Download Death mechanisms in epithelial cells following rotavirus infection

Journal of General Virology (2010), 91, 2007–2018
DOI 10.1099/vir.0.018275-0
Death mechanisms in epithelial cells following
rotavirus infection, exposure to inactivated rotavirus
or genome transfection
Peter Halasz,3 Gavan Holloway3 and Barbara S. Coulson
Correspondence
Department of Microbiology and Immunology, The University of Melbourne, VIC 3010, Australia
Barbara S. Coulson
[email protected]
Received 29 November 2009
Accepted 8 April 2010
Intestinal epithelial cell death following rotavirus infection is associated with villus atrophy and
gastroenteritis. Roles for both apoptosis and necrosis in cytocidal activity within rotavirus-infected
epithelial cells have been proposed. Additionally, inactivated rotavirus has been reported to
induce diarrhoea in infant mice. We further examined the death mechanisms induced in epithelial
cell lines following rotavirus infection or inactivated rotavirus exposure. Monolayer integrity
changes in MA104, HT-29 and partially differentiated Caco-2 cells following inactivated rotavirus
exposure or RRV or CRW-8 rotavirus infection paralleled cell metabolic activity and viability
reductions. MA104 cell exposure to rotavirus dsRNA also altered monolayer integrity. Inactivated
rotaviruses induced delayed cell function losses that were unrelated to apoptosis.
Phosphatidylserine externalization, indicating early apoptosis, occurred in MA104 and HT-29 but
not in partially differentiated Caco-2 cells by 11 h after infection. Rotavirus activation of
phosphatidylinositol 3-kinase partially protected MA104 and HT-29 cells from early apoptosis. In
contrast, activation of the stress-activated protein kinase JNK by rotavirus did not influence
apoptosis induction in these cells. RRV infection produced DNA fragmentation, indicating latestage apoptosis, in fully differentiated Caco-2 cells only. These studies show that the apoptosis
initiation and cell death mechanism induced by rotavirus infection depend on cell type and degree
of differentiation. Early stage apoptosis resulting from rotavirus infection is probably counterbalanced by virus-induced phosphatidylinositol 3-kinase activation. The ability of inactivated
rotaviruses and rotavirus dsRNA to perturb monolayer integrity supports a potential role for these
rotavirus components in disease pathogenesis.
INTRODUCTION
The cytocidal outcome following rotavirus infection of
enterocytes has been considered to be the cause of villus
atrophy associated with the induction of severe diarrhoea
and dehydration (Bishop et al., 1973; Burns et al., 1995;
Little & Shadduck, 1982; Snodgrass et al., 1979). Other
suggested diarrhoeal mechanisms involve non-replicating
rotavirus particles and toxin-like effects of the rotavirus
non-structural protein (NSP)4 (Ball et al., 1996; Berkova et
al., 2006; Seo et al., 2008; Shaw et al., 1995). Apoptosis of
jejunal cells in infant mice following murine rotavirus
infection has been identified through cleaved caspase-3
expression (Boshuizen et al., 2003). However, the mechanism of intestinal cell death following rotavirus infection,
including the role of viral-triggered pro-apoptotic and cell
survival signals, is not established.
Apoptosis refers to the programmed process of selfdestruction of damaged cells via a pre-determined
pathway. In contrast, necrosis is an unordered form of
3These authors contributed equally to this work.
018275 G 2010 SGM
cell death (Koyama et al., 2003). The extent to which
apoptosis and necrosis contribute to the death of rotavirusinfected cell monolayers has not been clearly determined.
Apoptotic features present following simian SA11 rotavirus
infection of the human enterocyte-like cell line HT-29 were
proposed to relate to the pathogenesis of rotavirus-induced
diarrhoeal disease (Superti et al., 1996). The monkey
kidney epithelial cell line MA104 also developed features of
apoptosis following infection with SA11 or porcine
rotavirus 1154 (Castilho et al., 2004). From the absence
of DNA cleavage in MA104 cells it was concluded that
necrosis was the major degenerative effect. DNA fragmentation was also absent following MA104 cell infection
with the porcine OSU rotavirus (Perez et al., 1998). From
these studies it was proposed that rotavirus-infected
MA104 cells die through oncosis, a form of necrosis.
However, an alternative assay showed enrichment of
nucleosomal DNA fragments starting 12 h after monkey
rotavirus RRV infection of MA104 cells, leading to
activation of Bax, a pro-apoptotic member of the Bcl-2
family (Martin-Latil et al., 2007). In fully differentiated
human intestinal Caco-2 cells, infectious but not inactivated
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P. Halasz, G. Holloway and B. S. Coulson
RRV induced DNA fragmentation indicative of apoptosis
(Chaibi et al., 2005).
Generally, the techniques used and the focus on late-stage
apoptotic markers and do not allow assessment of the
proportion of rotavirus-infected cells undergoing apoptosis, or the relative roles of apoptosis and necrosis. The
presence of necrosis could mask the detection of apoptotic
markers. Cells undergoing early apoptosis translocate the
membrane phospholipid phosphatidylserine from the
inner to the outer leaflet of the plasma membrane, which
can be detected using a fluorescent conjugate of Annexin V
(Vermes et al., 1995). Once externalized, this phospholipid
also acts as a ligand for phagocytic cells, leading to immune
response suppression (Balasubramanian & Schroit, 2003).
Demonstration of phosphatidylserine exposure by nonnecrotic cells during rotavirus infection would be required
to show the presence of early stage apoptosis.
The phosphatidylinositol 3-kinase (PI3K) pathway plays a
crucial role in the survival and differentiation of intestinal
epithelial cells. PI3K activation in human enterocytes
produces increased survival along the crypt–villus axis
(Laprise et al., 2004). Similarly, ageing of rat colonic
mucosa cells is associated with protection from apoptosis
through increased PI3K activity (Majumdar & Du, 2006).
Activation of the stress-activated protein kinase JNK is
linked to apoptosis induction (Shaulian & Karin, 2002).
The PI3K signalling pathway is a common activation target
for virus interference with apoptosis induction (Crusius
et al., 1998; Dawson et al., 2003; Lee et al., 2005; Portis &
Longnecker, 2004). Rotavirus infection activates kinase Akt
in a PI3K-dependent process early after infection of
MA104, HT-29 and Caco-2 cells (Dutta et al., 2009;
Halasz et al., 2008). This Akt activation is important for
elevated expression of the integrin a2b1 on rotavirusinfected intestinal cells, leading to increased cell survival
and rotavirus yields (Halasz et al., 2008). Many rotaviruses,
including RRV, utilize a2b1 as a cellular receptor or entry
co-factor (Graham et al., 2003). NSP4 has also been
reported to bind this integrin (Seo et al., 2008). Akt
activation and a2b1 upregulation are also induced by
rotaviruses that do not use a2b1 during cell entry, such as
the porcine CRW-8 strain (Halasz et al., 2008). This
indicates that Akt activation is independent of integrin
receptor usage. Rotavirus infection also induces JNK
activation, leading to AP-1-induced gene expression and
increased rotavirus replication (Holloway & Coulson,
2006). Studies of the ability of rotavirus to block cellular
apoptosis in infected cells have not been reported.
We investigated the relative roles of apoptosis and necrosis
in the death of rotavirus-infected MA104, HT-29 and
Caco-2 cells. Early stage apoptosis was specifically detected
by Annexin V staining. Late-stage apoptosis in rotavirusexposed cells was determined by analysis of cellular DNA
fragmentation or TdT-mediated dUTP nick-end labelling
(TUNEL) assay. In novel studies, replication-incompetent
and infectious RRV and CRW-8 were compared for their
2008
effects on cell monolayer integrity, metabolic activity and
viability. From our findings, rotaviruses induce early stage
apoptosis in MA104 and HT-29 cells that parallels rapid
loss of viability and metabolic activity, depends on virus
replication and is counter-balanced by virus-induced PI3K
activation. Late-stage apoptosis was detected following
RRV infection in differentiated Caco-2 cells only.
Inactivated rotaviruses induced a delayed loss of cell
viability, metabolic activity and morphological integrity,
which involved neither apoptosis nor a detectable secreted
soluble factor.
RESULTS
Cell monolayer integrity alterations following
treatment with inactivated rotavirus, rotavirus
infection or transfection of rotavirus dsRNA
Cell monolayer integrity determined by microscopy was
scored from 24 to 64 h after treatment with UV-psoralen
inactivated rotavirus (Table 1, Fig. 1a). Periods of longer
than 24 h were studied due to the expected slower rate of
effects induced by inactivated over infectious virus.
Rotavirus-infected cells were included for comparison.
Advanced monolayer damage, scored as 4 (as defined in
Methods), was observed in RRV- and CRW-8-infected
MA104 and HT-29 cells by 24 h after infection, and in
Caco-2 cells at 64 h after infection. This timing fully agrees
with previous studies of rotavirus-infected Caco-2 cells
(Dickman et al., 2000; Jourdan et al., 1997). Exposure to
inactivated RRV (I-RRV) or inactivated CRW-8 (I-CRW8) also induced monolayer integrity loss, scored as 2, in
MA104 and HT-29 cells after 24 h, and in Caco-2 cells after
40 h (Table 1, Fig. 1a). Scores reached 3 in all cell lines after
64 h of exposure. Using our sensitive flow cytometric assay
(Halasz et al., 2008), no cells expressing rotavirus antigen
were detected at 16 h after treatment with inactivated
rotavirus, demonstrating the absence of replication. The
effect of MA104 cell transfection with RRV dsRNA on
monolayer integrity was examined (Fig. 1b). This transfection also produced cell shrinkage and loss of cell-to-cell
contacts at 24 h after transfection (scored as 2), which was
similar to that observed 24 h after I-RRV treatment
(Fig. 1a). Transfection with cellular RNA produced no
alteration in cell monolayer integrity (Fig. 1b). No
monolayer integrity change was seen when RRV dsRNA
was added to cells without transfection reagent (data not
shown).
Rotavirus replication kinetics in MA104, HT-29
and Caco-2 cells
We showed previously that the proportions of live cells
infected by rotaviruses RRV and CRW-8 at an m.o.i. of 10
were 93–95 % (MA104), 78–83 % (HT-29) and 77–84 %
(Caco-2) at 16 h after inoculation (Halasz et al., 2008).
RRV replication was further investigated by generation of
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Cell death mechanisms of rotavirus
Table 1. Comparison of cell monolayer integrity alterations induced by infectious and inactivated rotaviruses
Inoculum*
Monolayer integrity scoresD for given cell line and time post-exposure (h)
MA104
RRV or CRW-8
I-RRV or I-CRW-8
HT-29
Caco-2
24
40
64
24
40
64
24
40
64
4
2
4
2
5
3
4
2
5
3
5
3
2
1
3
2
4
3
*Monolayer integrity changes induced by CRW-8 and I-CRW-8 were indistinguishable from those of RRV and I-RRV, respectively.
DThe scores of 1 (least damage) to 5 (greatest damage) are defined in Methods.
one-step growth curves in these cell lines (Fig. 1c). At this
high m.o.i., RRV yield peaked early (8 h after infection in
HT-29 cells; 16 h in MA104 and Caco-2 cells). Peak yields
[in fluorescent cell-forming units (f.f.u.) ml21] were similar
in MA104, HT-29 and Caco-2 cells, being 2.86108,
1.26108 and 1.26108, respectively. These yields are
similar to the peak RRV infectious yield of 16108 obtained
at 20 h after infection of fully differentiated Caco-2 cells at
the same m.o.i. (Chaibi et al., 2005).
Exposure to inactivated rotavirus or rotavirus
infection reduced cell viability and metabolic
activity
After treatment with I-RRV or I-CRW-8, cell viability
(detected by trypan blue exclusion) was maintained in
MA104 and HT-29 cells for up to 40 h, and in Caco-2 cells to
48 h, compared with diluent-treated (control) cells (Fig. 2).
At 48 h after treatment, 75–84 % of MA104 cells and 67–
69 % of HT-29 cells remained viable. By 64 h after treatment,
only 22–36 % of MA104 cells and 19–27 % of HT-29 cells
retained viability (Fig. 2a, b). The elevated viability loss in
HT-29 cells over MA104 cells correlated in time with their
more rapid monolayer integrity loss (Table 1). At 64 h after
treatment with I-RRV or I-CRW-8, 80–81 % of Caco-2 cells
remained viable (Fig. 2c). The more gradual loss of Caco-2
cell viability following treatment with inactivated rotavirus
paralleled their slower loss of monolayer integrity (Table 1).
Fig. 1. Changes in cellular monolayer integrity induced by
inactivated and infectious rotaviruses (a) and rotavirus dsRNA (b),
in relation to RRV replication kinetics (c). (a) Phase-contrast
microscopy images of confluent cell monolayers were obtained at
24 h after treatment. (b) Images were obtained at 24 h after MA104
cell transfection with purified RRV dsRNA or total RNA from
uninfected cells (ssRNA). One-step RRV growth curves in MA104,
HT-29 and partially differentiated Caco-2 cells are shown in (c). Bar,
SD. Most error bars are too small to be seen on the graph.
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Cell death also occurred earlier in MA104 and HT-29 cells
than in Caco-2 cells following rotavirus infection (Fig. 2).
At 16 h after RRV or CRW-8 infection, 60–77 % of MA104
and HT-29 cells and 96 % of Caco-2 cells remained viable.
At 24 h, no viable MA104 cells were detected, and only 14–
21 % of HT-29 cells were viable (Fig. 2a, b). At 64 h, 40–
45 % of Caco-2 cells remained viable (Fig. 2c). The viability
of diluent-treated (control) cells was .93 % at 48 h
(MA104) and .95 % at 64 h (HT-29 and Caco-2).
Although all cell lines were susceptible to staurosporine
(STS)-induced apoptosis (positive control), HT-29 cells
showed the most rapid decline with no viable cells by 48 h
after treatment (Fig. 2).
I-RRV- or I-CRW-8-treated MA104 and HT-29 cells
showed no significant change in metabolic activity at
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2009
P. Halasz, G. Holloway and B. S. Coulson
Fig. 2. The viability and metabolic activity of
MA104 (a), HT-29 (b) and partially differentiated Caco-2 (c) cells decreased following
treatment with inactivated rotavirus or rotavirus
infection. Cell viability is expressed as mean±
range and represents two independent experiments performed in duplicate. Cells treated
with 20 mM STS were included as a positive
control. Cellular metabolic activity was measured by the absorbance at 490 nm (A490),
which is directly proportional to the number of
living cells in the culture. The A490 value is
expressed as the mean±95 % CI and represents two independent assays performed in
triplicate. Acetone-fixed cells are included as a
negative control.
16 h after treatment, nor did Caco-2 cells at 40 h
(0.09¡P¡0.82; Fig. 2). However, treated MA104 and
HT-29 cells showed 20–23 % decrease (40 h) and 43–65 %
decrease (64 h) in metabolic activity (P,0.0001; Fig. 2a,
b). Caco-2 cells showed a 23–30 % reduction in metabolic
activity at 64 h (P,0.0001; Fig. 2c). RRV or CRW-8
infection reduced MA104 cell metabolic activity by 72–
75 % at 16 h and to background levels by 40 h, compared
with diluent-treated (control) cells (P,0.0001; Fig. 2a).
Infection reduced HT-29 cell metabolic activity by 49–55 %
at 16 h, and by 80–83 % at 40 h (P,0.0001; Fig. 2b). In
contrast, infection reduced the metabolic activity of Caco-2
cells at 16 h by only 6.8–11 % (0.11¡P¡0.12; Fig. 2c). By
64 h Caco-2 cell activity was reduced by 59 % compared
with control cells (P,0.0001).
These findings show that rotavirus replication was not
required for any decrease in cellular metabolic activity, as
inactivated rotavirus also produced this effect in MA104
and HT-29 cells. In contrast, Caco-2 metabolic activity loss
following infection or inactivated virus treatment was more
gradual. In all cell lines, replicating virus reduced cell
activity more rapidly than inactivated rotavirus, indicating
that increased cytocidal pressures were exerted by rotavirus
replication. The reduced cellular metabolic activity was
consistent with the observed rates of cell viability and
2010
monolayer integrity loss, and the timing of peak infectious
virus yields.
Early stage apoptosis was induced in MA104 and
HT-29 cells after rotavirus infection
Cells infected with RRV or CRW-8, or treated with IRRV, I-CRW-8 or STS, were stained with PE-labelled
Annexin V and 7-AAD from 6 to 24 h after treatment and
analysed by flow cytometry. The data obtained are
summarized in Table 2. Neither apoptosis nor necrosis
was induced in MA104 and Caco-2 cells at 6 h postinfection. Early stage apoptosis was detected in 21–22 % of
MA104 cells and 43 % of HT-29 cells infected with
RRV or CRW-8 for 11 h (Table 2). In contrast, rotavirus
infection of Caco-2 cells did not induce apoptosis
detected by Annexin V staining over the 24 h period.
The replication kinetics of RRV and the proportions of
RRV- and CRW-8-infected cells were similar between HT29 and Caco-2 cells (Fig. 1c) (Halasz et al., 2008), but only
HT-29 cells developed apoptosis. This validates comparison of the degree of apoptosis between these cell lines.
Similarly, both MA104 and HT-29 cells showed substantial degrees of apoptosis, so the slightly higher titre and
infection rate in MA104 cells did not affect the outcome
of these apoptosis studies.
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Table 2. Analysis of early apoptosis and necrosis caused by RRV and CRW-8 infection in epithelial cell lines
ND,
Not determined.
Cell line
Treatment
Cells (mean±range) undergoing early apoptosis and late apoptosis/necrosis at the given time (h) after treatment* (%)
6
MA104
HT-29
Caco-2
RRV
I-RRV
CRW-8
I-CRW-8
Mock
STS
RRV
I-RRV
CRW-8
I-CRW-8
Mock
STS
RRV
I-RRV
CRW-8
I-CRW-8
Mock
STS
11
12
14
ApoptoticD
Necroticd
Apoptotic
Necrotic
Apoptotic
Necrotic
1.4±0.3
3.1±0.5
ND
ND
ND
ND
ND
ND
4.2±0.8
2.3±0.8
ND
ND
2.5±0.1
2.6±0.3
10.1±3.8
19.2±1.4
ND
ND
ND
ND
ND
ND
ND
ND
2.2±0.8
4.1±1.2
2.4±2.1
2.2±1.1
ND
2.1±0.7
3.5±0.8
3.9±1.2
ND
2.5±1.2
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
37.4±0.1
23.0±1.5
ND
ND
ND
ND
ND
ND
ND
ND
4.0±1.5
3.3±1.2
ND
ND
ND
ND
34.5±3.8
25.5±0.6
ND
ND
ND
ND
ND
ND
ND
ND
1.8±0.3
2.8±1.2
1.5±0.9
2.7±0.9
ND
ND
3.7±1.7
3.5±1.2
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
1.7±1.1
3.1±1.1
10.6±2.0
3.1±1.4
10.9±0.6
2.8±0.3
4.2±1.9
5.1±1.5
6.4±1.7
1.5±1.0
7.7±0.9
1.8±1.1
3.3±1.8
4.1±1.1
0.2±0.1
0.2±0.0
0.2±0.1
0.2±0.1
0.2±0.0
1.4±1.0
20.2±2.3
ND
22.1±3.6
3.5±1.2
20.8±3.0
3.0±0.4
2.3±1.6
73.2±3.3
43.1±2.3
3.8±0.9
43.0±3.6
3.9±0.2
2.4±0.3
50.3±3.4
1.1±0.4
1.0±0.5
1.1±0.2
0.6±0.1
2.1±1.5
31.9±3.7
12.6±3.0
ND
5.1±1.5
3.1±1.1
ND
ND
3.1±0.1
18.3±0.2
ND
ND
0.5±0.1
0.2±0.1
ND
ND
5.0±1.6
2.7±1.3
ND
ND
2.6±0.6
18.4±3.1
ND
ND
2.5±0.7
2.7±0.4
ND
ND
2.9±1.6
2.3±0.9
ND
ND
Apoptotic
24
Necrotic
Apoptotic
Necrotic
ND
ND
0.2±0.1
0.3±0.1
ND
2.5±0.7
0.5±0.0
0.8±0.2
ND
4.9±0.6
1.5±0.1
4.1±0.2
ND
ND
ND
ND
ND
ND
2011
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Cell death mechanisms of rotavirus
*Cells were treated with RRV, I-RRV, CRW-8 or I-CRW-8, or mock-treated, and harvested at the given time after treatment. Cells treated with 20 mM STS were used as a positive control. Cells were
stained with Annexin V and 7-AAD and analysed by flow cytometry as described in Methods. Cell populations were identified as undergoing early apoptosis or necrosis as described in the legend to
Fig. 3. Data are given as the per cent±range of cells and were obtained from at least two independent experiments, each performed in duplicate.
DApoptotic cells are in the early stage of apoptosis.
dNecrotic cells are in the late apoptotic or necrotic stage.
P. Halasz, G. Holloway and B. S. Coulson
At 11 h after infection (Fig. 3), and at later times (Table 2),
the numbers of Annexin V-positive, 7-AAD-positive cells
also increased. These non-viable cells include both necrotic
and late apoptotic cells. These cells are excluded from the
early apoptotic cell population, so a decrease in early
apoptotic cell numbers was observed. This corresponds to
the timing of increased membrane permeability following
rotavirus infection of MA104 and HT-29 cells (Perez et al.,
1999), and consequently might have resulted in an
underestimation of early apoptotic cell numbers due to
their 7-AAD uptake. Rotavirus induction of apoptosis was
replication-dependent, as exposure to I-RRV or I-CRW-8
did not induce apoptosis in any cell line tested (Table 2,
Fig. 3). STS induced early apoptosis in 73 % of MA104
cells, 50 % of HT-29 cells and 32 % of Caco-2 cells at
11 h after exposure (Table 2), indicating that the intestinal cell lines were less susceptible to STS-induced early
apoptosis.
Inhibition of PI3K but not JNK increased the level
of rotavirus-induced early apoptosis in MA104
and HT-29 cells
Rotavirus infection strongly activates the PI3K-dependent
Akt signalling pathway in MA104, HT-29 and Caco-2 cells
(Dutta et al., 2009; Halasz et al., 2008). The involvement of
PI3K/Akt signalling in the apoptosis induced by rotavirus
infection was investigated. Baseline activated Akt levels are
similar in these three cell lines (Halasz et al., 2008).
Compared with diluent-treated (control) cells, the percentages of MA104 and HT-29 cells undergoing early stage
apoptosis following rotavirus infection increased by 1.7–
2.0-fold and 1.4–1.5-fold, respectively, in the presence of
the PI3K inhibitor LY294002 (Table 3). In contrast,
rotavirus infection of Caco-2 cells in the presence of
LY294002 produced no change in proportions of apoptotic
cells over diluent treatment (Table 3). This shows that
Caco-2 cells were not stimulated to undergo apoptosis
following rotavirus infection, even in the presence of an
inhibitor of the PI3K/Akt signalling pathway that plays an
important role in cell survival. These findings indicate that
rotavirus activation of PI3K partially protects MA104 and
HT-29 cells from early apoptosis following rotavirus
infection.
It has been suggested that the PI3K inhibitor LY294002
reduces the yield of infectious SA11 rotavirus produced in
MA104 cells (Dutta et al., 2009). The effect of LY294002 on
RRV cell entry and virus protein production was determined. Cells infected with RRV (m.o.i. 0.02) in the absence
or presence of LY294002 for 16 h were fixed and stained
for rotavirus antigen by indirect immunofluorescence.
Fig. 3. Rotavirus-infected MA104 and HT-29 cells underwent replication-dependent, early stage apoptosis. MA104, HT-29
and partially differentiated Caco-2 cells were treated with RRV, I-RRV, CRW-8 or I-CRW-8, or mock-treated, and harvested at
11 h after exposure. Early stage apoptosis was detected by flow cytometric analysis of cells stained with Annexin V and 7-AAD.
The lower left quadrant of the dot-plot indicates viable, non-apoptotic cells, the lower right quadrant indicates cells in early
apoptosis and the upper right quadrant indicates non-viable cells, including necrotic cells. Data are given as the per cent of cells
in each quadrant.
2012
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Cell death mechanisms of rotavirus
Table 3. PI3K inhibition increased early stage apoptosis in rotavirus-infected MA104 and HT-29 cells, whereas JNK inhibition did
not affect apoptosis in any rotavirus-infected cell line
Treatment
Mean of cells undergoing early apoptosis and late apoptosis/necrosis in the given cell line* (%)
MA104
RRV+LY294002
RRV+DMSO control for
LY294002D
RRV+SP600125
RRV+DMSO control for
SP600125D
CRW-8+LY294002
CRW-8+DMSO control
for LY294002
CRW-8+SP600125
CRW-8+DMSO control
for SP600125
Mock+LY294002d
Mock+SP600125d
HT-29
Caco-2
Apoptotic
Necrotic
Apoptotic
Necrotic
Apoptotic
Necrotic
45.1
26.2
16.3
16.0
76.4
52.6
8.0
10.4
2.2
2.7
2.3
1.7
23.8
23.3
10.3
10.1
47.3
49.1
7.2
6.1
1.3
1.1
0.4
0.3
43.9
21.9
15.9
14.1
71.0
53.2
8.2
7.7
2.8
2.4
2.3
2.8
22.6
22.7
10.3
11.9
47.9
48.2
9.1
7.3
1.8
1.4
0.3
0.4
5.1
2.1
1.2
3.1
2.7
2.7
5.5
2.2
2.6
1.5
1.8
0.8
*Cells were inoculated and treated, stained with Annexin V and 7-AAD at 11 h after inoculation, analysed by flow cytometry and identified as
(early) apoptotic or (late apoptotic) necrotic as described in Methods and the legend to Table 2.
DTreatment comprised the DMSO-containing diluent of each inhibitor, without added inhibitor.
dProportions of apoptotic and necrotic mock-infected cells in the presence or absence of the given inhibitor were indistinguishable.
The infectivity titres of RRV were unchanged by PI3K
inhibitor treatment. Respective titres (f.f.u. ml21) in diluentand inhibitor-treated cells were a mean±95 % CI of
1.7±0.26104 and 1.7±0.36104 in MA104 cells,
2.0±0.16104 and 2.0±0.46104 in HT-29 cells, and
1.6±0.26104 and 1.6±0.56104 in Caco-2 cells. These
data indicate that LY294002 did not have a noticeable effect
on the levels of RRV protein production.
Involvement of the JNK signalling pathway in the degree of
MA104, HT-29 and Caco-2 cell apoptosis following
rotavirus infection was examined. The proportion of
MA104, HT-29 and Caco-2 cells undergoing early stage
apoptosis was not affected by the presence of the JNK
inhibitor (Table 3). Following JNK inhibitor or diluent
treatment, 23–24 % (MA104) and 47–49 % (HT-29) of
CRW-8- and RRV-infected cells showed early stage
apoptosis. JNK inhibition during rotavirus infection of
Caco-2 cells did not affect the very low level of cell death
observed. These observations demonstrate that JNK
signalling did not play a role in rotavirus-induced
apoptosis in MA104 and HT-29 cells.
Late-stage apoptosis following rotavirus infection
was only detected in differentiated Caco-2 cells
Cellular DNA collected from rotavirus-infected cells from
16 to 64 h after infection at 8 h intervals was examined for
late-stage apoptosis in the form of DNA fragmentation. No
DNA fragmentation was detected during this period in
http://vir.sgmjournals.org
RRV- or CRW-8-infected MA104 or HT-29 cells, or in
Caco-2 cells cultured for 5 days prior to infection to
produce partial differentiation (Fig. 4). As a control, STStreated MA104 cells yielded lower molecular mass DNA
fragments typical of late-stage apoptosis. Several bands
present in the infected cell profiles in Fig. 4 correspond to
viral RNA bands. It has been reported that only fully
differentiated Caco-2 cells undergo apoptosis following
RRV infection (Chaibi et al., 2005). To confirm this, Caco2 cells cultured for 12 days to induce complete differentiation were infected with RRV or CRW-8. A high level of
apoptosis in the form of DNA fragmentation was detected
in these cells (Fig. 4), even though their membrane
integrity was reduced at a similar rate to that of the
infected, partially differentiated Caco-2 cells shown in
Fig. 1(a) and Table 1 (data not shown). This confirms
that Caco-2 cells require complete differentiation to be
susceptible to apoptosis following rotavirus infection.
These findings also demonstrate that both monkey and
porcine rotaviruses induce late-stage apoptosis in differentiated Caco-2 cells.
Cell monolayer integrity, viability and metabolic activity
changes following inactivated rotavirus treatment were less
extensive than those induced by infection, and occurred in
the absence of detectable early stage apoptosis (Fig. 3,
Tables 1 and 2). However, inactivated rotavirus may induce
detectable late-stage apoptosis. Rather than DNA fragmentation analysis, the potentially more sensitive TUNEL
assay for DNA-strand breaks was used to investigate
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P. Halasz, G. Holloway and B. S. Coulson
apoptosis in HT-29 and MA104 cells, but not in partially
differentiated Caco-2 cells.
Fig. 4. DNA fragmentation in MA104, HT-29 and Caco-2 cells
infected with RRV or CRW-8 (CRW). DNA was isolated at 16, 24,
36, 48 and 64 h after infection, or 24 h after 20 mM STS
treatment. The data shown (36 h) are representative of that
obtained at all times in two independent experiments. DNA
fragments were not detected in MA104, HT-29 or partially
differentiated Caco-2 (U-Caco-2) cells (cultured for 5 days), but
were present in differentiated Caco-2 (D-Caco-2) cells (cultured
for 12 days). Asterisks indicate rotavirus RNA segments. MW,
Molecular weight standard. Bands of .10 kbp are undigested
genomic DNA.
late-stage apoptosis in cells treated with I-RRV for 40, 44
and 48 h (Fig. 5). The proportions of TUNEL-positive
diluent-treated cells were 8.0–9.8 % (MA104), 6.3–9.1 %
(HT-29) and 6.9–9.8 % (Caco-2). I-RRV-treated cells
showed similar proportions of TUNEL-positive cells,
namely 8.1–9.3 % (MA104), 7.8–8.3 % (HT-29) and 6.7–
8.7 % (Caco-2). These data show that the proportion of
TUNEL-positive cells was unaltered by the treatment with
I-RRV. Overall, these studies indicate that losses in
monolayer integrity, cell viability and metabolic activity
following I-RRV treatment occurred in the absence of early
or late-stage apoptosis.
DISCUSSION
The effects of rotaviruses on MA104 cells have been
extensively studied due to their high degree of rotavirus
susceptibility. However, the permissive HT-29 and Caco-2
cells provide closer models of human enterocytes, and
Caco-2 cells spontaneously differentiate in culture (Chaibi
et al., 2005). We found that rotavirus infection was
associated with the loss of metabolic activity and viability
in each cell line, which correlated with the rate of
membrane integrity loss. HT-29 and MA104 cells lost
function much more rapidly than partially differentiated
Caco-2 cells, paralleling the development of early stage
2014
As end-stage apoptosis was not detected despite apoptosis
initiation, it is likely that HT-29 and MA104 cells died
through necrosis prior to the completion of the apoptotic
process. Consistent with these data, others have observed
morphological changes in the absence of DNA fragmentation in these rotavirus-infected cell lines (Castilho et al.,
2004; Perez et al., 1998; Superti et al., 1996). Our findings
also confirm the previous report that RRV infection
induces end-stage apoptosis in terminally differentiated
Caco-2 cells (Chaibi et al., 2005), and extend this to include
another rotavirus strain, CRW-8. The slower monolayer
integrity loss in these differentiated cells, probably
indicating low necrosis levels, would explain the detection
of end-stage apoptosis. The lack of monolayer disruption
and membrane damage in differentiated Caco-2 cells until
48 h post-infection also support this view (Jourdan et al.,
1997). This late-stage apoptosis is similar to that observed
in murine rotavirus-infected enterocytes lining intestinal
villi (Boshuizen et al., 2003), providing further evidence of
the utility of differentiated Caco-2 cells as a model of
rotavirus-triggered intestinal cell responses.
Overall, rotavirus-infected undifferentiated (HT-29) and
partially differentiated (Caco-2) intestinal epithelial cells
die from necrosis, whereas infected differentiated cells
(Caco-2) become apoptotic. The early apoptosis generated
by rotavirus infection in undifferentiated HT-29 cells
could represent an alternative mechanism by which
rotavirus impairs intestinal functionality. In addition, it
has been proposed that the exposed phosphatidylserine
enables viruses to evade immune recognition and reduce
inflammation (Soares et al., 2008). These authors showed
that a chimeric antibody targeted to this anionic lipid
has a potential as an antiviral agent. Studies of the roles
of exposed phosphatidylserine in rotavirus immune
evasion and as a target for rotavirus therapy would be
of interest.
The absence of early stage apoptosis in all cell lines
following treatment with I-RRV and I-CRW-8 is a strong
indication that initiation of apoptosis requires viral
replication. These observations extend the previous
demonstration that RRV replication is necessary to induce
apoptosis in differentiated Caco-2 cells (Chaibi et al.,
2005), by showing that apoptosis is not induced in partially
differentiated Caco-2 cells.
The slower development of monolayer disruption and
losses of viability and metabolic activity induced by
replication-deficient rotavirus were internal non-apoptotic
cell responses. Replicating virus might induce a similar
response more rapidly, by producing higher levels of a
trigger such as viral protein or nucleic acid. However, the
mechanisms responsible for loss of cell function due to
inactivated and infectious rotavirus may not be the same,
and require further study. As cells transfected with purified
RRV dsRNA exhibited similar changes to inactivated
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Cell death mechanisms of rotavirus
Fig. 5. Inactivated RRV did not induce latestage apoptosis in MA104 (a), HT-29 (b) or
partially differentiated Caco-2 (c) cells. Cells
treated with I-RRV or mock-treated were
processed for TUNEL assay at 40, 44 and
48 h after treatment. The per cent±range of
TUNEL-positive cells of the 10 000 collected
is indicated on each histogram. Data are
representative of two independent experiments.
rotavirus, cellular responses to viral dsRNA, possibly
mediated through Toll-like receptor-3, RNA helicases or
protein kinase R, might be responsible for the monolayer
integrity loss due to inactivated rotavirus (Hirata et al.,
2007; Randall & Goodbourn, 2008). RRV inactivated by the
same protocol used here causes diarrhoea in infant mice
(Shaw & Hempson, 1996; Shaw et al., 1995), so the cellular
effects of inactivated rotavirus exposure we observed could
relate to this diarrhoeal response. Determining the
molecular basis of this cytopathology may improve the
understanding of rotavirus diarrhoeal mechanisms.
Integrins play an important role in controlling programmed cell death (Frisch & Ruoslahti, 1997). However,
as both the integrin-utilizing RRV and integrin-independent CRW-8 rotavirus induced apoptosis to similar levels,
the signal for apoptosis is independent of integrin receptor
engagement by rotavirus. Although rotavirus activation of
Akt is also unrelated to integrin receptor usage (Halasz
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et al., 2008), sialic acid receptors used by rotaviruses might
play a role in these events (Haselhorst et al., 2009). Newly
formed rotavirus particles are released from the apical pole
of HT-29 and Caco-2 cells via the endoplasmic reticulum
through a vesicular vectorial transport prior to any cell lysis
(Chwetzoff & Trugnan, 2006; Jourdan et al., 1997). In
contrast, simian rotavirus SA11 was retained in the
endoplasmic reticulum of MA104 cells until cell lysis
(Altenburg et al., 1980; Musalem & Espejo, 1985). As HT29 and MA104 cells lost cellular activity and viability at a
similar rate after infection, it is unlikely that the prolonged
survival of rotavirus-infected Caco-2 cells was due to the
mechanism of virion release. However, signalling differences between HT-29 and Caco-2 cells following rotavirus
infection may affect their rate of viability loss. In particular,
the stress-activated p38 kinase, known to activate transcriptional factor AP-1, was activated in Caco-2 cells, but
not in HT-29 cells, after rotavirus infection (Holloway &
Coulson, 2006).
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P. Halasz, G. Holloway and B. S. Coulson
Levels of early apoptosis in rotavirus-infected MA104 and
HT-29 cells increased substantially when PI3K activity was
inhibited. This inhibitor does not affect basal activated Akt
levels during rotavirus infection (Dutta et al., 2009; Halasz
et al., 2008), so this increased apoptosis was associated with
the blockade of virus-induced Akt activation. These
findings suggest that rotavirus inhibits apoptosis in part
by PI3K/Akt activation. As PI3K/Akt blockade in infected,
partially differentiated Caco-2 cells was insufficient to
induce apoptosis, differentiation-related factors appear to
be required for rotavirus-induced apoptosis in Caco-2 cells.
Cellular heat-shock protein (hsp) 70 produced in response
to stress-related stimuli is protective against apoptosis
(Daugaard et al., 2007). For example, hsp70 protects cells
from apoptosis induced by the human immunodeficiency
virus type 1 protein R (Bukrinsky & Zhao, 2004). Hsp70
expression in Caco-2 cells is increased after rotavirus
infection and appears to negatively control infection by
targeting rotavirus proteins for degradation (Broquet et al.,
2007; Cuadras et al., 2002). In contrast, hsp90, a
constitutively expressed regulator of cell survival signalling
pathways including PI3K/Akt, has been associated with
rotavirus activation of Akt and is proposed to be a positive
regulator of rotavirus replication (Dutta et al., 2009). It is
likely that hsp70 and hsp90 are involved in the regulation
of rotavirus-induced apoptosis.
Our studies have advanced the understanding of processes,
leading to the death of rotavirus-exposed cells. Notably,
inactivated rotavirus hastens cell death through a nonapoptotic mechanism. Through PI3K/Akt activation and
apoptosis induction, rotavirus appears to strike a balance
between prolongation of host cell survival and limitation of
host inflammatory responses.
METHODS
Cell lines and rotaviruses. Caco-2, HT-29 and MA104 cells were
propagated as described previously (Halasz et al., 2008; Londrigan
et al., 2000). The origins of Rhesus monkey rotavirus RRV (P5B[3]G3)
and porcine rotavirus CRW-8 (P9[7]G3) have been described
previously (Coulson, 1993; Nagesha et al., 1989). Viruses were
cultivated in MA104 cells following trypsin activation, purified by
glycerol gradient ultracentrifugation and titres determined by indirect
immunofluorescent infectivity assay in MA104 cells, as described
previously (Hewish et al., 2000; Jolly et al., 2000). UV-psoralen
inactivation of purified rotaviruses was performed as described
previously (Groene & Shaw, 1992), and verified using infectivity
assays and ELISA as described previously (Halasz et al., 2008;
Holloway & Coulson, 2006). Cells were infected at an m.o.i. of 10
unless otherwise stated, in order to ensure maximum infection
efficiency (Halasz et al., 2008). The degree of cellular apoptosis has
been shown to depend on RRV m.o.i. (Chaibi et al., 2005). For onestep growth curves, rotavirus titres were determined in whole-cell
lysates harvested from 1 to 64 h after infection.
Cell monolayer integrity studies. MA104, HT-29 and Caco-2 cell
monolayers in 24-well trays were mock-infected, infected with RRV
or CRW-8, or treated with inactivated rotavirus at the equivalent
m.o.i. for the indicated times, as described previously (Halasz et al.,
2016
2008). Monolayer integrity was scored as 0, no cell rounding or loss of
cells; 1, no cells lost and ~25 % of cells rounded; 2, ~25 % of cells lost
and ~20–~80 % of remaining cells rounded; 3, ~50 % of cells lost and
~30–~80 % of remaining cells rounded; 4, ~75 % of cells lost and
.40 % of remaining cells rounded; and 5, ,10 % of cells remaining
in the monolayer.
MA104 cells at approximately 50 % confluence (1.56105 cells per
well) were transfected with 2 mg dsRNA, purified from RRV by
phenol extraction as described previously (Smith et al., 1980), using
5 ml Transit LT-1 (Mirus Bio). Control cells were treated with Transit
LT-1 and 2 mg total cellular RNA purified from MA104 cells using an
RNeasy kit (Qiagen). RNA was quantified by UV spectrophotometry
using a Nanodrop 1000 (Thermo Fisher Scientific). Images were
obtained at 24 h post-transfection by phase-contrast microscopy
using a DM IL inverted microscope (Leica) at 6200 magnification.
Assays of cellular metabolic activity and viability. Cells were
placed in suspension using trypsin-EDTA as described previously
(Halasz et al., 2008). Cell viability was determined by the trypan blue
dye exclusion assay and cells were counted in a haemocytometer.
Cellular metabolic activity was determined through mitochondrial
enzymic activity in the CellTiter 96 AQueous One Solution Cell
Proliferation assay (Promega) according to the manufacturer’s
protocol. Differences in metabolic activity were analysed by oneway ANOVA using GraphPad Prism Software with significance set at
P,0.05.
Flow cytometric detection of apoptosis. The Annexin V assay was
used (Vermes et al., 1995). Confluent cell monolayers were infected
with trypsin-activated rotaviruses or exposed to inactivated rotavirus
as described previously for analysis of rotavirus effects on cellular
protein expression (Halasz et al., 2008). Cells were treated with 20 mM
STS (Sigma), a broad-spectrum protein kinase inhibitor, as a positive
control (Meggio et al., 1995). In some experiments, chemical
inhibitors of PI3K (LY294002; 20 mM) and JNK (SP600125;
10 mM), purchased from Calbiochem, or matched DMSO control
solutions, were added to the cell culture medium for the infection
period. At the concentrations used, these inhibitors alone did not
inhibit cellular metabolic activity, as described previously (Halasz et
al., 2008; Holloway & Coulson, 2006). From 6 to 24 h after infection,
cells were placed in suspension as described previously (Halasz et al.,
2008), washed and incubated in binding buffer (10 mM HEPES,
pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) containing 5 % Annexin V-PE
(Pharmingen) and 10 % 7-AAD (Pharmingen) at room temperature
for 15 min. Samples were analysed by flow cytometry using FACSort
and CellQuest software (Becton Dickson). Viable cells were selected
by their exclusion of 7-AAD, which enters only membranecompromised cells and binds to DNA. Compensation quadrants
were set on cells stained with Annexin-V or 7-AAD alone. Cells
resuspended in binding buffer only acted as negative controls.
DNA fragmentation analysis. Confluent cell monolayers (36105
cells) in 24-well trays were infected with rotavirus or treated with
20 mM STS. Cells were placed in suspension using trypsin-EDTA,
washed and resuspended in lysis buffer (1 % NP-40, 150 mM NaCl,
50 mM Tris/HCl, pH 8.0; 5 mM EDTA) containing 50 mg Proteinase
K (Promega) ml21 and incubated for 1 h at 37 uC. Precipitated DNA
fragments were separated on a 2 % agarose gel containing 0.6 mg
ethidium bromide ml21, using a 1 kbp DNA ladder as a standard
(New England Biolabs). DNA fragments were visualized by UV
illumination.
TUNEL assay. Confluent cell monolayers were infected, placed into
suspension as above, fixed with 4 % paraformaldehyde (Sigma) in
PBS for 30 min at room temperature, washed and permeabilized by
resuspension in PBS containing 0.2 % Triton X-100 (Sigma) for
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Cell death mechanisms of rotavirus
2 min on ice. Washed cells were incubated with FITC-conjugated
dUTP (Promega) for 1 h at 37 uC, resuspended in PBS and analysed
by FACSort and CellQuest software as described above. Apoptotic
cells show increased fluorescence from binding of cleaved DNA 39OH
ends by FITC-conjugated dUTP.
Cuadras, M. A., Feigelstock, D. A., An, S. & Greenberg, H. B. (2002).
ACKNOWLEDGEMENTS
Dawson, C. W., Tramountanis, G., Eliopoulos, A. G. & Young, L. S.
(2003). Epstein–Barr virus latent membrane protein 1 (LMP1)
We are most grateful to C. Cheers and B. Gilbertson for reagents and
assistance with the TUNEL assay, and F. Carbone for provision of
STS. This work was supported by Project Grant 350252 and Research
Fellowship 350253 awarded to B. S. C. from the National Medical
Research Council of Australia.
activates the phosphatidylinositol 3-kinase/Akt pathway to promote
cell survival and induce actin filament remodeling. J Biol Chem 278,
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Gene expression pattern in Caco-2 cells following rotavirus infection.
J Virol 76, 4467–4482.
Daugaard, M., Rohde, M. & Jaattela, M. (2007). The heat shock
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Dickman, K. G., Hempson, S. J., Anderson, J., Lippe, S., Zhao, L.,
Burakoff, R. & Shaw, R. D. (2000). Rotavirus alters paracellular
permeability and energy metabolism in Caco-2 cells. Am J Physiol
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