Human herpesvirus 1 protein US3 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
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
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 proteins may traffic to mitochondria (Henkler et al.,
2001) and affect the function of mitochondrial components
to mitochondrial inner membranes, triggering apoptosis
(Gibbs et al., 2003; Yamada et al., 2004). However, 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 induces a rapid
inhibition of host-cell respiration and blocks electron tran-
sport between complexes II and III (Koundouris et al.,
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 respiratory chain.
Further investigation of the HHV-1-induced effect showed
that the virus targeted a site between complexes II and III
and that this was mediated by protein US3.
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
Meulen, Wu ¨rzburg, Germany) and coxsackievirus (strain
B3) were grown in HeLa cells; cytomegalovirus (strain Ad
169, kindly provided by Professor A. C. Minson) was culti-
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 (36106) 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 consumption was attributed entirely
to mitochondria, as inhibition of mitochondrial electron
transportbyantimycinA(3 mg ml21)abolishedmeasurable
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-infection
(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.
0008-1949 G 2006 SGMPrinted in Great Britain 2155
Journal of General Virology (2006), 87, 2155–2159
times. Inhibition of cell respiration occurred well before the
onset of cytopathic effect.
Influenza virus protein PB1-F2 is known to collapse
mitochondrial membrane potential (Gibbs et al., 2003),
which may be responsible for the effect observed. However,
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
harvestedasabove,washed twice withmitochondrial respir-
ation buffer (210 mM mannitol, 70 mM sucrose, 1 mM
EGTA, 5 mM HEPES, pH 7?1), resuspended in 0?4 ml
mitochondrial respiration buffer and transferred into the
OE. Digitonin (0?375 mg ml21) was added to permeabilize
the plasma membranes. Respiration was monitored fol-
lowing the addition of substrate electron donors feeding
electrons into known points in the electron-transport chain.
Whenappropriate, electron transportthroughspecificcom-
plexes was subsequently blocked by using specific inhibitors
to confirm observations. Substrates/inhibitors used were:
pyruvate(5 mM)plusmalate(5 mM),rotenone(3 mg ml21),
succinate(5 mM),antimycinA(3 mg ml21),tetramethyl-p-
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
and subsequently by the addition of rotenone 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-
(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
showed an increased rate of oxygen consumption (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 functional 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 b-
protein US3 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 US3
proteinand this was also consistent with the time of onset of
the effect, which in our hands occurred during the b phase,
well before the onset of synthesis of the gamma-phase
marker protein glycoprotein D. Consequently, we sought
to examine the role of US3 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 vitro. For this purpose, HeLa cells were
grown in 75 cm2tissue-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 US3 coding
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-CGAAGCTTCGA-
The US3 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-orientation 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 pSP64 Poly(A) for use as a control
to determine both transfection efficiency and the optimum
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
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
US3 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 US3 mRNA affected mitochondrial
respiration profoundly, resulting in a decrease in total cell
luciferase mRNA, which were indistinguishable from the
mock-transfected cells (Fig. 3a).
This analysis indicated clearly that protein US3 alone was
capable of inducing a reduction in respiration; it did not,
however, demonstrate whether US3 is the only virus protein
contributing to this effect. In order to investigate this
possibility, wemade useofawell-characterized US3deletion
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 US3, 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 exhibited
the inhibition of mitochondrial respiration that we had
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 US3 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.
US3 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; Geenenet al.,2005;Goshima et al.,1998; Jerome etal.,
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 increasingly
apparent that many viruses interact with mitochondria in
the infected cell, possibly in an attempt to modulate the
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
direct result of mitochondrial dysfunction (Okuda et al.,
2002). Hepatitis C virus core protein is known to traffic to
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 syndrome.
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Fig. 3. Effect of US3 protein expression on respiration. (a)
mRNAs for US3 and luciferase proteins were synthesized in vitro
and transfectedintoHeLa cells
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 US3 mRNA-transfected cells. Expression of
US3, but not luciferase, led to a profound decrease in cellular
respiration. (b) HeLa cells were mock-infected or infected with
US3 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.
by using Lipofectinas
2158Journal of General Virology 87
M. Derakhshan and others
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