Entry of Herpes Simplex Virus 1 in BJ Cells That Constitutively Express Viral Glycoprotein D Is by Endocytosis and Results in Degradation of the Virus
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JOURNAL OF VIROLOGY, Jan. 1988, p. 159-167 Vol. 62, No. 1
0022-538X/88/010159-09$02.00/0
Copyright © 1988, American Society for Microbiology
Entry of Herpes Simplex Virus 1 in BJ Cells That Constitutively
Express Viral Glycoprotein D Is by Endocytosis and Results in
Degradation of the Virus
G. CAMPADELLI-FIUME,l M. ARSENAKIS,2 F. FARABEGOLI,3 AND B. ROIZMAN2*
Section on Microbiology and Virology1 and Section on General Pathology,3 Department of Experimental Pathology, the
University of Bologna, Bologna, Italy, and the Marjorie B. Kovler Viral Oncology Laboratories, the University of
Chicago, Chicago, Illinois 606372
Received 28 July 1987/Accepted 23 September 1987
The BJ cell line which constitutively expresses herpes simplex virus 1 glycoprotein D is resistant to infection
with herpes simplex viruses. Analysis of clonal lines indicated that resistance to superinfecting virus correlates
with the expression of glycoprotein D. Resistance was not due to a failure of attachment to cells, since the
superinfecting virus adsorbed to the BJ cells. Electron microscopic studies showed that the virions are
juxtaposed to coated pits and are then taken up into endocytic vesicles. The virus particles contained in the
vesicles were in various stages of degradation. Viral DNA that reached the nucleus was present in fewer copies
per BJ cell than that in the parental BHKtk- cells infected at the same multiplicity. Moreover, unlike the viral
DNA in BHKtk- cells which was amplified, that in BJ cells decreased in copy number. The results suggest that
the glycoprotein D expressed in the BJ cell line interfered with fusion of the virion envelope with the plasma
membrane but not with the adsorption of the virus to cells and that the viral proteins that mediate adsorption
to and fusion of membranes appear to be distinct.
The initial interaction between viruses and cells takes the steps can be done by different proteins and if the attachment
form of binding of one or more viral surface proteins to a and fusion can each be blocked by antibody.
specific receptor on the surfaces of cells. The entry of The process of fusion of the virion envelope with the
enveloped viruses into cells requires the fusion of the virion plasma membranes of susceptible cells has as its model a
envelope with the plasma membrane, either at the cell mutation (syn) which causes infected cells to form polykar-
surface or in endocytic vesicles (for reviews, see references yocytes by fusion of plasma membranes of adjacent cells.
30, 47, and 51). In the case of herpes simplex virus 1 The use of this model system to identify the viral proteins
(HSV-1), electron microscopic studies have suggested that required for the fusion of the envelope with the plasma
both mechanisms of entry may be operative. Thus, early membrane presents three problems. Foremost, wild-type
studies have drawn attention to the presence of both en- viruses, while entering cells by fusion of their envelopes with
veloped virus particles in vesicles and of unenveloped cap- plasma membranes, do not fuse the plasma membranes of
sids in the cytoplasm. These studies have suggested that the infected cells, suggesting that this manifestation of viral
infective virus particles are released from endocytotic vesi- membrane proteins is specifically blocked in wild-type virus
cles. Subsequent studies demonstrating the fusion of the infection. Second, several loci which can mutate to yield
envelope with the plasma membrane have raised questions viruses with syn phenotype have been noted. These loci map
regarding the role of endocytosis of enveloped virus in the in physical domains of glycoprotein genes as well as in
process of penetration of virus leading to productive infec- domains in which no membrane protein gene has yet been
tion (for a review, see reference 42). In this report we mapped (13, 23, 44). One interpretation of this observation is
demonstrate that in cells expressing the HSV-1 glycoprotein that fusion is mediated by the structural malformation of a
D (gD1) endocytosis of enveloped virus particles occurs but complex as a consequence of mutations in any one of the
the productive infection does not ensue. several components of the complex (44). Even if polykar-
Relevant to this report is the role of HSV-1 proteins in the yocytosis were a valid model of fusion-mediated entry of
process of entry of the virus into susceptible cells. HSV-1 is virus into cells, the assignment of the fusion function could
known to specify at least seven surface proteins that are
glycosylated and designated glycoproteins (g) B, C, D, E, G, not be made on the basis of studies with the entire complex.
H, and I (1, 7, 9, 25, 29, 41, 43, 45, 46). Another set of The possibility that more than one viral membrane protein is
nonglycosylated membrane proteins has been postulated to involved in the fusion process also emerges from the obser-
exist, but none has been identified to date. The function of vation that antibody to at least three glycoproteins, gD (18),
the glycoproteins in the two critical steps of entry, i.e., gH (19), and gE (S. Chatterjee and R. J. Whitley, submitted
attachment and fusion of the viral envelope with the plasma for publication), have been shown to be able to block fusion
membrane, has not been identified. Inherent to the problem of the plasma membranes of infected cells. Last, the large
of assigning functions is the evidence that antibody to number of viral glycoproteins identified to date raises the
several of the glycoproteins (e.g., gD, gB, gH, and gC [19, question whether at least some of the viral glycoproteins are
34, 37]) neutralizes virus, but the value of neutralization in functionally redundant, at least for cells in culture. Thus, of
the assignment of function becomes less significant if both the seven glycosylated proteins, four (gC, gE, gG, and gI,
[25-27]) have been shown not to be required for infection,
maturation, release, and spread of virus from cell to cell.
*
Corresponding author. The failure to assign specific functions to each glycopro-
159160 CAMPADELLI-FIUME ET AL. J. VIROL.
tein raises two hypotheses. The critical complex hypothesis Fd69 for gI (25) and allowed to react for 3 h at 4°C. The
is that each of the components in a complex performs a immune precipitates were collected with protein A-Sepha-
unique function required for either attachment or fusion. The rose beads (Sigma) washed extensively with buffer A and
functional redundancy hypothesis is that the functionally finally with 0.015 M NaCl in 0.05 M Tris hydrochloride, pH
analogous step can be carried out by more than one viral 7.0, and released from the beads by being boiled for 5 min in
protein, but in different cells. Since conditional lethal muta- disruption buffer. Samples were subjected to electrophoresis
tions have been reported so far in only two glycoprotein in 8.5% acrylamide gels cross-linked with N-N'-diallyltartar-
genes (gB and gH [19, 28, 50]), one approach to assignment diamide, as previously described (32). Gels were fixed in
of functions to specific glycoproteins is to analyze the 10% acetic acid and 20% isopropanol and soaked for 15 min
process of infection in cells which constitutively express in Amplify (Radiochemical Center). Dried gels were exposed
individual or selected sets of the viral glycoproteins. In the to X-Omat film (Eastman Kodak Co., Rochester, N.Y.) for
accompanying article (2), we report on the construction of a fluorography.
cell line which constitutively expresses gDl. In this report Adsorption of HSV. To measure the adsorption of infec-
we describe studies on the infection of these cells with tious HSV-1(F) to cells, monolayers of BJ clonal lines in
HSV-1 and HSV-2. 50-cm2 glass flasks were infected with 10,000 PFU of HSV-
1(F) in 2 ml of medium. At 0, 30, 60, and 90 min, triplicate
MATERIALS AND METHODS samples of the medium of 50 RI1 each were withdrawn and
immediately plated on Vero cells for plaque assay. To
Viruses and cells. HSV-1(F) and HSV-2(G), the prototype measure the adsorption of [3H]thymidine-labeled HSV-1(F),
wild-type viruses used in our laboratories, have been previ- triplicate monolayers of BJ clonal lines in 24-well dishes
ously described (17). The BHKtk- cells (line B-1, were infected in the presence of 1 mM thymidine with
GM0348A; N.1.G.5. Human Genetic Mutant Cell Reposi- Dextran T10 gradient-purified [3H]thymidine-labeled HSV-
tory) were grown in Dulbecco modified Eagle medium 1(F) at an input multiplicity of 20 PFU per cell and a total of
supplemented with 10% fetal bovine serum. The BJ cell line, 105 cpm. At 0, 30, 60, and 90 min, 25 ,ul of duplicate medium
described in the accompanying article (2), was grown in the samples was withdrawn and assayed for residual radioactiv-
same medium supplemented with 440 nM methotrexate. The ity.
30 clonal cell lines derived from the BJ cell line were cloned Treatment of cells with cycloheximide and PAA. Cells were
by serial dilution c' the parental cells. exposed to cycloheximide (100 ,ug/ml) or phosphonoacetic
Solutions and buffers. Disruption buffer consisted of 0.05 acid (PAA) (300 ,ug/ml) 1 h before exposure to virus. The
M Tris hydrochloride (pH 7.0)-8.5% (wt/vol) sucrose-5% infected cells were maintained in the same drug concentra-
P-mercaptoethanol-2% (wt/vol) sodium dodecyl sulfate and tions until they were harvested.
was supplemented with 10-4 M Na-p-tosyl-L-lysine chloro- Determination of HSV-1 genome copy number. Infected or
methyl ketone (Sigma Chemical Co., St. Louis, Mo.) and mock-infected cells were rinsed three times with ice-cold
10-4 M L-1-tosylamide 2-phenylmethyl chloromethyl ketone phosphate-buffered saline containing Mg2 + and Ca2,
(Sigma). Phosphate-buffered saline consisted of 8.2 mM scraped off the flasks, pelleted, and allowed to swell for 10
Na2HPO4, 1.5 mM KH2PO4, 0.14 M NaCl, and 2.5 mM KCI. min in reticulocyte standard buffer. They were disrupted
Reticulocyte standard buffer consisted of 0.01 M Tris hydro- with 10 strokes of a Dounce homogenizer, and Nonidet P-40
chloride (pH 7.4), 0.01 M KCl, and 0.0015 M MgCl2. Buffer was added to yield a final concentration of 1% (vol/vol).
A consisted of 0.01 M Tris hydrochloride, pH 7.4, containing After 15 min at 4°C nuclei were pelleted by 6 min of
1% (vol/vol) Nonidet P-40, 1% (wt/vol) sodium deoxycho- centrifugation at 750 x g, suspended in 1 mM phosphate
late, and 10-4 M each of Na-p-tosyl-L-lysine chloromethyl buffer (pH 7.2) containing 0.25 M sucrose, and centrifuged
ketone and L-1-tosylamide 2-phenylmethyl chloromethyl ke- through a cushion of 1 M sucrose in the same buffer. Pelleted
tone. nuclei were suspended in phosphate-buffered saline. DNA
Infection and radiolabeling of cells. Cells were infected was isolated from the nuclear preparations by lysis in 0.5%
with HSV-1(F) or HSV-2(G) at an input multiplicity of 10 Nonidet P-40 in 10 mM Tris hydrochloride, pH 7.6, plus 1
PFU per cell, except when otherwise stated. To prepare mM EDTA and subsequent treatment with 0.1 mg of RNase
[3H]thymidine-labeled HSV-1(F), BHK cells grown in roller A (Sigma) per ml, followed by digestion with 0.5 mg of
bottles were infected with 5 PFU of HSV-1(F) per cell and proteinase K (Sigma) per ml in 0.5% sodium dodecyl sulfate
were labeled from 6 to 18-24 h postinfection in medium for 2 h at 37°C. The samples were then extracted twice with
containing [3H]methyl-thymidine (200 p.Ci/ml; Radiochemi- phenol equilibrated in 10 mM Tris hydrochloride, pH 7.6,
cal Center, Amersham, United Kingdom; specific activity, and 1 mM EDTA, followed by three extractions with water-
40 to 60 Ci/mmol, 1.5 to 2.2 TBq/mmol). The virus was saturated ether to remove residual phenol. The DNA was
purified in Dextran T10 gradients from the cytoplasm of ethanol precipitated and quantitated spectrophotometrically
infected cells, as described previously (48). For labeling of at 260 nm. For copy number determination, 10 pg of cell
proteins, infected or uninfected cells were labeled for 10 to DNA was digested with BamHI and Sall restriction endo-
12 h from the time of infection or mock infection in a medium nucleases. Reconstructions were done by hybridizing the
containing 1/10 the normal concentration of methionine and 6,050-base-pair BamHI-SaII subfragment of the HSV-1
50 to 100 p.Ci of [35S]methionine per ml of medium (specific BamHI G fragment cloned in pRB2017 (35) and labeled by
activity, 37 TBq/mmol; Radiochemical Center). nick translation with [32P]dCTP to a BamHI-SalI digest of
Immune precipitation, polyacrylamide gel electrophoresis, mixtures of 10 pLg of untransformed BHKtk- cell DNA and
and autoradiography. Infected or mock-infected cells were appropriate amounts of pRB2017 DNA electrophoretically
disrupted in buffer A, sonicated, and then clarified from separated in an 0.9% agarose gel and transferred to nitrocel-
particulate matter by centrifugation at 100,000 x g for 60 lulose. The conditions for hybridization were as previously
min. The supernatant fluids were mixed with H1380, a described (39).
monoclonal antibody type specific for gDl, or with mono- Electron microscopy. BJ clonal lines were infected with
clonal antibody H233 for gB, H600.1 for gEl (11, 37), or HSV-1(F), 100 or 500 PFU per cell at 37 or 4°C, respectively.VOL. 62, 1988 ENTRY OF HSV INTO CELLS THAT EXPRESS gD 161
At indicated times the cells were rinsed with phosphate- BJ clone i I k h I n 01 p I r I m
buffered saline containing Ca2 + and Mg2 + and fixed with 2% HSV-2 - + - + - t - + - + -- + + -
para-formaldehyde and 2% glutaraldehyde in Sorensen M.Ab. 1380 233 1380 233 1380 233 138D 233 1380 233 1380 233 1380 233 1380 233 1380 233
buffer, postfixed with osmium tetraoxide, dehydrated in gs -
ethanol, washed in propylene oxide, embedded in Epon, thin
sectioned, stained with uranyl acetate and lead citrate, and
examined in a Siemens 102 electron microscope.
RESULTS
FIG. 2. Autoradiographic image of electrophoretically separated
Selection and properties of BJ clonal cell lines. The accom- gDl and gB2 immune precipitated from BJ clonal cell lines, either
panying article reports that the BJ cell line constructed by uninfected (-) or infected with HSV-2(G) (+). The cells were
transfection of the BamHI J DNA fragment into BHKtk- labeled with [35S]methionine for 12 h from the end of mock or virus
cells expressed gD constitutively and was resistant to infec- adsorption. gDl was immune precipitated from uninfected cells with
tion by HSV-1 or HSV-2 (2). To study this phenomenon monoclonal antibody H1380. gB was immune precipitated from
HSV-2(G) infected clonal lines with monoclonal antibody H233.
further, 30 clonal cell lines were established from the paren-
tal BJ cells. To characterize these clones with respect to the
expression of the resident HSV-1 DNA fragment, each of the
clonal lines was exposed to HSV-2(G) and labeled with entry and gene expression were noted in cell lines BJ-j, BJ-o,
[35S]methionine from the end of the adsorption period until BJ-h, and BJ-p, which produced relatively small amounts of
the cells were harvested at 12 h postinfection. The constitu- gDl. Conversely, gB2 was not detected in cell lines BJ-l and
tive and induced expression of gDl encoded by the resident BJ-m, which produced relatively higher amounts of gDl.
BamHI J DNA fragment was measured by immune precipi- The experiments described below were done with the BJ-o,
tation of the glycoprotein from lysates of mock-infected and BJ-h, and BJ-l clonal cell lines, representing examples of
infected cells with monoclonal antibody H1380 specific for semipermissive and nonpermissive cell lines.
gDl. As illustrated in Fig. 1 with representative cell lines, all Assay of clonal cell lines for expression of gE and gI. Studies
30 clonal cell lines tested expressed gDl constitutively, but described in the accompanying article (2) show that the
the level of expression varied. After infection with HSV- parental BJ cells transcribe the truncated genes located at
2(G), the expression of gDl either remained constant or the extreme ends of the BamHI J fragment, i.e., the 3'
decreased. Rounding of cells by the superinfecting virus was domain of the US3 open reading frame specifying a protein
not detectable for all cell lines (data not shown). kinase and the 5' domain of US8 specifying glycoprotein E
Expression of HSV-2 gB gene in superinfected BJ cells. The (gEl) (31, 40). On the basis of studies of the deletion
objective of these experiments was to determine whether the mutants, the product, if any, of the truncated US3 gene
different clonal lines varied in their ability to support HSV-2 would not have protein kinase activity (40). The product of
infection and whether this feature correlated with extent of the truncated gE gene was not detected in immune precipi-
gDl expression. In these experiments, replicate cultures of tation tests with monoclonal antibody H600. This monoclo-
each of the clonal cell lines were mock infected or infected nal antibody reacts with the truncated gE protein induced by
with HSV-2(G) and labeled with [35S]methionine from the infection of the at4/BJ cell line (2). Furthermore, although
end of the adsorption period until the cells were harvested at transcripts of the US7 open reading frame (31) had not been
12 h postinfection. The expression of the resident gDl gene detected in BJ cells (2), since the completion of that study it
and that of HSV-2(G) virus was monitored by immune has been shown that US7 specifies gIl (25). Inasmuch as the
precipitation of gDl from the lysates of mock-infected cells clonal cell line derivatives differed significantly in the
and that of the HSV-2 gB (gB2) from the infected cell lysates amounts of gD they produced, it was of interest to determine
with monoclonal antibodies H1380 and H233, respectively. whether these cell lines expressed the products of the
The choice of gB2 synthesis as an indicator of HSV-2 gene truncated gEl or gIl. In these experiments, BHKtk- and the
expression was made on the basis of the observation re- clonal derivatives BJ-j, BJ-h, BJ-l, BJ-m, and BJ-o cell lines
ported in the accompanying article that virus-specific pro- were mock infected or infected with HSV-1(F) or HSV-2(G)
teins were barely visible on the background of continued and labeled with [35S]methionine from the end of the adsorp-
cellular protein synthesis in the infected BJ cells (2). The tion period until the cells were harvested at 12 h postinfec-
results of representative immune precipitations shown in tion. The autoradiographic images of the electrophoretically
Fig. 2 indicate that the production of gB2 was inversely separated immune precipitates illustrated in part in Fig. 3
related to the expression of the resident gDl gene. For indicated that none of the clones tested expressed detectable
example, moderate amounts of gB2 indicative of successful quantities of gIl or gE2, consistent with the previous stud-
ies.
Effect of increasing the multiplicity of infection on permis-
BJ o10 e b h dm | sivity of BJ cells. The parental BHKtk- and the clonal
Y 2
HSV- - + +
derivatives BJ-j, BJ-k, BJ-o, and BJ-l cell lines were infected
with HSV-2(G) at multiplicities of infection of 0, 3, 10, 30, or
gD 100 PFU per cell and labeled with [35S]methionine from the
end of the adsorption until the cells were harvested at 10 h
FIG. 1. Autoradiographic image of electrophoretically separated after infection. Cell monolayers in replicate cultures from
gDl immune precipitated from uninfected (-) or HSV-2(G)-infected the same experiment were reacted with monoclonal antibody
(+) clonal cell lines derived from the BJ cell line. The cells were H233 and stained with avidin-biotin-amplified immunoper-
labeled with [35S]methionine from the end of mock or virus adsorp- oxidase (3). The parental cell line produced the highest
tion for 12 h. Immune precipitation was performed with monoclonal amounts of gB2 at the lower multiplicities and drastically
antibody H1380 specific for gDl. decreased amounts at the highest multiplicity tested (100162 CAMPADELLI-FIUME ET AL. J. VIROL.
flasks were exposed to 2 ml of medium containing 104 PFU
HSV-HV2
2 BHK 0-
B-i Bi-k
Jk0- BJ-o B-l
plu ,l-. 0 10 30
|
100 0 3 10 30 100
|
D
|
3
|
30 0 10 30 100 0 10 30 100
of HSV-1(F). The amount of residual virus was measured by
independently titrating in Vero cell cultures three 50-,u
9 B- *3 4 portions removed from the inocula at 0, 30, 60, and 90 min
pgB'
postexposure of the virus tW6cells. In the second experiment,
r3tvt- 3 .3 .3ag3 - 1 ,1+2,* 3+ - f+ 2|l+3+1 - - f as
three replicate cultures of each of the cell lines in 24-well
dishes were each exposed to 20 PFU per cell of a total of 105
FIG. 3. Autoradiographic image of electrophoretically separated cpm of [3H]thymidine-labeled HSV-1(F) purified in a Dex-
gB immune precipitated from BJ clonal lines j, k, o, and 1, infected tran T10 gradient. The residual labeled virus was measured
with HSV-2(G) at increasing multiplicities of infection. Cells were in portions of the inocula removed at 0, 30, 60, and 90 min
infected with HSV-2(G) at the indicated multiplicities (PFU per cell) postexposure. Both the infectivity and the radioactivity were
and labeled with [35S]methionine for 12 h starting at the end of virus removed from the inoculum by all three cell lines (Fig. 5).
adsorption. gB was immune precipitated with monoclonal antibody The rates of removal of radioactivity and of infectious virus
H233. The cell monolayers were reacted with monoclonal antibody
H233 and stained with avidin-biotin-amplified immunoperoxidase. for each of the three cell lines were not significantly dif-
Immunoreactive cells were scored under a light microscope. The
scores of -, 1+, 2+, and 3+ indicate that the percentage of
immunoreactive cells was 0 to 10%t, 10 to 30%o, 30 to 60%o, and 60 to Time after exposure tovirus
90%, respectively.
(min)
PFU per cell) (Fig. 4). The amounts of gB2 produced by the
clonal derivatives of the BJ cell lines, with the exception of
that of BJ-l line, were related to the multiplicity of infection
and yielded significant amounts of the protein only at the
highest multiplicity. Moreover, there was a correlation be-
tween the amount of gB2 detected in these cells and the
relative fraction of cells producing gB2. The exception, the
BJ-I cell line, produced little or no detectable gB at any of the
multiplicities tested. These results indicate that the nonper-
missivity could be overcome by increasing the multiplicity of
infection but that, even in clonal cell lines that did express 0
viral genes, the amounts of the indicator gene product (gB2) EQ
I
0
were lower than those made in the parental BHKtk- cells. CL
01
Entry and fate of HSV-1(F) in BJ-I and BJ-o clonal cell lines: 0
adsorption of HSV-1(F) to BJ clonal cell lines. The hypothesis :0 _
that the failure to detect normal levels of the gene products a 'C
of the superinfecting virus in BJ-l clonal cell line was due to 'U
a failure of te virus to adsorb to these cells was tested in 'a
two series of experiments. Both experiments measured the 0
. 4,
rate of disappearance of virus from the inocula. a
In the first experiment, monolayers of the parental 0
0
BHKtk and clonal BJ-I and BJ-o cell lines in 50-cm2 glass CL
zo
la
0
e C
U
(A
A I C S. 0C
e
I nock HSV-1 l HSV- 2 oclk HSV-2 mock FI
HV-2
m0lse9D gE g_OD 9E g 9D GE gI gE gi gD Ig gi gE gE 91 gD| gB 901 Q a.
so
0
40
50-
690
0
a~- .
46
32_
FIG. 5. The fraction of infectious and [3H]thymidine-labeled
HSV-1(F) remaining in the inoculum as a function of time after
FIG. 4. Autoradiographic image of electrophoretically separated exposure of the parental BHKtk- (top panel), BJ-o (middle panel),
glycoproteins immune precipitated from lysates of BHKtk- cells or BJ-1 (lower panel) cells to unlabeled or labeled virus. The studies
and of clonal lines BJ-h and BJ-o. (A) BHKtk- cells mock infected on the adsorption of infectious virus were done in 50-cm2 dishes,
and infected with HSV-1(F) or HSV-2(G). Leftmost lane, Labeled and each point represents the average of residual infectivity in three
molecular weight (M.W.) markers, 103. (B) Mock-infected and independently assayed samples removed from the inoculum. In the
HSV-2(G)-infected clonal line Bj-h. (C) Mock-infected and HSV- studies on the adsorption of radioactive virus, each point represents
2(G)-infected BJ-o. gIl, gDl, gEl, and gB2 were immune precip- the average radioactivity in two samples removed from each of the
itated with monoclonal antibodies Fd69, HD1, H600, and H233, three replicate monolayer cultures in 24-well dishes and assayed
respectively. independently.VOL. 62, 1988 ENTRY OF HSV INTO CELLS THAT EXPRESS gD 163
ferent. These results do not support the hypothesis that (38). This suggests either that the DNA of the virus accessing
HSV-1(F) fails to adsorb to the BJ-l clonal cell line. the nucleus of the BJ-l cells was damaged or that the virion
Fate of viral DNA in BHKtk- and BJ clonal cell lines. The proteins (e.g., the virion component responsible for the
objectives of these studies was to measure the amount of induction of a genes) required for efficient expression of
viral DNA retained in nuclei of untreated, cycloheximide-, HSV functions (6, 36, 39) were degraded or unavailable.
or PAA-treated cells exposed to virus for 90 min at 37°C and Electron microscopic studies of the fate of infecting virus in
incubated for 1 or 5 h further in the presence or absence of BHKtk- and BJ cells. Two series of experiments were done.
the drugs. The DNA extracted from the nuclear fraction, as In the first, HSV-1(F) was adsorbed to BHKtk and BJ-o cells
described in Materials and Methods, was digested with at 4°C. The infected cells were then shifted up to 37°C and
BamHI and SalI and hybridized with a 32P-labeled 6,050- fixed either 5 or 20 min after shift up. In the second series,
base-pair BamHI-SalI subfragment from the HSV-1(F) the cells were infected at 37°C and fixed at 2 h postexposure
BamHI G DNA fragment. The results of these studies (Fig. to virus. The electron micrographs shown in Fig. 7 and 8
6) indicate the following. (i) Although the cells were infected illustrate the following. (i) The virus particles seen in
at equal multiplicities, the viral DNA copy number in BHKtk- cells early after shift up to 37°C were in three
BHKtk- cells at 1 h postinfection was significantly higher locations, i.e., enveloped virus particles juxtaposed to the
than that in BJ-l or BJ-o cells. (ii) At 5 h after infection there plasma membrane, unenveloped virus particles singly or in
was a dramatic increase in the viral DNA copy number in small clusters in vesicles, and unenveloped virus particles
BHKtk- cells, reflecting ongoing synthesis of viral DNA. scattered in the cytoplasm (Fig. 7A and B). At 2 h postin-
Conversely, there was a decrease in the viral DNA copy fection of BHKtk- cells, capsids devoid of DNA were
number in both BJ-1- and BJ-o-infected cell lines relative to present in juxtaposition to nuclear pores (Fig. 8A and B), as
the copy number detected at 1 h postinfection. (iii) A previously described (5). Occasionally, a few viral particles
dramatic decrease in the copy number of viral DNA was both with and without DNA were found in vesicles, but
seen in BHKtk- cells treated with PAA or cycloheximide, these were usually small and contained one to two enveloped
consistent with earlier observations (38) that HSV DNA may virus particles (Fig. 8C). (ii) Early after shift up of the BJ
undergo degradation in cells maintained in the presence of cells to 37°C, enveloped virus particles were found in asso-
inhibitors. These studies indicate that (i) the amount of DNA ciation with plasma membranes, particularly at coated pits
accessing the nucleus was smaller in the clonal derivatives of (Fig. 7C to E). The dominant feature of the BJ cells at 2 h
the BJ cells than in the parental BHKtk- cells exposed to postinfection was the presence of numerous vesicles show-
the same multiplicities of infection. (ii) The DNA sequences ing in cross section one to an average of five to six virus
reaching the nucleus of the clonal BJ-1 and BJ-o cell lines particles with apparently intact as well as partially degraded
were unstable and were further degraded, in a manner virus particles (Fig. 8D). Empty capsids were not seen at the
analogous to that of viral DNA in cells treated with inhibitors nuclear pores.
DISCUSSION
HSV-1(F)-infected cells
B H K B B J-o Reconstruction
The salient features of the studies presented in this report
hours p.i. 1 5 5 5 1 5 1 5 ceopies percell
are that cells expressing gDl take up infectious virus into
treatment -
Cyclo PAA
genome endocytotic vesicles but that productive infection does not
3 10 30
ensue. The sequence of events apparent from our studies is
that HSV attaches efficiently to BJ cell surfaces and is taken
up by endocytosis into vesicles, wherein it appears to be
degraded. Concomitantly, the DNA of the entering virus that
reaches the nucleus is small in amount and decreases with
time. The resemblance of the endocytosed particles seen in
this study very early after exposure of the virus to cells to
the enveloped particles contained in vesicles and reported in
earlier articles tends to indicate that the entry of the virus by
way of endocytosis leads to abortive infection. The obser-
vations reported here raise two issues, i.e., (i) the role of the
gD encoded by the HSV-1 BamHI J fragment in causing the
infection to abort in the clonal lines of BJ cells and (ii) the
significance of the finding of enveloped virus particles in
FIG. 6. Autoradiographic image of BamHI-Sall digests of DNAs
cytoplasmic vesicles with respect to the entry of the virus
extracted from BHKtk-, BJ-1, and BJ-o cells infected with HSV-
into cells.
1(F) and of plasmid pRB2O17 electrophoretically separated in an Nonpermissivity of BJ cell lines correlates with expression of
agarose gel, transferred to nitrocellulose, and hybridized with a gD encoded by the resident HSV-1 BamHI J DNA fragment.
12 P-labeled large BamHI-Sall
subfragment from the HSV-1(F) Extensive analyses of the viral RNA homologous to the
BamHI G fragment. The HSV-1 BamHI-Sall DNA fragment from BamHI J fragment contained in the BJ cells (2) have shown
plasmid pRB2O17 contains a sequence from the domain of the gBl that they express a truncated transcript homologous to the
gene. The fragment was mixed with BHKtk- cell DNA for copy
open reading frame US3 encoding the viral protein kinase
number reconstructions shown in the rightmost three lanes of this
figure. The cells were harvested at 1 or 5 h postexposure of cells to
(31, 40), a truncated transcript homologous open reading
virus. The adsorption interval was 1.5 h. The concentrations of
frame US8 encoding the gEl, and the domain of the gene
cycloheximide (cyclo) and PAA were 100 and 300 R±g/ml of medium,
encoding gDl (31). As noted in the Results, the residual
respectively. The cells were exposed to the drugs 1 h before sequences of the US3 (protein kinase) are unlikely to specify
infection, and the treatment was continued until the cells were a functional protein. Analyses of the clonally derived cell
harvested. lines reported here and of the parental BJ cell line in the164 CAMPADELLI-FIUME ET AL.
g'\q*,8*S1-}.%-tst;#dXlPe^rSo.s,.,2*aLR;>tgb;SsiFe|.eM$',l.*. i . . . .m . ;sjg!:. , '
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VOL. 62, 1988 ENTRY OF HSV INTO CELLS THAT EXPRESS gD 165
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45
FIG. 8. Electron micrographs of BHKtk- (A to C) and BJ-o (D) cells fixed 2 h after exposure at 37'C to 100 PFU of HSV-1(F) per cell.
In panels A and B, the arrows point to empty capsids at nuclear pores. In panel C, the arrow points to a small cluster of virus particles,
enveloped and unenveloped, in cytoplasmic vesicles. In panel D, the arrow points to a vesicle containing a partially degraded viral particle.
Magnification, x 25,000 for panels A, B, and C, and x 80,000 for panel D.166 CAMPADELLI-FIUME ET AL. J. VIROL.
presence of numerous particles attached to membranes both both positively and negatively? J. Virol. 62:148-158.
on the surface of the cell and in the intracytoplasmic 3. Arsenakis, M., J. Hubenthal-Voss, G. Campadelli-Fiume, L.
vesicles. This observation suggests that the BJ cells do Pereira, and B. Roizman. 1986. Construction and properties of a
contain receptors for attachment of virus to cells. cell line constitutively expressing the herpes simplex virus
(ii) The hypothesis that gD is involved in membrane fusion glycoprotein B dependent on functional cA protein synthesis. J.
Virol. 60:674-682.
is supported by two lines of evidence. First, antibody to gD 4. Balachandran, N., S. Bacchetti, and W. E. Rawls. 1982. Protec-
precludes the fusion of cells into polykaryocytes (33). Sec- tion against lethal challenge of BALB/c mice by passive transfer
ond, the BJ cells exhibit a higher incidence of spontaneous of monoclonal antibodies to five glycoproteins of herpes simplex
polykaryocytosis than do the parental BHKtk- cells and are virus type 2. Infect. Immun. 37:1132-1137.
readily fused by a brief exposure to polyethylene glycol 6000 5. Batterson, W., D. Furlong, and B. Roizman. 1983. Molecular
(G. Campadelli-Fiume, manuscript in preparation). We genetics of herpes simplex virus. VIII. Further characterization
should note that gD may be a major, but not the sole, virion of a temperature-sensitive mutant defective in release of viral
component involved in the fusion of membranes as evi- DNA and in other stages of the viral reproductive cycle. J.
Virol. 45:397-407.
denced by the observation cited earlier in the text that 6. Batterson, W., and B. Roizman. 1983. Characterization of the
antibody to gE also precludes cell fusion. However, the herpes simplex virion-associated factor responsible for the
observations that a class of viral mutations causing fusion of induction of a genes. J. Virol. 46:371-377.
infected cells (syn mutants) map in numerous loci in the HSV 7. Baucke, R. B., and P. G. Spear. 1979. Membrane proteins
genome do not obviate the role of gD in this process. The specified by herpes simplex viruses. V. Identification of an
model requires that gD or the complex responsible for Fc-binding glycoprotein. J. Virol. 32:779-789.
membrane fusion is activated by interacting viral glycopro- 8. Berman, P. W., D. Dowbenko, L. A. Laskey, and C. C. Simon-
teins after adsorption. Such activation could also occur by sen. 1983. Detection of antibodies to herpes simplex virus with
mutations in the cell membrane proteins with which the a continuous cell line expressing cloned glycoprotein D. Science
fusion complex interacts. 222:524-527.
9. Buckmaster, E. A., U. Gompels, and A. Minson. 1984. Charac-
Parenthetically, cell lines expressing HSV glycoproteins terization and physical mapping of an HSV-1 glycoprotein of
have not previously been shown to affect the entry of HSV approximately 115 x 103 molecular weight. Virology 139:408-
into cells (21), either because the level of expression was too 413.
low or, as in the case of the a4/c113/gB cells expressing g131 10. Chan, W. L. 1983. Protective immunization of mice with spe-
(3), because they do not effectively interfere with the entry cific HSV-1 glycoproteins. Immunology 49:343-352.
of virus into cells. 11. Cohen, G. H., V. J. Isola, J. Kuhns, P. W. Berman, and R. J.
(iii) The identity of the glycoproteins responsible for the Eisenberg. 1986. Localization of discontinuous epitopes of her-
attachment of the virus to cell surfaces is not yet known. The pes simplex virus glycoprotein D: use of a nondenaturing
recent availability of mutants lacking the genes specifying ("native" gel) system of polyacrylamide gel electrophoresis
coupled with Western blotting. J. Virol. 60:157-166.
gE, gG, and gI (25-27), coupled with the mutation in the 12. DeLarco, J., and G. J. Todaro. 1976. Membrane receptors for
HSV-1(MP) which precludes the synthesis of gC (20), should murine leukemia viruses: characterization using purified viral
enable a better definition of the function of the three glyco- envelope glycoprotein gp7O. Cell 8:365-371.
proteins, i.e., gB, gD, and gH, known to be essential for 13. DeLuca, N., D. J. Bzik, V. C. Bond, S. Person, and W. Snipes.
virus growth in cell culture. 1982. Nucleotide sequences of herpes simplex virus type 1
According to this model, the failure of HSV-1 and HSV-2 (HSV-1) affecting virus entry, cell fusion and production of
to productively infect the BJ cells is caused by unavailability glycoprotein B, gB (VP7). Virology 122:411-423.
of the putative cognate proteins which are predicted to 14. Dix, R., L. Pereira, and J. R. Baringer. 1981. Use of monoclonal
interact with the gD of the infecting virus and cause the antibody directed against herpes simplex virus glycoproteins to
fusion of the envelope with the plasma membrane. Conceiv- protect mice against acute virus-induced neurological disease.
Infect. Immun. 34:192-199.
ably, these proteins are sequestered by the gD made consti- 15. Eisenberg, R. J., C. P. Cerini, C. P. Heilman, A. D. Joseph, B.
tutively in these cells. The availability of the cell line and of Dietzschold, E. Gollub, D. Long, M. Ponce de Leon, and G. H.
serologic reagents to the glycoproteins should make identi- Cohen. 1985. Synthetic glycoprotein D-related peptides protect
fication of the host components possible. mice against herpes simplex virus challenge. J. Virol. 56:1014-
1017.
ACKNOWLEDGMENTS 16. Eisenberg, R. J., D. Long, M. Ponce de Leon, J. T. Matthews, P.
G. Spear, M. G. Gibson, L. A. Lasky, P. Berman, E. Golub, and
We thank Lenore Pereira and Richard J. Whitley for gifts of the G. H. Cohen. 1985. Localization of epitopes of herpes simplex
monoclonal antibodies. virus type 1 glycoprotein D. J. Virol. 53:634-644.
The studies done at the University of Bologna were aided by 17. Ejercito, P. M., E. D. Kieff, and B. Roizman. 1968. Characteri-
grants from Progetto Finalizzato Ingegneria Genetica Centro Nazio- zation of herpes simplex virus strains differing in their effects on
nale delle Ricerche (grant 86.00066.510) and from Associazione social behavior of infected cells. J. Gen. Virol. 2:357-364.
Italiana per la Ricerche sul Cancro. The studies done at the 18. Fuller, 0. A., and P. G. Spear. 1985. Specificities of monoclonal
University of Chicago were aided by Public Health Service grants and polyclonal antibodies that inhibit adsorption of herpes
CA 08494 and CA 19264 from the National Cancer Institute and by simplex virus to cells and lack of inhibition by potent neutral-
grant MV-2W the American Cancer Society. M.A. was a fellow of izing antibodies. J. Virol. 55:475-482.
the Leukemia Research Foundation. 19. Gombels, V., and A. Minson. 1986. The properties and sequence
of glycoprotein H of herpes simplex virus type 1. Virology
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