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Journal of Virology, December 1998, p. 10207-10212, Vol. 72, No. 12
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Localization of a Baculovirus-Induced
Chitinase in the Insect Cell Endoplasmic Reticulum
Carole J.
Thomas,1,2
Helen L.
Brown,2
Chris R.
Hawes,2
Bum Yong
Lee,3
Mi-Kyung
Min,3
Linda A.
King,2 and
Robert D.
Possee1,*
NERC Institute of Virology and Environmental
Microbiology, Oxford OX1 3SR,1 and
School of Biological and Molecular Sciences, Oxford
Brookes University, Oxford OX3 0BP,2 United
Kingdom, and
MOGAM Biotechnology Research Institute,
Yongin-Kun, Kyonggi-Do 449-910, Korea3
Received 17 March 1998/Accepted 10 September 1998
 |
ABSTRACT |
Confocal immunofluorescence microscopy was used to demonstrate that
the Autographa californica nucleopolyhedrovirus
(AcMNPV) chitinase was localized within the endoplasmic
reticulum (ER) of virus-infected insect cells. This was consistent with
removal of the signal peptide from the chitinase and an ER localization motif (KDEL) at the carboxyl end of the protein. Chitinase release from
cells, a prerequisite for liquefaction of virus-infected insect larvae,
appears to be aided by synthesis of the p10 protein. Deletion of
p10 from the AcMNPV genome delayed the
appearance of chitinase activity in the medium of virus-infected cells
by 24 h and also delayed liquefaction of virus-infected
Trichoplusia ni larvae by the same period.
| |
TEXT |
The DNA genome of Autographa
californica nucleopolyhedrovirus (AcMNPV) encodes a
chitinase protein similar to chitinase A of Serratia
marcescens (57% identity, 65% similarity [2,
6]). In virus-infected Spodoptera frugiperda cells,
the AcMNPV chitinase gene (chiA) is expressed as
a late protein (58 kDa) with endo- and exochitinase activities
(6). The AcMNPV chiA gene
can be deleted without affecting virus replication in cell culture or insects, but liquefaction or melting of larvae is abolished
(7). A similar effect is observed when the cathepsin gene is
deleted from the AcMNPV genome (18). We proposed
previously that cathepsin removes the protein from chitin in the insect
cuticle, thereby facilitating the digestion of exposed chitin by
chitinase (7). This mechanism, however, is at variance with
a number of observations. At least 90% of the chitinase activity
induced in AcMNPV-infected S. frugiperda cells in
culture remains intracellular (6). Further, immunofluorescence staining with a polyclonal chitinase-specific antiserum located the protein to the cytoplasm of virus-infected cells.
This was inconsistent with the presence of a eukaryotic signal peptide
at the N terminus of the chitinase protein (6), which should
have served to attach it to the endoplasmic reticulum (ER),
facilitating entry to the secretory pathway. Clearly, unless AcMNPV chitinase is released from cells in an inactive form,
there must be a block in the secretion of this protein. In this study, we have determined the precise location of the chitinase within virus-infected cells.
Localization of AcMNPV chitinase in insect cells.
Confocal laser scanning microscopy (CLSM) was used to detect chitinase
in AcMNPV-infected cells. One million S. frugiperda (Sf9
[19]) cells were seeded in 35-mm dishes which contained 13-mm sterile glass coverslips. The ce
ells were incubated for 1 h at ambient temperature with
AcMNPV C6 (17) (10 PFU/cell) or were mock
infected with medium. The cells were then incubated at 28°C for
48 h. The coverslips were removed from the dishes, and
immunofluorescence staining was performed as described previously (9). The antibodies employed were polyclonal serum raised
against AcMNPV chitinase (6) and fluorescein
isothiocyanate (FITC)-conjugated anti-guinea pig goat
immunoglobulin G (IgG). The preparations were examined
under a Zeiss LSM410 confocal laser scanning microscope with a
455-nm argon laser and appropriate filter sets (see Fig. 1).
Uninfected Sf9 cells (Fig. 1A)
showed no fluorescence when stained with the
chitinase-specific antibody. The image presented has been digitally
enhanced to reveal the outline of the cells. In an
AcMNPV-infected cell, the chitinase was located as a
reticulate staining pattern which radiated out from the
perinuclear region through the cytoplasm (Fig. 1B). The nuclear
membrane was also stained, but no fluorescence was observed at the
plasma membrane. This distribution of chitinase appeared to follow an
ER staining pattern. Characteristically, in virus-infected cells, the
ER becomes dilated due to the cytological effects of infection and
extends from the nucleus as an irregular reticulate structure. Figure 1C is a transmitted light view of an AcMNPV-infected Sf9
cell with a superimposed image of the immunostained chitinase. The polyhedra can be seen within the nucleus of the cell. Surrounding the
nucleus and extending from the nuclear membrane is the chitinase, which, in this image, is visualized in red to complement the green background.
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FIG. 1.
Immunofluorescent staining of S. frugiperda
cells. Sf9 cells were infected with AcMNPV (10 PFU/cell) or
were mock infected with medium. After 48 h, the cells were
immunostained as described in the text and examined with a confocal
laser scanning microscope. (A) Mock-infected cells stained sequentially
with guinea pig chitinase-specific antiserum (1:10,000) and FITC
conjugated with anti-guinea pig IgG polyclonal antiserum (1:40). (B)
AcMNPV-infected insect cells stained as described for panel
A. (C) Transmitted light and immunofluorescence image of an
AcMNPV-infected insect cell immunostained as described
for panel A. The pattern of immunostaining is depicted in
red. (D) Mock-infected insect cell stained with mouse anti-HDEL
monoclonal antibody (1:10) and FITC-conjugated goat anti-mouse IgG
(1:40). (E to G) Colocalization of chitinase and ER proteins detected
by double labelling of AcMNPV-infected Sf9 cells.
Antichitinase antiserum and anti-HDEL monoclonal antibody were used as
primary antibodies, followed by detection with, respectively, the
following FITC-conjugated or Texas red-conjugated secondary antibodies:
chitinase (E), HDEL (F), and dual staining (G). Bar = 5 µm.
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Unambiguous evidence for the ER localization of the virus chitinase was
provided by double labelling AcMNPV-infected cells with the
chitinase-specific antibody and a mouse monoclonal antibody to an HDEL
ER retention motif (12). The HDEL and KDEL motifs, located
at the carboxyl ends of proteins, are widely used as markers for the ER
in many cell types (14). HDEL is believed to be the principal ER retention sequence in insect cells (9), so the ER can be visualized by using an HDEL-specific antibody. Figure 1D
shows uninfected Sf9 cells which have been immunostained with the
HDEL-specific antibody and antimouse antibody conjugated with Texas
red. The native ER proteins were stained to permit visualization of the
characteristic reticulate network staining of this organelle. Figure 1E
to G shows AcMNPV-infected Sf9 cells which have been incubated with both chitinase- and HDEL-specific primary antibodies prior to dual staining with secondary antibodies conjugated to FITC or
Texas red, respectively. Figure 1E shows the
immunofluorescence-labelled chitinase within the
AcMNPV-infected Sf9 cells. The location of the labelled
chitinase in this cell is in agreement with the previous result (Fig.
1B). Figure 1F is a view of the same cells, this time showing the
immunolabelled HDEL to highlight the ER. Figure 1G shows the
colocalization of the chitinase- and HDEL-specific antibodies with
their red and green fluorescent secondary antibody conjugates depicted
in yellow where colocalized. This confirmed that the chitinase
was located in the ER of the AcMNPV-infected insect cells.
Similar results have been obtained with S. frugiperda Sf21
cells (data not shown). Insect cells infected with an AcMNPV recombinant (AcchiA
) lacking the complete
chiA gene (7) failed to produce a
chitinase-specific signal in immunofluorescence studies (data not shown).
To confirm that the ER localization of chitinase was not confined to
very late time points, Ac
MNPV-infected cells were examined
at 12, 24, and 48 h postinfection (p.i.) by CLSM (Fig.
2). Faint
ER staining with a
chitinase-specific antibody was observed at
12 h p.i. (Fig.
2A),
with stronger signals at 24 and 48 h p.i.
(Fig.
2B and C,
respectively). A strong ER staining pattern with
the same antibody was
also seen at 24 h p.i. in Ac
MNPV-infected
Trichoplusia ni (Tn368) cells (Fig.
2D). This indicated that
the
localization of chitinase in the ER was not cell-type specific.
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FIG. 2.
Temporal production of chitinase in
AcMNPV-infected insect cells. Sf9 cells were infected with
virus; stained with a primary guinea pig chitinase-specific antiserum;
FITC conjugated with anti-guinea pig IgG at 12 (A), 24 (B), and 48 (C)
h p.i.; and examined by CSLM as described in the text. T. ni
(Tn368) cells were similarly infected and stained at 48 h p.i.
(D). Mock-infected Sf9 cells were stained with the same antibodies
(E).
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The ER localization of chitinase did not appear to be associated with
proteolytic cleavage of the protein, since a 58-kDa
product was evident
in virus-infected cells between 12 and 96
h p.i. (Fig.
3). Enzyme assays using a range of
substrates (
6)
showed that the intracellular chitinase was
active in each of
the samples harvested over the time course (data not
shown).
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FIG. 3.
Immunoblot analysis of chitinase in
AcMNPV-infected cells. Sf9 cells in suspension culture were
mock infected (M) or inoculated with AcMNPV (10 PFU/cell).
Virus-infected cells were harvested between 12 and 96 h p.i., and
the pellets from 1-ml culture volumes were fractionated in a 12%
polyacrylamide gel before immunoblotting with antichitinase antiserum
(1/10,000) and alkaline phosphatase-conjugated goat anti-guinea pig IgG
polyclonal antiserum (1/1,000). The position of the 58-kDa chitinase
band is indicated with an arrow.
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Immunogold labelling of chitinase in AcMNPV-infected
cells.
Immunogold labelling and electron microscopy were used to
confirm the localization of chitinase in AcMNPV-infected
cells. The Sf9 cells were infected with either AcMNPV or
AcchiA or were mock infected with culture
medium. The cells were harvested at 48 h p.i. and fixed, and
ultrathin sections were cut as described previously (3, 20).
The sections were incubated successively with antichitinase antiserum
(1:8,000) for 16 h at 4°C and then with anti-guinea pig
antiserum (1:20) conjugated to 10-nm colloidal gold for 1 h at
ambient temperature. After immunolabelling, sections were poststained
with 2% aqueous uranyl-acetate (5 min) and lead citrate (10 min)
before examination with a JEOL 1200EXII transmission electron microscope.
In cells infected with wild-type Ac
MNPV, polyhedra and
nonoccluded virus particles were observed within the nuclei (Fig.
4A).
Gold particles were observed around
the nuclear membrane and also
within vacuole-like structures which were
apparent throughout
the cytoplasm (Fig.
4A and B). In some vesicles,
the concentration
of gold particles was very high (Fig.
4B). The origin
of the vacuoles
was uncertain, but they were observed only in
Ac
MNPV-infected
cells. They most probably correspond to
areas of degenerate or
vacuolated ER. In Fig.
4C, note the outer
nuclear membrane, which
appears to be continuous with the membrane
encompassing the vacuole
containing gold particles. Virtually no gold
particles were detected
in the region of the plasma membrane in
Ac
MNPV-infected cells
(data not shown). In cells infected
with Ac
chiA, no immunogold labelling was
detected in either the nucleus or
cytoplasm, and the perinuclear region
was completely devoid of
gold particles (Fig.
4D). The vacuole-like
structures observed
in Ac
MNPV-infected cells were absent.
There was no labelling in
mock-infected cells or in
Ac
MNPV-infected cells in which the primary
antibody
treatment was omitted (data not shown).
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FIG. 4.
Immunogold staining of virus-infected insect cells with
chitinase-specific antiserum. Sf9 cells were infected (10 PFU/cell)
with AcMNPV (A to C) or AcchiA (D).
Scale bars = 200 nm. (A) Polyhedra (P) can be seen within the
nuclear membrane (arrowheads). Gold particles are visible around the
nuclear membrane and within a vacuole-like structure (V) in the
cytoplasm. (B) Dense gold staining was observed in the cytoplasmic
vacuole and also in a perinuclear distribution following the nuclear
membrane (arrowheads). (C) The vacuoles frequently appeared to be
continuous with the outer nuclear membrane (black arrowheads), with the
inner nuclear membrane remaining intact (white-bordered arrowheads).
(D) The nucleus (N) is bounded by a normal membrane lacking attached
vacuolar structures.
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N-terminal protein sequence of the AcMNPV
chitinase.
The ER location of chitinase suggested cleavage of the
putative signal peptide. Whole-cell extracts were prepared from
AcMNPV-infected Tn368 cells; the chitinase was
isolated by high-performance liquid chromatography, and its identity
was confirmed by Western blot analysis (data not shown). The N-terminal
amino acid sequence of the protein was determined in triplicate. The
data showed that the signal peptide was cleaved from the rest of the
protein after the alanine residue (Fig.
5). The signal peptide sequence for AcMNPV has little similarity with the prokaryotic signal
peptide for S. marcescens chitinase A (Fig. 5). Figure 5
also shows putative signal peptides for four other baculovirus
chitinases: Bombyx mori NPV (BmNPV) (10),
Orgyia pseudosugata MNPV (OpMNPV)
(1), Heliothis zea SNPV
(HzSNPV) (8), and Choristoneura
fumiferana MNPV (CfMNPV). The signal
peptides for BmNPV, OpMNPV, and CfMNPV are
similar to the AcMNPV sequence, but the HzSNPV
sequence is very different. It is probable that these sequences would
also be cleaved from the nascent protein in virus-infected cells.
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FIG. 5.
Alignment of the signal peptides and carboxyl termini of
the predicted amino acid sequences of the chitinases from S. marcescens (16), AcMNPV (6),
OpMNPV (1), BmNPV (10),
CfMNPV (U72030), and HzSNPV (8). The
prokaryotic signal peptide for S. marcescens comprises the
first 23 residues. The eukaryotic signal peptide of AcMNPV
chitinase is underlined, and the cleavage site of the signal peptidase
is indicated by the vertical arrow. The corresponding sequences in the
BmNPV, OpMNPV, CfMNPV, and HzSNPV
chitinases are shown. The KDEL (RDEL for BmNPV and HNEL for
HzSNPV) putative ER retention signals are underlined and
shown in boldface type. The length of each protein is indicated at the
end of each line.
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Identification of ER retention signals in baculovirus
chitinases.
The association of the AcMNPV chitinase
with the ER of virus-infected cells and cleavage of the signal peptide
suggested that secretion of chitinase was inhibited. This indicated
that a specific signal may be preventing transport of the chitinase out
of the ER. Analysis of the amino acid sequence of the AcMNPV
chitinase revealed a KDEL motif at the C terminus of the protein (Fig.
5). This tetrapeptide sequence motif is a known ER retention-retrieval signal (11).
The Op
MNPV and Cf
MNPV chitinase proteins also
possess KDEL sequences at their C termini, but the BmNPV chitinase has
an RDEL
sequence motif (Fig.
5). This is consistent with published
observations
that the tetrapeptide sequence is not strictly observed
and that
certain changes to the motif still enable retention of
proteins
in the ER (
15). Therefore, it appears that the KDEL
motif (or
a closely related sequence) is present in several baculovirus
chitinases. The exception to this observation is the chitinase
of
Hz
SNPV, which has HNEL at the C terminus. This partial
conservation
may be sufficient to serve the same role as the KDEL
motif. The
Hz
SNPV chitinase also has a 13-residue extension
at this end of
the protein, relative to the Ac
MNPV
chitinase. The significance
of this is unknown.
S. marcescens chitinase A does not possess
a KDEL motif at its C
terminus (Fig.
5).
Release of chitinase from virus-infected cells.
Our model for
AcMNPV-induced liquefaction of virus-infected insect larvae
requires chitinase to be released from cells to attack the cuticular
chitin (7). It was reported that Sf21 cells infected with
AcMNPV mutants lacking an intact p10 gene did not progress
to cell lysis (23). We examined the release of active
chitinase from Sf9 cells in a suspension culture infected with
AcMNPV (10 PFU/cell) or a mutant (AcUW1.p10)
lacking an intact p10 gene (22).
Figure
6 shows that at 24 h p.i.,
there was approximately sixfold more chitinase in the medium supporting
the growth of Ac
MNPV-infected
cells than in that of
AcUW1.p10
-infected cells. Only background levels of
chitinase activity
were recorded in the latter samples. Between 48 and
96 h p.i.,
however, similar levels of exo- and endochitinase were
in the
media of Ac
MNPV- and AcUW1.p10
-infected
cell cultures. The viabilities of both virus-infected
cell cultures
declined from nearly 100% at 24 h p.i. to 0% at
96 h p.i.
However, differences between the Ac
MNPV- and
AcUW1.p10
-infected cell cultures were observed when the
total numbers of
cells at each time point were assessed. For
Ac
MNPV, an initial
concentration of 1.1 × 10
6 cells/ml remained static at 24 h p.i., declined
slightly by 72
h p.i. (10
6 cells/ml), and then was
reduced to about 2 × 10
5 cells/ml by 96 h p.i.
In contrast, the concentration of cells
in cultures infected with
AcUW1.p10
slightly increased to 1.5 × 10
6 cells/ml by 48 h p.i. and declined only to 8 × 10
5 cells/ml at 96 h p.i.
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FIG. 6.
Chitinase activities in the media of virus-infected
cells and total cell numbers. Suspension cultures of Sf9 cells
(106 cells/ml) were mock infected or inoculated with
AcMNPV (A) or AcUW1.p10 (B) (10 PFU/cell).
Medium from each culture was harvested at the times indicated and
assessed for exo- and endochitinase activities (6). The cell
concentration at each time point was also determined (C).
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Infection of second-instar
T. ni larvae with
Ac
MNPV polyhedra resulted in host liquefaction at 5 days
p.i. This process, however,
was delayed by 1 day in insects infected
with AcUW1.p10
.
Generally, proteins that are located in the organelles of the secretory
pathway encode signals for their retention at the
correct location.
Proteins resident in the lumen of the ER usually
carry a signal motif
(KDEL or a closely related sequence) at their
carboxy terminus. The
sequence KDEL is predominantly found in
mammalian cells
(
11), HDEL is found in the yeast
Saccharomyces cerevisiae (
13), and (H/K/R)DEL is found in plants
(
21). The
precise sequence of the motif varies, although
generally conservative
changes are seen (R for K or D for E)
(
15). The demonstration
of similar protein retention signals
in widely divergent species
suggests that it is a universal feature of
eukaryotes (
15).
The signal serves to retain proteins in the
ER lumen of mammalian
(
11), yeast (
4), and plant
(
5) cells. Proteins escaping
the ER interact with a KDEL
receptor in the
cis-Golgi and are
returned via a retrograde
vesicle-mediated pathway to the ER.
If the sequence is deleted, or if
it is extended by the addition
of other amino acids, the protein is
secreted from the cell instead
of remaining in the lumen of the ER.
Conversely, if the tetrapeptide
sequence is added to the C terminus of
non-ER resident proteins
such as lysozyme, a known secretory protein,
the enzyme is retained
in the ER lumen instead of being secreted
(
11).
This study has described the identification of a KDEL motif at the C
terminus of the chitinase protein of Ac
MNPV. This sequence
motif is not observed in the
S. marcescens chitinase, but
has
been identified as KDEL in the chitinase proteins of
Op
MNPV and
Cf
MNPV and as RDEL in BmNPV. Our data
do not prove that the KDEL
motif in chitinase is solely
responsible for retaining the protein
in the ER, and we will have to
perform site-directed mutagenesis
on the chitinase gene to alter this
sequence and determine the
effect on protein
location.
The retention of chitinase within virus-infected cells is surprising
given that in the insect larva, our model for the mechanism
of
liquefaction requires that the enzyme should be extracellular
to attack
cuticular chitin (
7). We have not examined virus-infected
cells from larvae to determine if the chitinase is also retained
within
the ER. It may be advantageous for the virus to delay liquefaction
of
the host until the maximum yield of polyhedra has been attained.
If
chitinase were secreted from virus-infected cells as soon as
it was
synthesized (7 to 10 h p.i. [
6]), the insect
might disintegrate
too rapidly and reduce polyhedron production. The
eventual release
of chitinase probably occurs after cell lysis, which,
at least
in part, is mediated by production of the p10 protein. Cells
infected
with a virus unable to synthesize p10 released active
chitinase
24 h later than did Ac
MNPV-infected controls
and remained intact
for longer. The same p10 deletion mutant virus also
induced liquefaction
1 day later in insects. Although we need to
examine cell lysis
in virus-infected insects, these preliminary
observations suggest
that chitinase release is also associated with p10
production
in vivo. The fact that insects infected with a p10 deletion
mutant
virus eventually liquefy suggests that this protein is not the
only factor with a role in the process. Attempts to measure chitinase
activity in extracts from virus-infected insects have been made
difficult by the high levels of host chitinases present in these
samples and in uninfected
controls.
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ACKNOWLEDGMENTS |
We thank Barry Martin for expert assistance with electron microscopy.
This study was supported by an NERC grant awarded to R.D.P. and L.A.K.
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FOOTNOTES |
*
Corresponding author. Mailing address: NERC Institute
of Virology and Environmental Microbiology, Mansfield Rd., Oxford OX1 3SR, United Kingdom. Phone: 44 1865 281663. Fax: 44 1865 281696. E-mail: rpossee{at}worf.molbiol.ox.ac.uk.
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Journal of Virology, December 1998, p. 10207-10212, Vol. 72, No. 12
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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