It contains a double stranded circular DNA [5]. HBV belongs to the hepadnaviridae virus family. It is the major etiological agent for hepatitis. A chronic infection with HBV is the major risk factor for hepatocellular carcinoma (HCC), the most common malignant liver tumor with the highest mortality rate [6].

The HBV genome is about 3.2 kb, and it encodes 4 overlapping open reading frames designated as S (pre-S1, pre-S2 and S region), C (pre-core and core region), P and X [7]. HBx protein, encoded by HBX, contains 154 amino acids [8]. HBx has been closely associated with HBV-induced oncogenesis in the host cells in multiple ways including the regulation of transcription, signal transduction, cell cycle progress, protein degradation, apoptosis and chromosomal stability [9, 10].

HBx protein targets to the mitochondrial outer membrane [11]. The seven amino acid residues at the c-terminal, and Cys115 in particular, are essential for such translocation [12]. The integration of HBx into the mitochondrial outer membrane induces ROS overproduction and mtDNA oxidative damage [13]. In addition, HBx binds cytochrome c oxidase III (COX III) at the inner mitochondrial membrane [14], and up-regulates the expression of COX III [15]. HBV also activates mitochondrial fission, mediated by stimulating phosphorylation of dynamin-related protein (Drp1) at Ser616, and leads to mitochondrial perinuclear clustering [16].

Mitochondrial antiviral signaling protein (MAVS), located at the mitochondrial outer membrane, is an adaptor molecule downstream of RIG-I and MDA5, which detect intracellular dsRNA produced during viral replication, to coordinate pathways leading to induction of antiviral cytokines, and is thus critical for the innate immunity [17]. Alternatively, MAVS can also cause cell death by increasing the level of voltage-dependent anion-selective channel protein 1 (VDAC1), promoting the release of cytochrome c from mitochondria, and eventually inducing cell apoptosis [18]. HBx weakens the antiviral response of the innate immune system by enhancing ubiquitination of MAVS lysine at position 136 [19].

The mitochondrial haplotype constitutes the genetic background for host cells invaded by HBV. A comprehensive study on peripheral blood, tumor and/or adjacent non-tumor tissue from 49 HBV-HCC patients and 38 normal people revealed that people with mtDNA haplogroup M may have increased likelihood of onset of HCC [20].

On the other hand, it has been demonstrated that high cytosolic calcium is essential for HBV DNA replication [21], which involves HBx-mediated activation of Pyk2/ FA kinase and JNK- and MAPK-associated signal transduction pathways [22]. Mitochondrial calcium uptake plays an important role in sustaining elevated levels of cytosolic calcium [23, 24]. In association with mitochondrial permeability transition pore, HBx elevates mitochondrial dependent calcium signaling and stimulates HBV replication [25, 26].

2. HCV

Hepatitis C virus (HCV) is a positive-sense single-stranded RNA virus with a diameter of 55–65 nm. It belongs to the family of flaviviridae [27]. It is estimated that 3% of the world’s population is infected with HCV, and most of them are unidentified [28]. Like HBV, HCV is also a major risk factor for HCC. The incidence of HCC with HCV is increasing in many countries including the United States [29].

HCV has a 9.6 kb genome, composed of 5’ and 3’ non-translated regions flanking an open reading frame (ORF) encoding a large polyprotein. The HCV genome encodes 10 individual membrane-associated viral proteins which are divided into structural proteins including core protein, envelope 1 (E1) and envelope 2 (E2), and non-structural proteins, including p7 polypeptide, NS2, NS3, NS4A, NS4B, NS5A and NS5B proteins [30]. HCV core protein, a component of the viral nucleocapsid, plays a critical role in virus growth and differentiation [31]. NS3 complex with NS4A, in the form of the NS3–4A polyprotein, acts as a cofactor of the proteinase. NS4B protein induces the formation of a membranous web which facilitates the replication, assembly and release of HCV. NS5A was reported to cause aberrant and persistent G2/M-phase entry, thereby sustaining proliferative signaling [32].

The HCV core protein localizes both to ER and the mitochondrial outer membrane via a region spanning amino acids 112 to 152 [33]. The core protein induces a specific inhibition of complex I, which adapts mitochondria for hypoxia and enhances ROS production at the same time [34]. Meanwhile, the core protein facilitates ER Ca²⁺ release and increases mitochondrial Ca²⁺ uptake. Such calcium signaling modulation increases mitochondrial ROS production and mitochondrial permeability transition as well as decreasing MMP [35].

HCV infection has also been shown to stimulate mitophagy through up-regulating Parkin and PINK1 proteins. It also triggers Parkin translocation to mitochondria and induces mitochondrial perinuclear clustering [36].

The NS3 and NS4A complex NS3–4A protein, a serine protease, inhibits MAVS by cleaving it from the outer mitochondrial membrane and preventing the formation of MAVS signaling complex for antiviral functions [37]. In addition, HCV NS4B initiates the disruption of MMP and release of cytochrome c which in turn activates the caspase cascade and induces apoptosis [38]. NS5A modulates apoptosis by regulating cytochrome c and Bax [39].

3. HPV

The Human Papillomavirus (HPV) is small, double stranded circular DNA virus with a diameter of 45–55 nm [40]. It has been well established that genital tract epithelial infection with high-risk types of HPV causes cervical cancer (CC) [41]. At the same time, high-risk HPV DNA has also been detected in 99.7% of CC patients [42]. HPV belongs to the papillomavirus family, of which over 170 genotypes have been characterized [40]. HPV16 and 18 is the most common high-risk HPV [43, 44, 45].

The HPV genome is about 8.0 kb, and it encodes 3 overlapping open reading frames designated as long control region (LCR), early genes region (encoding early regulatory proteins, such as E2, E4, E6, and E7), and late genes region (encoding capsid proteins L1 and L2). E6 [46] and E7 [47] are essential for the production of HPV DNA synthesis proteins. In HPV-associated CC, E6 and E7 are always expressed [48, 49], while E2 and E4 are not expressed [50, 51]. There is also a spliced mRNA, E1Ê4, which encodes five amino acids from the E1 ORF spliced to the protein encoded by the E4 ORF, which binds and collapses the cytokeratin network and facilitates release of the virus from cells [52].

Mitochondrial transcription factor A (TFAM) is essential for mitochondrial transcription and replication [53, 54]. E2 protein could enhance mitochondrial biogenesis by up-regulating TFAM interacting protein P32 or gC1qR [55, 56]. P32/gC1qR, which localizes in the mitochondrial matrix near the nucleoid associated with TFAM, is also an essential RNA-binding protein for mitochondrial translation [56]. It was also reported that HPV 18 E2 interacted with repiratory chain directly and increased release of mitochondrial ROS, which correlateed with stabilization of HIF-1α and increased glycolysis. [57].

The impact of mtDNA haplogroup on CC has been investigated with 187 CC patients and 270 normal people in Mexico City. It was reported that people carrying mtDNA haplogroup B2 exhibited an increased risk of CC among the Mexican population [58].

HPV16 E1Ê4 protein binds to mitochondria, especially in cells lacking cytokeratin. E1Ê4 induces the detachment of mitochondria from microtubules, establishing a single large mitochondrial cluster adjacent to the nucleus. The translocation of mitochondria results in reduction of MMP and induces apoptosis [59]. Meanwhile, sustained over-expression of E6 in cervical carcinoma cells increases ROS production and mitochondrial membrane polarization, leading to apoptosis [60].

4. HIV

The human immunodeficiency virus (HIV), an RNA lentivirus, belongs to a subgroup of retroviruses, and is composed of two copies of positive single-stranded RNA with a diameter of about 120 nm. HIV infects the human immune system, in particular helper T cells, macrophages, and dendritic cells, establishing immunity destruction, promoting a variety of dysfunctions and the indefinite proliferation of cells [61]. The average survival time after HIV-1 infection is about 10 years and long-term HIV infections can give rise to shingles, tuberculosis, pneumonia, encephalitis and various types of cancers, leading to AIDS [62]. There are two major species of HIV, HIV-1 and HIV-2. HIV-1 is more virulent, and is the leading cause of HIV infections globally [63].

The HIV-1 genome is about 9.7 kb which carries 9 genes that encode 19 proteins. These genes include 3 encoding structural proteins gag, pol, and env, 3 regulatory genes tat, rev, nef and 3 proteins controlling virus maturation, assembly and release such as vif, vpu and vpr [64]. HIV-1 surface envelope glycoprotein 120 (gp120) encoded by env, binds to the CD4 glycoprotein and chemokine receptors on the host cell surface, initiating fusion of the virus and the host cell membrane [65]. The Vpr protein arrests cell division at G2/M [66]. In addition, the long terminal repeats (LTRs), a promoter region that mediates the expression of almost all of the HIV-1 proteins, is indispensable for HIV-1 expression and infection [67].

The MPTP consists of 3 major components including the voltage dependent anion channel (VDAC) in the mitochondrial outer membrane, cyclophilin D in the mitochondrial matrix and adenine nucleotide translocase (ANT) in the mitochondrial inner membrane [68]. Upon entry into the target cell, the C-terminal peptides of Vpr from HIV-1 bind to ANT, converting MPTP from a normal transporter into a pro-apoptotic pore. This transition is achieved by reducing MMP via Ca²⁺ influx into mitochondria, thus releasing apoptogenic proteins such as cytochrome c [69]. Mitochondria constantly undergo fusion and fission to maintain their proper function and morphology. Mitofusin-2 (Mfn2), a GTPase embedded in the outer mitochondrial membrane, is an essential component for mitochondrial fusion [70]. Vpr also targets Mfn2 and reduces its protein level. The reduction of Mfn2 damages the integrity of the outer mitochondrial membrane, and induces a progressive loss of MMP and mitochondrial deformation [71].

Cxc Chemokin Receptor 4 (CXCR4), a member of the G protein-coupled receptor family, mediates HIV infection of CD4⁺ T cells [72]. HIV gp120 protein binds CXCR4 to form the complex gp120-CXCR4, and this complex in turn binds mitochondria to initiate mitochondrial membrane depolarization and release of cytochrome c from the mitochondria, thus setting off a cascade of caspase activation [73]. It was also reported that HIV-1 infection blocked the expression of complex I subunit NDUFA6 protein, and thereby decreased the activity of complex I directly. The inhibition of complex I in turn enhanced ROS production and decreased MMP and ATP production as well [74].

A study of 1833 patients infected with HIV-1 revealed that mtDNA haplogroup J and U5a posed the most significant risk for AIDS. On the other hand, mtDNA haplogroup Uk, H3 and IWX were identified as being protective against infection of AIDS [75].

As for mitochondrial participation in HIV-1 carcinogenesis, it has been shown that stimulating generation of ROS activates NF-kappa B which further upregulates the HIV-1 related gene expression through HIV-1-LTRs. As a result, various oncogenic changes accumulate which in turn triggers proliferation of infected cells [76]. On the other hand, the high level of ROS in the mitochondrial matrix significantly raises the mutation rate of mtDNA. Over-production of ROS has also been implicated in the activation of mitochondrial glutaminase, and induction of the glutamate-mediated apoptosis pathway in the neuronal system [77].

5. EBV

Epstein-Barr virus (EBV) is a double helix DNA virus with a diameter of 122–180 nm which was first identified in Burkitt’s lymphoma cells [78]. EBV belongs to the human herpes virus type 4 (HHV-4), and B lymphocytes and epithelial cells are the main targets for its infection [79]. There are many EBV associated diseases, such as infectious mononucleosis, lymphoma, Burkitt’s lymphoma and nasopharyngeal carcinoma (NPC) [80]. Among these, the most common malignant otolaryngic tumor is NPC, which has the highest mortality rate. The first indication of the implication of EBV in NPC was detection of its significantly high antibody titer in NPC patients [81]. Later, EBV DNA was also found in NPC patients [82]. Now clinically, EBV DNA is widely used to monitor the progression and recurrence of NPC [83, 84], and it is also recognized as a screening tool in research as well as a risk stratification marker in development of therapies [85].

EBV has a double-stranded, circular 172 kb genome which encodes more than 90 proteins [79]. Among these, the latent membrane protein 1 (LMP1) plays an indispensable role in oncogenesis. LMP1 has been associated with a variety of important cancer-related pathways, involving NF-kB [86, 87, 88], AP-1 [88], STAT [86] and JNK [89]. Latent membrane protein 2A (LMP2A) induces a number of pathways that promote malignant cell growth through promoting metastasis and inhibiting differentiation [90]. Zta (BZLF1 or EB1) is an immediate-early protein which is expressed at the early stage of EBV infection. It is a DNA binding protein belonging to the basic leucine zipper transcription factor family [91]. It has been shown that Zta regulates the expression of transforming growth factor and fatty acid synthase genes [92, 93] and is essential for cell cycle progression [94].

mtDNA replication is initiated by transcription-mediated priming facilitated by mitochondrial single-stranded DNA binding protein (mtSSB). mtSSB is a tetramer composed of four 16 kDa subunits and binds to mitochondrial DNA at the transcription and replication initiation area [95]. Zta targets mtSSB and mediates the translocation of mtSSB from mitochondria into the nuclear compartment. It thus inhibits mtDNA replication, and decreases mtDNA copy number [96]. The mitochondrial genome is also considered to be one of the genetically susceptible areas in NPC [97, 98]. Patients with NPC showed a high frequency of mutation at mtDNA np16362 [99].

An epidemiologic study was carried out in south China where the NPC incidence is much higher than in the rest of the world [100]. The impact of mtDNA haplogroup on NPC incidence was investigated in 201 NPC patients with matched controls. It was reported that patients with haplogroup R9, and its sub-haplogroup F1 in particular, exhibited the most aggressive progression of NPC [101].

EBV infection enhances the production of ROS. ROS in turn regulate cytoskeleton rearrangements and induce mitophagy and autophagy, and such regulation is essential for tumorigenesis and metastasis. Peroxiredoxin-3 (PRDX3) is an antioxidant mitochondrial protein [102]. Inhibiting PRDX3 expression enhances metastasis with an increased mobility potential [103]. In addition, LMP2A-mediated Notch pathway enhances mitochondrial fission by elevating dynamin-related protein 1 (Drp1), which also promotes cellular migration [104].

6. Summary

It is well-accepted that tumorigenesis including viral tumorigenesis including ten important hallmarks: evading growth suppressors, activating invasion & metastasis, genome instability & mutation, inducing angiogenesis, enabling replicative immortality, sustaining proliferative signaling, resisting cell death, reregulating cellular energetics, avoiding immune destruction, and tumor promoting inflammation [3]. As we discussed in previous sections, mitochondria were reported involved in at least seven of them in 5 common oncoviruses covered in this review (Figure 1).

Figure 1. Mitochondria may mediate the pathogenesis of viral oncogenesis.

Virally induced cell responses are color coded according to the hallmarks of cancer to which they correspond. The figure is adapted from the review of Hanahan and Weinberg (2011) [3]. We summarize the recent studies which implicated mitochondria in multiple aspects of viral tumorigenesis, including activating invasion & metastasis, genome instability & mutation, enabling replicative immortality, sustaining proliferative signaling, resisting cell death, deregulating cellular energetics, and avoiding immune destruction. Mitochondrial influence on other three viral tumorigenesis processes have not yet reported in these common viruses discussed here.

Mitochondria are ubiquitous organelles in eukaryotic cells whose primary role is to generate energy supplies in the form of ATP through oxidative phosphorylation. Recent studies have also shown that mitochondria play a central role in modulating cell growth, host immune response and apoptosis [105, 106, 107]. They are in essence the major cellular hub for bioenergetics, biosynthesis and signal transduction. Human viral oncogenesis often involves persistent infections and overcoming resistance by the host’s immune reactions. Taking control of mitochondria and then integrating mitochondrial pathways are the central goals to achieve, first for viral survival and replication and ultimately for viral oncogenesis. We have discussed viral-mitochondrial interactions with 5 major oncoviruses as summarized in Figure 2. Such interactions certainly go beyond these common viruses. With human T lymphotropic virus type 1 (HTLV-1), p13II protein targets the mitochondrial inner membrane where it produces a membrane potential-dependent influx of potassium, leading to mitochondrial swelling and fragmentation, and altered mitochondrial calcium uptake [108]. The C-terminal peptides of F1L protein from vaccinia virus (VACV or VV) bind to mitochondria and interfere with apoptosis by inhibiting the loss of MMP [109] and inhibiting apoptosis [110]. The vMIA protein of Cytomegalovirus (CMV) binds Bax to form a vMIA-Bax complex which blocks Bax-mediated mitochondrial membrane permeabilization [111]. The UL12.5 protein of Herpes simplex virus-1 (HSV-1) targets onto mitochondria to induce the rapid and complete degradation of host mitochondrial DNA [112].

Figure 2. Virial regulation on mitochondria leading to tumorigenesis.

Mitochondrial dysfunction happens when viruses invade the host cells, which cause a series of events, ultimately induce tumorigenesis.

Although the roles of viral infection in mitochondrial modulation of cancer occurrence and development have been recognized for long time, investigation of the interactions between oncoviruses and mitochondria is still in its infant stage. This is partially due to the limited animal models and unsophisticated analysis systems presently available for such studies. The lack of synergetic discussions among scientists in the relevant fields of mitochondrial biology, virology and cancer biology also hinders the scientific progress. With successful overcoming these obstacles, we anticipate the following progresses in the near future: 1) a comprehansive understanding how mitochondrial DNA haplogroup contribute to the occurrence and development of oncoviral tumroigenesis; 2) identification of mitochondrial biomarkers for oncoviral oncogenesis in serveral prevalent cancers; 3) development of effective medicines for oncoviral oncogenesis targeting direct or indirect on mitochondrial biogenesis, mitochondrial dynamics, mitochondrial mediated apoptosis, mitophagy and mitochondrial mediated immunity. A better understanding of interactions between mitochondria and oncoviruses in the context of tumorigenesis will centainly provide unique opportunities for prevention and intervention of oncogenesis.

Highlights:

• Oncoviruses regulate the mitochondrial function of infected cells.

• Mitochondria modulate oncoviral oncogenesis.

• Exploring interactions between viruses and mitochondria will provide novel insights into mitochondrial biology and oncoviral oncogenesis.