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Journal of Virology, October 1999, p. 8750-8761, Vol. 73, No. 10
Institute of Molecular Biology, Academia
Sinica, Nankang, Taipei, Taiwan, Republic of China
Received 27 May 1999/Accepted 12 July 1999
We previously showed that an envelope A27L protein of intracellular
mature virions (IMV) of vaccinia virus binds to cell surface heparan
sulfate during virus infection. In the present study we identified
another viral envelope protein, D8L, that binds to chondroitin sulfate
on cells. Soluble D8L protein interferes with the adsorption of
wild-type vaccinia virions to cells, indicating a role in virus entry.
To explore the interaction of cell surface glycosaminoglycans and
vaccinia virus, we generated mutant viruses from a control virus,
WR32-7/Ind14K (A27L+ D8L+) to be defective
in expression of either the A27L or the D8L gene (A27L+
D8L Vaccinia virus is a large enveloped
DNA virus that belongs to the poxvirus family. It is the prototypic
member of the orthopox viruses and is known to infect cells of many
different origins. Although the viral genome has been sequenced, the
nature of the broad host range remains to be explored. Vaccinia virus
also contains three different forms of infectious virions:
intracellular mature virus (IMV), intracellular enveloped virus (IEV),
and extracellular enveloped virus (EEV) (1, 17, 48). These
different forms of vaccinia virus virions contain different membrane
structures and are responsible for various routes of infection (1,
30, 43). Of the three forms, IMV is the most abundant, and many of its surface envelope proteins have been identified (20,
47). Several proteins have been suggested to be involved in virus
entry. For example, A27L and D8L proteins bind to the cell surface
(22-24). A27L protein is required for fusion of
virus-infected cells (12, 34, 38, 50). Another envelope
protein, L1R, may play a role in virus penetration, since a monoclonal
antibody (MAb) recognizing L1R protein blocked virus entry at a
postbinding step (18, 19, 32, 51). Other proteins such as
A14L and A17L also interact with A27L protein, but their roles in virus
entry have yet to be determined (34, 39, 40).
Our previous data showed that viral A27L protein mediates IMV binding
to cell surface glycosaminoglycans (GAGs), mainly heparan sulfate (HS)
(6). The N-terminal region of A27L protein contains a
cluster of positively charged amino acids that are important for
binding to HS (16). In addition, binding to HS is essential for fusion of virus-infected cells, suggesting that conformational changes triggered after HS-A27L protein interaction initiate membrane fusion (16). To further clarify the mechanism of vaccinia
virus entry, we wished to identify other viral proteins that may also participate in the entry process. In this report we identified D8L
protein as the second envelope protein that binds to GAGs on cells.
However, D8L protein binds to chondroitin sulfate (CS) on cells,
revealing a different type of virus-GAG interaction.
Mutant viruses with defective A27L or D8L gene expression were also
constructed in order to determine whether virus growth in cell cultures
was affected by the impairment of GAG binding. These mutant viruses
were purified, and their growth in cell cultures, virion morphology,
and kinetics of binding to cells were analyzed. The data revealed a
role of D8L protein in the binding of vaccinia virus virions to cells.
Reagents and viruses.
Soluble heparin (HP), CS, and dermatan
sulfate (DS) were purchased from Sigma Inc. Spurr resin was purchased
from Electron Microscopy Sciences. An antiserum against TrpE-D8L
protein, D8-1, was obtained from E. Niles (26). L, gro2C,
and sog9 cells were obtained from F. Tufaro (2, 13).
Wild-type vaccinia virus (WR strain) was grown in BSC40 cells. A
recombinant vaccinia virus, WR32-7/Ind14K, was obtained from G. Smith
(36, 37). IMV virions were purified by centrifugation of
cell lysates through a 36% sucrose layer, and the pellets were saved
as virus stocks (21). EEV was collected from fresh medium of
the infected cell cultures and was used directly without freezing.
Protein expression and purification.
For expression of
soluble D8L, two primers were made for PCR amplification. The 5' primer
(5'-AAAGAATTCATGCCGCAACAACTATCT) and the 3' primer
(5'-AAAAAGCTTTGAAAAACATGTCTCTCT) were used with a
vaccinia virus DNA template in PCR amplification with a program of 94°C for 1 min, 42°C for 1.5 min, and 72°C for 1.5 min for 25 cycles. The amplified DNA fragment contained sequences corresponding to
D8L amino acids 1 to 264. The DNA fragment was digested with EcoRI and HindIII and cloned into pET21a
(Novagen). The resulting plasmid expressed an ectodomain with a T7 tag
peptide at the N terminus for easy identification and hexahistidine
sequences at the C terminus for purification with a nickel column.
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Vaccinia Virus Envelope D8L Protein Binds to Cell
Surface Chondroitin Sulfate and Mediates the Adsorption of
Intracellular Mature Virions to Cells
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
or A27L D8L+) or both
(A27L D8L). The A27L+
D8L+ and A27L D8L+ mutants grew
well in BSC40 cells, consistent with previous observations. However,
the IMV titers of A27L+ D8L and
A27L D8L viruses in BSC40 cells were
reduced, reaching only 10% of the level for the control virus. The
data suggested an important role for D8L protein in WR32-7/Ind14K virus
growth in cell cultures. A27L protein, on the other hand, could not
complement the functions of D8L protein. The low titers of the
A27L+ D8L and A27L
D8L mutant viruses were not due to defects in the
morphogenesis of IMV, and the mutant virions demonstrated a brick shape
similar to that of the control virions. Furthermore, the infectivities of the A27L+ D8L and A27L
D8L mutant virions were 6 to 10% of that of the
A27L+ D8L+ control virus. Virion binding assays
revealed that A27L+ D8L and
A27L D8L mutant virions bound less well to
BSC40 cells, indicating that binding of viral D8L protein to cell
surface chondroitin sulfate could be important for vaccinia virus entry.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
-D-thiogalactoside (IPTG) for 4 h at
37°C and then harvested. The bacterial pellets were sonicated, and
the supernatant recovered after centrifugation was loaded onto a nickel column as recommended in the pET system manual (Novagen). The column
was washed, and the bound protein was eluted with 0.3 M imidazole and
dialyzed against phosphate-buffered saline (PBS) at 4°C overnight as
described elsewhere (6).
Rabbit serum production and neutralization assays. New Zealand White rabbits were immunized subcutaneously with 750 µg of soluble D8L protein and subsequently boosted three times, at 2-week intervals, with another 500 µg of protein before being sacrificed. One rabbit serum, C8, was used in neutralization assays. For neutralization assays, preimmune or postimmune sera at various dilutions (1:100, 1:500, and 1:1,000) were mixed with 150 PFU of wild-type vaccinia virus at 4°C for 30 min. The mixtures were added to BSC40 cells in 60-mm dishes in duplicate and incubated at 37°C for another hour. Cells were then washed with PBS and overlaid with 1% agar. Plaque numbers were determined after 3 days.
Biotinylation of D8L protein and cell binding assays. D8L protein was biotinylated with an ECL biotinylation system purchased from Amersham Life Science, Inc. In brief, 1 mg of purified D8L protein was mixed with 40 µl of the biotinylation reagent N-hydroxysuccinamide ester in 40 mM bicarbonate buffer (pH 8.6) at room temperature for 1 h according to the manufacturer's recommendation. The biotinylated mixture was then loaded on a 9-ml Sephadex G-25 column previously equilibrated with PBS. Biotinylated D8L protein was collected as 500-µl aliquots from fractions 7 to 9, and the extent of biotinylation was confirmed by Western blot analysis with horseradish peroxidase-conjugated streptavidin as described elsewhere (6).
For cell binding assays, BSC40 cells (106) were washed with cold PBS and incubated with biotinylated D8L protein (10 µg/125 µl) in staining medium (PBS-4% fetal bovine serum-10 mM HEPES [pH 7.2]). In some experiments, different GAGs (10, 100, or 1,000 µg/ml) were also added as competitors. The mixture was incubated at 4°C for 60 min with gentle rotation. Cells were subsequently washed with cold PBS, and phycoerythrin-conjugated streptavidin (1:100) was added for another 60 min at 4°C. Cells were again washed three times with cold PBS and analyzed with a fluorescence-activated cell sorter (FACS) (excitation wavelength, 488 nm; emission wavelength, 578 nm) as described elsewhere (16).Virion binding assays. To test if soluble D8L protein blocked vaccinia virus infection at the binding step, BSC40 cells were incubated first with various amounts of soluble D8L protein (0, 1, 10, or 50 µg in 200 µl) at 4°C for 30 min. The cultures were subsequently infected with wild-type vaccinia virus at a multiplicity of infection (MOI) of 10 PFU per cell at 4°C for another 30 min. After a wash, these cells were immediately harvested, and cell lysates were freeze-thawed three times, sonicated, and used for plaque assays on BSC40 cells. The number of plaques obtained from cells infected in the absence of D8L protein was taken as 100%.
To measure viral early gene expression, BSC40 cells were infected with vMJ360, which expresses lacZ from an early promoter, at an MOI of 10 PFU per cell. The infected cells were harvested at 2 h postinfection (p.i.) for-galactosidase (-Gal) assays as
described elsewhere (16).
To determine the IMV binding kinetics of WR32-7/Ind14K or
D8L WR32-7/Ind14K virus, BSC40 cells were infected with
each virus at an MOI of 5 PFU per cell and incubated at 4°C for
various times (0, 1, 3, 4, and 5 h). At each time point, cells
were washed three times with cold PBS and lysates were harvested. The
virions were released from cell lysates by freeze-thawing three times
and were sonicated, and aliquots were removed for plaque assays on
BSC40 cells in the presence of 5 mM IPTG.
Construction of mutant vaccinia viruses defective in A27L and D8L gene expression. Plasmid pSC11-5 was digested with SalI and PstI to release a DNA fragment containing a lacZ gene regulated by the p11K promoter (5). The lacZ cassette was blunt ended and cloned into a filled-in BssHII site of plasmid p770 so that the lacZ cassette was flanked by D8L sequences (26). The lacZ gene is transcribed in the same direction as the D8L gene.
The resulting plasmid was transfected into CV-1 cells and infected with WR32-7/Ind14K at an MOI of 0.1 PFU per cell in the presence of 5 mM IPTG as described previously (37). The lysates were harvested after 3 days, and the viruses were titered on agar containing 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal) (300 µg/ml) and 5 mM IPTG. The recombinant virus that expresses -Gal, D8L WR32-7/Ind14K, was isolated after three
rounds of plaque purification. To facilitate plaque picking, IPTG was
always added to the agar so that large plaques were formed, and
subsequently isolations were repeated until pure plaques were obtained.
To confirm that we did not introduce extra mutations during recombinant
virus isolation, we obtained two independent D8L
WR32-7/Ind14K virus isolates. Viral DNA was purified from these clones,
and restriction digestions were performed to confirm that the
lacZ gene was inserted into the D8L locus. Since these two virus isolates behaved indistinguishably in cell cultures, we present
data obtained from experiments with one isolate.
A27L+ D8L+, A27L+
D8L, A27L D8L+, and
A27L D8L virus stock preparations.
In
order to make virions of four different phenotypes, A27L+
D8L+, A27L+ D8L,
A27L D8L+, and A27L
D8L, BSC40 cells were infected with WR32-7/Ind14K or
D8L WR32-7/Ind14K at an MOI of 0.05 PFU per cell in
duplicate sets. After infection, one set of cells infected by
WR32-7/Ind14K or D8L WR32-7/Ind14K was maintained in
medium with 5 mM IPTG for 3 days, and the cell lysates were harvested
and prepared as A27L+ D8L+ and
A27L+ D8L virus stocks, respectively. Another
set of infected cultures was maintained in medium without IPTG for 3 days and harvested as A27L D8L+ and
A27L D8L virus stocks. All these IMV stocks
were partially purified with 36% sucrose cushions as previously
described (21).
Determination of virion infectivity by confocal microscopy.
IMV virions of A27L+ D8L+, A27L+
D8L, A27L D8L+, and
A27L D8L viruses were sonicated, and virus
stocks were serially diluted. One part of each dilution was used for
plaque determination on BSC40 cells. Another part of the dilution was
spotted onto glass slides and fixed for confocal microscopy analysis
with an antiserum (1:500) against IMV as described previously
(49). Images of fluorescent virion particles were
photographed from three randomly selected fields. The virion particle
numbers were counted, and averages of three fields per sample are
presented in Table 2.
One-step growth curve analysis.
BSC40 cells were infected
with A27L+ D8L+, A27L
D8L+, A27L+ D8L, or
A27L D8L virus, respectively, at an MOI of
5 PFU per cell. Cells were cultured in the presence (for
A27L+ D8L+ and A27L+
D8L viruses) or absence (for A27L
D8L+ and A27L D8L viruses) of 5 mM IPTG and were harvested at various times (0, 2, 4, 8, 19, and
24 h) p.i. Virus IMV titers of each lysate were determined on
BSC40 cells in the presence of 5 mM IPTG.
Electron microscopy of virus morphogenesis.
BSC40 cells were
infected with A27L+ D8L+, A27L
D8L+, A27L+ D8L, or
A27L D8L virus, respectively, at an MOI of
20 PFU per cell. These cells were fixed at 24 h p.i. in 2.5%
glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2) at room
temperature for 1 h and rinsed in three changes of the same buffer
for 15 min each time. Cells were dehydrated with an ethanol series, and
Spurr's resin was used for infiltration and embedding (45).
Sectioning was done with an Ultracut E ultramicrotome. Thin sections of
90 nm were stained with uranyl acetate and lead citrate and were
subsequently analyzed with a Zeiss 902 transmission electron microscope
(33).
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Soluble vaccinia virus D8L protein bound to cell surface CS and blocked IMV binding to cells. The extracellular domain region of D8L protein from amino acid (aa) 1 to aa 264 was cloned into a bacterial expression vector so that the fusion protein, soluble D8L, contains a T7 tag at the N terminus for identification and hexahistidine sequences at the C terminus (Fig. 1A). The construct was expressed in bacteria, and soluble D8L protein was purified through a nickel column (Fig. 1B). This soluble D8L protein was used for generation of a rabbit serum, C8. We obtained another antiserum, D8-1, previously raised from a denatured fusion protein, TrpE-D8L, that contains D8L sequences of aa 77 to 294 (26). Both rabbit sera, C8 and D8-1, were tested for neutralization activity in vaccinia virus infections (Fig. 1C). The C8 serum readily neutralized vaccinia virus infection of BSC40 cells, whereas preimmune and D8-1 sera had no effect. The lack of neutralization ability of D8-1 serum was probably due to the fact that it recognized only denatured epitopes. Thus, the results indicated that the C8 serum recognized a native structure of D8L protein and blocked virus infections.
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Construction of a double-mutant vaccinia virus defective in
expression of both the A27L and the D8L protein.
Mutant viruses
defective in A27L or D8L gene expression have been described previously
(26, 36, 37, 41). A recombinant A27L virus,
WR32-7/Ind14K, that expresses A27L protein under IPTG regulation
exhibited a small-plaque phenotype in the absence of IPTG (36,
37). Despite the difference in the plaque size, the titers of IMV
produced by WR32-7/Ind14K were not affected by IPTG (37).
Recombinant D8L viruses have also been reported, and
these mutant viruses formed normal plaques and exhibited no loss of IMV
infectivity in cell cultures (26, 41). These data indicated
that A27L and D8L proteins are dispensable for IMV growth in cell
cultures. However, since vaccinia virus has two (and perhaps more)
envelope proteins for binding to cell surface GAGs, the contribution of
an individual protein in virus entry is difficult to assess by
single-gene mutation due to functional redundancy. The absence of any
one protein on virions does not eliminate functional virus attachment;
instead, both proteins must be impaired in order to render the virus
defective in binding to cell surface GAGs. Based on the above
rationale, we constructed a mutant virus that is defective in both A27L
and D8L gene expression in order to study the IMV entry process.
D8L+).
Despite the small-plaque phenotype, the A27L
D8L+ virus showed normal growth characteristics, similar to
those of the A27L+ D8L+ virus, with no loss of
IMV infectivity in cell cultures (37).
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(number of
plaques obtained with serum/number of plaques obtained without serum)] × 100. Results shown here are averages from four experiments.
WR32-7/Ind14K, expresses only A27L protein in the
presence of IPTG (A27L+ D8L) and expresses
neither protein (A27L D8L) without IPTG.
Expression of A27L protein was compared in BSC40 cells infected with
WR32-7/Ind14K and D8L WR32-7/Ind14K viruses (Fig. 4B).
Cells infected by either virus expressed A27L protein when IPTG was
added to the medium (Fig. 4B) (37). Cells infected by the
same viruses and cultured in the absence of IPTG expressed very little
A27L protein (Fig. 4B).
Expression of D8L protein was also investigated in these cells.
Full-length D8L protein of 32 kDa was detected in cells infected by the
parental WR32-7/Ind14K virus, which contains an intact D8L gene (Fig.
4B). At the same time, a smaller, truncated D8L protein was detected in
cells infected by D8L WR32-7/Ind14K virus (Fig. 4B).
Because the lacZ cassette was inserted into the
BssHII site of the D8L gene, which is at position 736, the
open reading frame upstream of the BssHII site presumably could encode a protein of 28 kDa. However, this truncated D8L protein
does not contain the transmembrane region located downstream of the
BssHII site and thus would not be expressed on the virion surface. This observation is similar to that described in a previous report when a D8L mutant virus (v-
BssHII), in which
the BssHII site was also used to generate a frameshift of
the D8L gene, expressed a smaller, 28-kDa D8L protein (26).
However, this altered D8L protein was not functional and did not affect
the mutant phenotype as well (26).
To be certain that these viruses indeed exhibit the respective mutant
phenotypes as we expected, we performed neutralization assays with
antisera specific to the A27L and D8L proteins (Fig. 4C). Preimmune
sera had no effect on titers of any of the four viruses (data not
shown). A postimmune serum specific to A27L protein neutralized virions
of the A27L+ D8L+ and A27L+
D8L phenotypes (16). Mutant viruses harvested
in the absence of IPTG did not express A27L protein and thus were
resistant to this serum (A27L D8L+ and
A27L D8L). At the same time, a postimmune
serum specific to D8L protein, C8, readily neutralized
A27L+ D8L+ and A27L
D8L+ viruses. Viruses expressing the truncated D8L protein
(A27L+ D8L and A27L
D8L) were resistant to the C8 serum, as expected. In
contrast to A27L expression, which was regulated by IPTG, the
neutralization effect on D8L protein was independent of IPTG. In
summary, the sensitivities of these viruses to serum neutralization
were consistent with the expression profile of the A27L and D8L
envelope proteins.
Inactivation of D8L protein from vaccinia virus WR32-7/Ind14K
reduced virus titers in cell cultures, and further removal of A27L
protein revealed no additional effects.
To monitor the growth
characteristics of A27L and D8L mutant viruses in cell cultures, BSC40
cells were infected with each of the four viruses at an MOI of 5 PFU
per cell. These infected cells were cultured in appropriate media as
described in Materials and Methods to ensure that virion progenies
maintained their respective phenotypes. The lysates were harvested
after a single round of virus infection and were titrated for IMV
production on BSC40 cells (Fig. 5A). The
titers of A27L+ D8L+ and A27L
D8L+ viruses at 24 h p.i. were comparable, suggesting
that inactivation of A27L protein expression had no effect. This was
consistent with the previous report (37). However, the
titers of A27L+ D8L virus were 1 log unit
lower than those of the A27L+ D8L+ control
virus. This was surprising to us because in previous reports
D8L mutant viruses yielded progeny titers similar to
those of wild-type viruses in cell cultures (26, 41). We
have independently isolated another D8L WR32-7/Ind14K
mutant virus clone, and the results were identical (data not shown).
Thus, it is unlikely that the difference in titers was due to extra
mutations generated during recombinant virus isolation. Finally, the
titers of A27L D8L virus appeared to
overlap those of the A27L+ D8L virus, with no
further attenuation.
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Reduction of A27L+ D8L and
A27L D8L virus IMV titers was not due to a
blockage of the virion assembly process.
There are two possible
explanations for the significant reductions in IMV titers produced by
A27L+ D8L and A27L
D8L viruses. It is possible that A27L+
D8L and A27L D8L mutant
viruses were defective at some stages in cell cultures, such as
morphogenesis. Alternatively, A27L+ D8L
mutant viruses may have grown well but produced virion particles with
lower infectivities than A27L+ D8L+ and
A27L D8L+ viruses. To differentiate between
these two possibilities, we first tested if the titer reduction
resulted from abnormal virion morphogenesis in these cells. Cells
infected by the A27L+ D8L and
A27L D8L mutant viruses at 24 h p.i.
often contained mature IMV virions that appeared compact, with dark
staining (Fig. 5B). Immature virion structures were also observed in
some of these infected cells, but the distribution of these
intermediate virion structures was comparable to those seen in cells
infected by A27L+ D8L+ and A27L
D8L+ viruses (data not shown). Furthermore, IMV virions
were purified from these infected cells and directly analyzed by
electron microscopy. The A27L+ D8L+ virions had
a brick-like shape and a width of roughly 200 nm. The other three
mutant virions, A27L+ D8L, A27L
D8L+, and A27L D8L, also had
comparable shapes and sizes (data not shown). We therefore concluded
that the reductions in the titers of A27L+
D8L and A27L D8L viruses were
not due to IMV assembly defects.
Reductions in A27L+ D8L and
A27L D8L IMV titers were not due to
EEV.
Although it is unlikely, EEV released from the infected cells
could have reinfected the same cultures and influenced the IMV titers
we observed in Fig. 5. We therefore collected the medium at 24 h
p.i. and determined the titers of EEV produced from these infected
cells (Table 1). The EEV titers produced
from cells infected by A27L+ D8L+ and
A27L+ D8L viruses were highest, roughly two-
to threefold higher than those for A27L D8L+
virus and sixfold higher than those for A27L
D8L virus. The reduction in EEV titers from cultures
infected by A27L D8L+ and A27L
D8L viruses was consistent with a role for A27L protein
in EEV wrapping (36). Most importantly, the titers of IMV
from these four cultures did not correlate with the respective titers
of EEV. For example, A27L+ D8L virus produced
high titers of EEV, similar to those with A27L+
D8L+ virus, but the IMV yields were 10-fold lower. We
therefore concluded that the EEV produced from these cultures did not
contribute to the IMV yield difference.
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A27L+ D8L and A27L
D8L viruses produced IMV of low infectivity.
The
above results raised the possibility that the low IMV titers of
A27L+ D8L and A27L
D8L viruses produced in one-step growth analysis could
reflect progeny particles with low infectivity. We directly compared
the infectivities of these mutant IMV virions. All four IMV virions
were purified, and the numbers of virion particles were determined by
particle counting under a confocal microscope (49). The
biological titers of infectious units of these viruses were
simultaneously determined by plaque assays on BSC40 cells. Comparison
of the number of virion particles required to form a plaque provides an
index of infectivity of these IMV virions. We have assigned the control
A27L+ D8L+ virus infectivity a value of 1, and
the relative infectivities of the other three viruses are shown in
Table 2. Analysis by confocal microscopy
showed that A27L+ D8L+ virus, which had higher
biological titers, contained fewer particles. A27L
D8L+ mutant viruses had intermediate infectivity, with a
slight, threefold reduction. A27L+ D8L and
A27L D8L mutant viruses, on the other hand,
contained more virion particles and had the lowest titers. The relative
infectivities of A27L+ D8L and
A27L D8L mutant virions were only 7 to 10%
of that of A27L+ D8L+ virions, i.e., an average
10- to 14-fold reduction in virion infectivity. These results suggested
that D8L protein acts as an important determinant for virion
infectivity.
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D8L protein affects the infectivity of WR32-7/Ind14K IMV at the
cell binding step.
To investigate the mechanism that renders these
IMV particles less infectious, we performed virion binding assays with
these mutant viruses (Fig. 6). The
abilities of these mutant viruses to bind to cells differed. Both
A27L+ D8L+ and A27L
D8L+ virions bound to cells to a comparable extent.
However, they both bound to cells better than A27L+
D8L and A27L D8L virions. The
binding data were all consistent with the infectivity assay results and
indicated an important role of D8L protein in virion adsorption. There
was a slight increase in virion binding to cells when A27L protein was
expressed on virions, but the difference was not significant,
consistent with all previous data (34, 35). In summary, the
data suggested that the loss of D8L protein reduced virion adsorption
to cells and, consequently, resulted in virions of low infectivity.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The amino acid sequences of D8L protein show homology with
cellular carbonic anhydrase, although D8L protein does not contain any
detectable enzymatic activity (24). In addition, a region with short homology to the attachment glycoprotein VP7 of rotaviruses exists on D8L protein near the C-terminal transmembrane region (26). However, the significance of these homologies is not
clear. In addition, D8L protein is dispensable for cell culture, since D8L mutant virus forms plaques of normal size (26,
41). These data indicated that D8L protein plays no important
role in cell culture systems.
In the present study, we found that vaccinia virus D8L protein binds to cell surface CS and is involved in virus entry. The evidence came from several experiments. First of all, binding of soluble D8L protein to cells was competed by CS. In addition, D8L protein bound poorly to mutant cells defective in CS expression. Furthermore, soluble D8L protein blocked the binding of vaccinia virus to cells. Finally, inactivation of D8L expression by vaccinia virus WR32-7/Ind14K resulted in a 10-fold reduction in IMV titers in cell culture growth. The reduction in IMV titers was a specific effect of D8L protein, since removal of A27L protein expression by withdrawal of IPTG from the culture medium did not change IMV titers.
In one-step growth experiments as described above, BSC40 cells were
infected with each of four mutant viruses at identical MOIs of 5 PFU
per cell so that the cell populations in all four situations were
equally infected, regardless of how many particles were initially put
on cells at the beginning of infection. After entering cells, all four
mutant viruses finished one round of the life cycle and produced
comparable amounts of IMV progeny particles. When these particles were
harvested and titered on BSC40 cells, the resulting plaques of
D8L WR32-7/Ind14K viruses, including A27L+
D8L and A27L D8L viruses,
were 10 times fewer than those of A27L+ D8L+
and A27L D8L+ WR32-7/Ind14K viruses,
suggesting that D8L WR32-7/Ind14K virus required more
particles to initiate a plaque formation. Indeed, this interpretation
was confirmed by the infectivity analysis for which results are shown
in Table 2, which demonstrated that D8L WR32-7/Ind14K
mutant viruses were 10 times less infectious than their
D8L+ counterparts. In summary, these results revealed that
D8L protein is important for IMV virion infectivity in BSC40 cells.
Since GAGs are ubiquitously expressed in vitro and in vivo, we may
expect that the results obtained with BSC40 cells will be applicable to
other cell lines.
We demonstrated that the low infectivity of D8L
WR32-7/Ind14K IMV virions was partly due to inefficient binding of
virions to BSC40 cells. Since we detected only a threefold difference in virion binding, these mutant virions may have additional defects in
postbinding fusion that account for the remainder of the infectivity loss. Resolution of this issue awaits further investigation.
The effect of D8L protein on IMV virion infectivity that we obtained is
different from the findings of previous studies, which indicated that
inactivation of D8L protein had no effect on IMV infectivity (26,
41). Previous studies introduced a frameshift mutation into the
open reading frame of the D8L gene, whereas we inserted a
lacZ cassette into the D8L locus. The direction of
lacZ transcription was the same as that of the endogenous
D8L gene in order to allow minimal perturbation of gene alignment on
the viral genome. It is possible that insertion of the lacZ cassette somehow exerted a polar effect on the expression of other genes adjacent to the D8L locus, such as D7R and D9R (11).
However, D7R encodes a subunit of viral RNA polymerase, and a mutation of D7R was reported to be lethal, which does not explain the phenotype of our D8L mutants (7). Thus, we think it
unlikely that the phenotype difference was due to technical problems.
Alternatively, the phenotype difference could be due to genetic
deletions of virus genomes. Previous D8L mutants were
generated from wild-type vaccinia virus, whereas the D8L
mutant we worked with was from a different parental virus,
WR32-7/Ind14K (36, 37). WR32-7/Ind14K was derived from
vaccinia virus 48-7, which was reported to have some deletions in the
left end of the viral genome (29, 36). Initially, we were
not concerned about the difference because the published results
indicated that the deleted region was nonessential for vaccinia virus
growth in cell cultures (28, 29). In addition, the growth
characteristics and A27L expression of WR32-7/Ind14K virus were
indistinguishable from those of the wild-type virus (36,
37). Only after we inactivated expression of the D8L gene of
WR32-7/Ind14K virus did we find that the resulting D8L
mutant behaved differently from the same D8L mutant built
on a wild-type virus background. This surprising result raised the
interesting possibility that the deleted viral genomic sequences in
WR32-7/Ind14K encode a protein that also binds to CS. This hypothesis
of gene redundancy would posit that the D8L mutant
generated from the wild-type virus backbone remained fully infectious
because of functional complementation. However, when these sequences
were deleted from the WR32-7/Ind14K virus, inactivation of D8L
expression seriously impaired virus infectivity, since no other gene
could compensate for D8L functions. Similar observations were reported
for the HS-binding proteins, gB and gC, on HSV (4, 14). gC
is the principal HS-binding protein on HSV-1, whereas binding of the
virus to cells became gB dependent only when gC was inactivated
(4). Another possible mechanism of gene redundancy is that
the deleted region encodes an HS-binding protein that normally binds
more strongly to cells and "masks" CS interactions. This is based
on previous studies of HSV-1 that bound to cell surface GAGs; binding
of HSV through CS became evident only when the HS was removed
(3). Regardless which mechanism is operative, D8L WR32-7/Ind14K mutant virus revealed a previously
unknown situation in which D8L protein becomes important in vaccinia
virus entry. Currently, boundaries of these deletions in WR32-7/Ind14K
are not molecularly defined, and at least the HindIII C
fragment appeared to be missing (data not shown). Precise mapping of
the deleted regions and identification of viral genes within those
regions will help us clarify the difference in the future.
Even with a 10-fold drop in IMV infectivity, the D8L
WR32-7/Ind14K mutant viruses (both A27L+ D8L
and A27L D8L phenotypes) were nevertheless
viable. This finding indicated that, besides A27L and D8L proteins,
other viral proteins exist to mediate virus-cell interactions. Other
viruses, such as HSV, are known to contain two proteins, gB and gC, for
GAG binding (15, 25, 44, 46). Considering the broad host
range of vaccinia virus, it would not be surprising to discover that
vaccinia virus has evolved multiple envelope proteins for interaction
with different GAGs on cells, including HS and CS.
In summary, for the understanding of IMV entry into mammalian cells, identification of viral and cellular components important for vaccinia virus binding and fusion is only the beginning. Further exploration of the biochemical interactions of these molecules will allow us to discover the mechanisms and obtain new insights into vaccinia virus entry.
| |
ACKNOWLEDGMENTS |
|---|
We thank E. Nile for plasmid p770, rabbit serum D8-1, and helpful suggestions on our manuscript; G. Smith for the virus WR32-7/Ind14K; and F. Tufaro for L, gro2C, and sog9 cells. We also thank Sue-Ping Lee for excellent techniques in electron microscopy.
This work is supported by grants from Academia Sinica and the National Science Council (NSC88-2311-B-001-103) of the Republic of China.
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Institute of Molecular Biology, Academia Sinica, #128 Sec. 2, Yen Chiu Yuan Rd., Nankang, Taipei 11529, Taiwan, Republic of China. Phone: 886-2-2789-9230. Fax: 886-2-2782-6085. E-mail: mbwen{at}ccvax.sinica.edu.tw.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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