Journal of General Virology (2014), 95, 442–452
DOI 10.1099/vir.0.057059-0
Bovine adenovirus 3 core protein precursor pVII
localizes to mitochondria, and modulates ATP
synthesis, mitochondrial Ca2+ and mitochondrial
membrane potential
Sanjeev K. Anand,1,2 Amit Gaba,1,2 Jaswant Singh3
and Suresh K. Tikoo1,2,4
Correspondence
Suresh K. Tikoo
[email protected]
1
Vaccine & Infectious Disease Organization – International Vaccine Center (VIDO-InterVac),
University of Saskatchewan, Saskatoon, Canada
2
Veterinary Microbiology, University of Saskatchewan, Saskatoon, Canada
3
Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Canada
4
Vaccinology & Immunotherapeutics Program, School of Public Health, University of Saskatchewan,
Saskatoon, Canada
Received 10 July 2013
Accepted 10 October 2013
Viruses modulate the functions of mitochondria by translocating viral proteins to the mitochondria.
Subcellular fractionation and sensitivity to proteinase K/Triton X-100 treatment of mitochondrial
fractions of bovine adenovirus (BAdV)-3-infected/transfected cells suggested that core protein
pVII localizes to the mitochondria and contains a functional mitochondrial localization signal.
Moreover, mitochondrial localization of BAdV-3 pVII appears to help in the retention of
mitochondrial Ca2+, inducing a significant increase in the levels of ATP and maintaining the
mitochondrial membrane potential (MMP) in transfected cells. In contrast, mitochondrial
localization of BAdV-3 pVII has no significant effect on the levels of cytoplasmic Ca2+ and
reactive oxygen species production in the transfected cells. Consistent with these results,
expression of pVII in transfected cells treated with staurosporine decreased significantly the
activation of caspase-3. Our results suggested that BAdV-3 pVII localizes to mitochondria, and
interferes with apoptosis by inhibiting loss of the MMP and by increasing mitochondrial Ca2+ and
ATP production.
INTRODUCTION
Mitochondria are vital organelles of the cell that regulate
cellular functions and generate energy for all molecular
processes (Hackenbrock, 1966; Mannella, 2006; Rapaport,
2003). In addition to energy production, mitochondria also
play a central role in Ca2+ buffering, supply of metabolites,
regulation of apoptotic factors, ageing and development
(Chan, 2006; Hollenbeck & Saxton, 2005). Many viruses
alter the structure and function of the mitochondria (Ohta
& Nishiyama, 2011), inducing oxidative stress (Huh &
Siddiqui, 2002; Machida et al., 2010), the mitochondrial
membrane potential (MMP) and the production of ATP
(Chang et al., 2009; Monné et al., 2007; Su & Hong, 2010)
by translocating their proteins to the mitochondria.
Although adenovirus replicates in the nucleus of the cell,
the possibility of its dependence on mitochondria appears
logical. However, little is known about the role of
mitochondria in adenovirus infections. Human adenovirus
Supplementary material is available with the online version of this paper.
442
(HAdV) has been reported to localize to the mitochondria
in cells infected with high-titre virus (Alesci et al., 2007),
inducing damage to the mitochondrial architecture. Adenoviral early proteins localize to the mitochondria and either
prevent or induce apoptosis (Degenhardt et al., 2000;
Lomonosova et al., 2005). Adenovirus protein V interacts
with p32 and localizes to the mitochondria (Matthews &
Russell, 1998). Adenovirus death protein encoded by the E3
region of HAdV-5 induces oxidative stress and helps in the
release of virus progeny from the virus-infected cell
(Tollefson et al., 1996). A recent report suggested that
HAdV-5 releases cathepsin B activity in the cytoplasm due to
endosomal membrane rupture leading to the production of
reactive oxygen species (ROS) (McGuire et al., 2011).
Bovine adenovirus (BAdV)-3, a member of the genus
Mastadenovirus, is a non-enveloped icosahedral virus,
which is being developed and evaluated as a vaccine
delivery vector for animals (Zakhartchouk et al., 1999) and
humans (Rasmussen et al., 1999). The complete DNA
sequence and the transcription map of BAdV-3 genome
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BAdV-3 pVII and mitochondria
have been reported (Reddy et al., 1998, 1999). Since
mitochondria are major cellular organelles performing
various functions, study of the mitochondria/virus interaction may provide new insights into the viral/host
interactions. Adenovirus core protein VII is found as a
precursor (pVII) in infected cells and as a mature protein
(VII) in progeny virions (Hindley et al., 2007). Previous
studies suggested that adenovirus core protein VII is
involved in the import of the adenovirus genome into the
nucleus (Wodrich et al., 2006), protecting it from the
Mre11–Rad50–Nbs1 complex at early times after infection
(Karen & Hearing, 2011), and may have a role in the
(a)
Mito
U
I
Cyto
Nucl
U
I U
I
Controls
U 24 48
Anti-COX-1
Anti-fibrillarin
Anti-ERK
Anti-Hsp70
(b)
Mito
U
I
Cyto
U I
Nucl
U
I
Controls
U
24
Anti-pVII
packaging of DNA in capsids (Zhang & Arcos, 2005). Here,
we demonstrated that BAdV-3 pVII localized to mitochondria and appeared to play a beneficial role by
modulating mitochondrial functions, including apoptosis.
RESULTS
Isolation of mitochondria from bovine cells
To determine the localization of BAdV-3 proteins to
mitochondria, the mitochondria-rich fraction was purified
from mock- or BAdV-3-infected Madin–Darby bovine
kidney (MDBK) cells and analysed for purity by Western
blotting. Equal amounts of proteins from the mitochondrial fraction, cytoplasmic fraction and nuclear fraction of
mock- or BAdV-3-infected cells were separated using 10 %
SDS-PAGE, transferred to nitrocellulose membranes and
probed with protein-specific antibodies (Fig. 1a). Anticytochrome oxidase (COX)-1 serum detected a specific
protein in the mitochondrial fraction but not in the
nuclear or cytoplasmic fractions of mock- or BAdV-3infected cells. Anti-extracellular signal-regulated kinase
(ERK) serum detected a specific protein in the nuclear
fraction and cytoplasmic fractions of mock- or BAdV-3infected cells but not in the mitochondrial fraction.
However, anti-fibrillarin serum detected a specific protein
Infected cell total lysate
Infected cell Mito. fraction
Proteinase K treatment
Triton X-100 treatment
(a)
–
+
–
–
–
+
+
–
–
+
+
+
+
–
–
–
Anti-hexokinase
Anti-IVa2
Anti-cll
Anti-penton
(b)
Anti-hexon
Anti-hexon
Fig. 1. Western blot analysis of cellular fractions. Proteins from
lysates of the indicated cellular fraction isolated from mock- or
BAdV-3-infected MDBK cells were separated by SDS-PAGE,
transferred to nitrocellulose membranes and probed by Western
blotting. (a) Anti-cytochrome oxidase (COX)-1 serum (mitochondrial marker), anti-fibrillarin serum (nuclear marker), anti-extracellular signal-regulated kinase (ERK) serum (cytoplasmic marker)
and anti-heat shock protein 70 (Hsp70) serum (loading control).
(b) Anti-pVII serum, anti-IVa2 serum, anti-penton serum and antihexon serum. Mitochondrial (Mito), cytosolic (Cyto) and nuclear
(Nucl) fractions from uninfected (U) or infected (I) cells. Uninfected
MDBK cells (Controls) and BAdV-3-infected MDBK cells collected at 24 or 48 h post-infection (24 or 48).
http://vir.sgmjournals.org
Anti-pVII
Anti-penton
Anti-IVa2
Fig. 2. Analysis of mitochondrial (Mito.) fractions from BAdV-3infected MDBK cells. Proteins from the lysates of cellular fractions
isolated from BAdV-3-infected cells were treated as indicated and
separated using 10 % SDS-PAGE, transferred to nitrocellulose
membranes, and probed by Western blotting with (a) antihexokinase mAb and anti-cII mAb, and (b) anti-pVII serum, antihexon serum, anti-penton serum and anti-IVa2 serum.
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443
S. K. Anand and others
(a)
pVIIMLS
1
54
pcDNA.pVII
pEY.VIImls
EYFP
Infected cell total lysate
Transfected cell Mito. fraction
–
+
–
+
–
+
+
–
Proteinase K treatment
–
+
+
–
Triton X-100 treatment
–
–
+
–
pcDNA3.pVII
Anti-pVII
pEY.VIImls
Anti-pVII
(b)
pVIIMLS
1
54
EYFP
pEY.VIImls
Oct
DsRed
MLS
pOCT.DsRed
EYFP-VIImls
DAPI
OCT-DsRed
Merge
(c)
1
*; **; **. ; *; ***;
54
; .
.
;
*** *;
*; *;
**
Fig. 3. Analysis of the mitochondria-rich fraction from transfected cells. (a) Schematic diagram of plasmids. The names of the
plasmids are given on the left of the panel. Proteins from the lysates of the mitochondrial (Mito.) fraction isolated from cells
infected with WT BAdV-3 or transfected with indicated plasmid DNA were treated as indicated, and separated using 10 %
SDS-PAGE, transferred to nitrocellulose membranes and probed by Western blotting with protein-specific serum. (b) Vero cells
co-transfected with pEY.VIImls and pOCT.DsRed (Harder et al., 2004) were visualized directly under a confocal microscope 48 h
after transfection. Nuclei were stained with DAPI. The merger of EYFP-VIImls, OCT-DsRed and DAPI staining is shown.
444
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Journal of General Virology 95
BAdV-3 pVII and mitochondria
Fig. 3. cont. (c). Amino acid homology of BAdV-3 pVII-like proteins. Alignment of deduced amino acid sequences of BAdV-3
pVII homologues with those of porcine adenovirus (PAdV)-3 (GenBank accession no. BAA76967), ovine adenovirus (OAdV)A (GenBank accession no. AP_000010), canine adenovirus (CAdV)-1 (GenBank accession no. AP_000055), HAdV-5
(GenBank accession no. AP_000207), HAdV-F (GenBank accession no. NP_040858), HAdV-52 (GenBank accession no.
ABK35040), simian adenovirus (SAdV)-48 (GenBank accession no. ADZ39809), HAdV-A (GenBank accession no.
YP_002640218), Gorilla gorilla adenovirus B7 (GenBank accession no. ADQ38367) and HAdV-31 (GenBank accession no.
CAO78634.1). Identical, strongly conserved and weakly conserved residues are indicated by an asterisk (*), semi-colon (;)
and full point (.), respectively. The vertical arrow indicates a potential protease cleavage site.
in the nuclear fraction but not in the cytoplasmic or
mitochondrial fractions of mock- or BAdV-3-infected cells.
Anti-heat shock protein 70 (Hsp70) serum detected
specific protein(s) in all fractions of mock- or BAdV-3infected cells and was used as a loading control. These
results suggested that the mitochondrial fractions purified
from mock- or BAdV-3-infected cells were enriched highly
in mitochondria.
BAdV-3 proteins associate with mitochondria
To determine if BAdV-3 proteins localize to the mitochondria, proteins from different cellular (mitochondrial,
nuclear and cytoplasmic) fractions purified from mockor BAdV-3-infected MDBK cells were separated using
10 % SDS-PAGE and analysed by Western blotting with
selected BAdV-3 protein-specific antibodies (Fig. 1b). As
seen in Fig. 1(b), anti-IVa2 serum, anti-penton serum
and anti-hexon serum recognized specific proteins in
BAdV-3-infected cells or purified mitochondrial, cytoplasmic and nuclear fractions of BAdV-3-infected cells. No
such proteins could be detected in uninfected cells or
purified mitochondrial, cytoplasmic and nuclear fractions
of uninfected cells. Similarly, anti-pVII serum recognized
specific proteins in BAdV-3-infected cells or purified
mitochondrial and nuclear fractions of BAdV-3-infected cells.
No such proteins could be detected in purified cytoplasmic
BAdV-3-infected cells, uninfected cells or purified mitochondrial and nuclear fractions of uninfected cells.
BAdV-3 pVII localizes to mitochondria in BAdV-3infected cells
Preliminary studies could not discriminate if the viral
proteins localize to the mitochondria on their own due to
the presence of the mitochondrial localization signal (MLS;
integral membrane proteins and soluble proteins located in
the inter-membrane space or matrix) or if they are loosely
attached to outer mitochondrial membranes. However,
analysis of BAdV-3 protein sequences (GenBank accession
no. AF030154) by PSORT and WolfPSORT (Nakai & Horton,
1999), SherLoc (Shatkay et al., 2007) PreDator (Rost et al.,
2004), TargetP (Emanuelsson et al., 2007) and MitoProt
(Claros & Vincens, 1996) software identified a potential
MLS in pVII and pIVa2 but not in hexon or penton.
To resolve the issue, mitochondria-rich fractions
from BAdV-3-infected MDBK cells were treated with
proteinase K and analysed by Western blotting using
http://vir.sgmjournals.org
protein-specific antibodies. Proteinase K treatment
degrades hexokinase protein (inserted in the outer
mitochondrial membrane) but does not degrade complex
II protein (inserted in the inner mitochondria membrane). As seen in Fig. 2(a), anti-hexokinase serum
detects hexokinase protein in untreated mitochondria or
BAdV-3-infected cells but not in proteinase K-treated
mitochondria (Fig. 2a). As expected, anti-cII serum
detects a complex II protein in untreated mitochondria,
BAdV-3-infected cells and also in proteinase K-treated
mitochondria (Fig. 2a). To demonstrate that the
isolation procedure did not damage mitochondrial
integrity, the purified mitochondria were treated with
both proteinase K and 0.1 % Triton X-100, and analysed
by Western blotting using protein-specific antibodies.
This treatment renders proteins contained within the
mitochondria (inner membrane and matrix) susceptible
to protease treatment (Sardanelli et al., 2006). As
expected, anti-cII serum did not detect a complex II
protein in proteinase K/Triton X-100-treated mitochondria (which degrades proteins inserted both in the outer
and inner membranes). These results reconfirmed and
established that proteinase K treatment degrades the
proteins exposed on the outer mitochondrial membrane
but has no effect on the proteins inside the outer
mitochondrial membrane. Next, the mitochondrial
fraction isolated from BAdV-3-infected cells was treated
with proteinase K in the absence or presence of Triton X100. Treated and untreated mitochondria were analysed
by Western blotting using BAdV-3 protein-specific
antibodies. As seen in Fig. 2(b), anti-pVII serum detected
pVII in proteinase K-treated but not in proteinase K/
Triton X-100-treated mitochondria. In contrast, antihexon serum, anti-penton serum or anti-IVa2 serum did
not detect protein-specific bands in proteinase K- or
proteinase K/Triton X-100-treated mitochondria. Taken
together, these results suggested that whilst IVa2 protein
is exposed to the cytosol, pVII is contained within the
mitochondria (inner membrane and/or matrix).
BAdV-3 pVII localizes to mitochondria in
transfected cells
To determine if pVII could localize independently to the
mitochondria, Vero cells were transfected with 0.4 mg
cm22 of the indicated plasmid DNA. After 48 h of
transfection, mitochondria-rich fractions isolated from
the transfected cells were treated with proteinase K or
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S. K. Anand and others
BAdV-3 pVII contains a functional MLS
Previous protein analysis predicted the presence of a
potential MLS at the N-terminus of pVII (aa 1–54) (Fig.
3c). To determine if the MLS was targeting pVII to
mitochondria, the DNA encoding the potential pVII MLS
(aa 1–54) was fused in-frame to EYFP to create pEY.VIImls
(Fig. 3). The mitochondria-rich fractions were isolated
from the cells transfected with indicated plasmid DNA,
treated with proteinase K or proteinase K/Triton X-100
and analysed by Western blotting using protein-specific
antibodies. As seen in Fig. 3(a), both pVII and EY.VIImls
fusion protein were resistant to proteinase K treatment but
degraded after proteinase K/Triton X-100 treatment. These
results suggest that targeting pVII to mitochondria involves
the N-terminal 54 aa. Secondly, Vero cells were co-transfected
with indicated plasmid DNA and analysed 48 h posttransfection by confocal microscopy. As seen in Fig. 3(b), a
portion of recombinant EYFP co-localized with recombinant
OCT-DsRed (which localizes only to mitochondria; Harder
et al., 2004) in cells co-transfected with pEY.VIImls and
pOCT.DsRed, suggesting that recombinant EYFP was
localized to the mitochondria of the transfected cells.
Interestingly, analysis of the N-terminal 54 aa of BAdV-3
pVII showed significant homology to corresponding proteins
of other adenoviruses (Fig. 3).
the cells expressing pVII showed a significant increase in
mitochondrial Ca2+ levels, whereas the cells expressing the
BAdV-3 protein 100K did not show any significant increase
in mitochondrial Ca2+ buffering activity. Thapsigargin
treatment of the cells results in a global and transient
increase in cytosolic calcium levels (Ong & Hausenloy,
2010), which allowed us to examine the ability of mitochondria to effectively uptake and sequester Ca2+.
Following incubation of cells in thapsigargin, Ca2+ is
released from the endoplasmic reticulum and is available
for uptake by all organelles. In the absence of any stimulus,
homeostasis directs Ca2+ back to normal depots (Fig. 5b).
Observations were recorded at 100 s intervals to determine
if released Ca2+ was taken up by the mitochondria of
transfected cells. In normal cells, thapsigargin induces
release of Ca2+, which subsides over time (homeostasis).
The cells expressing pVII showed a significant sequestration and retention of mitochondrial Ca2+ even after
20 min post-treatment with thapsigargin (1 mM), whereas
the cells expressing 100K showed no significant effect in
mitochondrial calcium uptake post-treatment with thapsigargin (1 mM) (Fig. 5b). In addition, Vero cells expressing
pVII or 100K (Fig. 5c) and exposed to 1 mM thapsigargin
(Fig. 5d) showed no significant change in cytosolic Ca2+
(a) 10000
b
8000
ATP (c.p.s.)
proteinase K/Triton X-100 and analysed by Western
blotting using protein-specific antibodies. As seen in Fig.
3(a), pVII was detected in proteinase K-treated mitochondria but not in proteinase K/Triton X-100-treated mitochondria. These results confirmed earlier observations and
suggested that BAdV-3 pVII localizes inside the mitochondria (inner membrane or matrix) independently of any
other viral protein.
6000
4000
a
a
2000
BAdV-3 pVII regulates ATP production
Next, we determined if localization of pVII to mitochondria
had any effect on the production of ATP in the transfected
cells. To observe this, Vero cells were transfected with
indicated plasmid DNA and ATP production was measured
48 h post-transfection. ATP concentration was found to be
significantly higher in the cells transfected with pcDNA3.pVII
DNA compared with that in the cells transfected with
pcDNA3 or pcDNA3.100K (Fig. 4a) DNA. This indicated that
pVII induced ATP production in the cells and played some
role in ATP synthesis.
BAdV-3 pVII regulates mitochondrial Ca2+ levels
Next, we examined if pVII could regulate the Ca2+
buffering ability of mitochondria of transfected cells.
Vero cells were transfected with individual plasmid
DNAs, and mitochondrial and cytosolic Ca2+ levels were
measured 48 h post-transfection using Fluo-4 AM and
Rhod-2 AM, which are highly specific indicators of cellular
and mitochondrial Ca2+, respectively. As seen in Fig. 5(a),
446
0
pcDNA3
pcDNA3.pVII pcDNA3.100K
(b)
Anti-β-actin
Anti-100K
Anti-pVII
Fig. 4. ATP production in transfected cells. (a) Vero cells were
transfected with individual plasmid DNAs and ATP production
measured 48 h post-transfection using the ATPLite 1step kit and a
multi-label reader. Measurements are given in arbitrary units
[counts per second (c.p.s.)]. Data represent the mean±SEM of
two independent experiments, each with three replicates. Means
with a different letter are significantly different. *P,0.0001. (b)
Proteins from lysates of these transfected cells were also
separated using 10 % SDS-PAGE, transferred to nitrocellulose
membranes, and probed by Western blotting with anti-b-actin
mAb, anti-100K serum and anti-pVII serum.
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Journal of General Virology 95
BAdV-3 pVII and mitochondria
(a)
1200
1100
500
1100
1200
1000
–500
900
0
800
pcDNA3.100K
1000
700
pcDNA3 pcDNA3.pVII
1500
600
100
900
pcDNA3
pcDNA3.pVII
pcDNA3.100K
500
200
1000
800
700
600
500
400
300
Time post-treatment (s)
(d) 2000
300
0
–500
pcDNA3 pcDNA3.pVII pcDNA3.100K
400
400
0
300
500
a
200
(c)
Cytosolic Ca2+ (c.p.s.)
0
a
500
100
1000
1000
200
2000
pcDNA3
pcDNA3.pVII
pcDNA3.100K
100
b
Cytosolic Ca2+ (c.p.s.)
Mitochondrial Ca2+ (c.p.s.)
3000
Mitochondrial Ca2+ (c.p.s.)
(b) 1500
4000
Time post-treatment (s)
(e)
Anti-β-actin
Anti-100K
Anti-pVII
Fig. 5. Mitochondrial Ca2+ in transfected cells. (a) Vero cells were transfected with individual plasmid DNAs. At 48 h posttransfection, the transfected cells were treated with Rhod-2 AM (Molecular Probes) and analysed for fluorescence using a
PerkinElmer multi-label reader (left). (b) The same cells were treated with 1 mM thapsigargin for 30 min and fluorescence
measurements were taken for 1200 s post-treatment at 100 s intervals. Measurements are given in arbitrary units (c.p.s.). Data
represent the mean±SEM of two independent experiments, each with three replicates. Means with a different letter are
significantly different. Means with the same letter are not significantly different. *P,0.0001. (c) Vero cells were transfected with
individual plasmid DNAs and cytosolic Ca2+ was measured 48 h post-transfection; the cells were treated with Fluo-4 AM
(Molecular Probes) and analysed for fluorescence using a PerkinElmer multi-plate reader. (d) The same cells were treated with
1 mM thapsigargin for 30 min and fluorescence measurements were taken for 1200 s post-treatment at 100 s intervals (right).
Measurements are given in arbitrary units (c.p.s). Data represent the mean±SEM of two independent experiments, each with
three replicates. Means with the same letter are not significantly different. *P,0.0001. (e) Proteins from the lysates of these
transfected cells were also separated using 10 % SDS-PAGE, transferred to nitrocellulose membranes, and probed by
Western blotting with anti-b-actin mAb, anti-100K serum and anti-pVII serum.
levels over the period of treatment. This showed that
expression of pVII induced mitochondria to sequester and
retain Ca2+ compared with those expressing 100K.
BAdV-3 pVII regulates the MMP
To verify if alterations in mitochondrial Ca2+ caused any
changes in the MMP, we measured the MMP in transfected
cells using tetramethylrhodamine methyl ester (TMRM).
Vero cells were transfected with indicated plasmid DNA
and MMP changes were measured 48 h post-transfection
(Fig. 6a). TMRM fluorescence levels decreased significantly
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in cells expressing 100K but not in the cells expressing
pVII (Fig. 6a). To further confirm the role of pVII in
maintaining the MMP, we performed a thapsigargin
treatment experiment. As Ca2+ helps to maintain the
MMP, thapsigargin treatment should not cause loss of
Ca2+ in pVII-expressing cells and thus no loss of Ca2+dependent MMP in these cells. As seen in Fig. 6(b), after
thapsigargin treatment there was significant loss of the
MMP in cells expressing 100K but not in cells expressing
pVII. This indicated that expression of BAdV-3 pVII
helped the cells to maintain the MMP, whereas 100K had
little or no effect on the maintenance of the MMP.
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S. K. Anand and others
(a) 1000
b
(b) 150
pcDNA3
pcDNA3.pVII
a
a
400
200
50
1200
1100
900
1000
800
700
600
(c)
–50
500
pcDNA3.pVII pcDNA3.100K
400
pcDNA3
300
0
200
0
pcDNA3.100K
100
100
600
MMP (c.p.s.)
MMP (c.p.s.)
800
Time post-treatment (s)
Anti-β-actin
Anti-100K
Anti-pVII
Fig. 6. MMP in transfected cells. (a) Vero cells were transfected with individual plasmid DNA, and the MMP was measured 48 h
post-transfection using TMRM and a multi-label reader. (b) The same cells were treated with 1 mM thapsigargin for 30 min and
measurements were taken for 1200 s post-treatment at 100 s intervals. Data represent the mean±SEM of two independent
experiments, each with three replicates. Means with a different letter are significantly different. *P,0.0001. (c) Proteins from
these transfected cells were also separated using 10 % SDS-PAGE, transferred to nitrocellulose membranes, and probed by
Western blotting with anti-b-actin mAb, anti-100K serum and anti-pVII serum.
BAdV-3 pVII and ROS production in Vero cells
To assess mitochondrial function in the cells expressing
BAdV-3 pVII or 100K, we measured mitochondrial ROS
production in Vero cells transfected with indicated plasmid
DNA. At 48 h post-transfection, the cells were incubated
with dichlorofluorescein diacetate (DCF-DA) and the
fluorescence was quantified (Fig. 7a). The cells transfected
with pcDNA3.100K DNA showed a significant increase in
the level of ROS production compared with those
transfected with pcDNA3 or pcDNA3.pVII, indicating that
expression of pVII did not increase oxidative stress levels in
Vero cells.
BAdV-3 pVII inhibits caspase-3 activation
To confirm if pVII induces or inhibits apoptosis, a caspase3 assay was performed. Caspase-3 is an active cell death
protease involved in the execution phase of apoptosis (Zou
et al., 1997). The caspase-3 assay employs a specific
caspase-3 substrate, N-Ac-DEVD-N9-AFC, which upon
cleavage by active caspase-3 generates a highly fluorescent
product that can be measured using excitation and
emission wavelengths of 400 and 505 nm. Vero cells were
transfected with indicated plasmid DNA. After 48 h posttransfection, designated cells were treated with staurosporine (a caspase-independent direct activator known to
induce apoptosis in cells by blocking the activity of
kinases) for 4 h before measuring caspase-3 activity. The
data were normalized by measuring the luciferase activity
448
in each sample. As seen in Fig. 8(a), staurosporine
treatment of cells transfected with pcDNA3 DNA showed
significant activation of caspase-3. Interestingly, expression
of pVII did not lead to the activation of caspase-3. In
contrast, expression of pVII reduced significantly the
activation of caspase-3 in staurosporine-treated cells,
suggesting an anti-apoptotic role for pVII.
DISCUSSION
A number of viruses target mitochondria during the
infection process and alter their functions (Ohta &
Nishiyama, 2011). This involves usually the transport of
specific viral proteins to the mitochondria, leading to the
modulation of mitochondrial functions. We observed
previously that BAdV-3 interacted with the mitochondria
and altered mitochondrial structure during the course of
infection. In the present study, we demonstrated that core
protein pVII localizes into the mitochondria and modulates the mitochondrial physiology.
Like other viruses, BAdV-3 infection appears to affect
mitochondrial functions, including ATP production,
mitochondrial Ca2+ concentrations and the MMP. As
localization of viral proteins to mitochondria may alter
their function, it is tempting to speculate that one or more
BAdV-3 protein(s) may be involved in interactions with
the mitochondria to help in inducing these processes.
Previous amino acid sequence analysis identified that pVII
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Journal of General Virology 95
BAdV-3 pVII and mitochondria
(a) 400
(a) 100 000
b
60 000
a
a
40 000
20 000
0
pcDNA3
pcDNA3.pVII
pcDNA3.100K
(b)
b
Caspase-3 cleavage (c.p.s.)
ROS (c.p.s.)
80 000
300
200
c
100
Anti-β-actin
a
0
Anti-pVII
pcDNA3
Anti-pVII
d
pcDNA3
+
S
pcDNA3.pVII pcDNA3.pVII
+
S
(b)
Anti-β-actin
Fig. 7. Induction of ROS in transfected cells. (a) Vero cells were
transfected with individual plasmid DNAs. At 48 h post-transfection, the cells were treated with DCF-DA (Molecular Probes)
and analysed for fluorescence using a multi-label reader.
Measurements are given in arbitrary units (c.p.s). Data represent
the mean±SEM of two independent experiments, each with three
replicates. Means with a different letter are significantly different.
Means with the same letter are not significantly different.
*P,0.0001. (b) Proteins from the lysates of these transfected
cells were also separated using 10 % SDS-PAGE, transferred to
nitrocellulose membranes, and probed by Western blotting with
anti-b-actin mAb, anti-100K serum and anti-pVII serum.
contains a potential MLS. However, Western blot analysis
of the mitochondrial fraction isolated from infected cells
suggested that the all tested proteins appeared to be
associated with the mitochondria. It is possible that some
of these proteins associate non-specifically with mitochondria due to the effect of virus replication on the
distribution of mitochondria in the infected cells.
Electron microscopy analysis of infected cells at 12 h
post-infection showed the presence of mitochondria in the
close vicinity of protein synthesis factories in the infected
cells (S. K. Anand & S. K. Tikoo, unpublished observations). These protein(s)-synthesizing factories might be
synthesizing viral proteins, which might have been purified
with the mitochondrial fraction during the purification
process.
Of the four BAdV-3 proteins that were found to be
associated with mitochondria, only pVII appeared not to
be attached loosely, but localized to the mitochondria.
Several lines of evidence support the suggestion that pVII
localizes to the mitochondria due to the presence of a
http://vir.sgmjournals.org
Anti-pVII
Fig. 8. Caspase-3 assay in cells expressing pVII. (a) Vero cells
were transfected with individual plasmid DNAs. At 48 h posttransfection, the indicated cells were treated with staurosporine
(S) and cleavage of caspase-3 was measured 53 h posttransfection using a multi-label reader. Data represent the
mean±SEM of two independent experiments, each with three
replicates. Means with a different letter are significantly different.
*P,0.0001. (b) Proteins from these transfected cells were also
separated using 10 % SDS-PAGE, transferred to nitrocellulose
membranes, and probed by Western blotting with anti-b-actin
mAb and anti-pVII serum.
functional MLS. (1) pVII-specific proteins could be
detected in Western blots of mitochondrial fractions of
infected/transfected cells treated with proteinase K. (2) A
potential MLS (aa 1–54) could localize the cytoplasmic
protein EYFP to the mitochondria of transfected cells. (3)
EYFP-VIImls fusion proteins could be detected in Western
blots of mitochondrial fractions of transfected cells treated
with proteinase K.
Localization of viral proteins in mitochondria (Nakai &
Horton, 1999) has been implicated in altering various cellular
processes, including subverting the host defence mechanisms
to establish themselves and replicate (Castanier & Arnoult,
2011), altering Ca2+ homeostasis (Zhou et al., 2009), cellular
metabolism (Maynard et al., 2010) and apoptosis (Danthi,
2011). Mitochondrial localization of BAdV-3 pVII induces a
significant increase in the levels of ATP, indicating a positive
role played by this protein during the course of infection.
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S. K. Anand and others
Increased ATP is involved in the maintenance of ion gradients
and thus the MMP across the mitochondrial membranes
(Hollenbeck & Saxton, 2005). pVII also appears to help in
the retention of mitochondrial Ca2+ and consistent ATP
generation, which help maintain the MMP (Agudo-López
et al., 2011; Halestrap, 2009, 2010). As Ca2+ is a physiological
stimulus for ATP synthesis and is one of the positive effectors
of oxidative phosphorylation (Balaban, 2009), it is conceivable that mitochondrial Ca2+ retention helps cells to
maintain a steady supply of ATP, thus helping to maintain
the MMP. Although pVII helps in the increase and retention
of mitochondrial Ca2+, it has little or no effect on cytosolic
Ca2+. This may be due to the fact that other Ca2+ stores such
as the endoplasmic reticulum, which acts as a main storehouse
of Ca2+ in cells, may be releasing enough Ca2+ to maintain
cytosolic levels in spite of significant portions of Ca2+ being
retained by the mitochondria.
Viruses have devised different strategies, including inhibition of cell apoptosis, to facilitate their replication in
infected cells. Proteins encoded by several DNA viruses,
including vaccinia virus FIL (Wasilenko et al., 2003),
Kaposi sarcoma herpesvirus K7 (Wang et al., 2002) or
human cytomegalovirus vMIA (Goldmacher et al., 1999),
block apoptosis by inhibiting the MMP at the mitochondrial level. Based on our observations, pVII appears to be
an anti-apoptotic protein and may help in prolonging the
life of the cells, thus helping BAdV-3 to complete its life
cycle. Consistent with earlier observations is the fact that
(1) pVII has little or no effect on ROS generation in the
cells and thus may act as proapoptotic, and (2) expression
of pVII in cells treated with staurosporine decreases
significantly the activation of caspase-3.
Adenovirus mature core protein VII imports viral DNA in
the nucleus (Hindley et al., 2007; Wodrich et al., 2006) and
protects virion DNA in the nucleus from the DNA damage
response at early times after infection (Karen &Hearing,
2011), As a potential MLS of pVII appears to be located at aa
1–54, the predicted mature core protein VII (aa 25–171;
Reddy et al., 1998) may contain the MLS (Y. Zhao & S. K.
Tikoo, unpublished observations). We speculate that during
early stages of infection when the virus needs viable cells,
BAdV-3 pVII of infecting virus may localize to mitochondria and help to maintain the life of the cells.
An earlier report suggests that adenovirus pVII condenses viral
DNA during progeny virus assembly in the nucleus (Hindley
et al., 2007). Here, we demonstrate that BAdV-3 pVII also
localizes to mitochondria, and appears to be involved in
enhancing vital mitochondrial processes and prolonging the
longevity of the cell at later stages of virus infection. Thus, pVII
appears to be a multifunctional protein, which may be
involved in different aspects of adenovirus infection.
METHODS
Reagents. Lipofectamine 2000 (Invitrogen), the mitochondria
isolation kit for mammalian cells (Pierce), BCIP/nitro blue tetrazolium
450
(NBT) reagent and staurosporine (Sigma), the Dual-Luciferase
Reporter Assay System (Promega), ATPLite 1step kit reagents
(PerkinElmer), Rhod-2 AM, Fluo-4 AM and TMRM (Molecular
Probes), minimal essential medium (MEM; Invitrogen), and
Dulbecco’s modified Eagle’s medium (DMEM; Sigma) were used as
described by the manufacturer’s. Assays used either a multi-label
counter (Victor X3; PerkinElmer) or confocal microscope (TCS-SP5;
Leica).
Cell lines and virus. MDBK cells were grown in MEM supplemented
with 10 % heat-inactivated FBS. WT BAdV-3 (WBR-1 strain) was
propagated in MDBK cells in MEM supplemented with 2 % FBS
(Reddy et al., 1998). Vero cells were propagated in DMEM
supplemented with 10 % FBS.
Antibodies. Anti-penton and anti-hexon sera detect proteins of 62
and 98 kDa, respectively, in BAdV-3-infected cells (Kulshreshtha et al.,
2004). Anti-pVII serum recognizes two proteins of 22 and 20 kDa in
BAdV-3-infected cells (Paterson, 2010). Anti-IVa2 serum recognizes a
protein of 55 kDa in BAdV-3-infected cells (A. Gaba & S. K. Tikoo,
unpublished observations). Anti-100K serum recognizes a protein of
130K in BAdV-3-infected cells (Kulshreshtha & Tikoo, 2008). mAb
specific to hexokinase and polyclonal antibodies specific to ERK2 and
fibrillarin were purchased from Santa Cruz Biotechnology. mAb
specific to COX-1 was purchased from Invitrogen, mAb specific to
Hsp70 was purchased from Stressgen, mAb specific to mitochondrial
complex II subunit was purchased from Mitosciences. mAb specific to
b-actin was purchased from Sigma. Alkaline phosphatase (AP)conjugated goat anti-rabbit IgG and AP-conjugated goat anti-mouse
antibody were purchased from Jackson ImmunoResearch.
Plasmid construction. pOCT.DsRed (Harder et al., 2004) was a gift
from Dr H. McBride (University of Ottawa). The construction of
pcDNA.100K has been described previously (Kulshreshtha & Tikoo,
2008). The other plasmids (supplementary File S1, available in JGV
Online) were constructed using standard procedures.
Isolation of mitochondria. MDBK cells were infected with BAdV-3
at a m.o.i. of 5. Vero cells were transfected with individual plasmid
DNAs (0.4 mg cm22) using Lipofectamine 2000. At indicated times
post-infection or transfection, the cells were collected and used for
isolation of mitochondria using the mitochondria isolation kit for
mammalian cells as per the manufacturer’s instructions with some
modifications. Approximately 26107 MDBK cells (mock or infected)
or Vero cells (mock or plasmid-transfected) were Dounce homogenized and pelleted at 300 g to collect the cell debris and nuclei.
Supernatant 1 was collected, and the pellet containing the cell debris
and nucleus was dissolved in nucleus isolation buffer [10 mM KCl,
10 mM MgCl2, 10 mM Tris/HCl (pH 7.4) and 10 mM DDT], further
homogenized in a Dounce homogenizer, and finally centrifuged at
212 g to obtain the nuclear fraction. Supernatant 1 was centrifuged at
3200 g to pellet the mitochondria-enriched fraction. The resulting
supernatant 2 left after isolation of the mitochondria-enriched
fraction was used as the cytoplasmic fraction.
Western blot analysis. MDBK cells infected with WT BAdV-3 at an
m.o.i. of 5 were harvested at indicated times post-infection. Vero cells
were transfected with individual plasmid DNA (0.4 mg cm22) using
Lipofectamine 2000 and harvested at indicated times post-transfection. Proteins from the lysates of cells, mitochondria, cytoplasmic or
nuclear fractions were separated using 10 % SDS-PAGE and analysed
by Western blotting as described previously (Kulshreshtha & Tikoo,
2008).
Proteinase K treatment. The isolated mitochondria were dissolved
in buffer C of the mitochondria isolation kit with or without Triton
X-100 containing proteinase K at a final concentration 150 mg ml21.
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Journal of General Virology 95
BAdV-3 pVII and mitochondria
The treated samples were incubated for 30 min on ice before
centrifugation at 6700 g for 15 min at 4 uC (Huh & Siddiqui, 2002).
The pellet fraction(s) was subsequently analysed by Western blotting
using protein-specific antibodies.
SAS) for effect of treatment (DNA transfection). P.0.05 was
considered non-significant. Tukey’s post-hoc tests for multiple
comparisons were performed if the main effect was significant
(P¡0.05). The values are expressed as mean±SEM.
Cellular ATP. Vero cells grown in 96-well plates were transfected
with indicated plasmid DNA (0.2 mg per well) using Lipofectamine
ACKNOWLEDGEMENTS
2000. At indicated times post-infection or transfection, the cells were
treated with ATPLite 1step kit reagents. The emitted light, which is
proportional to the ATP concentration, was recorded using a multilabel counter.
Mitochondrial and cytosolic Ca2+. Vero cells grown in 96-well
plates were transfected with indicated plasmid DNA (0.2 mg per well)
using Lipofectamine 2000. At indicated times post-infection or
transfection, the cells were incubated with 5 mM mitochondrial Ca2+sensitive dye Rhod-2 AM or 10 mM cytosolic Ca2+-sensitive dye
Fluo-4 AM for 30 min at 37 uC. The cells were washed three times in
Ca2+-free PBS or KRH buffer [129 mM NaCl, 5 mM NaHCO3,
4.8 mM KCl, 1.2 mM KH2PO4, 1 mM CaCl2, 1.2 mM MgCl2,
2.8 mM glucose and 10 mM HEPES (pH 7.4)] and equilibrated for
10 min. Fluorescence signals were detected using a multi-label
counter using a 480/31 nm filter to excite the Fluo-4 AM fluorescence
and 531 nm filter to excite Rhod-2 AM fluorescence.
The signals were detected at 535 (Fluo-4 AM) and 572 nm (Rhod-2
AM).
MMP. Vero cells grown in 96-well plates were transfected with 0.2 mg
per well of indicated plasmid DNA using Lipofectamine 2000. At
indicated times post-infection or transfection, the cells were incubated
for 30 min with 100 nM TMRM in KRH-glucose buffer containing
0.02 % pluronic acid, then washed and allowed to equilibrate for
20 min. Fluorescence signals were measured using a multi-label
counter with a 531 nm excitation and 572 nm emission filter.
Mitochondrial ROS. Vero cells grown in 96-well plates were
transfected with 0.2 mg per well of indicated plasmid DNA using
Lipofectamine 2000. At indicated times post-transfection, the cells
were incubated with 10 mM DCF-DA (Degli Esposti, 2002) and
incubated for 30 min in KRH buffer. Finally, the cells were washed
three times in KRH buffer and equilibrated for 10 min. Fluorescence
signals were measured using a multi-label counter with 480/31 nm
excitation and 535 (DCF-DA) and 580 nm emission filters.
The authors thank members of the Tikoo laboratory for their
suggestions and staff of the Animal Care Unit with production of
antiserum. Published as VIDO article no. 635. The work was
supported by a Discovery Grant from the Natural Sciences and
Engineering Research Council of Canada to S. K. T.
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