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Medical Studies around Oxygen

Oxygen and viruses: a breathing story

Introduction
Louis Pasteur’s work on bacterial fermentation in 1861 and
the creation of the McIntosh–Fildes anaerobic jar in 1916

are pioneering works examining the effect of oxygen ten-
sion on bacterial metabolic regulation. It was only at the

beginning of the 20th century that some reports on the
interaction between oxygen and viruses emerged. In 1993,
Cipolleschi et al. published an article on the existence of
an oxygen gradient within the haematopoietic niche,
which is related to our current work on human parvovirus
B19 (B19), which displays a sharp tropism for erythroid
progenitors (Cipolleschi et al., 1993). Concurrently, we

were also intrigued by the fact that some rodent parvo-
viruses were reported to be oncolytic for solid tumours

which exist under hypoxic conditions (Dupressoir et al.,
1989). These data prompted us to study the impact of

oxygen tension levels on B19 replication in primary ery-
throid progenitors, which led to the finding that hypoxia

(1% O2) enhanced viral gene expression (Pillet et al.,
2004). This seminal observation contributed to the ongoing
debate on the physiological relationship between oxygen
and viruses.
The aim of this insight review is to introduce the non-specialists
to this novel aspect of viral physiology. For further details on
this topic outside the scope of this review, please see our
mini-review published in 2013 (Morinet et al., 2013).
Cell biology and oxygen
Cell biologists and virologists have examined the effects of
pH and the concentrations of amino acids and/or growth
factors in cell culture for decades. However, the role of

oxygen tension levels has been neglected, despite the find-
ing that hypoxia downregulates influenza virus replication

in vitro The oxygen tension level is 20% in ambient air

(21.3 kPa) but is only 2–9% (1.9–8.7 kPa) in peripheral tis-
sues; thus, human cells subsist under chronic physiological

hypoxia in vivo. In the haematopoietic niche, the partial
pressure of oxygen (pO2) oscillates between 1 and 6%.

Such a gradient is beneficial to stem cells since it limits oxi-
dative stress and consequent risk of mutations, and also

aids in the maintenance of totipotency. In addition, the
culture of haematopoietic stem cells at low oxygen tension
levels permits a better engraftment into mice models
(Speth et al., 2014).
It is a well-known observation that solid tumours grow under
hypoxic conditions. Notably, hypoxia-induced resistance
to chemo- and radiotherapies led some researchers to use
hyperbaric oxygen chambers to treat solid tumours, but
without success. Two putative reasons for the observation of
chemoresistance in hypoxic tumour cells are: (i) they localize
far from blood vessels, and thus the drug fails to perfuse the
tumour adequately; and (ii) they are resistant to p53-mediated
apoptosis.
Hypoxia-inducible factor 1a (HIF-1a)
In 1992, Semenza and Wang described a transcription
factor that bound the erythropoietin enhancer in response
to hypoxia (1% O2), which was termed hypoxia-inducible
factor (HIF) (reviewed by Morinet et al., 2013) The HIF

heterodimer comprises two subunits: an oxygen-labile a-
subunit (HIF-1a) and a constitutively expressed b-subunit

(HIF-1b). Recently, two additional labile subunits have
Journal of General Virology (2015), 96, 1979–1982 DOI 10.1099/vir.0.000172

000172 G 2015 The Authors Printed in Great Britain 1979

been described: HIF-2a and HIF-3a. All three isoforms
contain an oxygen-dependent degradation domain
(ODD). HIF-1a regulates glycolysis and angiogenesis,
both of which promote tumour progression, whereas
HIF-2a stimulates erythropoiesis. At present, the function
of HIF-3a remains poorly defined. The hypoxia response
element (HRE) HIF-binding motif is 59-RCGTG-39 (R is
either A or G). Under normoxic conditions, HIF-1a is

hydroxylated on two proline residues (Pro-402 and Pro-
564) located in its ODD by a family of dioxygenase

enzymes called prolyl hydroxylases (PHDs), leading to its
interaction with the von Hippel–Lindau (VHL) tumour
suppressor E3 ligase complex. This interaction with VHL
leads to Lys-48-linked polyubiquitination of HIF-1a and
its degradation by the 26S proteasome. Consequently,
HIF-1a is constitutively repressed under normoxia.
In contrast, increases in nitric oxide and reactive oxygen
species are known to stabilize HIF-1a via PHD inhibition
(Zepeda et al., 2013). In hypoxia, PHDs are inactivated,
leading to HIF-1a stabilization and migration into the

nucleus, where it can associate with HIF-1b and other tran-
scriptional factors to facilitate its binding to HREs in the

promoters of target genes.
Cell respiration, oxygen and viruses

One of the hallmarks of cancer is a tendency to ferment glu-
cose to lactate, even when sufficient oxygen is available to

support mitochondrial oxidative phosphorylation. This is
known as the Warburg effect; it is less efficient in terms of
ATP generation (2 vs 36 ATPs), but enables glucose to be
used as a carbon source for the synthesis of nucleotides,
amino acids and lipids for cell growth and division (Ngo
et al., 2015). Some viruses can reprogramme cell metabolism
to assist virus replication. For instance, it has been shown
that adenoviral infection decreases cellular respiration and
induces a rapid Warburg-like shift to glycolysis (Thai et al.,
2014). The early adenoviral gene product E4ORF is sufficient
to promote this increased glucose metabolism via the
enhancement of Myc transcriptional activity, consequently
inducing optimal conditions for adenovirus replication.

In a similar manner, human cells infected with cytomegalo-
virus exhibit alterations in glucose metabolism, including

increased glucose uptake, glycolysis, and the redirection of
glucose carbon to support biomolecule synthesis (Yu et al.,
2014). For vaccinia virus, the viral protein C16 binds

PHD2 and prevents it from hydroxylating HIF-1a, thus sta-
bilizing HIF-1a under normoxic conditions (Mazzon et al.,

2013), and can also contribute to the reprogramming of cel-
lular energy metabolism (Mazzon et al., 2015). Notably, the

effective inhibition of glutamine metabolism blocks vaccinia
virus synthesis (Fontaine et al., 2014). The role of HIF-1a in
these metabolic changes remains ill-defined; however, it was
reported recently that the E6 oncogenic protein of human
papillomavirus type 16 contributes to the Warburg effect
by precluding the binding of VHL to HIF-1a, thus enhancing
HIF-1a-induced glycolysis (Guo et al., 2014). In addition, it

was reported that the expression of Kaposi’s sarcoma-associ-
ated herpesvirus (KSHV) microRNAs within the oncogenic

cluster induces the Warburg effect in primary endothelial

cells, resulting in reduced oxygen consumption and mito-
chondrial biogenesis, while increasing glucose metabolism

and lactate secretion. Moreover, the expression of these
miRNAs also leads to stabilization and activation of

HIF-1a (Yogev et al., 2014). Thus, it can reasonably be sus-
pected that viral RNAs able to modify host cell metabolism

were selected for during evolution in order to favour virus
replication.
Hypoxia and viruses
For many human DNA viruses, HIF-1a activity is responsible
for the hypoxia-mediated upregulation of the neurotropic JC
polyomavirus and KSHV (reviewed by Morinet et al., 2013).
The key latent antigen latency nuclear antigen (LANA),
encoded by KHSV, cooperates with HIF-1a to induce lytic

gene expression. In addition, LANA can target the hypoxia-
sensitive chromatin remodelling protein KAP1, a member

of the TRIM (tripartite motif) protein family; however, the
detailed mechanism underlying the involvement of KAP1
in the hypoxic response remains unclear (Zhang et al., 2014).
In contrast, hypoxia results in downregulation of the E1A
adenovirus type 5 mRNA (Chang et al., 2014). Hypoxic cells
switch from eukaryotic initiation factor 4E (eIF4E)- to
eIF4E2-dependent translation to synthesize a portion of their
proteins (Uniacke et al., 2014). We have also suggested that

the inefficient translation of the E1A mRNA might be prefer-
ably dependent on eIF4E under hypoxic conditions.

For human RNA viruses, hypoxia induces an upregulation
of hepatitis C virus and human immunodeficiency virus 1

(HIV-1) virus replication; however, the function of HIF-
1a in these mechanisms remains obscure (reviewed by

Morinet et al., 2013). Furthermore, hypoxia has no effect

on rubella virus expression and is known to suppress influ-
enza virus replication (Kilburn & van Wezel, 1972). These

more recent data are in agreement with the fact that influ-
enza virus exhibits a higher replication rate in vivo in the

top of the lung, which has the best ventilation.
However, whether HIF-1a always regulates viral expression

in response to hypoxia remains uncertain, as other tran-
scription factors have also been implicated in mediating

these effects. For example, STAT5A but not HIF-1a was
implicated in the hypoxia-induced enhanced production
of human parvovirus B19 virions (Morinet et al., 2013).
Hypoxia and virotherapy
The idea of using viruses to lyse tumour cells dates back to the
1950s (Russell et al., 2012). In human cancers, the animal H1
parvovirus is a candidate for oncolysis (Geletneky et al., 2015)

and can effectively decrease HIF-1a protein levels in pancrea-
tic cancer cells under hypoxia (Cho et al., 2015). In 1998, it was

F. Morinet and others

1980 Journal of General Virology 96

published that reoviruses usurped the Ras signalling pathway
to facilitate their replication, leading to the hypothesis that
these viruses might be used to lyse Ras-dependent tumours,
including colorectal cancer (Zenonos & Kyprianou, 2013).
The interest in the use of reoviruses as anti-cancer agent is
heightened by the fact that these viruses are lytic in vitro for
hypoxic tumour cells via a caspase-dependent mechanism
(Figova ́ et al., 2013). In the same way, Newcastle disease
virus induces oncolysis under hypoxic conditions in the
human renal clear cell carcinoma cell line 786-O, which is
defective for the tumour suppressor gene VHL (Ch’ng et al.,
2013). These promising results encouraged researchers to

use viruses to kill highly resistant tumours, such as glioblas-
toma. An oncolytic herpes simplex virus (oHSV) C101

strain was deleted of its neurovirulence gene c134.5, which is
essential for replication in quiescent normal cells but not in
tumour cells. Several human glioma xenolines were infected
with this strain under hypoxic conditions, but hypoxia
failed to enhance, and even decreased, oHSV infectivity,
virus replication and cytotoxicity (Friedman et al., 2012).
These disappointing results strongly emphasize that oxygen
tension must be considered when preparing next-generation
viruses for clinical application.
Perspectives
In our view, the regulation of virus replication by oxygen
prompts three remarks.
First, it is necessary to classify viruses according to their

response to hypoxia – some viruses are strictly aerobic (influ-
enza viruses), some aero-anaerobic (HIV) and others strictly

anaerobic. This would be particularly relevant for viruses of
the digestive tract, where there is a steep, radial gradient of
O2 (Robinson & Pfeiffer, 2014) owing to mucosal-associated
microbes, as they quickly consume O2 that has diffused

across the intestinal wall, resulting in extremely low concen-
trations within the lumen (v133 Pa) (Albenberg et al., 2014).

Secondly, related to this observation, we cannot exclude that

cultivation of virological samples at a different oxygen ten-
sion level will permit the detection of unusual viruses.

Finally, it remains unclear whether hypoxia-induced viral gene
regulation is intrinsic to the virus or the viral strain, or if it is cell
specific. The benefit of HIF-1a stabilization by different viruses

with respect to their replication or the metabolic reprogram-
ming of infected cells merits further investigations. Very

recently, Cheng et al.(2014) suggested that induction of aerobic
glycolysis through an AKt–mTOR–HIF-1a pathway represents
the metabolic basis of virus-induced trained immunity.
Acknowledgements

This work was supported partly by grants from the Assistance Publi-
que-Hopitaux de Paris and the Centre National de la Recherche

Scientifique (CNRS).

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