Trends in

Microbiology OPEN ACCESS

Review

Mitochondria-mediated oxidative stress during

viral infection
Jonathan Foo, 1,2 Gregory Bellot, 3 Shazib Pervaiz, 4,5 and Sylvie Alonso 1,2,
*

Through oxidative phosphorylation, mitochondria play a central role in energy Highlights

production and are an important production source of reactive oxygen species Many viruses manipulate mitochondrial
(ROS). Not surprisingly, viruses have evolved to exploit this organelle in order respiratory and apoptotic functions to
ensure a successful intracellular life cycle.
to support their infection cycle. Beyond its role in the cellular antiviral response,
induction of oxidative stress has emerged as a common strategy employed by Viral interactions with mitochondrial
many viruses to promote their replication. Here, we review the key molecular membranes and other mitochondria-
mechanisms employed by viruses to interact with mitochondria and induce associated components lead to in-
creased production of reactive oxygen
oxidative stress. Furthermore, we discuss how viruses benefit from increased species (ROS).
ROS levels, how they control ROS production to maintain a favorable redox
environment, and how they cope with ROS-mediated cell death. Virus-induced mitochondrial ROS
(mtROS) benefit viral replication through
modulation of host pathways and cova-
lent changes in viral components.
Introduction
Viruses are obligate acellular parasites that exploit their host cell’s molecular machineries for Viruses control the oxidative status of the
host cell during the course of infection.
genome replication, protein translation, and post-translational modifications, virion assembly
and virus particle release. For optimal outcome, viruses have evolved strategies to interfere Manipulation of mtROS-induced apo-
with host antiviral defenses and subvert host cellular pathways for their own benefit. Within the ptosis (intrinsic apoptosis) represents an
cell, mitochondria (Box 1) play a key role in energy production and are involved in diverse cellular important viral strategy for optimal intra-
cellular viral replication and timely release
processes such as metabolism, production of reactive oxygen species (ROS) (see Glossary),
of viral progeny.
and apoptotic regulation [1]. As such, mitochondria are critically involved in the maintenance of
cellular homeostasis, whereby altered mitochondrial function(s) have been linked to pathologies Antioxidant treatments have emerged as
(often age-associated) of metabolic, cardiovascular, cancer, and neurological nature [2]. Furthermore, a promising antiviral strategy, although
further understanding of the complex in-
mitochondria contribute to the antiviral innate immunity of the host via the mitochondrial antiviral terplay between viruses and the cellular
signaling (MAVS) pathway which modulates the type I interferon response [3]. Thus, it is not surprising redox response is needed.
that viruses have evolved strategies to escape, exploit, or manipulate mitochondrial functions.
Notably, interactions between viral proteins and mitochondrial membranes promote ROS production.
Although oxidative stress triggers the host antiviral innate response, an increasing number of in vitro
and in vivo studies have described that increased ROS levels actually favor viral replication. 1
Infectious Diseases Translational
As such, modulation of the cellular redox status, in particular that which is mitochondria-driven, Research Programme, Department of
has emerged as an important underlying strategy employed by many viruses to thrive in their Microbiology and Immunology, Yong
Loo Lin School of Medicine, National
host. This, however, represents a delicate balancing act, as viruses have to deal with oxidative University of Singapore, Singapore
stress-related detrimental outcomes, including increased antiviral immunity, oxidative structural 2
Immunology Programme, Life Sciences
changes, and cell death. In this review, we discuss the molecular mechanisms involved in virus- Institute, National University of
Singapore, Singapore
induced oxidative stress in a mitochondria-dependent manner, and the consequences for 3
Department of Hand and Reconstructive
both the host and the pathogen. The latter includes the molecular mechanisms by which viruses Microsurgery, University Orthopedic,
(i) benefit from increased ROS levels, (ii) modulate ROS production to maintain a favorable redox Hand, and Reconstructive Microsurgery
Cluster, National University Health
environment, and (iii) gain control of oxidative stress-induced intrinsic cell death. System, Singapore
4
NUS Centre for Cancer Research
Oxidative phosphorylation and oxidative stress (N2CR) and Department of Physiology,
Yong Loo Lin School of Medicine,
ATP is synthesized as electrons shuttle through the mitochondrial electron transport chain (ETC), National University of Singapore,
which consists of multimeric protein complexes, I–IV, located on the inner mitochondrial Singapore

Trends in Microbiology, July 2022, Vol. 30, No. 7 https://doi.org/10.1016/j.tim.2021.12.011 679

© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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5
Box 1. Structure and functions of a mitochondrion National University Cancer Institute,
National University Health System,
Mitochondria are membrane-bound organelles found inside most eukaryotic cells. Referred to as the powerhouse of the
Singapore
cell, the primary function of a mitochondrion is to generate energy in the form of ATP by oxidative phosphorylation
(OXPHOS). Structurally, mitochondria have two distinct membranes, namely outer and inner membranes, that define three
compartments. The inner most compartment or mitochondrial matrix is where numerous enzymatic reactions occur,
including replication and transcription of mitochondrial DNA (mtDNA), protein translation, the citric acid cycle, and fatty acid
catabolism. The inner mitochondrial membrane (IMM) is permeable only to oxygen, carbon dioxide, and water. The IMM
forms cristae that invaginate into the matrix, and contains the electron transport chain (ETC) and the ATP synthase, *Correspondence:
responsible for OXPHOS. During this process, electrons are shuttled from the initial electron donor (NADH or FADH2) micas@nus.edu.sg (S. Alonso).
through a series of electron carriers to the final acceptor, molecular oxygen (O2), eventually resulting in the production of
ATP. The electron carrier proteins are embedded in the IMM; they consist of four protein complexes (complexes I–IV),
forming the ETC. Movement of electrons through the ETC during redox reactions is coupled to the pumping of protons
out of the mitochondrial matrix across the IMM and into the mitochondrial intermembrane space, thereby creating potential
energy in the form of a pH gradient and electrical potential across the IMM. Protons accumulated in the intermembrane
space are then channelled back to the matrix across the IMM through the ATP synthase, which uses this energy to make
ATP from ADP and inorganic phosphate (Pi). At the end of the ETC, electrons combine with molecular oxygen and protons
to make water.

The outer mitochondrial membrane (OMM) is a phospholipid bilayer freely permeable to small molecules and harboring
porins that allow bigger molecules to be transported across – among which are the mitochondrial antiviral-signaling protein
(MAVS), and the pore-forming voltage-dependent anion channel (VDAC), which is a major OMM trafficking protein.
Mitochondria are also involved in the storage and release of calcium ions (Ca2+). In mitochondria, Ca2+ are important for
energy production, signaling events, and opening of the mitochondrial permeability transition pore (mPTP). The interplay
between mitochondria and the endoplasmic reticulum (ER), another important calcium storage site, ensures the calcium
homeostasis of the cell. Ca2+ is transported across the OMM to the mitochondrial intermembrane space through VDAC,
then across the IMM to the matrix through specialized transporters.

membrane (IMM) in a process known as oxidative phosphorylation (OXPHOS) (Figure 1). During
this process, 1–2% of the electrons inevitably leak out of the ETC and reduce surrounding molec-
ular O2 to ROS superoxide (O2•–) [4]. Electron leakage occurs primarily at complexes I and III of the
ETC and is enhanced by saturation with electrons fed from the redox cofactor NADH or when
reverse electron transport occurs due to high proton motive force within the mitochondria during
respiration [5]. Notably, inhibition of electron transfer from complexes I and III to subsequent
electron carriers within the ETC by rotenone and antimycin A, respectively, can lead to accumu-
lation and leakage of electrons from these complexes [5,6]. While ROS can be produced through
various mechanisms, the ETC represents a major source of ROS within the cell. At steady state,
redox homeostasis is maintained through a series of reactions involving the dismutation of O2•–
to hydrogen peroxide (H2O2) by superoxide dismutase enzymes [SOD1 and SOD3 (Cu/ZnSOD;
cytosolic and extracellular) and SOD2 (MnSOD; mitochondrial)], which can be further detoxified
by a number of antioxidant enzymes such as catalases, peroxiredoxins, and glutathione/
glutathione peroxidase systems [7] (Figure 1). Oxidative stress occurs when the rate of
intracellular ROS production outweighs the antioxidant capacity of the cell. This situation
may result from either increased ROS generation and/or disabled antioxidant defenses.
However, challenging the popular dogma that ROS indiscriminately damage cellular macro-
molecules, these reactive molecules have been increasingly recognized and described as
secondary messengers that play a critical role in cell signaling by modulating gene transcription
as well as protein structure, stability, and function [8].

Molecular interactions between viruses and mitochondria leading to increased

ROS production
Increased mitochondrial ROS (mtROS) production, resulting from direct interactions between
viral proteins and mitochondrial membranes, and leading to respiratory dysfunction, has been
abundantly reported (Figure 1). In addition, viral interactions with other mitochondria-associated
components could also result in mtROS production.

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Viral interactions with mitochondrial membranes Glossary

Viral interactions with mitochondrial membranes lead to depolarization of the mitochondrial trans- Antioxidants and ROS scavengers:
membrane potential (MMP), ensuing ROS generation. Stable expression of the hepatitis B virus X antioxidants are defined as substances
protein (HBx) in hepatoma HepG2 cells resulted in downregulation of mitochondrial proteins or molecules that either delay or prevent
the oxidation of a substrate. Antioxidants
within the respiratory complex, thereby increasing sensitivity of the mitochondria to MMP act either by preventing the formation of
depolarizing insults [9]. HBx was also reported to interact with voltage-dependent anion channel ROS or by capturing and neutralizing
(VDAC) 3, a class of outer mitochondrial membrane (OMM) porin and a constituent of the mito- ROS (scavengers). The former involves
chondrial permeability transition pore (mPTP) [10]; dysregulation of mPTP has been associated molecules that negatively regulate
ROS-producing events and reactions.
with increased conductance of mitochondrial membranes leading to MMP depolarization and For example, glutathione peroxidase 4
ROS production. HBx localization to mitochondria has also been associated with increased (GPx4) suppresses the release of
mitochondrial Ca2+ uptake through the mPTP, and subsequent increase in cytosolic Ca2+ via cytochrome c from mitochondria,
thereby limiting ROS production.
store-operated calcium entry [9,11]. However, a functional link between VDAC–HBx interaction,
Scavenger molecules have distinct
mPTP dysregulation, and increased Ca2+ uptake has yet to be established. Also, whether HBx- modes of action. Examples of
mediated OXPHOS and mPTP dysregulation events are codependent remains unclear and endogenous scavengers include
further studies are required to establish a more cohesive model. enzymes such as superoxide
dismutase, catalase, and glutathione
peroxidase, and substances such as
Hepatitis C virus (HCV) is another example of viruses that induce mtROS generation through in- glutathione, proteins (ferritin, transferrin,
teractions with mitochondrial membranes. HCV Core, E1, E2, and NS3 proteins were found to ceruloplasmin, albumin) and
be associated with mitochondria and they inhibit electron transfer from respiratory complex I to low-molecular-weight molecules like uric
acid, coenzyme Q, and lipoic acid.
the next electron carrier, resulting in MMP depolarization and ROS production [12].
Glutathione (GSH): GSH is an
antioxidant molecule naturally found in
ROS production arising from MMP depolarization has also been documented during HIV animals, plants, and certain
infection, with Tat protein translocating from the nucleus to mitochondria [13]. A separate study microorganisms. GSH is a modified
tripeptide (glycine, cysteine, and
attributed Tat-mediated loss of membrane potential to the dysregulation of anionic channels glutamic acid) with a gamma peptide
[14]. The viral protein R (Vpr) is another HIV protein that was found to destabilize the mitochondrial linkage that reduces ROS through
inner membrane through its direct interaction with adenine nucleotide translocator (ANT) [15]. disulfide bond formation (GSSG). The
ratio of GSH to GSSG defines the
oxidative status of a cell, with GSH
Our group has also reported the association between enterovirus A71 (EV-A71) replication representing 90% of the total glutathione
complexes and mitochondria-bound prohibitin1 (PHB1) in murine motor neuron cells [16]. This pool in healthy cells. Recycling of GSSG
neurotropic RNA virus causes hand, foot and mouth disease (HFMD) in young children and is back to GSH involves NADPH and a
glutathione reductase. GSH has been
associated with potential neurological complications [17]. Mitochondrial PHB is involved in the
commercialized as an antioxidant
maintenance of inner membrane cristae structure, thus contributing to ETC activity [18]. It is supplement. However, due to poor oral
thus plausible that interaction between one or several EV-A71 proteins and PHB1 at the bioavailability, few clinical benefits have
mitochondrial membrane results in ETC dysfunction and ROS production, although this remains been reported.
Hypoxia-inducible factor 1α
to be experimentally demonstrated.
(HIF-1α): a master transcriptional
regulator of the adaptive response to
Viral interactions with other mitochondria-associated components hypoxia. HIF-1 consists of a
Viral interference with mitochondrial matrix proteins has been described to indirectly perturb constitutively expressed subunit,
HIF-1β, and a subunit HIF-1α whose
mitochondrial functions and leads to increased mtROS levels. In addition to its direct interaction
stability and activity are modulated by
with mitochondrial membranes, the HIV Vpr protein was found to indirectly trigger MMP loss oxygen. In response to low oxygen
through reduced levels of mitochondrial fusion protein mitofusin 2 (Mfn2), the latter involving the tension, stabilized HIF-1 upregulates
VprBP–DDB1–CUL4A ubiquitin ligase complex [19]. In a similar fashion, the interaction between genes involved in glycolysis, allowing
ATP production in an
HCV Core protein with heat-shock protein 60 (Hsp60) in the mitochondrial matrix triggered oxygen-independent manner. In
ROS production, presumably by interfering with the protein’s chaperone activity and leading to addition, under oxidative stress, HIF-1α
mitochondrial respiratory dysfunction [20]. relocates to the mitochondria and
protects against ROS-induced
apoptosis, independently of its role in
Furthermore, virus-induced endoplasmic reticulum (ER) stress (Box 2) is another indirect way transcription.
to increase mtROS levels. The latter stimulates calcium efflux from the ER and leads to mtROS Nuclear factor erythroid 2-related
production, conceivably as a result of Ca2+ uptake by mitochondria in the vicinity [21]. Such a factor 2 (Nrf2): a redox-sensitive
transcription factor that contributes to
scenario has been described during hepatitis B virus (HBV) infection, where accumulation of

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the hepatitis B surface (HBsAg) and core (HBcAg) antigens in the ER led to ER stress and was redox homeostasis by modulating
antioxidant response element
associated with ROS production [22]. (ARE)-dependent transcription and the
expression of antioxidant defense
How ROS production benefits viruses enzymes.
Despite the fact that virus-induced increased ROS levels trigger innate antiviral immunity, studies Reactive oxygen species (ROS):
oxygen species formed by the partial
have increasingly reported that ROS production benefits viruses. These claims are mostly based reduction of oxygen, including radicals
on the observation that antioxidant treatments resulted in lower viral titers in vitro in the culture su- such as superoxide anion (•O−2),
pernatant, and improved disease outcome in animal models. While the latter often results from peroxide radicals (•O–22 ), hydroxyl

preventing detrimental organ and tissue damage linked to excessive production of ROS, the for- radicals (HO•), hydroxyl ion (OH–), as
well as non-radicals such as hydrogen
mer suggests a more direct role of ROS during the virus intracellular life cycle. However, fewer peroxide (H2O2), singlet oxygen (1O2),
studies have pinpointed the molecular mechanisms involved, and this may be due to the pleiotro- and ozone (O3). ROS alter biomolecules'
pic effects of ROS within the cell. The mechanisms by which viruses benefit from increased ROS redox status, including proteins, lipids,
and nucleic acids, thereby affecting their
levels during infection can be broadly categorized into two classes, namely direct and indirect
functions. Generation of ROS has been
mechanisms. The former involves oxidative changes of viral protein activities while the latter con- recognized as an important mechanism
sists of ROS-mediated modulation of host signaling pathways. that regulates signaling events inside the
cell (also known as redox signaling),
while excessive levels of ROS can lead to
Oxidative modification of viral proteins
cell and tissue damage.
Oxidative stress has been known to induce covalent modifications of biological molecules, includ- Redox homeostasis: refers to the
ing DNA, protein, lipids, and glycans, resulting in loss or gain of function [23]. Naturally, viral mol- endogenous capacity of cells to maintain
ecules are also expected to undergo redox-mediated changes, which modulate their function a physiological redox status, essential for
the proper functioning of cellular
and activity. Consistently, the guanylyltransferase activity of alphavirus nsP1 and flavivirus NS5 processes and signaling events.
proteins was found to be enhanced under oxidative conditions, resulting in increased RNA cap-
ping activity and increased RNA genome replication [24]. The redox status was also predicted to
significantly impact binding affinity of the severe acute respiratory syndrome coronavirus 2
(SARS-Cov2) Spike protein to its receptor angiotensin-converting enzyme 2 (ACE2) through
influencing disulfide bond formation in both proteins [25]. Furthermore, SUMOylation is a post-
translation modification that is modulated by oxidative stress [26], and viruses have exploited
this activity. During infection with infectious salmon anemia (ISA) virus, nucleocapsid (NP)-induced
ROS results in SUMOylation of this protein that correlated with improved virus output [27]. SUMO
modification of the RNA-dependent RNA polymerase 3D and protease 3C from EV-A71 was also
reported to modulate their activity [28,29].

Other evidence for redox regulation of viral protein activity is illustrated by the observation that
glutathionylation of cysteine residues Cys67 and Cys95 modulates HIV-1 protease activity [30].
Redox regulation of HIV-1 protease activity is believed to allow timely virus maturation and pre-
vents premature host cell death, given the ability of this viral protein to cleave host prosurvival
Bcl-2 protein [31]. Interestingly, the presence of these cysteine residues at the dimerization inter-
face of other retroviral proteases from feline immunodeficiency virus (FIV) and Simian immunode-
ficiency virus (SIV) suggests that this redox-mediated modulation of viral protease activity may be
conserved among retroviruses [32].

Modulation of host signaling pathways

ROS-mediated modulation of host signaling pathways has been exploited by viruses to thrive in
their host. EV-A71 is a prime example. In recent years, evidence has indeed emerged that EV-A71
exploits the oxidative stress response for its replication and release of its viral progeny [33,34].
Both cellular ROS via the EGFR/integrin-β1/Rac-1/NADPH oxidase axis [35], and mtROS
through morphological changes and MMP depolarization [33] contribute to increased ROS levels
during EV-A71 infection. These increased ROS levels were found to promote EV-A71 replication,
which was associated with decreased sirtuin1 (SIRT1) expression [35], decreased expression of
nuclear factor erythroid 2-related factor 2 (Nrf2) [36], increased NF-κB [37] and JNK [38]

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Trends in Microbiology

Figure 1. Interactions between viral proteins and components of the electron transport chain (ETC). (A) Viral proteins such as HBsAg and HBcAg from
hepatitis B virus (HBV) induce stress in the endoplasmic reticulum (ER) and calcium efflux; the calcium can be taken up by mitochondria, leading to the production of
reactive oxygen species (ROS). (B) HBx protein interacts with voltage-dependent anion channel 3 (VDAC3) and promotes influx of mitochondrial calcium. (C) Hepatitis
C virus (HCV) E1, E2, and NS3 proteins inhibit complex I of the ETC, resulting in the production of ROS. (D) Enterovirus A71 (EV-A71) replication complexes interact
with mitochondrial prohibitin 1 (PHB1), which may alter mitochondria integrity and function. (E) Human immunodeficiency virus Vpr protein increases mitochondrial
membrane permeabilization by interacting with adenine nucleotide translocator (ANT) of the mitochondrial permeability transition pore (mPTP), leading to efflux of
apoptogenic factors. (F) HIV Tat protein dysregulates anionic channels, leading to loss of mitochondrial transmembrane potential (MMP).

signaling, and induction of the ER stress/autophagy signaling pathway [39], all of which could be
abrogated by ROS scavengers, with a resultant decrease in viral replication. Modulation of
these pathways during infection has been associated with timely induction of apoptosis for
optimal viral replication within the host cell.

Protein translation is another ROS-modulated activity that EV-A71 exploits. Virus-induced

increased mtROS production was shown to correlate with decreased phosphorylation of eukary-
otic translation initiation factor 2-alpha (eIF2α), which drives cap-dependent protein translation,
thereby resulting in preferential translation of EV-A71 cap-independent internal ribosome entry
site (IRES)-containing mRNA (Box 3) [33].

HCV is another example of a virus that benefits from increased ROS levels through modulation
of signaling pathways. HCV NS5A protein has been reported to induce expression of Death
Receptor 6 (DR6) through ROS-mediated activation of NF-κB signaling, which could be
abrogated by antioxidant N-acetylcysteine (NAC) and NF-κB inhibitor, SN50 [40]. Interaction
between NS5A and DR6 was then found to be critical for production of infectious virus, through

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Box 2. ER stress
The ER is the site where membrane and secreted cellular proteins are synthesized and folded. This implies a close
monitoring of the protein flux through the ER, to avoid build-up of misfolded proteins, thereby causing ER stress that
may impair cellular functions and homeostasis [99]. An elaborate array of signaling pathways from the ER to the cytosol
and nucleus allow the cell to detect and respond to the presence of misfolded proteins within the ER. Such response is
known as the unfolded protein response (UPR). The UPR starts with ER-resident proteins that are able to sense ER stress
through their domains protruding within the ER lumen and activate a cascade of signaling events via their cytosolic effector
domains. The initial goal of the UPR is to restore cellular function and it does so by downregulating protein translation,
degrading misfolded proteins, and increasing the production of chaperones involved in protein folding. Should this outcome
not be achieved by a certain timeline, the UPR triggers apoptosis. In addition, ER stress is associated with Ca2+ release from
the lumen into the cytosol, and Ca2+ eventually accumulates in the mitochondrial matrix (see Box 1 in the main text), which,
beyond a critical threshold, triggers proapoptotic mitochondrial outer membrane permeabilization (MOMP).

Chronic ER stress and sustained over-activation of the UPR have been increasingly recognized as key players in diseases
such as cancer, metabolic (diabetes) and neurodegenerative diseases, and inhibiting the UPR represents a potential
therapeutic strategy for these diseases. ER stress also occurs during viral infection, where large amounts of viral proteins
are being produced and saturate the ER.

dampening the ability of DR6 to suppress AKT-mediated survival signaling, thereby allowing HCV
to replicate without triggering cell death [40]. Besides NF-κB, HCV-induced ROS production also
activates signal transducer and activator of transcription (STAT)-3 signaling [41], leading to increased
expression of the long noncoding RNAs, lnc-7SK and lnc-IGF2-AF, which promote HCV replication,
and this was associated with upregulation of phosphatidylinositol 4-phosphate (PI4P) [42]. While the
proviral role of PI4P during HCV infection remains poorly defined at the molecular level, other studies
have reported its involvement in HCV replication and secretion [43,44].

Oxidative stress-mediated stabilization of hypoxia-inducible factor 1α (HIF-1α) subunit activity

has also been described to benefit viruses. A recent study has reported that mtROS production is
critical to sustain SARS-CoV2 replication in human monocytes, through the stabilization of HIF-1α
subunit activity, which in turn leads to enhanced HIF-1-mediated transcription of the IL-1β-
encoding gene and glycolytic genes [45]. Likewise, stabilization of HIF-1α activity in an mtROS-

Box 3. Cap-dependent and cap-independent initiation of protein translation

Eukaryotic messenger RNAs (mRNAs) contain a modified guanosine [7-methyl-G (5')ppp(5')N, where N is any nucleotide],
at their 5′ end, named cap structure. Initiation of translation of these capped mRNAs starts in the nucleus with the binding
of the small ribosomal 40s subunit and translation initiation factor 4E (eIF4E) to the cap structure, facilitating nuclear export
of the mRNA. In the cytoplasm, the eIF4E-cap complexes associate with two other translation initiation factors, eIF4G and
eIF4A, forming the eIF4F complex, which scans the RNA molecule until it finds the start codon at which point the large 60S
ribosomal subunit joins the 40S subunit for translation elongation to occur.

Cap-independent translation initiation was first described in picornaviruses, whose viral RNA genome 5′ end is uncapped,
and for which translation is initiated at highly structured untranslated regions (UTRs) called internal ribosome entry sites
(IRESs) where ribosomes, translation initiation factors (IFs), and other factors known as IRES-transacting factors (ITAFs)
are directly recruited. Since then, cap-independent initiation of translation has been described for many RNA viruses
[109]. Viral IRESs can be categorized into four groups depending on their requirements in IFs and ITAFs, with the idea that
the more compact IRES needs less of these auxiliary proteins. Cap-independent initiation of translation also occurs in
eukaryotes, mostly for genes involved in response to cellular stresses such as viral infection, during which cap-dependent
translation is shut down by various viral proteolytic activities. Cellular IRESs are less structured than their viral counterparts
with little sequence and structure conservation among them.

Furthermore, other non-IRES cap-independent mechanisms of translation initiation have also been reported, and these
mechanisms involve cap-independent translation elements (CITEs) that can be found in the 3′ UTR of many plant RNA
viruses with various structures and sequences.

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dependent manner was found to be responsible for enhanced HIV replication in CD4+ helper T
cells and secretion of extracellular vesicles [46]. Further studies have shown that ROS-
dependent stabilization of HIF-1α during HIV infection resulted in enhanced expression of
CXCR4 and glucose transporter (Glut), thereby resulting in increased HIV entry and glucose
uptake, respectively [47–50]. These studies thus indicated that the ROS/HIF-1α/Glut/CXCR4
axis is exploited by HIV to support its replication and facilitate infection of neighboring cells.

How viruses control ROS levels

As excessive production of ROS will inevitably lead to detrimental outcomes for both the virus and
the host, viruses have naturally evolved mechanisms to control ROS levels or counteract ROS-
mediated effects in order to keep the cellular environment favorable for viral replication. However,
description of such mechanisms is relatively scarce compared with the abundant literature on
virus-induced oxidative stress.

Increase in the antioxidant cell capacity and control of lipid peroxidation

One viral approach to control excessive production of ROS is to increase the antioxidant capacity of
the cell. Increased catalase expression and activity were measured in cells stably transfected with
human papillomavirus (HPV) E7 protein, making the cells more resistant to H2O2-induced cell
death [51]. Likewise, HCV protein NS5A was found to be involved in increased Mn-SOD expression
through activation of p38 MAPK, JNK, and AP-1 [52]. During infection of blooms of the phytoplank-
ton Emiliania huxleyi with the E. huxleyi virus (EhV), a double-stranded DNA coccolithovirus of the
family Phycodnaviridae, a concomitant rise in H2O2 and glutathione (GSH) concentrations was
observed during the late stage of viral infection, implying that a fine balance between induction of
oxidative stress and antioxidant cellular response is needed for the timely release of viral progeny
[53]. In addition, the increase in GSH metabolism may also be needed to convert ribonucleotides
to deoxyribonucleotides to support synthesis and replication of the EhV large genome [53].
However, whether this increase in GSH metabolism during EhV infection is a cell response or is
actively triggered by some viral components remains to be addressed.

A number of DNA viruses from the families of poxviruses, baculoviruses, mimiviruses, and
phycodnaviruses have also been documented to encode a copper–zinc superoxide dismutase
(Cu–Zn SOD)-like protein, although the enzymatic activity and/or the role of these proteins during
the virus life cycle remain to be experimentally demonstrated [54]. Functionality of the Cu–Zn SOD
produced by Paramecium bursaria chlorella virus 1 (PBCV-1) of the family Phycodnaviridae, genus
Chlorovirus, has however been reported [54]. The authors proposed that the enzyme is packaged
within the virion and may contribute to reducing ROS levels during the early phase of virus infection.

Furthermore, some viruses have evolved strategies to counteract the potential negative outcome
of oxidative-stress-induced lipid peroxidation on their own components. Lipid peroxidation
results in formation of lipid peroxides and reactive aldehydes that are cytotoxic, leading to
membrane dysfunction and altering DNA and protein structure and functions [55]. NS5A from
HCV was found to induce the production of glutathione peroxidase 4 (GPx4), which reduces
lipid peroxides, and therefore helps to limit incorporation of lipid peroxides into newly formed
virions that may otherwise alter their fusiogenic activity [56]. Furthermore, the discovery of residues
in HCV NS4A and NS5B that are resistant to lipid peroxidation suggests another mechanism by
which viruses are able to cope with oxidative-stress-induced lipid peroxidation [57].

Biphasic modulation of ROS production

A biphasic modulation of ROS production has been reported as yet another viral strategy to
harness the redox cell response. For example, human cytomegalovirus (CMV) has been shown

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to induce ROS production during the early phase of infection [58], but it increases GSH synthesis
afterwards to antagonize oxidative stress, thereby maintaining mTORC1 activity, which is critical
for cell growth and proliferation [59]. Similarly, in an HCV infection model using the human hepa-
tocyte Huh 7.5 cell line, increased oxidative stress was associated with high HCV replication
activity and cellular apoptotic activity, followed by increased expression of metabolic enzymes
involved in de novo synthesis and regeneration of GSH, resulting in lower HCV replication activity,
thereby favoring viral persistence [60]. This finding thus suggested that HCV controls its
replication activity, and hence the infection status (acute or chronic), through modulation of the
redox environment. The increased levels of GSH may result from increased expression of Nrf2,
a transcription factor that drives GSH de novo synthesis. The same study also showed that
dormant HCV infection in Huh 7.5 cells could be reactivated by addition of the pro-oxidant
drug, auranofin, leading to higher viral replication activity and apoptosis [60].

A biphasic activity was also observed during influenza A virus (IAV) infection where MAPK
activation peaked during virus entry and at late stages of IAV infection when nuclear export of
IAV nucleoprotein (NP) is facilitated via ERK phosphorylation of its Ser392 residue [61,62]. Given
the known role of ROS in regulating MAPKs activity [63], it is plausible that IAV actively manipulates
the host redox status throughout its replication cycle to ensure a successful outcome.

Control of ROS production through metabolic switch

Metabolic reprogramming has long been known as a viral strategy to generate all the molecules
and metabolites necessary for optimal virus production [64]. More specifically, several viruses,
such as HBV, HCV, HIV-1, and SARS-CoV2, have been reported to induce a metabolic switch
to aerobic glycolysis in a manner reminiscent of the Warburg effect observed in cancer [65–68].
The metabolic switch to aerobic glycolysis has been described as an important underlying mech-
anism to ensure the generation of anabolic intermediates required for proliferation of cancer cells,
and likely serves a similar function during viral infection for the synthesis of viral progeny. In addition,
one dares to argue that a switch to aerobic glycolysis may also represent a strategy by which
viruses avoid excessive production of mtROS by diverting fuel away from oxidative phosphorylation.
Supporting this idea, knockdown of the key glycolytic enzyme lactate dehydrogenase A (LDHA) led
to accumulation of pyruvate in mitochondria and promoted oxidative stress [69]. In addition, upreg-
ulation of key glycolytic enzymes has been reported in human fibroblasts in response to sublethal
doses of H2O2, highlighting glycolysis as an important mechanism to counteract oxidative stress
[70]. Further studies are nevertheless needed in order to establish a link between metabolic
reprogramming and control of the oxidative stress response during viral infection, which may
open new therapeutic avenues to influence the redox status through metabolic manipulation.

How viruses take control of mtROS-induced cell death

Oxidative stress has been well established as one of the stimuli that initiate the intrinsic apoptotic
pathway. In this process, mitochondrial outer membrane permeabilization (MOMP) is triggered,
leading to the release of cytochrome C, second mitochondria-activator of caspases/direct IAP
binding protein with low pl (SMAC/Diablo), and high temperature requirement serine peptidase
A2 (HtrA2/Omi) (Figure 2) [71]. Cytochrome c binds and activates apoptotic proteinase-activating
factor 1 (Apaf-1) in an ATP-dependent manner. The resulting apoptosome complex promotes
autocleavage of pro-caspase-9, which activates downstream caspases, such as caspase 3,
leading to apoptosis. SMAC/Diablo and HtrA2/Omi antagonize inhibitor of apoptosis proteins
(IAPs), relieving inhibition of caspase activity, thereby reinforcing cell death signals.

The release of proapoptotic factors as a result of MOMP is regulated by Bcl-2 family proteins [72],
which can be categorized into antiapoptotic members [including Bcl-2, B cell lymphoma-extra-

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Trends in Microbiology

Figure 2. Viral modulation of the intrinsic apoptotic pathway. Viruses can inhibit cell apoptosis by directly regulating caspase activity (A), or by antagonizing (B) or
inhibiting (C) proapoptotic Bcl-2 proteins. Alternatively, a virus can induce cell apoptosis by promoting activation of BAK and BAX (D), or by promoting mitochondria
permeabilization and release of cytochrome c (E). A virus may also increase apoptotic sensitivity by relieving Apaf-1 inhibition (F), or by increasing host translation of
proapoptotic factors such as Apaf-1 (G). Abbreviations: ADV, adenovirus; CMV, cytomegalovirus; EV-A71, enterovirus A71; hnRNPA1, heterogeneous nuclear
ribonucleoprotein A1; IAP, inhibitor of apoptosis proteins; IAV, influenza A virus; IRES, internal ribosome entry site; MOMP, mitochondrial outer membrane
permeabilization; SMAC, second mitochondria-activator of caspases; VACV, vaccinia virus.

large (Bcl-xL), induced myeloid leukemia cell differentiation protein (Mcl-1)], and proapoptotic
members [such as Bcl-2-associated X protein (Bax), Bcl-2 homologous antagonist killer (Bak),
Bid, and Bad] (Figure 2).

Since virus-induced ROS promotes cell death, viruses have evolved strategies to interact with
mitochondria in order to take control of the intrinsic apoptotic pathway and counteract undesirable
or untimely cell death. Indeed, while preventing cell host apoptosis allows viruses to complete their
intracellular life cycle, inducing apoptosis represents a means for some of them to exit the host cell
and release their viral progeny.

Viral antagonism of the intrinsic apoptotic pathway

One notable and conserved strategy employed by many viruses to prevent premature apoptosis
of their host cell is to antagonize proapoptotic Bcl-2 proteins (Figure 2), which could represent an
interesting therapeutic target space.

E1B 19K from adenovirus represents one of the earliest viral Bcl-2 antagonists discovered [73]. In
spite of limited sequence similarity to mammalian Bcl-2, E1B 19K functionally complements
human Bcl-2 and inhibits p53-dependent apoptosis through interaction with proapoptotic
protein Bak, preventing Bax–Bak assembly on the mitochondrial membrane [74]. E1B 19K also
binds to Bcl-2 interacting killer (Bik/Nbk), which disrupts Bak/Mcl-1and Bak/Bcl-xL interactions,
further dampening mitochondria-dependent intrinsic apoptosis. The antagonistic function of E1B

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19K towards proapoptotic proteins prolongs the viability of host cells, thereby allowing sufficient
time for virus maturation to complete.

Vaccinia virus of the family Poxviridae also encodes a Bcl-2-antagonizing protein, named F1L
[75]. F1L also lacks discernible sequence homology with mammalian Bcl-2, localizes to the
mitochondrial membrane, and inhibits apoptosis through interaction with proapoptotic Bim and
Bak proteins [76]. F1L also inhibits apoptosis by preventing mitochondrial translocation and
oligomerization of Bax on the mitochondrial membrane in Bak-deficient cells; however, this is
likely to occur via an indirect mechanism, as interaction between F1L and Bax has not been
reported [76], possibly involving the inhibition of Bim upstream [77].

Other viral strategies to prevent premature apoptosis of the host cell have been reported
(Figure 2). Epstein–Barr nuclear antigen (EBNA) 3A and 3C proteins were found to downregulate
the expression of proapoptotic protein Bim, thus promoting survival of EBV-infected B cells [78].
The UL36 gene product from CMV suppresses caspase 8 proteolytic activation by binding to its
pro-domain [79]. Of note, while caspase 8 is an initiator of extrinsic apoptosis, its inhibition also
prevents the activation of proapoptotic Bid, thereby inhibiting further amplification of apoptotic
signal through the mitochondrial pathway [80]. CMV can also directly act on mediators of intrinsic
apoptosis Bax and Bak through viral mitochondrial localized inhibitor of apoptosis (vMIA), a gene
product of UL37 [81–83]. In addition, CMV produces antiapoptotic noncoding miRNAβ2.7,
which binds and stabilizes complex I of the ETC, preventing release of the GRIM19 subunit,
thereby maintaining MMP and respiration in CMV-infected cells and improving cell viability
when challenged with metabolic stressors [84].

Virus-induced apoptosis
A number of viruses rely on cell lysis to release viral progeny in the external environment and infect
neighboring cells. Consistently, viral induction of the intrinsic apoptotic pathway has been
described. Two such prominent examples include EV-A71 [85] and IAV [86] for which a variety
of mechanisms have been reported (Figure 2).

Nonstructural protein 2B (NS2B) of EV-A71 signals Bax translocation to the mitochondrial outer
membrane and induces its conformational activation, resulting in formation of the apoptotic pore
[85]. One might dare to conjecture that an increase in intracellular ROS may represent a probable
signal for the mitochondrial recruitment and oligomerization of Bax, which further fuels mtROS
generation and apoptosis. EV-A71 3C protease has also been implicated in apoptosis induction
through cleavage of host protein heterogenous ribonucleoprotein A1 (hnRNPA1), which relieves
its binding to the IRES sequence in Apaf-1 mRNA and allows IRES-dependent synthesis of
Apaf-1, thereby resulting in downstream caspase activation and apoptosis [87]. Increased
IRES-dependent translation of Apaf-1 is further contributed to by EV-A71 NS2A-mediated cleav-
age of eukaryotic initiation factor 4G1 (eIF4G1), a key factor for cap-dependent mRNA translation
[88]. Downstream cytochrome c release, both M1 and NP proteins from IAV have also been im-
plicated in the upregulation of Apaf-1. M1 binds to Hsp70 and reduces its interaction with Apaf-1,
allowing apoptosome assembly [89]. Conversely, NP downregulates the expression of host ap-
optosis inhibitor 5 (API5), a protein that antagonizes E2F transcription factor 1 (E2F1), which
leads to upregulation of its gene target Apaf-1 [90]. Furthermore, PB1-F2 protein from IAV local-
izes to the mitochondria and increases permeability of the mitochondrial membrane by interacting
with ANT3 and VDAC1 of the mPTP, resulting in MMP loss and increased efflux of cytochrome c
[91]. In addition, IAV-induced apoptosis likely involves activation of proapoptotic Bax/Bak pro-
teins and downregulation of antiapoptotic proteins Bcl-xL and Mcl-1 [92]. Activation of MAPKs
p38 and ERK1/2 during IAV infection could also promote host cell apoptosis through their

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Box 4. Antioxidant treatment as a promising antiviral strategy? Outstanding questions

Observation of the close interplay between viruses and mitochondrial redox metabolism has prompted the idea that anti-
How does oxidative stress modulate
oxidant interventions could have potential clinical benefits in viral infections, through direct impact on viral protein activity
viral protein activity, and how do
and function, and modulation of host signaling pathways. While this idea is not new, the coronavirus disease 2019
viruses protect their components from
(COVID-19) pandemic has certainly provided an opportunity to bring antioxidant treatments to the forefront, at a time when
oxidative damage?
specific antiviral drugs have been lacking. Modulation of cellular glutathione (GSH) levels as a therapeutic approach to fight
viral infections has recently been reviewed [100]. Melatonin and vitamin D have also been proposed to be potentially Do viruses modulate ROS production
beneficial in the overall management strategy of COVID-19 patients, and this effect is mechanistically associated with the main- through manipulation of miRNAs?
tenance of mitochondrial integrity and function [101,102]. A bioinformatics approach using network pharmacology has also
identified resveratrol, a natural polyphenol long known for its antioxidant and anti-inflammatory properties [103], as a potential How do viruses interfere with, and
therapeutic option to treat COVID-19 patients, predicting its targets to be part of the host inflammatory response [104]. manipulate, the ferroptosis pathway?
Furthermore, the clinical benefits of treatment by NAC, a well-known precursor of GSH, in COVID-19 and IAV-infected
patients has recently been reviewed [105]. A recent clinical trial has also explored NAC treatment in tuberculosis–HIV coinfected Can antioxidant–antiviral combination
patients and found that the NAC-treated arm displayed increased GSH levels and improved total antioxidant status that was therapies provide clinical benefits?
accompanied by reduced lipid peroxidation compared with the control arm [106]. Furthermore, long-term dietary antioxidant
supplementation has been shown in animal models to effectively mitigate viral infections such as influenza [107].

However, while clinical benefits of antioxidant treatments in the context of viral infections have been reported, this approach is
unlikely to be sufficient to effectively protect against explosive, acute viral infections. Combining antioxidants with antiviral
drugs instead may represent a promising therapeutic approach [108]. In addition, a deeper understanding of the contribution
of oxidative stress during viral infections may still be required to harness fully the therapeutic potential of antioxidants.

associated signaling targets, namely Bcl-2 and p53 [93,94]. Activation of these MAPKs was ob-
served during the late phase of the IAV replication cycle, which is believed to assist in the release
of viral particles via apoptosis [61,94].

Concluding remarks
Viruses have devised sophisticated and well-orchestrated strategies to interact with multiple
mitochondrial components in order to support their intracellular life cycle. This includes timely
manipulation of the redox metabolism, and mitochondrial integrity and function. A dichotomy of
functional outcomes vis-à-vis redox modifications from the standpoint of cell fate determination
illustrates the remarkable complexity of intracellular perturbations triggered during the acute
and chronic phase of a viral infection. While much knowledge has been generated over the past
decades, and has shed light on the molecular interactions between viruses and mitochondria,
the pleiotropic effects of these interactions has made it very challenging to apprehend fully the
implications of these interactions. More specifically, our incomplete understanding of the molecular
mechanisms and dynamics of redox modulation during viral infection has limited our ability to
harness fully the therapeutic potential of antioxidants (Box 4).

Furthermore, a number of areas have remained underexplored (see Outstanding questions).

For example, a greater understanding on how the redox environment modulates viral protein
activities and how viruses protect their components from detrimental oxidative changes may
open novel therapeutic avenues. Additionally, the dependence on redox-regulated pathways
for viral activities may pave the way for repurposing existing drugs that target these pathways
in the treatment of viral diseases. Another potential area of further investigation is the crosstalk
between ROS and miRNAs during viral infections. Indeed, manipulation of miRNAs by viruses
to support their infection cycle has been well established, typically through dampening of the
cellular antiviral responses [95]. Separately, the crosstalk between miRNAs and oxidative
stress in the context of noncommunicable diseases such as cancer and diabetes [96,97] has
also been well established whereby expression of some miRNAs is modulated by ROS levels,
while some other miRNAs modulate the oxidative response. It is therefore conceivable
that some viruses may modulate ROS production through the manipulation of miRNAs, but
such a scenario has yet to be reported. Finally, ferroptosis is another area worth looking into.
Ferroptosis has recently been identified as a distinct iron- and ROS-induced cell death pathway

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involving mitochondria and lipid peroxidation. Whether and how viruses interfere with, and
manipulate, this pathway remains to be investigated [98].

Declaration of interests
No interests are declared.

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