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http://www.abbs.info e-mail:[email protected] ISSN 0582-9879 ACTA BIOCHIMICA et BIOPHYSICA SINICA 2002, 34(6): 707-711 CN 31-1300/Q |
Apoptosis
of Spodoptera litura Cells Induced by AcMNPV ie-1 Gene
(
State Key Laboratory for Biocontrol, Zhongshan University, Guangzhou 510275,
China;
1Virology
Research Unit, Fudan University, Shanghai 200433,
China )
Both
time and level of expression of immediate-early genes are important in the
initiation of apoptosis[5]. ie-1 is the gene encoding
transactivator IE1，a
multifunctional protein acting as a strong transcriptional activator in
transient-expression assays[6,7]. The promoter and upstream
regulators of ie-1 can be active in non-infected cells. In
transient-expression assay, AcMNPV ie-1 induces the apoptosis of Sf-21
cells[8], even when the p35 gene expression level is reduced
by partial expression of ie-1[9]. This suggests that IE1
protein may regulate Sf-21 apoptosis, but the mechanism involved is still not
clear.
Previous
studies showed that wild-type AcMNPV induces apoptosis in Sl-zsu-1 cells 8 to
12 h after virus infection[10], suggesting that one of the stimuli
should be the transcriptional or translation product of the immediate-early or
early gene. Because Sl-zsu-1 is not a permissive cell line to AcMNPV, this
apoptosis model is quite different from those p35 mutant-induced cases.
So in this study, we tried to explain the role of BV entry in apoptosis, ie-1
gene transcription after AcMNPV infection, ie-1 gene regulation, the
impact of viral replicative events on apoptosis and the apoptotic-inducing
effect of ie-1 gene expression (including level and time) on Sl-zsu-1
cells.
1.1
Materials
1.1.1 Cells and viruses Spodoptera litura cells (designated as
Sl-zsu-1)[11] and Sf-21 were propagated in TC-100 medium (Gibco
Laboratories) supplemented with 10% heat-inactivated fetal bovine serum (Gibco
Laboratories) and 2.6 mg of tryptose broth per ml. Sl-zsu-1 is a nonpermissive
cell line to AcMNPV.
Wild-type
AcMNPV (L1) was a gift from University of California at Riverside, and the tsB821
mutant of AcMNPV from University of Georgia[12]. The tsB821
mutant was derived from 5-bromodeoxyuridine mutagenesis of wild-type
AcMNPV(L1). The ts virus was propagated and titered at a permissive
temperature, 23 ℃
and phenotypically characterized at the restrictive temperature of 33 ℃
in Sf-21 cells.
1.1.2 Chemicals
25 mmol/L ammonium chloride (Sigma) was prepared in TC-100 medium and
adjusted to pH 6.4 with KOH. Cytochalasin D (Sigma) and aphidicolin (Sigma)
were both initially suspended in dimethyl sulfoxide(DMSO) at 5 g/L then further
diluted (1/1 000, 5 mg/L) in culture medium.
1.2
Methods
1.2.1 Plasmid construction The plasmid pAcie-1/HC containing the
AcMNPV ie-1 gene in the ClaI-HindIII fragment(94.7 to 96.9
m.u.) was cloned in pBluescriptⅡKS(+).
1.2.2 Transient-expression assaysSl-zsu-1 cells ( 2.0×105
cells per dish) were seeded into 35-mm-diameter dishes. Cell monolayers were
washed twice with serum-free TC-100 medium. Transfections were conducted with
Cellfectin Reagent (Gibco BRL) according to recommendations of the
manufacturer. 2 mg
pAcVie-1 DNA were mixed with Cellfectin and sterile H2O, added to a
final volume of 1 ml. The suspension was added dropwise to cell monolayers.
After 4 h incubation at 27 ℃,
the Cellfectin solution was removed and replaced with fresh TC-100 medium
supplemented with 10% fetal bovine serum. For control, 2.0×105
cells were transfected with equimolar amount of pBluescript II KS(+). After
tranfection, cells were checked every 6 h and counted after staining with 0.08%
trypan blue.
1.2.3 Cytochalasin D (CD) inhibition assay Sf-21 cells were infected
with wild-type AcMNPV (MOI=10) using the methods of Volkman et al.[13]
with modifications. Cultures treated with CD were exposed from 30 min
preinoculation until the virus was harvested at 36 h p.i.(post infection). The
control cells were virus-inoculated and maintained in normal medium minus CD.
Then the two supernatants were collected separately by centrifugation and
seeded with Sl-zsu-1 cells (at the same time CD was added to the supernatant in
the control). 1 h after inoculation, cells were washed twice with phosphate
buffered saline and then transferred to fresh TC-100 medium.
1.2.4 Ammonium chloride inhibition assay The inhibition assay was conducted as described before[14]
using ammonium chloride as the lipophilic amine. Wild-type (wt) AcMNPV were
inoculated in Sl-zsu-1 cells monolayers at 4 ℃
for 1 h, then incubated at 27 ℃
with TC-100 medium containing 25 mmol/L ammonium chloride.
1.2.5 Aphidicolin inhibition assay This assay was conducted as described before[4].
Sl-zsu-1 cells were treated with TC-100 medium containing aphidicolin from 1 h
preinoculation until the end of experiment.
1.2.6 Reverse transcriptase (RT) PCR Sl-zsu-1 cells were infected at a MOI of 10 with
wild-type AcMNPV and harvested at 1 h p.i. mRNA for synthesizing cDNA was
isolated using the mRNA Capture Kit(Boehringer Mannheim) , and the products
were used for PCR. PCR was performed for 41 cycles (cycle one: 94 ℃
for 5 min,50 ℃
for 2 min, 72 ℃
for 3 min; cycles 2 up to 40: 94 ℃
for 1 min, 50 ℃
for 2 min, 72 ℃
for 3 min; the last cycle: 94 ℃
for 1 min, 50 ℃
for 2 min, 72 ℃
for 10 min) with two synthetic primers: a 5′
primer in the sense orientation: (5′-ACTGGTTATTACATGT-TTGTGGTT)
and a 3′ primer
in the antisense orientation: (5′-GTGCAATGTAGTGCTCTCTCT-TCG)
near the 3′
terminal of the ie-1 transcript. The product was then dissolved in a 1%
agarose gel together with the PCR products of AcMNPV DNA with the above primers.
1.2.7 Extraction of fragmented DNA Low-molecular weight DNA was isolated as
described by Herrmann et al.[15], with modifications. Cell pellets were
treated longer (for 2 min) with lysis buffer (1%NP-40 in 20 mmol/L EDTA; 50
mmol/L Tris-HCl, pH 7.5), less than 10 mol/L NaAc was added for digestion and
1.5% agarose gel (instead of 1.0%) was used in electrophoresis.
2.1
AcMNPV-BV entry was required to induce apoptosis in Sl-zsu-1 cells
In
CD inhibition assay, the noninfectious BV of AcMNPV lacking nucleocapsids were
produced from infected cells grown in the presence of CD. When Sl-zsu-1 cells
were inoculated with this noninfectious culture supernatant, no apoptosis was
observed[Fig.1(A)], whereas in the control，apoptosis
was induced in cells inoculated with infectious BV [Fig.1(B)].
Ammonium
chloride inhibition assay showed that after infection, the virus entry was
blocked in cells with the medium containing ammonium chloride although receptor
binding and internalization of BV might have occurred. In Sl-zsu-1 cells treated
with ammonium chloride, the infection did not trigger apoptosis [Fig.1(C)],
whereas the control cells did [Fig.1(B)].
Fig.1
Microphotographs of Sl-zsu-1 cells
(A) Cells infected by supernatants
collected from AcMNPV infected Sf-21 in the presence of cytochalasin D, 48 h
p.i.; (B) Cells infected by untreated AcMNPV BVs, 48 h p.i.; (C) Cells infected
with AcMNPV BVs under ammonium chloride, 48 h p.i.
RT-PCR
using ie-1 primers detected a specific fragment (Fig.2) in the cells
infected by wt AcMNPV 1 h p.i., while not in the mock-infected cells.
Fig.2 Agarose gel electrophoresis showing
RT-PCR result
M, marker; 1, using mock-infected cells
cDNA as template; 2, using AcMNPV-infected cells cDNA as template; 3, using
AcMNPV DNA as template.
2.2
The ie-1 gene product is capable of inducing apoptosis in
Sl-zsu-1 cells
Transfected
with pAcie-1, Sl-zsu-1 cells underwent drastic changes both
morphologically and biochemically. At 12 h post transfecting, extensive cell
surface blebbing appeared and cell death began; after 48 h, apoptosis peaked
with 50%-60%
cell mortality，whereas
the control cells remained quite normal [Fig.3(A)].
Cellular
total DNAs extracted from cells transfected with pAcie-1 had shown a
characteristic ladder upon electrophoresis[Fig.3(B)]. In contrast, DNAs
extracted from control cells did not form such a pattern. It can be concluded
therefore，that
IE1 protein encoded by pAcie-1 is capable of inducing apoptosis in
Sl-zsu-1 cells.
Fig.3
Transfection assay
(A) Microphotographs of Sl-zsu-1 cells;
(B) Agarose gel electrophoresis showing extract from Sl-zsu-1 cells.1，
cells transfected with pAcie-1, 48 h; 2, cells transfected with
pBluescript II KS(+), 48 h.
2.3
Effect of aphidicolin on apoptosis induction
To
examine the effect of viral replicative late events on cell death signalling,
Sl-zsu-1 cells were infected with AcMNPV in the presence of DNA polymerases
inhibitor aphidicolin. The result showed that Sl-zsu-1 cells apoptosis
occurred: apoptotic bodies appeared p.i., and a DNA ladder was detected (figure
not shown).
2.4
Effect of ie-1 expression on tsB821-infected Sl-zsu-1 cells
When
Sl-zsu-1 cells were infected with tsB821 (MOI=10) at a permissive
temperature of 23 ℃,
apoptotic bodies appeared at 12 h p.i., and peaked at 36 h p.i. with 95%-100%
cell death[Fig.4(A)]. The result was well matched with wt AcMNPV infection at
23 ℃. When the cells were infected with tsB821
at a restrictive temperature of 33 ℃,
no cells showed any sign of apoptosis. The cellular total DNAs were extracted
and separated by electro-phorosis[Fig.4(B)]. The result coincided with the
observation under microscopy: DNAs extracted from AcMNPV-infected cells at 23 ℃,
33 ℃ and from tsB821-infected at 23 ℃
showed a ladder, whereas those from tsB821-infected cells at 33 ℃
did not show this pattern.
Fig.4
Results of tsB821 infection
(A) Microphotographs of Sl-zsu-1 cells.
(1) cells infected by tsB821 at 23 ℃,
36 h p.i.; (2) cells infected by tsB821 at 33 ℃,
36 h p.i.(B) Agarose gel electrophoresis showing the extract from Sl-zsu-1
cells. M, marker; 1, untreated cells; 2, cells infected by AcMNPV at 23 ℃,
36 h p.i.; 3, cells infected by AcMNPV at 33 ℃,
36 h p.i.; 4, cells infected by tsB821 at 23 ℃,
36 h p.i.; 5, cells infected by tsB821 at 33 ℃,
36 h p.i..
3
Discussion
In
this study, we tried to explore the apoptosis signaling mechanism of Sl-zsu-1
cells induced by AcMNPV infection. Firstly, we have shown that in AcMNPV
infection of Sl-zsu-1 cells, the entry of BV is essential for the induction of
apoptosis. Cytochalasin B (CB) and cytochalasin D (CD) interfere with
microfilament-dependent functions in Sf-21 and the production of infectious
budded particles are inhibited[16]. In the CD inhibition assay, we
found that Sl-zsu-1 cells were unaffected by AcMNPV budded particles collected
from medium containing CD, demonstrating that apoptosis could not be triggered
by binding process alone. Lysosomotropic reagents (ammonium chloride) prevented
the acidification of the endosome when added to cells so that virus entry
blocked, and again there was no apoptosis.
The
entry of infectious BV has now paved the way for the transfection of ie-1
gene resulting in triggering apoptosis of Sl-zsu-1 cells. The product of ie-1
gene, IE1 protein, is multifunctional: transactivating transcription of other
viral early genes and host cell genes[6,7], and regulating the
replication of viral DNA[8,9,17]. In this study, we are interested
to note that apoptosis can be induced by the ie-1 gene alone (50%-60%
apoptotic cells). Prikhod′ko
et al.[17] reported that cotransfection of AcMNPV pe38
and ie-1 genes could augment IE1-induced apoptosis in Sf-21 cells, but
not by pe38 alone. There was also the possibility that more genes could
be involved. We ruled out the possibility that late events, such as DNA
replication and late genes' expression, were involved in apoptosis signaling
because the inhibition of DNA replication had no effect on this cell death
process. This result was contrary to that reported by Clem et al.[4].
We concluded from these results that Sl-zsu-1 cell apoptosis was triggered
sometime during the period from viral nucleocapsids being released into
cytoplasm to DNA replication, in which IE1 played a vital role probably by
directly disturbing host cell cycles. The mechanism of apoptosis regulation
differs between IE1 in Sl-zsu-1 and in Sf-21, although transfection of ie-1
gene alone induced partial apoptosis of both Sl-zsu-1 and Sf-21 in vitro
assays. Different responses between cell lines indicate that apoptosis is a
species-specific response.
Regulation
of apoptosis induction can be studied by using the temperature sensitive AcMNPV
mutant tsB821. tsB821 was blocked at very early stage of virus
infection resulting in viral DNA replication and budded virus production being
delayed[12,18]. Infection of Sl-zsu-1 cells with tsB821 at
the permissive temperature of 23 ℃
induced apoptosis because the ie-1 gene was transcribed and expressed
just like wt ie-1 gene before those suppressors of apoptosis function.
Gershburg et al.[19] reported that AcMNPV-induced SL2 cell
apoptosis was inhibited by p35 overexpression but not when p35
was not sufficiently expressed. On the other hand, when Sl-zsu-1 cells were infected
with tsB821 at a restrictive temperature of 33 ℃,
only a small amount of functional IE-1 was produced, with the result that those
suppressors present fully functionally at late stage to inhibit the apoptosis.
This result suggests that AcMNPV-induced Sl-zsu-1 cell apoptosis can be rescued
by AcMNPV apoptosis suppressor (p35) if ie-1 is expressed in a
delayed, low-level manner.
The
cell line from AcMNPV non-permissive host Spodoptera litura suppressed
viral propagation via rapid apoptosis, which also provided evidence for the
hypothesis that apoptosis be a factor of anti-viral system of insects. We
concluded that ie-1 gene was a key factor in AcMNPV-induced apoptosis of
Sl-zsu-1 cells. However, the role of other relative genes triggering this
process is not clear. Experiments to find the exact factors and their roles in
this apoptosis signaling process are ongoing and should help explain the
apoptosis molecular mechanisms.
Acknowledements We thank Dr. Friesen PD for
the gift of AcMNPV mutant tsB821 and Dr. Rae DJ for her comments on the
manuscript.
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Received：May
8, 2002 Accepted：June
24, 2002
This
work was supported by grants from the National Natural Science Foundation of
China (No.39730030， No.39800092)
*Corresponding
author: Tel, 86-20-84113860; Fax, 86-20-84037472; e-mail, [email protected]