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Indian J Med Res 129, May 2009, pp 579-586

Influenza a virus induced apoptosis: Inhibition of DNA laddering &


caspase-3 activity by zinc supplementation in cultured HeLa cells

Vikram Srivastava, Shweta Rawall, V.K. Vijayan* & Madhu Khanna

Departments of Respiratory Virology & *Respiratory Medicine, V.P. Chest Institute, University of Delhi
Delhi, India

Received January 10, 2008

Background & objectives: The pathogenesis of influenza virus infection involves virus replication in
epithelial cells of the respiratory tract and the consequent degeneration of infected cells. Influenza virus
induces cellular degeneration following infection of cultured cells in vitro, and the cytopathic effect (CPE)
occurs principally through apoptotic cell death. This study was undertaken to find out the effect of zinc
on influenza virus induced apoptosis in cultured HeLa cells.
Methods: The sub-confluent monolayer HeLa cells were used to study the effect of zinc on influenza
virus induced apoptosis. The apoptotic markers viz., caspase-3 activity, phagocytic index, morphological
changes, and DNA fragmentation were assayed.
Results: When HeLa cells were infected with a cell adapted pathogenic strain of influenza A (A/Udorn/
317/72H3N2) virus, DNA fragmentation was observed in virus infected cells by 24 h post infection and
caspase-3 activity was maximum at 4 h post infection after which it reached to plateau. Treatment of cells
with 0.1 5mM concentration of zinc till 8 h post infection inhibited DNA fragmentation and also caspase
3 activity was decreased significantly up to 2 h post infection.
Interpretation & conclusions: When the infected HeLa cells were incubated with adherent macrophages,
efficient phagocytosis occurred and the release of virus into the culture medium was inhibited. These
results suggested that inhibitory effect on influenza virus induced apoptotic death of cultured cells can
be determined at an early stage of the infection by treatment of zinc.

Key words Apoptosis - caspase activity - DNA fragmentation - influenza virus

Apoptosis is a programmed cell death in which the significance of apoptosis in determining the outcome
cell actively participates in its own destructive process1. of virus infections3. The ability of viruses to control
In addition to its physiologic roles, apoptosis plays an the apoptotic response of their host can influence the
important role in the pathogenesis of infectious diseases pathogenicity of the virus. The virulent influenza A
caused by viruses, bacteria and prions2. Many viruses viruses produce devastating disease, and experimental
carry genes, which directly influence the apoptotic infection by these viruses is frequently used as a model
ability of their host cell thus changing the view about system to examine the mechanism of disease4,5.
579
580 INDIAN J MED RES, MAY 2009

The damage caused by influenza virus infection in originally propagated in embryonated chicken eggs
cultured cells, which permit high levels of replication and further grown in cultured HeLa cells. When HeLa
of influenza A virus exhibited several features cell cultures were 85-90 per cent confluent, the cultures
characteristic of programmed cell death6-8. Depletion of were pretreated with 199 medium (Sigma, USA) and
lymphocytes due to apoptosis has also been described inoculated with influenza virus at a multiplicity of
in mice infected with a highly virulent influenza A virus infection of approximately ten, 50 per cent tissue
(H5N1) isolated from humans9-11. All mammalian and culture infectious doses (TCID50) per cell for 45 min at
avian influenza A and B virus strains induce apoptosis 37°C. Control HeLa cells were mock infected with 199
in many different cell types, including lymphocytes medium alone. The mock and IV-infected HeLa cells
and human monocytes12. However, the consequence of were incubated at 37°C in 199 medium with 0.25 per
this activation for virus replication or host cell defense cent BSA (Sigma, USA) and 1 mg/ml L-1-tosylamido-
is not well established12. 2-phenylethyl chloromethyl ketone-trypsin (Sigma,
Zinc supplementation prevents apoptosis induced USA) for various times before analysis. The amount of
by a variety of agents in both, in vitro and in vivo virus released into the culture medium was determined
models13. The protective effect of zinc has generally by haemagglutination test and plaque forming unit
been attributed to inhibition of Ca2+ and Mg2+ (PFU) assays.
dependent endonucleases, thereby causing inhibition Plaque forming unit (PFU): Virus particles in HeLa
of DNA fragmentation, a terminal step and hallmark cell culture suspensions following IV infection
of apoptosis14. However, many studies have also were quantified by PFU enumeration using standard
identified caspase-3 as a site of zinc inhibition in procedures as described by Kurokawa et al19. Briefly,
the apoptotic death14-16. This protease is frequently confluent monolayers of Madin-Darby canine kidneycells
activated in mammalian cell apoptosis to cleave key were incubated with the culture supernatants collected
cellular proteins, leading to completion of the apoptotic at various time points after virus infection serially
process once the cell has been committed to die15,17,18. diluted in phosphate buffer saline (PBS) containing 1
The exact role of zinc in the apoptotic pathway of per cent bovine serum albumin ((pH-7.2) for 1 h at room
influenza A virus infected cells has not been fully temperature. The cells were then overlaid with Eagle’s
established. minimal essential medium (EMEM) supplemented
This study was undertaken to characterize the with 0.2 per cent bovine serum albumin, 0.1 per cent
mechanism of cell damage caused by influenza virus DEAE Dextran, 1 µg trypsin/ml and 0.8 per cent agar,
infection in cultured HeLa cells; an extensive time- and maintained in a humid atmosphere containing 5 per
interval study was performed to elucidate the sequence cent CO2 for 3 days. The agar media were then removed
of hallmark events in apoptosis. The effect of zinc and the cells were fixed with 5 per cent formalin solution
was investigated on both the early and late phase of and stained with 0.03 per cent methylene blue solution.
apoptosis in a time dependent manner in cultured cells Visualized plaques were counted and the virus titre was
and confirmed that caspase-3 was a novel and proximal expressed as log10 pfu/ml.
site of Zn2+ inhibition in the apoptotic pathway. Cell viability assays: Cells were transferred to a slide
Material & Methods and stained with 0.5 per cent trypan blue. After 1 min
staining, cells were observed under the microscope
Virus and infection of HeLa cells: HeLa cells obtained
(Nikon E600, Japan). Dead cells were stained
from National Centre for Cell Sciences (NCCS), Pune,
blue, whereas living cells remained unstained. The
India, were cultivated in either 35-mm cell culture
percentage of cell death was obtained by calculating
dishes or 25 or 75 cm2 culture flasks (Greiner, Germany)
the ratio of dead cells to total cells.
with Eagle’s minimal essential medium (Sigma, USA)
containing 10 per cent foetal bovine serum (Sigma, DNA fragmentation assay: To assess the fragmentation
USA) and antibiotics (100 U/ml penicillin, 100 mg/ of cellular DNA into the characteristic apoptotic ladder,
ml streptomycin, 0.25 mg/ml fungizone, 25 mg/ml IV-infected HeLa cells were pelleted at 6, 12, 18 and
gentamicin; all from BioWhittaker, USA) at 37°C 24 h post infection (pi) and mock infected cells were
in a 5 per cent CO2 incubator. A pathogenic strain pelleted at 24 h pi. DNA samples were extracted using
of influenza virus (IV), A/Udorn/317/72(H3N2), a similar method as described by Gong et al 20 with
obtained from CDC, Atlanta in lyophilized form was slight modifications. Briefly, HeLa cells were lysed in
SRIVASTAVA et al: INFLUENZA VIRUS INDUCED APOPTOSIS IN HELA CELLS 581

ice-cold cell lysis buffer (10 mM Tris, pH 7.5; 1 mM old BDF1 mice obtained from the animal house of
EDTA; 0.5% Triton X-100). Samples were then treated V.P. Chest Institute, Delhi23. The macrophages were
with 0.1 per cent sodium dodecyl sulphate (SDS) maintained in RPMI 1640 (Sigma, USA), 10 per cent
(GIBCO-BRL, USA) and 300 mg/ml of Proteinase K foetal bovine serum22. 1x106 HeLa cells infected with
(Sigma, USA) at 56°C overnight. Lysates were phenol- influenza A virus were collected at 0, 6, 9, 12, 18, 24
chloroform extracted and the DNA samples were h post infection, labelled with biotin and added to the
cold ethanol precipitated in the presence of 300 mM macrophage culture at a ratio of one HeLa cell to two
sodium acetate ((pH 5.2). DNA was further treated with macrophages. The mixed culture was incubated for 1 h
RNase A (Sigma, USA) (1 mg/ml) for 1 h at 37°C and at 37°C, washed extensively by pipetting with PBS and
subjected to electrophoresis (Submarine System, Sub then with trypsin (0.5 µg/ml) to remove HeLa cells free
Cell GT, Bio-Rad, USA) using 1.8 per cent agarose from or lightly attached to macrophages.The remaining
in Tris-EDTA buffer. DNA was visualized by staining cells were further fixed with PBS containing 2 per cent
with ethidium bromide and photographed in gel paraformaldehyde, 0.5 per cent glutaraldehyde, and
documentation system (AlphaImagerTM 2200, USA)7. 0.05 per cent Triton X-100 and then supplemented with
FITC-conjugated avidin (Vector Laboratories, USA).
Caspase-3 assay: The Caspase-3 assay was performed
The number of macrophages containing engulfed cells
in a multiwell plate using a kit obtained from BD
was determined using fluorescence and phase-contrast
Biosciences Pharmingen (USA). Cell lysates (1x106
microscopy (Nikon E600, Japan) and expressed
cells/ml) were prepared at 0, 1, 2, 3, 4, 5, 6, and 7
relative to the total number of macrophages, this ratio
h post infection. For each reaction, 5µl fluorigenic
was termed the phagocytic index24, 25.
substrate, Ac-DEVD-AMC (acetyl-Asp-Glu-Val-
Asp/7-amino-4-methylcoumarin) was added to a well Effect of zinc on DNA fragmentation: To assess the
containing 0.2 ml of 1X HEPES buffer; 50 µl of cell ability of Zn2+ ions to block endonucleolytic DNA
lysate was added to each well containing HEPES cleavage ZnSO4 (Sigma, USA) (0.15 mM,) was added
buffer. Caspase-3 blocking assays were set up in to the infected monolayer cultures at 0, 4, 8, 12, 16,
parallel to each reaction mixture by adding 5 µl Ac- and 20 h post infection. At 24 h pi, cellular DNA was
Asp-Glu-Val-Asp-CHO. The reaction mixtures were extracted from virus infected control, mock infected
incubated for 1 h at 37˚C. The amount of fluorescence and infected cells with treatment and analyzed by gel
emitted was measured using a spectrofluorometer with electrophoresis26.
an excitation wavelength of 380 nm and an emission
Effect of zinc on caspase-3 activity: Infected HeLa
wavelength range of 440 nm21.
cells were treated with 0.15 mM of ZnSO4 and the
Annexin V assay: Detection of flipping of caspase-3 activity was observed at 0, 1, 2, 3, 4, 5 and 6
phosphatidylserine was done by annexin V assay h post infection. The activity at 4 h p.i. was compared
by using a commercially available TACSTM Annexin to that of mock infected treated and untreated infected
V-FITC Apoptosis Detection kit (R & D, USA)20. At HeLa cells at 4 h26.
0, 3, 6, 9, 12, and 15 h pi, HeLa cells were pelleted
Statistical analysis: The data were analyzed with one
and specific binding of annexin V was achieved by
and two way analysis of variance (ANOVA) followed
incubating 106 HeLa cells in 60 µl of binding buffer
by bonferroni’s multiple comparison tests. All the
with a saturated concentration of annexin V for 15
results were expressed as mean ± SEM.
min at 4°C in the dark. To discriminate between early
apoptosis and necrosis, the cells were simultaneously Results
stained with annexin V and propidium iodide (PI) Effect of influenza virus infection on cell death: The
before analysis. The binding of FITC-annexin V inoculated plates were subjected to virus titration at 6
(FL1) and PI (FL2) to the cells was measured by flow hourly intervals and it was observed that the virus titre
cytometry (FACSCalibur; BD Biosciences, USA) using increased with time. A significant increase in the virus
Cell Quest software from BD Biosciences, (USA). At titre was observed at 12 h ((P<0.05) post infection.
least 10,000 cells were counted in each sample22. At 24 h pi, the maximum virus titre was observed
Phagocytosis of influenza virus- infected cells by after which it reached a plateau. At 4 hourly intervals
macrophages: Macrophages were isolated from post-infection, the infected HeLa cells were stained
peritoneal fluids of thioglycollate-treated eight weeks with trypan blue and assessed for cell viability. It was
582 INDIAN J MED RES, MAY 2009

observed that the percentage of cell viability decreased Kinetics of activation of cysteinyl proteases; caspase-3
as the time of infection increased and at 24 h post on influenza virus infection in cultured cells: To study
infection, it was less than 10 per cent. the activation of caspase-3 time-interval study was
performed, cell lysates were prepared at various time
Detection of DNA laddering in influenza virus infected
points after inoculation of virus i.e., 0, 1, 2, 3, 4, 5,
HeLa cells: DNA laddering is commonly accepted as
6, and 7 h respectively and the amount of AMC (7-
an important biochemical hallmark of apoptosis. Thus
amino-4-methylcoumarin) liberated from Ac-DEVD-
it was critical to test whether or not influenza virus
AMC was measured using a spectrofluorometer.
infection induced DNA laddering in cultured HeLa
Apoptotic cell lysates containing active caspase-3 were
cells. Agarose gel electrophoresis analysis of DNA
estimated by a considerable emission as compared to
extracted from virus infected cells showed distinct
non-apoptotic cell lysates. A significant increase in
laddering of DNA fragments increasing in size by
fluorescence occurred 2 h after infection ((P<0.001).
multiples of 180 bp started to appear at 12, 18 and the
Maximum caspase-3 activity was observed at 4 h post-
classical ladder pattern of apoptosis was observed at 24
infection (Fig. 2).
h post infection whereas no DNA fragmentation was
shown in DNA from the 6 h pi and mock infected cells.
The results suggested activation of endonuclease by
influenza virus infection (Fig. 1).

Fig. 2. Caspase -3 activity during influenza virus induced cell


death in mock infected, infected, infected with treatment and
mock infected with treatment HeLa cells. Values are the means ±
SD of three separate experiments. The level of significance was
**P<0.05 and ***<0.001, NS-not significant (Two-way ANOVA,
Bonferroni post tests).
Counts

Fig. 1. DNA ladder assay showing virus-induced DNA fragmentation.


HeLa cells were infected with A/Udorn/317/72 (H3N2). Equal
numbers of total cells, harvested at 6, 12, 18 and 24 h p.i. examined Annexin V
for apoptotic DNA. Mock infected cells (MO) used as controls.
DNA fragments were visualized by ethidium bromide staining on Fig. 3. Flipping of plasma membrane by annexin V staining of
1.8 per cent agarose gels. Lane 1 - DNA ladder; Lane 2 - DNA from phosphatidylserine (PS) externalization at 0, 3, 6, 9, 12, 15 h p.i. (a,
Mockinfected cells ; Lane 3-6 - DNA ladder at 6, 12, 18, 24 h p.i. b, c, d, e and f respectively).
SRIVASTAVA et al: INFLUENZA VIRUS INDUCED APOPTOSIS IN HELA CELLS 583

Flipping of plasma membrane in response of virus infection effectively blocked the DNA fragmentation
induced cell death: Monolayers of HeLa cells were in HeLa cells. The inhibition of DNA fragmentation
infected with the virus for various time periods (0, 3, was still evident when ZnSO4 was added at 3 and 6 h
6, 9, 12, 15 h respectively), and cells were examined p.i. It was also observed that lower concentration of
for flipping of plasma membrane by measuring zinc i.e. 0.10 mM and higher concentration 0.25 mM
phosphatidylserine (PS) externalization. Annexin V were unable to cause inhibition of DNA fragmentation
assay at various time points showed that maximum (data not shown here). To study the effect of zinc
phosphatidylserine externalization occurred after 9 h treatment on DNA fragmentation with time, 0.15 mM
of infection (Fig. 3). zinc concentration was used and it was observed that
treatment of zinc till 8 h p.i. completely protected cells
Phagocytosis of infected HeLa cells: The extent of
from DNA fragmentation (Fig. 5).
phagocytosis of normal HeLa cells and of HeLa
cells that had been infected with influenza virus for Effect of zinc on caspase-3 activity during influenza
various periods (0, 6, 9, 12, 18, 24 h respectively) virus infection in cultured cells: The cells treated with
was determined. It was observed that phagocytosis 0.15 mM zinc showed significantly less fluorescence
became evident at significant levels at 9 h post ( <0.05) in contrast to the untreated cells at 2 h and the
(P
infection, and the phagocytic index continued to maximum decrease in fluorescence after zinc treatment
increase thereafter (Fig. 4). At 18 h p.i., more than was observed at 4 h p.i. ((P<0.001) (Fig. 2).
30% of the macrophages appeared to incorporate
virus-infected cells, whereas these did not react with
normal uninfected cells.
Effect of zinc supplementation on DNA fragmentation
in influenza virus infected HeLa cells: To determine
the effect of zinc on influenza A/Udorn/317/72 (H3N2)
virus infected HeLa cells, 0.15 mM, 0.2 mM, and 0.25
mM of ZnSO4 was added to the infected monolayer
cultures at 0, 3, 6, 9, 12, and 18 h post infection and
cellular DNA was analyzed by gel electrophoresis. It
was observed that ZnSO4 at a concentration of 0.15
mM, and 0.2 mM added to the medium just after

Fig. 5. DNA ladder assay showing inhibition of virus-induced


DNA fragmentation by zinc treatment. HeLa cells were infected A/
Udorn/317/72 (H3N2). Cells were treated by zinc at 0, 4, 8, 12, 16,
20 h p.i harvested at 24 h p.i., were examined for apoptotic DNA
laddering pattern. Mock infected cells (MO) and virus infected
cells without treatment (MT) used as negative and positive controls
Fig. 4. Phagocytic index of infected HeLa cells: phagocytic of apoptosis. DNA fragments were visualized by ethidium bromide
macrophage is represented relative to total macrophages. Error staining on 1.8 per cent agarose gels. Lane 1 - Mock infected cells
bars indicate standard deviations. The level of significance was (MO); Lane 2-7 - DNA ladder at 0,4,8,12,16 and 20 hr p.i.; Lane
**P <0.05 and ***<0.001, compared with NS- not significant 8 - Virus infected cells without treatment (MT); Lane 9 - DNA
(Two-way ANOVA, Bonferroni post tests). ladder (M).
584 INDIAN J MED RES, MAY 2009

Discussion 3 activity observed in uninfected cells. Concurrent


with earlier reports by using various influenza virus
A current challenge in the field of influenza A
strains and cell types, it was confirmed that caspase-3
virus pathogenesis is to understand how virus infection
activation at the onset of apoptosis was a crucial event
leads to specific tissue damage and disease or death
during influenza A virus induced apoptosis in cultured
of the host. A few previous studies, have shown that
cells30,31.
all mammalian and avian influenza A strains induce
apoptotic death of cultured cell lines as well as that The flipping of some components of the plasma
of tissues in infected animals27,28. In this in vitro study membrane principally phosphatidylserine, from the
cultured HeLa cells were used, which permitted low inside surface to the outside surface is a fairly early
levels of replication of influenza A/Udorn/317/72 event in apoptosis22. Annexin V acts as a positive
(H3N2) virus and show strong evidence for the influenza identification tag for cells undergoing apoptosis.
virus induced apoptosis in cultured HeLa cells. Virus Assay of annexin V in the infected cells at various
infection resulted in condensation of cytoplasm, time periods indicated that membrane phospholipid
DNA fragmentation and nuclear disintegration and phosphatidylserine (PS) appeared on the surface
collapse. All these cellular changes are stereotypic of virus-infected cells 9 h post infection. This
hallmarks of apoptosis in both animal and plant cells. externalization of phosphatidylserine induces the
The virus titre of the culture medium of infected HeLa phagocytosis of infected cells, which are committed
cells determined by pfu enumeration at various time to apoptosis. The extent of phagocytosis was also
intervals post infection indicated that about 100 per determined using HeLa cells that had been infected
cent infection was achieved and that virus release with influenza A virus for various time periods. The
began at approximately 6 h post-infection, and it results suggested that the virus together with its host
reached a maximum at 24 h of infection. However it is cells was digested in macrophages. These findings add
reported that the completion of apoptosis as determined to our understanding that apoptosis of influenza virus-
by various biochemical assays, required almost 36 infected cells induces inhibition of virus growth and
h29,6. This suggested that peak of virus replication limits the progression of disease by playing an initial
was preceding the arrest of host cell macromolecular defense against viral invasion. However, findings
synthesis due to apoptosis. from the in vivo studies showed that virus infection
invokes hyperactivation of immune response which
A biochemical confirmation of the occurrence of
in turn induces apoptosis in bystander uninfected cells
apoptosis due to influenza A virus infection was done
and also contributes to the inciting the disease11. It is
by DNA fragmentation assay. The assay revealed that
thus anticipated that apoptosis of the influenza virus-
DNA fragmentation began at 12 h p.i. and classical
infected respiratory epithelial cells controls the tissue
fragmentation occurred at 24 h p.i. The uninfected
load of cells at the inflamed site by eliminating the
cells, however, did not exhibit fragmentation of
virus infected cells thereby playing a very important
chromosomal DNA. Takizawa et al 6 reported that
role in the pathogenesis of disease.
chromosomal DNA in influenza A virus-infected
MDCK cells exhibited fragmentation by 6 h p.i. The Pandey et al32,33 found that DNA fragmentation in
delayed appearance in HeLa cells may reflect a slower nuclei isolated from 5123 tc rat hepatoma cells required
progression and lower degree of cell lysis than that of the presence of calcium and magnesium ions. Zinc
MDCK cells. supplementation has been known to inhibit apoptosis
induced by a variety of agents both in vitro and in vivo
The study was further extended to understand the
models34,35. The inhibition of influenza virus induced
kinetics of the phenomenon. The central component
DNA laddering by zinc supplementation in our study is
of the apoptotic machinery is a proteolytic system
probably based on the same mechanism. These results
consisting of a family of cysteinyl proteases, termed
led to a postulation that in HeLa cells the terminal step
caspases. The activity of caspase-3, where both the
of the apoptotic pathway, that is DNA fragmentation,
pathways, extrinsic and intrinsic converge in the
is one of the sites on which zinc exerts inhibitory effect
infected HeLa cells was measured at various time
possibly via preventing activation of endonuclease.
points and a significant increase in the activity was
observed after 2 h infection with the maximum activity However, David et al15 have identified caspase-3
at 4 h p.i. There was no significant increase in caspase- as one of the site of zinc inhibition in the apoptotic
SRIVASTAVA et al: INFLUENZA VIRUS INDUCED APOPTOSIS IN HELA CELLS 585

pathway. When infected HeLa cells were treated with infected with a highly virulent influenza A (H5N1) virus
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Acknowledgment apoptosis induced by glucocorticoid of thymocytes in vitro.
Biol Trace Elem Res 2005; 105 : 215-27.
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Reprint requests: Dr Madhu Khanna, Reader, Department of Respiratory Virology, V.P. Chest Institute, University of Delhi
Delhi 110 007, India
e-mail: madhukhanna@hotmail.com

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