Human herpesvirus 1 protein US3 induces an inhibition of mitochondrial electron transport
Previous studies have identified virus proteins that traffic to mitochondria and may affect mitochondrial function. Here, it is reported that Human herpesvirus 1 (HHV-1, herpes simplex virus 1) and influenza virus reduced mitochondrial respiration, whilst Measles virus, cytomegalovirus, coxsackievirus B4 and Feline calicivirus did not. The inhibition of total cellular respiration was caused by a block in the mitochondrial electron-transport chain. This effect occurred during beta-phase protein synthesis and the inhibition of mitochondrial respiration could be reproduced by ectopic expression of the beta-phase protein US3. An HHV-1 mutant lacking this protein failed to inhibit oxygen consumption in infected cells relative to controls. It was concluded that US3 was mediating the suppression of mitochondrial respiration following HHV-1 infection. The integrity of the electron-transport chain in HHV-1-infected cells was analysed further and the site of the block in electron transport was located between complexes II and III, a site previously shown to be affected by Poliovirus.
Human herpesvirus 1 protein U
3 induces an
inhibition of mitochondrial electron transport
Mohammad Derakhshan, Margaret M. Willcocks, Michael A. Salako,3
George E. N. Kass and Michael J. Carter
Michael J. Carter
School of Biomedical and Molecular Sciences, University of Surrey, Guildford GU2 7XH, UK
Received 14 February 2006
Accepted 30 March 2006
Previous studies have identiﬁed virus proteins that trafﬁc to mitochondria and may affect
mitochondrial function. Here, it is reported that Human herpesvirus 1 (HHV-1, herpes simplex virus
1) and inﬂuenza virus reduced mitochondrial respiration, whilst Measles virus, cytomegalovirus,
coxsackievirus B4 and Feline calicivirus did not. The inhibition of total cellular respiration was
caused by a block in the mitochondrial electron-transport chain. This effect occurred during
b-phase protein synthesis and the inhibition of mitochondrial respiration could be reproduced by
ectopic expression of the
b-phase protein U
3. An HHV-1 mutant lacking this protein failed to inhibit
oxygen consumption in infected cells relative to controls. It was concluded that U
3 was mediating
the suppression of mitochondrial respiration following HHV-1 infection. The integrity of the
electron-transport chain in HHV-1-infected cells was analysed further and the site of the block in
electron transport was located between complexes II and III, a site previously shown to be affected
Many viruses affect mitochondria: morphological altera-
tions are induced by human immunodeﬁciency virus
(Radovanovic et al., 1999), human T-cell leukemia virus
type 1 (D’Agostino et al. , 2002) and Rubella virus (Lee et al.,
1999), and changes in location are observed in infection by
Human herpesvirus 1 (HHV-1, herpes simplex virus 1) and
Hepatitis B virus (Murata et al., 2000; Takada et al., 1999).
Virus protei ns may trafﬁc to mitochondria (Henkler et al.,
2001) and affect the function of mito chondrial components
(Rahmani et al., 2000). Inﬂuenza virus protein PB1-F2 binds
to mitochondrial inner membranes, triggering apoptosis
(Gibbs et al., 2003; Yamada et al., 2004). How ever, relatively
little is known about how such processes might affect the
basic mitochondrial function of energy generation. Norkin
(1977) demonstrated that total cellular respiration was
reduced by simian virus 40 infection of CV-1 cells, although
levels of ATP were maintained. Poliovirus induce s a rapid
inhibition of host-cell respiration and blocks electron tran-
sport between complexes II and III (Koundouris et al.,
2000). In this case too, cellular ATP levels remain unaffected.
In contrast, ATP levels decline during HHV-1 infection,
which has been attributed to mitochondrial dysfunction
(Murata et al., 2000). We hypothesized that such effects
might be common in virus infection and have tested
different viruses. We found that HHV-1 and inﬂuenza
virus both inhibited the mitochondrial respirato ry chain.
Further investigation of the HHV-1-induced effect showed
that the virus targeted a site between complexes II and III
and that this w as mediated by pro tein U
Cellular respiration was measured by using a Clark-type
oxygen electrode (OE) (Hansatech Instruments). Cells were
infected with 10 p.f.u. per cell by using the following virus–
cell combinations: HHV-1 (strain HFEM , kindly provided
by Professor A. C. Minson, Cambridge, UK), Measles virus
(Edmonston strain, kindly provided by Professor V. ter
rzburg, Germany) and coxsackievirus (strai n
B3) were grown in HeLa cells; cytomegalovirus (strain Ad
169, kindly provided by Professor A. C. Minson) was culti-
vated in MRC-5 cells; Feline calicivirus (strain F9) was grown
in CRFK cells and inﬂuenza virus (strain A/Puerto Rico/8/
34, kindly provided by Dr P. Digard, Cambridge, UK) was
grown in MDCK cells. Both mock- and virus-infected cells
were recovered by scraping and their viability was deter-
mined by dye exclusion. Cells (3610
) were washed with
Dulbecco’s PBS and resuspended in 0?4 ml Dulbecco’s PBS
for transfer to the OE for measurement of oxygen con -
sumption. Oxygen consumpti on was attributed entirely
to mitochondria, as inhibition of mitochondrial electron
transport by antimycin A (3
) abolished measurable
Only HHV-1 and inﬂuenza virus (Fig. 1a and b) suppressed
cellular respiration. Feline calicivirus, Measles virus, cyto-
megalovirus and coxsackievirus B4 had no detectable effect
(data not shown). HHV-1 was the more potent, reducing
oxygen consumption rate by 31 % at 6 h post-infectio n
(p.i.), 54 % at 12 h p.i. and 60 % at 18 h p.i. (Fig. 1a).
Inﬂuenza virus reduced consumption by 30 % at 6 h p.i.
and by 45 % at 12 h p.i. (Fig. 1b). The viabilities of infected
and uninfected cells were not signiﬁcantly different at these
3Present address: Cancer Research UK, London EC1M 6BQ, UK.
2006 SGM Printed in Great Britain 2155
Journal of General Virology (2006), 87, 2155–2159 DOI 10.1099/vir.0.81949-0
times. Inhibition of cell respiration occurred well before the
onset of cytopathic effect.
Inﬂuenza virus protein PB1-F2 is known to collapse
mitochondrial membrane potenti al (Gibbs et al., 2003),
which may be responsible for the effect observed. However,
the genesis of this effect by HHV-1 was unknown. Therefore,
we analysed the integrity of the electron-transport chain in
infected cells to locate the site of any blockage. HHV-1-
infected (12 h p.i.) or mock-infected HeLa cells were
harvested as above, washed twice with mitochondrial respir-
ation buffer (210 mM mannitol, 70 mM sucrose, 1 mM
EGTA, 5 mM HEPES, pH 7?1), resuspended in 0?4ml
mitochondrial respiration buffer and transferred into the
OE. Digitonin (0?375 mg ml
) was added to permeabilize
the plasma membranes. R espiration was monitored fol-
lowing the addition of substrate electron donors feeding
electrons in to known points in the electron-transport chain.
When appropriate, electron transport through speciﬁc com-
plexes was subsequently blocked by using speciﬁc inhibitors
to conﬁrm observations. Substrates/inhibit ors used were:
pyruvate (5 mM) plus malate (5 mM), rotenone (3
succinate (5 mM), antimycin A (3
phenylenediamine (TMPD) (0?5 mM) plus ascorbate
(1 mM), and sodium azide (5 mM). The results are pre-
sented in Fig. 2. Complex I was assessed by the addition of
pyruvate plus malate (P/M) to donate electrons to complex I
and subsequently by the addition of rote none to prevent
their onward transmission to complex II. We detected no
increase in oxygen consumption following pyruvate and
Fig. 1. Effect of virus infection on total cell respiration. In each
case, cells were infected or mock-infected with virus and
harvested at the indicated times p.i. Total cell respiration was
determined in infected and control cells by using an OE. (a)
HHV-1 infection of HeLa cells. $, Mock-infected cells; #,
virus-infected cells. (b) Inﬂuenza virus infection of MDCK cells.
Filled columns, mock-infected cells; shaded columns, virus-
infected cells. Asterisks indicate signiﬁcant divergence from the
control values: *, P<0?05; **, P<0?01; ***, P<0?001.
Fig. 2. Analysis of mitochondrial respiratory-chain integrity.
Mitochondrial respiration was determined in mock-infected (a)
and HHV-1-infected (b) cells with permeabilized plasma mem-
brane to permit the addition of electron donors to selected
parts of the respiratory chain. Respiration was monitored in real
time and the resulting traces are presented, with the times of
addition of the various compounds indicated by arrows above
the line. Downward deﬂection indicates increased use of oxygen
following electron-donor addition. Any such effects were con-
ﬁrmed by addition of the appropriate inhibitor to block induced
ﬂow and before the next complex in the chain was analysed.
Compound additions were as follows: addition of digitonin (Dig)
to permeabilize the cells; pyruvate/malate (P/M) (for complex I);
succinate (Suc) (for complex II) and TMPD/ascorbate (T/A)
(electron donation to cytochrome c). The inhibitors used were
rotenone (Rot) (blocks complex I), antimycin A (AA) (blocks
between complexes II and III) and sodium azide (Az) (blocks
complex IV). Note the differential effect of succinate addition
between infected and mock-infected cells. Each trace is repre-
sentative of at least three independent experiments.
2156 Journal of General Virology 87
M. Derakhshan and others
malate addition, even in the controls, probably because
endogenous substrates were present in excess, saturating
ﬂow through this complex. Assessment of complex II by the
addition of succinate clearly stimulated oxygen consump-
tion in mock-infected cells. This was blocked by antimycin A
(Fig. 2a), demonstrating that electron transfer from com-
plex II to oxygen via complexes III and IV was responsible.
This stimulation was absent in HHV-1-infected cells
(Fig. 2b) and antimycin A had no inhibitory effect. This
conﬁrmed that electrons donated by the succinate were not
able to ﬂow on to complex III and located an infection-
speciﬁc respiratory-chain block between complexes II and
III. We then investigated electron ﬂow post-cytochrome c
to complex IV by using TMPD/ascorbate. Following the
addition of TMPD/ascorbate, both control and infected cells
showed an increased rate of oxygen con sumption (Fig. 2a,
b) and thus electrons were able to ﬂow from cytochrome c
to complex IV. As expected, in all cases, sodium azide
blocked respiration completely. These data conﬁrmed that
complex IV was fun ctional in both infected and mock-
infected cells. We concluded that the most likely site of the
virus-induced electron-transport block was between com-
plexes II and III, although an additional block at complex I
could not be excluded.
One possible explanation for this effect could be that viruses
synthesize proteins that interact with mitochondria and
modulate the organelle’s activity: the pseudorabies virus
3 is known to trafﬁc to the mitochondria (Calton
et al., 2004) and work by Reynolds et al. (2002) has
demonstrated that the equivalent protein made by HHV-1
concentrates in the perinuclear area, the same site to which
mitochondria are known to migrate in the infected cell
(Murata et al., 2000). Suspicion thus fell on the HHV-1 U
protein and this was also consistent with the time of onset of
the effect, which in our hands occurred during the
well before the onset of synthesis of the gamma-phase
marker protein glycoprotein D. Consequently, we sought
to examine the role of U
3 directly by speciﬁc expression of
this protein inside uninfected cells. In order to avoid effects
caused by any carrier or polymerase-expressing viruses, we
decided against vaccinia T7-based or baculovirus expres-
sion systems and selected instead direct transfection using
mRNA produced in vitr o. For this purpose, HeLa cells were
grown in 75 cm
tissue-culture ﬂasks, infected with HHV-1
at an m.o.i. of 10 p.f. u. per cell and harvested when a
cytopathic effect was visible. Total RNA was extracted by
using RNAzol B (Biotecx Laboratories) and the U
sequence was produced by RT-PCR ampliﬁcation from total
RNA. The following primers were designed to introduce
unique restriction sites at each end (HindIII and XbaI) for
ease of subsequent cloning (underlined): reverse (antisense)
CGG-59, and forward (sense) primer, 59-CG
3 PCR-ampliﬁcation products were cloned into
pGEM-T Easy vector (Promega), veriﬁed by sequencing
and subsequently excised from the vector by using HindIII
and SpeI for forced-orientati on ligation into the pSP64
Poly(A) vector (Promega) from which mRNA transcripts
could be prepared in vitro using an Ambion mMESSAGE
mMACHINE SP6 transcription kit. The gene for luciferase
was similarly cloned into pSP6 4 Poly(A) for use as a control
to determi ne both transfection efﬁciency and the optimum
time of protein expression following transfection. Both tran-
scripts were veriﬁed as active by translation of the mRNA to
be used in transfection in vitro. In both cases, products of
the expected size were derived (data not shown). HeLa cells
were transfected with 2
mg mRNA prepared in vitro by using
Lipofectin (Invitrogen). Expression of transfected mRNA in
the cells was con ﬁrmed by reference to the control luciferase,
detected by luminometry. Peak expression occurred after
6 h. Transformation efﬁciency was determined as 80 % by
immunostaining of transfected cells with anti-luciferase
antibody (Promega). In order to determine the effect of the
3 gene product on mitochondrial respiration, we used
both mRNAs in separate transfections and compared these
with a ‘no RNA’ control. At 6 h post-transfection, we
measured total HeLa cell respiration in all samples in the
OE. Transfection with U
3 mRNA affected mitochondrial
respiration profoundly, resulting in a decrease in total cell
respiration of 47 % in comparison with cells transfected with
luciferase mRNA, which were indistinguishable from the
mock-transfected cells (Fig. 3a).
This analysis indicated clearly that protein U
3 alone was
capable of inducing a reduction in respiration; it did not,
however, demonstrate whether U
3 is the only virus protein
contributing to this effect. In order to investigate this
possibility, we made use of a well-characterized U
mutant (and its wild-type progenitor), kindly provided by
Professor B. Roizman (University of Chicago, IL, USA) and
reported previously (Purves et al., 1987). This mutant
expresses only the ﬁrst 68 residues of U
3, with aa 69–357
deleted, including all motifs associated with protein kinase
function; residues beyond the deletion are frame-shifted.
This mutant (termed R7041 in the original report) lacks
detectable protein kinase activity, a phenotype that is
reversed when the deletion is reverted (Purves et al., 1987).
R7041 was constructed in HHV-1 (strain F) virus and it
was necessary ﬁrst to establish that this strain also exh ibited
the inhibition of mitochondrial respiration that we had
observed in strain HFEM. We found that the time of onset of
cytopathic effect in both wild-type and mutant virus was
indistinguishable from that observed using strain HFEM,
and HHV-1 strain F wild-type virus clearly induced an
identical effect on respiration, establishing a 49 % reduction
by 12 h p.i. In comparison, the U
3 deletion mutant failed
to inhibit oxygen consumption and respiration was indis-
tinguishable from that of mock-infected cells (Fig. 3b). As
the function of US3 had already been established, it was not
necessary to consider the revertant virus in this experiment.
3 clearly has a role in pre- and post-mitochondrial
modulation of apoptosis in HHV-1-infected cells (Asano
Herpesvirus mitochondrial respiration inhibition
et al., 1999, 2000; Benetti & Roizman, 2004; Cartier et al.,
2003; Geenen et al., 2005; Goshima et al., 1998; Jerome et al.,
1999; Leopardi et al., 1997; Munger & Roizman, 2001; Ogg
et al., 2004). Here, we have also shown an effect on the
mitochondria themselves. It is becoming increasing ly
apparent that many viruses interact with mitochondria in
the infected cell, possibly in an attempt to modulate the
induction of apoptosis in the host. Such protein interactions
may have an effect on mitochondrial function, whether or
not this is their primary role. Mitochondrial dysfunction
may be relatively insigniﬁcant in acute infection unless it
reduces energy supply sufﬁciently to impair virus replica-
tion. However, effects such as these may gain in impor-
tance in the context of longer-term persistent or chronic
infections, where the cell’s energy balance may be subtly
altered, perhaps affecting cell function. For example,
following infection with Hepatitis C virus, oxidative injury
occurs through the generation of reactive oxygen species as a
direct result of mitochondrial dysfunction (Okuda et al.,
2002). Hepatitis C virus core protein is known to trafﬁc to
mitochondria (Schwer et al., 2004) and persistent expression
of this protein in transgenic mice leads to alterations in
mitochondrial appearance and, ultimately, damage to heart
muscle and cardiomyopathy (Omura et al., 2005). Similar
effects might provide a common pathway for host-cell
functional impairment operated by diverse viruses in differ-
ent host cells and should be considered where virological
explanations for cellular dysfunction are suspected, but
evidence for causation by one particular type of virus is
weak. This may be relevant for instance in suspected viro-
logical aetiology of chronic fatigue syndrom e.
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Fig. 3. Effect of U
3 protein expression on respiration. (a)
mRNAs for U
3 and luciferase proteins were synthesized in vitro
and transfected into HeLa cells by using Lipofectin as
described in the text. Six hours later, cells were harvested and
total cellular respiration was measured. First column, respiration
from control (non-transfected cells); second column, respiration
from luciferase (Luc) mRNA-transfected cells; third column,
respiration from U
3 mRNA-transfected cells. Expression of
3, but not luciferase, led to a profound decrease in cellular
respiration. (b) HeLa cells were mock-infected or infected with
3 deletion-mutant virus or its wild-type progenitor and cell
respiration was determined by using an OE as described in the
text. Asterisks indicate samples with signiﬁcant divergence from
control values: ***, P<0?001.
2158 Journal of General Virology 87
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