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Journal of Virology, September 1998, p. 7563-7568, Vol. 72, No. 9
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Incorporation of the Green Fluorescent Protein
into the Herpes Simplex Virus Type 1 Capsid
Prashant
Desai* and
Stanley
Person
Virology Laboratories, Department of
Pharmacology and Molecular Sciences, Johns Hopkins University
School of Medicine, Baltimore, Maryland 21205
Received 26 January 1998/Accepted 28 May 1998
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ABSTRACT |
The herpes simplex virus type 1 (HSV-1) UL35 open reading frame
(ORF) encodes a 12-kDa capsid protein designated VP26. VP26 is located
on the outer surface of the capsid specifically on the tips of the
hexons that constitute the capsid shell. The bioluminescent jellyfish
(Aequorea victoria) green fluorescent protein (GFP) was
fused in frame with the UL35 ORF to generate a VP26-GFP fusion protein.
This fusion protein was fluorescent and localized to distinct
regions within the nuclei of transfected cells following infection with
wild-type virus. The VP26-GFP marker was introduced into the HSV-1
(KOS) genome resulting in recombinant plaques that were fluorescent. A
virus, designated K26GFP, was isolated and purified and was shown to
grow as well as the wild-type virus in cell culture. An analysis of the
intranuclear capsids formed in K26GFP-infected cells revealed that the
fusion protein was incorporated into A, B, and C capsids. Furthermore,
the fusion protein incorporated into the virion particle was
fluorescent as judged by fluorescence-activated cell sorter (FACS)
analysis of infected cells in the absence of de novo protein synthesis. Cells infected with K26GFP exhibited a punctate nuclear fluorescence at
early times in the replication cycle. At later times during infection a
generalized cytoplasmic and nuclear fluorescence, including
fluorescence at the cell membranes, was observed, confirming visually that the fusion protein was incorporated into intranuclear capsids and mature virions.
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TEXT |
The physical structure required for
transporting the herpes simplex virus type 1 (HSV-1) genome is an
icosahedral proteinaeous capsid (26). Three types of
capsids, termed A, B, and C can be isolated from HSV-1-infected cells
(10). The A and C capsids are similar in protein content,
but only the C capsid contains a genomic equivalent of DNA and matures
into the infectious virion (reviewed in references
19 and 21). The B capsid
comprises seven proteins. Their designations, and the open
reading frames (ORF) encoding the proteins (in parentheses), are
VP5 (UL19), VP19C (UL38), 21 (UL26), 22a (UL26.5), VP23 (UL18),
VP24 (UL26), and VP26 (UL35) (4, 10, 11, 14).
VP26 is the smallest capsid protein (12 kDa) and is encoded by the UL35
ORF (6, 15). It is expressed late in the infectious cycle
after the onset of DNA replication and has been shown to be present
multiple phosphorylated forms (16). Reports that show that
VP26 localizes to the infected cell nucleus (16) and requires the presence of VP5 for this localization in transfected cells
(18) have been published. There are approximately 900 copies
of this molecule, and they occupy the tips of the hexons but not the
pentons, both of which are composed of VP5 (1, 24, 27, 28).
Although it interacts with VP5, it is not required for capsid formation
in a baculovirus expression system (22, 23). Studies in our
laboratory have shown that VP26 is not required for growth in cell
culture; however, it is important for the production of infectious
virus in trigeminal ganglia (8).
The green fluorescent protein (GFP) of the bioluminescent jellyfish
(Aequorea victoria) has been extensively used, both as a
genetic reporter and to monitor the cellular locations of proteins fused to it (2). GFP absorbs UV or blue light and emits
green fluorescence. It does not require any other cofactors or
substrates or additional A. victoria gene products for this
activity. Consequently, detection of GFP can be performed in
heterologous systems and in living cells or tissues (reviewed in
reference 3). Variants of the GFP chromophore that
improve the use of the GFP reporter have been generated. One such
variant, termed EGFP, contains amino acid substitutions that allow it
to fluoresce 35-fold more intensely than wild-type GFP when excited
with blue light, and therefore it is useful for analysis using
fluorescence microscopy and flow cytometry (5).
Furthermore, this variant contains several silent base changes that
correspond to optimal human codon usage for better expression in
eukaryotic systems. This modified version of GFP (Clontech) was
used for the experiments described below.
The aim of the experiments described below was to incorporate the GFP
into the HSV-1 capsid. A tagged nucleocapsid structure should be useful
for the investigation of the early events in the uncoating of the virus
particle and for monitoring the virus nucleocapsid during replication
and transport in cell culture and in vivo. The rationale behind this
approach was to utilize the VP26 polypeptide, which is located on the
outer surface of the capsid shell. A fusion between the VP26 and GFP
polypeptides was generated, and it was hoped that this fusion form of
VP26 would still be capable of interaction with VP5 and would
incorporate the GFP polypeptide onto the capsid structure.
Therefore, the nucleocapsid and consequently the mature virion
would be tagged with a fluorescent marker that is activated by light.
Construction of a VP26-GFP fusion protein.
The goal of the
molecular manipulations described below was to fuse the GFP ORF with
that of VP26. An XhoI restriction enzyme site was introduced
into the UL35 ORF (Fig. 1) by overlap
extension PCR assays. The methodology of this assay is described in
greater detail elsewhere (8, 12). The restriction site
overlaps codons 5 to 7 of the UL35 ORF. The aim was to introduce the
GFP ORF at this position to generate a VP26-GFP fusion
construct. This was achieved by the introduction of an
oligonucleotide duplex into the XhoI site which
specified NcoI and BsrGI restriction sites (Fig. 1). The GFP ORF was derived from pEGFP-N1 (Clontech) as an
NcoI-to-BsrGI fragment (716 bp) and cloned
into the same sites in UL35. This plasmid was designated pK26GFP.
The GFP ORF was fused in frame at the sequence corresponding to
the VP26 N terminus to the codons for the first 4 authentic residues of
VP26 and to the remaining portion of the VP26 coding sequences (108 residues) at the sequence corresponding to the C terminus.
Therefore, expression of the GFP fusion polypeptide should be mediated
by the UL35 promoter sequences and translation initiation should
occur at the VP26 ATG codon.
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FIG. 1.
Construction of a VP26-GFP fusion protein. The 5.2-kb
EcoRI L fragment of HSV-1 strain KOS was cloned into pUC19.
The EcoRI L fragment contains UL35 (VP26) and the
C-terminal-encoding sequences of the UL34 and UL36 genes
(14). A shortened version of the EcoRI L clone
spanning the EcoRI and NotI restriction sites was
used for subsequent manipulations. An XhoI restriction site
which spans residues 5 and 7 of the UL35 ORF was created by overlap
extension PCR assays (8, 12). This plasmid was cleaved with
XhoI, and an oligonucleotide duplex specifying
NcoI and BsrGI restriction sites was annealed at
this position. The GFP ORF derived from pEGFP-N1 (Clontech) as an
NcoI-BsrGI fragment was cloned into this plasmid
to generate pK26GFP. UL, unique long; US,
unique short.
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Transient transfection assays were then carried out to determine
whether the GFP fusion construct was fluorescent.
Briefly,
Vero cell monolayers were transfected with either
pK26GFP or pEGFP-N1.
The latter plasmid contains the GFP
sequences under the control
of the constitutive human cytomegalovirus
immediate-early promoter.
Twenty-four hours after transfection the
cells were either mock
infected or infected with KOS at a multiplicity
of infection (MOI)
of 10 PFU per cell. Cells were then examined under a
fluorescence
microscope at various times after infection. Whereas
fluorescence
was detected in pEGFP-N1-transfected cells, in both mock-
and
KOS-infected samples, cells transfected with pK26GFP only
displayed
fluorescence upon superinfection (data not shown).
Fluorescence
in pK26GFP-transfected cells was observed as early as
4 h after
infection and was distributed throughout the cell, as
observed
for the pEGFP-N1-transfected cells (Fig.
2A). However, at later
times
(12 h) during infection, a majority of the cells displayed
a
fluorescence pattern in which the fluorescence was primarily
within the
nucleus and in distinct locations within this structure
(Fig.
2B).
Therefore, the VP26-GFP fusion protein was fluorescent
and was
localized to the nucleus, as would be expected during
the
assembly of the capsid structure. The VP26-GFP fusion protein
was
localized to the nucleus in punctate regions only in cells
infected
with KOS (Fig.
2B) or the VP26-null mutant (
8), K
26Z
(data not shown), both of which assemble capsids in the nuclei
of
infected cells. This pattern of nuclear fluorescence was not
observed
when cells were infected with viruses that do not assemble
capsids,
such as the VP5-null mutant K5
Z (
7) (Fig.
2C).
Therefore,
the VP26-GFP fusion protein was biologically
functional in the
context of capsid assembly.
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FIG. 2.
Transfection-infection assays with pK26GFP. Vero cells
seeded at 3.5 × 104 cells/cm2 in culture
dishes were transfected approximately 16 h later with 5 µg each
of plasmids pEGFP-N1 (A) or pK26GFP (B and C) (CellPhect; Pharmacia
Upjohn). Twenty-four hours after transfection the cells were infected
with either KOS (B) or K5Z (C) at an MOI of 10 PFU/cell or were mock
infected (A). Cells were visualized live 12 h postinfection with
an Olympus BH-2 fluorescence microscope (×20 objective).
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Transfer of the VP26-GFP marker into the virus genome.
The
next step was to introduce the VP26-GFP marker into the virus genome.
This was carried out by standard marker transfer procedures in Vero
cells. Cell monolayers were cotransfected with KOS viral DNA and
linearized pK26GFP plasmid DNA. Forty-eight hours after transfection
lysates were prepared and the transfection progeny were diluted and
plated on Vero cell monolayers. The plaques that formed were visualized
in a fluorescence microscope to detect recombinant viruses.
Fluorescent plaques indicative of recombinant viruses were observed,
and these were marked and picked. Two independent isolates were
purified through two further cycles of limiting dilution, and the
viruses were designated K26GFP-1 and K26GFP-2. When plaques of K26GFP
(either isolate) were examined in a fluorescence microscope, all the
plaques were found to be fluorescent, indicative of the purity of
the virus stocks. A single fluorescent plaque of K26GFP-1 is shown in
Fig. 3A. Southern blot analysis was
performed on DNA extracted from both KOS- and K26GFP-infected
cells to confirm the genotypes of the recombinant viruses (Fig. 3B).
Viral DNA was either digested with EcoRI (lanes 1 to 3) or
double digested with NcoI and BsrGI
(lanes 4 to 6), and the resulting fragments, following transfer
to nitrocellulose, were hybridized to 32P-labeled
pK26GFP. The probe hybridized to the 5.2-kb EcoRI L fragment of KOS DNA (lane 1); this fragment exhibited a decreased mobility in K26GFP-1 (lane 2) and K26GFP-2 (lane 3) DNA due to the
insertion of the 716-bp GFP coding sequence. In the case of the
digestion with NcoI and BsrGI the probe
hybridized to a 3.9-kb fragment in wild-type DNA (lane 4), which was
cleaved in K26GFP-1 (lane 5) and K26GFP-2 (lane 6) DNA into three
fragments of 3.0, 0.9, and 0.7 kb due to the introduction of
NcoI and BsrGI sites into UL35 followed by the
insertion of the 0.7-kb GFP sequence. Weak hybridization of the probe
to a 2.0-kb NcoI fragment, observed in lanes 4 to 6, was due
to the presence of sequences in the probe that correspond to DNA
spanning the NcoI and EcoRI sites at the 3' end
of the EcoRI L fragment. This analysis confirmed the
introduction of the ORF for the VP26-GFP fusion construct into the
virus genome. Since both isolates were phenotypically and genotypically
identical, the experiments presented below were carried out with only
one of the isolates, now referred to as K26GFP.
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FIG. 3.
Marker rescue of VP26-GFP into the virus genome. Vero
cells seeded at 3.5 × 104 cells/cm2
in culture dishes were transfected approximately 16 h later with
KOS viral DNA (2 µg) and linearized pK26GFP (5 µg) (CellPhect;
Pharmacia Upjohn). The transfection progeny was harvested 2 days later
and plated for single plaques. Plaques were visualized in a
fluorescence microscope, and the fluorescent plaques were picked
and purified further. (A) One such virus, designated K26GFP-1,
was plated on Vero cell monolayers, and a single plaque was
photographed upon visualization in a fluorescence microscope (×10
objective). (B) Two micrograms of KOS (lanes 1 and 4), K26GFP-1 (lanes
2 and 5), and K26GFP-2 (lanes 3 and 6) was digested with
EcoRI (lanes 1 to 3) or double digested with NcoI
and BsrGI (lanes 4 to 6), the restriction fragments
were resolved by agarose gel electrophoresis and transferred to
nitrocellulose (Schleicher and Schuell), and the filters were probed
with 32P-labeled pK26GFP. The diagram below the blot shows
the EcoRI L region of HSV-1 strain KOS. The probe, pK26GFP,
is shown below this. Relevant restriction sites are indicated: E,
EcoRI; Nc, NcoI; and B, BsrGI. For
details of autoradiograph scanning, see the legend for Fig. 4.
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A null mutation in the UL35 ORF resulted in a twofold decrease in
the virus yield from infected cells compared to that for
wild-type
virus, as judged by single-step growth assays (
8).
Similar
assays using the K26GFP virus revealed that the recombinant
GFP
virus replicated at levels comparable to that of the wild-type
virus (data not shown). Therefore, the VP26-GFP fusion protein
retained
biological activity during the virus replication cycle.
VP26-GFP is incorporated into the capsid shell and the
mature virion.
The next set of experiments was carried out
to determine the composition of the capsids formed in
K26GFP-infected cells. HEL cell monolayers were infected with KOS and
K26GFP at an MOI of 10 PFU/cell and metabolically labeled with
[35S]methionine from 8 to 24 h postinfection.
Prepared nuclear lysates were sedimented through sucrose gradients, and
the fractions corresponding to A, B, and C capsids were analyzed
by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) (Fig. 4A). The VP26 and GFP
polypeptides are 12 and 27 kDa in size, respectively; therefore, a
VP26-GFP fusion protein would be expected to have a mobility of 39 kDa in SDS-PAGE gels. For KOS (lanes 1) capsids (A, B, or C) the 12-kDa VP26 polypeptide was evident. In the K26GFP (lanes 2) capsid fractions the 12-kDa band was absent and a novel polypeptide of approximately 39 to 40 kDa was present. This was presumably the VP26-GFP fusion protein.
The VP26-GFP fusion protein was obscured in B capsids by the presence
of the abundant 22a polypeptide, which is similar in size. Therefore,
the VP26-GFP fusion protein was capable of interacting with the major
capsid protein (VP5) and was incorporated into the capsid shell.
Immunoprecipitation assays were carried out with
[35S]methionine-labeled infected cell extracts
and a peptide antibody specific to the C terminus of VP26. The 12-kDa
VP26 polypeptide was precipitated from KOS-infected cell extracts
(Fig. 4B, lane 1), and a polypeptide of approximately 40 kDa was
observed in extracts of K26GFP-infected cells (lane 2). This
polypeptide had the same mobility as the VP26-GFP fusion protein
observed in capsids.
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FIG. 4.
Sedimentation analysis of K26GFP intranuclear capsids.
(A) HEL cells seeded at 6.4 × 104
cells/cm2 and infected approximately 16 h later with
KOS (lanes 1) and K26GFP (lanes 2) at an MOI of 10 PFU/cell were
metabolically labeled with
[35S]methionine from 8 to 24 h postinfection
(17). Nuclear lysates were prepared and sedimented in
20 to 50% sucrose gradients (7). The
radioactivity in each fraction collected was determined by
liquid scintillation counting. The peak fractions containing A
(fraction 10), B (fraction 8), and C (fraction 4)
capsids were precipitated with trichloroacetic acid and
analyzed by SDS-PAGE (17% acrylamide). The positions of the capsid
proteins are indicated on the left of the figure, and those of
VP26 and VP26-GFP are indicated on the right. (B) Vero cells seeded at
1.1 × 105 cells/cm2 and infected
approximately 16 h later with KOS (lane 1) and K26GFP (lane 2) at
an MOI of 10 PFU/cell and labeled with
[35S]methionine as described for panel A. Infected
cell extracts were prepared and were precipitated with a peptide
antibody to VP26, and the resulting precipitates were
resolved by SDS-PAGE (17% acrylamide). Shown to the left of the figure
are the relative mobilities of protein molecular weight standards. The
autoradiographs in both panels were scanned on a Umax Powerlook II
scanner. The images were scanned at 300 dots per inch into
Adobe Photoshop 3.0 and were transported as PICT files into Microsoft
Powerpoint for figure presentation and printing.
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In order to determine whether the GFP tag in the virus was still
fluorescent, experiments were carried out with a fluorescence-activated
cell sorter (FACS), an EPICS profile analyzer (Coulter Corp.).
Vero
cell monolayers were infected with KOS and K26GFP at an MOI
of 250 PFU/cell or were mock infected. The cells were incubated
prior to and
during the infection period in cycloheximide to prevent
de novo
protein synthesis. Cells were harvested at 4 h
postinfection,
resuspended in phosphate-buffered saline at
10
6 cells/ml, and subjected to FACS analysis using the FL1
emission
channel. Background levels of fluorescence were
determined with
mock-infected cells. The majority of KOS-infected
cells (99.1%)
exhibited background levels of fluorescence,
whereas only 1.7%
of K26GFP-infected cells displayed background
fluorescence. For
the remainder of the K26GFP-infected cells, 98.3%,
there was a
shift in peak detected mean fluorescence. The mean detected
fluorescence
levels were 1.8 and 2.1 for mock- and KOS-infected
cells, respectively,
whereas the mean detected fluorescence
level for K26GFP-infected
cells was 28.4. Therefore, the
VP26-GFP fusion protein incorporated
into the virus capsid retained
fluorescent activity.
Localization of VP26-GFP in infected cells.
The next
experiment was performed to monitor the localization of the VP26-GFP
fusion protein and its incorporation into the virus capsid. Vero cells
were infected with K26GFP and visualized "live" at various times
after infection throughout the virus replication cycle (Fig.
5). Early in infection (6 and 8 h;
Fig. 5A and B) the fusion protein localized to distinct regions within
the nucleus of the infected cell. This punctate pattern was similar to
that reported by others (25) and probably corresponds to
compartments within the nucleus in which capsids are assembled.
There was either very little or undetectable cytoplasmic
fluorescence, which indicated that VP26 was sequestered in the
nucleus with VP5. When the VP26-GFP marker was introduced into a virus
genome that specified a null mutation in VP5 (K5Z), the fluorescence
observed in those infected cells was uniformly distributed throughout
the cell (data not shown) and was similar to that observed in Fig. 2C.
At 10 and 12 h (Fig. 5C and D) after infection the intensity of
the fluorescence as well as the number of fluorescent cells increased.
At later times during infection with K26GFP, the punctate nuclear
fluorescence became less intense and was replaced with a more
generalized nuclear fluorescence and an increase in cytoplasmic
fluorescence was observed. There was also an accumulation of
fluorescence at the plasma membrane. This can dramatically be seen in
Fig. 5E to H. Presumably the change in the fluorescence pattern
reflects virus assembly in the nucleus and its transport through the
cytoplasm to the plasma membrane. Fluorescence at the plasma membrane
reflects the accumulation of viruses at the cell surface. In addition
to being able to trace the path of the capsid from its origin in the
nuclear compartment to the cytoplasm, the accumulation of fluorescence
at the plasma membrane may allow one to study the release of virus from
cells, as well as the spread of virus from cell to cell.
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FIG. 5.
Localization of VP26-GFP in infected cells. Vero cells
seeded at 3.5 × 104 cells/cm2 in culture
dishes were infected at an MOI of 10 PFU/cell. Cells were visualized
live at various times after infection with an Olympus BH-2 fluorescence
microscope (×40 objective). Photographs were taken at 6 (A), 8 (B), 10 (C), 12 (D), 16 (E and F), 20 (G), and 24 (H) h postinfection.
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Discussion.
GFP, by virtue of its properties, is a useful tag
that can be used to monitor the fate of the tagged protein. In this
case, by being fused to HSV-1 capsid protein VP26, it is incorporated into the capsid structure on the outer surface of the shell and therefore allows one to follow the fate of that particle in the course
of the virus life cycle. Using antibodies to the capsid proteins,
immunofluorescence assays have determined the location of the capsid
upon entry into the cell (20). However, problems of antibody
reactivity, nonspecific cross-reactivity, and the fixation procedures
involved can limit the usefulness of these assays. With GFP as a
marker, entry and uncoating can be monitored in living infected cells
without the need to fix cells or to rely on the availability of
antibodies. GFP has been used as a reporter in recombinant pseudorabies
virus (13) and herpesvirus saimiri (9); however,
this is the first report of using a GFP fusion protein to incorporate
this molecule into the virion structure. This is therefore a useful
reagent for the analysis of HSV-1 biology, in particular, the analysis
of virus replication and transport both in cell culture and in the
trigeminal ganglia of mice.
An interesting outcome of this project is the ability to fuse a
relatively large sequence (27 kDa) to the smallest capsid
protein, VP26
(12 kDa), and still retain the ability of VP26 to
interact with
VP5 and become incorporated into the capsid shell.
The VP26-GFP fusion
protein retained the structural activity,
as judged by capsid assembly,
and the biological activity, as
judged by infectious virus progeny, of
the wild-type VP26 protein.
Potentially, this technique can be utilized
to tag the capsid
shell with other small molecular sequences or
proteins.
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ACKNOWLEDGMENTS |
This work was supported by Public Health Service grant AI33077
(S.P.) from the National Institutes of Health.
We gratefully acknowledge discussions of the data with Wade Gibson,
Mike Baxter, Mary-Elizabeth Harmon, and Scott Plafker. We also
acknowledge Richard Hampton and Zhaohao Liao for assistance with the
FACS analysis.
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FOOTNOTES |
*
Corresponding author. Mailing address: Virology
Laboratories, Department of Pharmacology and Molecular Sciences, Johns
Hopkins University School of Medicine, Baltimore, MD 21205. Phone:
(410) 614-1581. Fax: (410) 955-3023. E-mail:
pdesai{at}welchlink.welch.jhu.edu.
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Journal of Virology, September 1998, p. 7563-7568, Vol. 72, No. 9
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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