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Journal of Virology, May 1999, p. 4110-4119, Vol. 73, No. 5
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Live-Cell Analysis of a Green Fluorescent
Protein-Tagged Herpes Simplex Virus Infection
Gillian
Elliott* and
Peter
O'Hare
Marie Curie Research Institute, The Chart,
Oxted, Surrey RH1 0TL, United Kingdom
Received 28 December 1998/Accepted 9 February 1999
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ABSTRACT |
Many stages of the herpes simplex virus maturation pathway have not
yet been defined. In particular, little is known about the assembly of
the virion tegument compartment and its subsequent incorporation into
maturing virus particles. Here we describe the construction of a herpes
simplex virus type 1 (HSV-1) recombinant in which we have replaced the
gene encoding a major tegument protein, VP22, with a gene expressing a
green fluorescent protein (GFP)-VP22 fusion protein (GFP-22). We show
that this virus has growth properties identical to those of the
parental virus and that newly synthesized GFP-22 is detectable in live
cells as early as 3 h postinfection. Moreover, we show that GFP-22
is incorporated into the HSV-1 virion as efficiently as VP22, resulting
in particles which are visible by fluorescence microscopy.
Consequently, we have used time lapse confocal microscopy to monitor
GFP-22 in live-cell infection, and we present time lapse animations of
GFP-22 localization throughout the virus life cycle. These animations
demonstrate that GFP-22 is present in a diffuse cytoplasmic location
when it is initially expressed but evolves into particulate material
which travels through an exclusively cytoplasmic pathway to the cell
periphery. In this way, we have for the first time visualized the
trafficking of a herpesvirus structural component within live, infected cells.
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INTRODUCTION |
The herpesvirus particle is made up
of four concentric compartments, namely, the DNA core, the capsid, the
tegument, and the envelope (4, 24). The tegument is the
least understood compartment of the virion in relation to the functions
of its individual constituents, its role in virus entry, and its
mechanism of assembly and incorporation into the maturing virion. In
particular, the site of tegument assembly within the cell remains to be
determined. While it is well established that during virus replication
genomic DNA is packaged into assembling capsids within the nucleus
(25), the subsequent sites of tegument assembly, capsid
envelopment, and virion maturation are somewhat controversial. Early
electron microscopy studies showing the thickening of nuclear membranes
next to assembled capsids (17, 18, 27) were interpreted to
mean that the virion envelope was acquired at the inner nuclear
membrane and that maturation of envelope glycoproteins occurred as
vesicles containing these virions moved through the secretory pathway.
Nonetheless, this model has always been complicated by the presence of
numerous unenveloped capsids in the cytoplasm of infected cells
(33), a feature which has previously been suggested to
reflect a "dead-end" pathway of terminal de-envelopment
(2). However, several recent reports on the localization and
processing of virus glycoproteins provide evidence that these naked
capsids may be on the true pathway of virus assembly, whereby virions
budding through the nuclear membrane would proceed through a further
stage of de-envelopment at the outer nuclear membrane and acquire their
final envelope downstream in the secretory pathway (1, 3, 29,
33). It is therefore likely that the identification of the
cellular site of tegument assembly will help to address the issues
surrounding the herpesvirus maturation pathway(s).
The herpes simplex virus type 1 (HSV-1) protein VP22 is a major
tegument component of the virus particle (11, 13, 28). While
the exact role of VP22 during virus infection remains unclear, we have
shown that VP22 exhibits several fascinating properties. During
expression in tissue culture cells by either transient transfection or
virus infection, VP22 exhibits the property of intercellular spread,
which is so efficient that an individual cell expressing the protein
can deliver it to as many as 200 surrounding cells (8).
Furthermore, we have recently shown that in cells which actively
synthesize VP22, during either transient transfection or virus
infection, the protein reorganizes and stabilizes the cellular
microtubule network, and as such VP22 is the first animal virus-encoded
protein shown to possess the properties of a cellular microtubule-associating protein (9). Thus, not only is VP22 a major structural component of the virus particle, but it also exhibits several interesting cellular interactions which may be important to the virus replication cycle.
We have previously shown that a green fluorescent protein (GFP)-VP22
fusion protein (GFP-22) is competent for both intercellular movement
and interaction with microtubules (8), suggesting that the
addition of GFP onto the VP22 open reading frame has no effect on VP22
activities within the cell. In this report we describe the construction
of an HSV-1 recombinant in which we have exchanged the single copy of
the VP22 open reading frame in the HSV-1 genome, gene UL49
(10), with a gene encoding GFP-22. Surprisingly, this virus
is fully viable and exhibits growth kinetics similar to those of its
parental virus. Moreover, GFP-22 is incorporated into the virus
particle with the same efficiency as VP22. The presence of GFP-22 in
the virion results in fluorescent particles which are readily
visualized with a light microscope. Furthermore, we show that newly
synthesized GFP-22 is detectable as early as 3 h after infection
at a high multiplicity, allowing the direct visualization of GFP-22
within live cells. As a consequence of such sensitive detection of
GFP-22 throughout infection, we have been able to use time lapse
confocal microscopy to monitor the trafficking of GFP-22 within
individual cells, at both high and low multiplicities of infection, the
results of which we present as time lapse animations. Thus, we have
generated a reagent which will enable the visualization of several
aspects of HSV-1 infection in live cells, including virus entry,
assembly, trafficking, and egress.
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MATERIALS AND METHODS |
Cells and virus infections.
Vero cells, COS-1 cells, and
BHK-1 cells were maintained in Dulbecco's modified minimal essential
medium containing 10% newborn calf serum. The parental virus used in
this study was HSV-1 strain 17.
Virions were purified from extracellular virus released into the
infected cell medium. Approximately 5 × 108 BHK cells
were infected at a multiplicity of 0.05. Four days later, the infected
cells were harvested into the cell medium and centrifuged for 30 min at
3,000 rpm in a Sorvall GS3 rotor. The supernatant was removed and
centrifuged for a further 90 min at 10 K to pellet the extracellular
virus. The resulting crude virus pellet was resuspended in 1 ml of
phosphate-buffered saline (PBS) and laid onto a 12-ml 5 to 15% Ficoll
gradient, which was centrifuged at 12,000 rpm in a Sorvall TH641 rotor
for 2 h. The virion band was harvested through the side of the
tube, diluted in 10 ml of PBS, and pelleted for 1 h at 25,000 rpm
in a Sorvall TH641 rotor. Following resuspension in 0.5 ml of PBS, the
virion stock was placed at 
70°C.
Infectious virus DNA was also produced from extracellular virus. BHK
cells were infected as described above, and the crude
extracellular
virus pellet was resuspended in 5 ml of 10 mM Tris-HCl
(pH 7.6)-1 mM
EDTA-1% sodium dodecyl sulfate (SDS)-50 µg of proteinase
K/ml.
After gentle mixing the suspension was incubated at 50°C
for 4 h, extracted twice with phenol and twice with phenol-chloroform,
and
ethanol
precipitated.
Construction of GFP-22 virus.
The construction of the
GFP-22-expressing virus is detailed in Fig.
1. The two 400-bp flanking sequences of
the HSV-1 UL49 gene (Fig. 1, diagram 3) were amplified together by PCR
from purified genomic DNA to construct a single 800-bp fragment
incorporating an EcoRI site at one end, an XbaI
site at the other, and a BamHI site engineered in place of
the UL49 gene (Fig. 1, diagram 3). This was inserted into plasmid pSP72
(Promega) as an EcoRI/XbaI fragment to produce
plasmid pGE120 (Fig. 1, diagram 3). A GFP-UL49 cassette contained on a
BamHI fragment was then inserted into the BamHI
site of pGE120 to produce plasmid pGE166 (Fig. 1, diagram 4), which
consisted of GFP-UL49 surrounded by the UL49 flanking sequences and
hence driven by the UL49 promoter.
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FIG. 1.
Construction of the GFP-22-expressing virus. (1)
Schematic representation of the HSV-1 genome. (2) The region of the
genome which contains the UL49 gene (shaded). The arrow indicates the
direction of transcription. (3) The EcoRV/BamHI
fragment which contains the UL49 gene and its flanking sequences. The
400-bp sequences on either side of the UL49 gene were amplified by PCR
with EcoRI and XbaI sites at either end, and a
BamHI site in place of the UL49 gene (4). The
EcoRI/XbaI fragment was inserted into
EcoRI/XbaI-digested pSP72 to make pGE120. A
GFP-VP22 BamHI cassette was inserted into the
BamHI site of pGE120 to make pGE166. (5) The structure of
the genome resulting from recombination of plasmid pGE166 with HSV-1
DNA.
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Equal amounts (2 µg) of plasmid pGE166 and infectious HSV-1 strain 17 DNA were transfected into 10
6 COS-1 cells grown in a
60-mm-diameter dish by using the calcium
phosphate precipitation
technique modified with BES
[
N,
N-bis(2-hydroxyl)-2-aminoethanesulfonic
acid]-buffered saline in place of HEPES-buffered saline. Four
days
later, the infected cells were harvested into the cell medium
and
subjected three times to freeze-thawing, and the resulting
virus was
titrated on Vero cells. Around 6,000 plaques were then
plated onto Vero
cells and screened for possible recombinants
by GFP
fluorescence.
Virus genomic DNA screening.
Virus DNA for restriction
digestion was purified from 5 × 107 infected BHK
cells. Infected cell pellets were washed in cold PBS and resuspended in
5 ml of 10 mM Tris-HCl (pH 7.5)-2 mM MgCl2-10 mM
NaCl-0.5% Nonidet P-40. After vortexing, the cells were left on ice
for 5 min and vortexed again. Nuclei were pelleted out at 2,000 rpm in
a benchtop centrifuge, and 0.5% SDS and 10 mM EDTA were added to the
supernatant. This was extracted twice with phenol-chloroform and once
with chloroform and then was ethanol precipitated. After pelleting, the
nucleic acid was resuspended in 100 ml of H2O and digested
overnight with EcoRV in the presence of RNase A. Electrophoresis was carried out in a 0.8% agarose gel for 8 h at
100 V, and the gel was transferred to a nylon membrane by standard
procedures. The Southern blots were then hybridized with a
32P-labelled DNA probe synthesized by the random priming of
fragments specific for either UL49 or GFP.
SDS-PAGE and Western blot analysis.
Solubilized proteins
were subjected to SDS-polyacrylamide gel electrophoresis (PAGE), and
the gels were either stained with Coomassie blue or transferred to
nitrocellulose filters and reacted with the appropriate primary
antibody. A horseradish peroxidase-linked secondary conjugate was used,
and reactive bands were visualized with the enhanced chemiluminescence
(ECL) detection reagents (Amersham).
The polyclonal anti-VP22 antibody, AGV30, has been described previously
(
8). Monoclonal antibodies against
tubulin and
acetylated tubulin were obtained from Sigma. The monoclonal anti-GFP
antibody was obtained from Clontech. Antibodies against IE110
(11060),
thymidine kinase (TK), and VP16 (LP1) were kindly provided
by Roger
Everett, David Gower, and Tony Minson,
respectively.
Live-cell microscopy and time lapse analysis.
Cells for
short-term live analysis of GFP expression were plated onto 24-mm
coverslips placed in six-well trays (Costar). At the specified time of
analysis, the coverslip was transferred to a 35-mm Attofluor cell
chamber (Molecular Probes), 2 ml of fresh medium was added, and the
cells were examined with a Zeiss LSM 410 inverted confocal microscope.
Resulting images were processed by using Adobe Photoshop software.
Cells for long-term time lapse analysis were plated onto 42-mm
coverslips contained in 60-mm dishes. Prior to analysis, the
coverslip
was transferred to a Bachhoffer POC-chamber (obtained
from Carl Zeiss)
in open cultivation mode. This chamber was placed
on a Saur heated
frame (obtained from Carl Zeiss) on the microscope
and covered with a
Perspex lid through which a constant supply
of 5% CO
2 was
fed. XYZT software from Zeiss was used to collect
a Z series of images
for each point in the time series, and these
were then merged to
produce an individual Z image for each time
point. Animation of the
time series was carried out by using NIH-Image
software, and each
series was saved as a Quicktime video. The
animations accompanying Fig.
7 and
8 are located at
http://mc11.mcri.ac.uk/mov/figure7.mov and
http://mc11.mcri.ac.uk/mov/figure8.mov.
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RESULTS |
Construction of an HSV-1 recombinant virus expressing GFP-22.
The HSV-1 structural protein VP22 is encoded by the UL49 gene
(10) located in the Bam F restriction fragment of
the unique long region of the genome (Fig. 1, diagrams 1 and 2). UL49
was replaced with the gene encoding GFP-22, as described in detail in
Materials and Methods. Briefly, plasmid pGE166, consisting of the
GFP-22 open reading frame surrounded by UL49 flanking sequences (Fig.
1, diagram 4) was cotransfected into COS-1 cells together with purified
HSV-1 strain 17 genomic DNA and incubated for 4 days until cytopathic
effect was present in all cells. Virus was harvested, and around 6,000 plaques were plated onto Vero cells and screened by using GFP
fluorescence to identify potential recombinants. Two green plaques were
detected and plaque purified a further two times. One of these viruses
was chosen for further analysis and was designated 166v (Fig. 1,
diagram 5).
To ensure that recombination had taken place in the correct location on
the genome and that the endogenous copy of the VP22
gene had been
replaced by the GFP-22 gene, genomic DNA was purified
from both the
wild-type (WT) S17 virus and the 166v virus and
was subjected to
restriction digestion with
EcoRV (Fig.
2). Incorporation
of GFP-22 into the
genome should result in an increase in the
size of the
EcoRV
K fragment of the genome from 5.55 to 6.3 kb
(Fig.
1; compare diagrams
2 and 5). The restriction pattern of
EcoRV-digested virus
DNA shows the loss of the 5.55-kb K fragment
in the recombinant virus
and the appearance of a larger fragment
of 6.3 kb (Fig.
2, stained
gel). Southern blotting carried out
on the gel by using both a UL49
probe and a GFP probe (Fig.
2)
indicated that this new larger fragment
hybridized with both sequences,
confirming the presence of the GFP-22
gene in the
EcoRV K fragment.
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FIG. 2.
Southern blotting of virus DNA confirms the presence of
the GFP-22 cassette in the HSV-1 genome. Viral DNA purified from cells
infected with either S17 or 166v was digested overnight with
EcoRV and electrophoresed in a 0.8% agarose gel containing
ethidium bromide. The gel was photographed (stained gel), transferred
to a nylon membrane, and hybridized with a probe specific for the UL49
gene. The same membrane was stripped and rehybridized with a probe
specific for GFP. Arrow points to the WT EcoRV K fragment.
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166v has the same growth characteristics as WT HSV-1.
To
ascertain the growth properties of the GFP-22 virus, a time course of
infection was carried out for both WT and 166v viruses. Vero cells were
infected at a multiplicity of 10 and harvested every 3 h
postinfection up to 24 h. Total-cell lysates were analyzed by
SDS-PAGE and Western blotting, and the kinetics of synthesis of several
virus proteins were assessed (Fig. 3).
Western blotting with both the anti-VP22 polyclonal antibody AGV30 and
the anti-GFP monoclonal antibody (Fig. 3A) indicated that 166v
synthesized a GFP-22 fusion protein of the correct size, 65 kDa,
confirming the loss of the endogenous VP22 gene from the 166v virus.
Moreover, the GFP-22 fusion protein remained intact throughout the
course of the infection, as judged by both the VP22 and the GFP Western blotting (Fig. 3A). In addition, the overall kinetics of VP22 and
GFP-22 synthesis were very similar, with both VP22 and GFP-22 first
being detected 6 h postinfection (Fig. 3A). The kinetics of
synthesis of three other virus proteins representing different classes
of genes, namely, the immediate-early gene IE110, the early gene TK,
and the late gene VP16, were identical for both the viruses (Fig. 3B).
These results suggest that the 166v virus enters cells and initiates
virus replication as efficiently as the WT virus.
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FIG. 3.
The GFP-22 virus replicates as efficiently as the WT
virus. Vero cells infected with either the WT virus or 166v at 10 PFU
per cell were harvested every 3 h after infection, up to 24 h, as indicated above the gels. Equal amounts of total cell lysates
were analyzed by SDS-PAGE followed by Western blotting with antibodies
against VP22 and GFP (A); IE110, TK, and VP16 (B); or tubulin and
acetylated tubulin (C).
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We have recently shown that a feature of VP22 in both transfection and
virus infection is its ability to stabilize the cellular
microtubule
network (
9). One consequence of such microtubule
stability
is an increase in the level of acetylation present on
tubulin
(
21), a modification which is detectable with an antibody
specific for the acetylated form of
tubulin. In agreement with
our
previous results, Western blotting of the same time course
samples used
above with antibodies specific for both
tubulin
and acetylated
tubulin (Fig.
3C) showed an increase in acetylated-tubulin
levels in
the WT virus infection in comparison to overall
tubulin
levels,
correlating with the synthesis of VP22 (Fig.
3C). Moreover,
the 166v
virus was also able to induce the acetylation of
tubulin
as
efficiently as the parental virus (Fig.
3C). Therefore, it
appears that
166v virus infection increases the stability of cellular
microtubules
as efficiently as WT virus
infection.
While these results demonstrated that virus gene expression was similar
in 166v- and WT virus-infected cells, it was possible,
since VP22 is a
structural component of the virus particle, that
the virus could be in
some way restricted for assembly and/or
release from the cell. To
assess the rate of virus assembly and
egress, one-step growth curves
were carried out for both the 166v
and the WT virus (Fig.
4). Vero cells were infected at a
multiplicity
of 10 and harvested every 3 h for 24 h, and both
total-virus yield
and extracellular-virus yield were calculated for
both viruses.
The results show that the growth curves for total and
released
virus from WT virus and 166v infections were very similar
(Fig.
4), implying that the rates of both virus assembly and virus
egress
from the cell were the same for the two viruses.
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FIG. 4.
One-step growth curves for WT and 166v viruses. Vero
cells infected with either the WT virus or 166v at 10 PFU per cell were
harvested every 3 h after infection up to 24 h and were
titrated onto Vero cells. Total, virus yield from intra- and
extracellular virus. Released, virus yield from extracellular virus.
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GFP-22 is efficiently incorporated into 166v virus particles.
VP22 is a major component of the tegument of the virus particle.
However, it has not yet been determined if VP22 is essential for virus
assembly, and it was therefore of interest to determine if GFP-22 was
incorporated into virus particles produced during 166v infection.
Virions of both the 166v and the parental virus were purified by
pelleting extracellular virus released into infected cell medium and
purifying the particles on a 5 to 15% Ficoll gradient. Approximately
equivalent numbers of particles were analyzed by SDS-PAGE followed
either by Coomassie blue staining or by Western blotting (Fig.
5A). The total virion protein profiles
showed that the 38-kDa VP22 species present in the WT profile had been
lost from the 166v profile and that a new, 65-kDa species, the correct size for GFP-22, was present (Fig. 5A, left panel). Western blotting with both the anti-VP22 and anti-GFP antibodies confirmed that this new
virion component represented the GFP-22 fusion protein (Fig. 5A, right
panels). It is noteworthy that, in spite of the fact that GFP-22 is
almost twice the size of VP22, the levels of GFP-22 and VP22
incorporated into their respective particles were roughly equivalent
when compared to a VP16 loading control (Fig. 5A, right panels).
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FIG. 5.
Incorporation of GFP-22 into virus particles. (A)
Purified WT and 166v virions were solubilized and analyzed by SDS-PAGE
on a 9% acrylamide gel, followed by either Coomassie blue staining
(left) or Western blotting with antibodies against VP22, GFP, or VP16
(right). VP22 and GFP-22 species are indicated. (B) 166v virions are
fluorescent. Approximately 10 PFU of purified 166v virions per cell was
laid onto a monolayer of Vero cells maintained at 4°C. Thirty minutes
later the cells were examined live by both phase-contrast (Phase) and
fluorescence (GFP-22 fluorescence) microscopy.
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The inclusion of GFP-22 into the virions of 166v raised the possibility
that these particles would be detectable by fluorescence.
To determine
if this was the case, gradient-purified virions were
laid onto a
coverslip of Vero cells maintained at 4°C at a multiplicity
of 10, and the cells were examined 30 min later by fluorescence
microscopy. A
typical field, showing both GFP fluorescence and
the phase image of the
Vero cells, is shown in Fig.
5B. Strikingly,
fluorescent particles were
readily visualized on the outer surfaces
of these cells (Fig.
5B, left
panel).
Detection of newly synthesized GFP-22 in infected cells.
One
of the major possibilities for a virus expressing a fluorescent
structural protein would be the ability to localize that protein within
the cell, to follow its trafficking as infection progresses, and
ultimately to visualize the pathway of virus assembly in live cells. To
determine the stage of infection at which newly synthesized GFP-22
could initially be detected by fluorescence, and to analyze its
localization in live cells at various stages of infection, Vero cells
were infected with the 166v virus at a multiplicity of 10, and the
cells were examined for GFP fluorescence every hour over a period of
14 h. Representative images collected at each time point are shown
in Fig. 6. Notably, GFP-22 was first observed in the majority of cells as early as 3 h postinfection, at which time the protein was localized in a diffuse cytoplasmic pattern (Fig. 6). By 4 h
postinfection, GFP-22 was visible in most cells, in the same diffuse
cytoplasmic location, but with a small number of additional intensely
fluorescent spots situated throughout the cytoplasm (Fig. 6). Over the
next 4 h the diffuse intensity of GFP-22 fluorescence in the
cytoplasm increased, while a new form of particulate GFP-22 started to
accumulate at the edge of the nucleus (Fig. 6). This material increased
in intensity and localized to other regions of the cytoplasm, and by
10 h a similar form of GFP-22 was present at the peripheries of
the cells (Fig. 6). At 14 h, fluorescent particles, presumably
virions, were visible outside the cells (Fig. 6). After 18 h, the
cells were intensely fluorescent, and it became difficult to discern any more information from them. These preliminary results suggest that
throughout infection GFP-22 is concentrated primarily in the cytoplasm
of the cell, where it exhibits distinctive patterns of localization,
ranging from an initial diffuse pattern to an accumulation around the
nucleus, followed by a cell periphery location.
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FIG. 6.
Live-cell analysis of GFP-22 localization during a
high-multiplicity infection of 166v. Vero cells were infected with 166v
at 10 PFU per cell and were examined every hour up to 14 h
postinfection (14 h.p.i.) for GFP-22 fluorescence. The same settings
for the confocal microscope were used at each time point. Extracellular
fluorescent particles can be seen in the image taken at 14 h
postinfection (arrow).
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Time lapse analysis of virus infection.
The ultimate elegance
of a GFP-incorporating virus is the potential for monitoring virus
infection in individual living cells. To determine the optimum
conditions for the long-term maintenance of cells for confocal
microscopy, so that time lapse analysis could be carried out, we
examined a variety of growth conditions. The final conditions used were
as follows. Cells were grown on a glass coverslip, which was then
sealed into our microscope chamber at the required time. Standard
Dulbecco's minimal essential medium was added to the cells, and the
entire chamber was maintained at 37°C by placing it on a heated
platform seated on the microscope. With the provision of a constant
supply of 5% CO2, the cells could be maintained on the
microscope as long as 48 h. To generate time lapse data on cells
infected with the GFP-22-encoding virus, Vero cells on a coverslip were
infected at a multiplicity of 10 and placed in the chamber, and a
single field was chosen for time lapse analysis beginning at 5 h
postinfection. Images were collected every 5 min for a further 12 h to form the time series represented as both an animation
(http://mc11.mcri.ac.uk/mov/figure7.mov) and the static images of
hourly intervals shown in Fig. 7. At the
first time point (5 h postinfection), the bright fluorescent spots
observed previously (at 4 h postinfection) (Fig. 6) were also
observed in the cytoplasm of these infected cells (Fig. 7). As the
infection progressed over the next few hours, these spots were engulfed
in the mass of particulate GFP-22 which formed at the edge of the
nucleus (Fig. 7, 10 h postinfection). This same material then
travelled through the cytoplasm from the edge of the nucleus to the
cell periphery, and in particular to the extremities of the cells (Fig.
7, 14 h postinfection).
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FIG. 7.
Time lapse analysis of GFP-22 trafficking in a
high-multiplicity 166v infection. Vero cells were infected with 166v at
10 PFU per cell and were transferred to the heated chamber 5 h
postinfection (5 h.p.i.). A single field was chosen for analysis, and
images were collected every 5 min for a further 15 h. A time point
representing each hour is shown, and the corresponding animation can be
found at http://mc11.mcri.ac.uk/mov/figure7.mov.
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It was clear from the evolving localization patterns observed for
GFP-22 over the 16 h of a high-multiplicity infection (Fig.
6 and
7) that after the initiation of VP22 synthesis its localization
altered
rapidly, and detailed trafficking of the protein may therefore
be
difficult to assess. Thus, to slow down the infection process
and
enable GFP-22 trafficking to be examined more precisely, Vero
cells
were infected at a low multiplicity so that approximately
1 cell in 50 was infected. Eight hours postinfection it was possible
to detect
individual cells which were expressing GFP-22 in its
early diffuse
cytoplasmic pattern. Two such cells were chosen
for further analysis by
time lapse confocal microscopy (Fig.
8),
and images of these cells were
collected every 5 min over a period
of 15 h. The resulting time
series of 150 images was animated
to produce a time lapse analysis of
infection progressing in these
two cells
(
http://mc11.mcri.ac.uk/mov/figure8.mov), with hourly
images presented
as a gallery in Fig.
8. Between 8 and
12 h postinfection,
the diffuse GFP-22 evolved into particulate
GFP-22 in a manner
similar to that seen during the high-multiplicity
infection (Fig.
8), with the exception that none of the bright
fluorescent spots
which had been seen at the earlier stages of the
high-multiplicity
infection (Fig.
6,
4 and
5 h postinfection) were
observed during
this low-multiplicity infection. The particulate
material localized
initially around the nucleus (Fig.
8, e.g., 9 h
postinfection),
but over the next few hours it appeared in other
regions of the
cytoplasm (Fig.
8, e.g., 13 h postinfection). As
more of this
particulate material appeared in the cytoplasm of these
two cells,
it began to move to the periphery of the cell (Fig.
8,
15 h postinfection;
seen clearly in the right-hand cell) and in
particular to the
apices of the cell. This trafficking continued for
the rest of
the time course. It can be noted from this time lapse, and
in
particular from the animation, that at the later stages of infection
there was continued movement of the cell membranes and that projections
containing a high concentration of GFP-22 were actively thrown
out from
the cytoplasm. Thus, we suggest that these processes
may represent
tracks along which virus egress takes place and
that in the future it
may be possible to visualize the progression
of individual
GFP-22-containing vesicles along these projections.
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FIG. 8.
Time lapse analysis of GFP-22 trafficking in a
low-multiplicity 166v infection. Vero cells infected with 166v at 0.02 PFU per cell were transferred to the heated chamber 8 h
postinfection (8 h.p.i.). A single field of cells was chosen for
further analysis, and images were collected every 5 min for a further
14 h. A time point representing each hour is shown, and the
corresponding animation can be found at
http://mc11.mcri.ac.uk/mov/figure8.mov.
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DISCUSSION |
While certain features of the herpesvirus replication
cycle are now broadly understood, the fine details of virion
trafficking and maturation remain to be elucidated. One of the major
obstacles to our full understanding of virus assembly and egress has
been the inability to analyze and follow virus trafficking at the
single-cell level. The development of GFP technology has recently begun
to overcome problems associated with the study of protein trafficking in several aspects of cell biology, such as endoplasmic
reticulum-to-Golgi apparatus transport (22, 26), the
cytoskeleton (16, 20, 30), and cell division (6,
12). Furthermore, the use of time lapse analysis of cells
expressing GFP fusion proteins has for the first time enabled the
tracking of individual proteins over time at the subcellular level
(15, 19, 32). In this report we present the first time lapse
images of virus infection in live cells captured by the use of a
herpesvirus incorporating a GFP-tagged protein into its structure. This
virus produces fluorescent particles which can be readily resolved by
fluorescence microscopy and can therefore be traced through the
different stages of virus replication. Thus, we have generated an
extremely powerful tool for the analysis of herpesvirus infection.
The protein to which we have fused GFP for incorporation into the
virion is the major tegument protein VP22. Although we have recently
identified several unusual properties of this protein (8,
9), the exact role of VP22 in virus infection is still unclear.
We have previously shown that the fusion of GFP to VP22 has little
effect on any of the properties of VP22 which we have identified by
transient transfection. Strikingly, we show here that the addition of
the 27-kDa GFP protein onto the VP22 open reading frame also has little
effect on any aspect of virus infection, including efficiency of virus
entry, the rate of virus assembly, and the rate of release from the
cell. Moreover, the tegument of the virus particle appears flexible
enough to accommodate the same number of GFP-22 molecules as VP22
molecules, in spite of the near-doubling in size of the protein, with
no apparent increase in size of the individual particles, as judged by
electron microscopy (data not shown), and with no obvious changes in
the ratio of other tegument proteins within the virions. This suggests
that the tegument compartment exhibits some flexibility in its
structure and is capable of incorporating fusion proteins or, as
previously shown, excess copies of at least some of the constituent
proteins (14). Interestingly, it has recently been shown
that the HSV-1 capsid can also accommodate a GFP-fusion protein in the
form of GFP-VP26, which is located at the tips of the capsid hexons
(5).
Our preliminary analysis of the 166v infection demonstrated that newly
synthesized GFP-22 was detectable in live cells as early as 3 h
postinfection. This sensitivity of GFP-22 fluorescence suggested that
it may be possible, in combination with time lapse imaging of
individual cells, to analyze the trafficking of GFP-22, and hence of
HSV-1 virions, dynamically in live cells. We have therefore produced
time lapse animations of 166v infections and have subsequently built up
an overall picture of GFP-22 trafficking during infection. These
animations show us that when GFP-22 is initially synthesized, it
appears in a diffuse cytoplasmic pattern. However, as infection
progresses, the GFP-22 molecules take on a more distinctive
pattern, concentrated in particles to one side of the nucleus, in a
pattern reminiscent of the Golgi apparatus. This material then travels
towards the cell periphery and eventually appears extracellularly as
individual fluorescent particles. Thus, we would suggest that this
pathway reflects GFP-22 incorporation into assembling virions, followed
by virion trafficking and egress from the cell. Moreover, further
studies on the relative proportion of infected-cell VP22 which is
incorporated into virions should help to address the relevance of this pathway.
Central to the debate concerning the herpesvirus maturation pathway(s)
is the identification of the tegument assembly site within the cell. It
has recently been demonstrated that at least two HSV-1 tegument
proteins localize in the nucleus close to putative sites of capsid
assembly, termed assemblons (31), an observation taken to
mean that tegument proteins may assemble into the virus particle in the
nucleus. However, we have previously shown by immunofluorescence
studies that VP22 and another tegument protein, VP16, colocalize in the
cytoplasm rather than the nucleus of both infected and transfected
cells (7), suggesting that during coexpression these
proteins are targeted to the cytoplasmic compartment. Moreover, it has
been demonstrated that the tegument can assemble independently of the
capsid (23). Our results presented here demonstrate that
throughout the course of infection, GFP-22 appears almost entirely
cytoplasmic. While we cannot exclude the possibility that GFP-22
travels through the nucleus so rapidly and efficiently that it is never
detected there, or that GFP-22 fluorescence varies depending on its
cellular location, the animations provide strong evidence that this
tegument protein is incorporated into the virion at a stage downstream
of capsid translocation through the nuclear envelope. Consequently,
these results would suggest that the final envelope of the mature
virion is acquired at a cytoplasmic location, further along the
exocytotic pathway from the site of VP22 inclusion into the virion.
The development of a GFP-labelled herpesvirus and the demonstration
that the incorporation of GFP-22 into the virus particle is in no way
detrimental to virus replication open up a wide range of applications
for such a reagent. Purified virions could be used to analyze virus
entry, and in particular the fate of the tegument after the envelope
has fused to the cell membrane. Moreover, detailed studies on the
kinetics of virus egress could be conducted to assess the effect of
virus infection on the exocytotic pathway. The GFP-22 virus could be
combined with mutations in other genes to uncouple the individual steps
of virus maturation as we now observe it in live cells. Thus, with the
advent of GFP technology, it may now be possible to address many of the
issues surrounding herpesvirus morphogenesis by the use of viruses
incorporating GFP into the various compartments of the virion.
| |
ACKNOWLEDGMENTS |
We thank Tony Minson for antibody LP1, Roger Everett for
antibody 11060, and David Gower for the anti-TK antibody.
This work was funded by Marie Curie Cancer Care.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Marie Curie
Research Institute, The Chart, Oxted, Surrey RH1 0TL, United Kingdom.
Phone: 01883 722306. Fax: 01883 714375. E-mail:
g.elliott{at}mcri.ac.uk.
| |
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Journal of Virology, May 1999, p. 4110-4119, Vol. 73, No. 5
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
