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Journal of Virology, December 1998, p. 9479-9490, Vol. 72, No. 12
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
p53 Status Does Not Determine Outcome of E1B
55-Kilodalton Mutant Adenovirus Lytic Infection
Felicia D.
Goodrum and
David A.
Ornelles*
Molecular Genetics Program and Department of Microbiology
and Immunology, Wake Forest University School of Medicine, Wake
Forest University, Winston-Salem, North Carolina 27157
Received 16 June 1998/Accepted 20 August 1998
 |
ABSTRACT |
The ability of the adenovirus type 5 E1B 55-kDa mutants
dl1520 and dl338 to replicate efficiently and
independently of the cell cycle, to synthesis viral DNA, and to lyse
infected cells did not correlate with the status of p53 in seven cell
lines examined. Rather, cell cycle-independent replication and
virus-induced cell killing correlated with permissivity to viral
replication. This correlation extended to S-phase HeLa cells, which
were more susceptible to virus-induced cell killing by the E1B 55-kDa
mutant virus than HeLa cells infected during G1. Wild-type
p53 had only a modest effect on E1B mutant virus yields in H1299 cells
expressing a temperature-sensitive p53 allele. The defect in E1B 55-kDa
mutant virus replication resulting from reduced temperature was as much as 10-fold greater than the defect due to p53 function. At 39°C, the
E1B 55-kDa mutant viruses produced wild-type yields of virus and
replicated independently of the cell cycle. In addition, the E1B 55-kDa
mutant viruses directed the synthesis of late viral proteins to levels
equivalent to the wild-type virus level at 39°C. We have previously
shown that the defect in mutant virus replication can also be overcome
by infecting HeLa cells during S phase. Taken together, these results
indicate that the capacity of the E1B 55-kDa mutant virus to replicate
independently of the cell cycle does not correlate with the status of
p53 but is determined by yet unidentified mechanisms. The
cold-sensitive nature of the defect of the E1B 55-kDa mutant virus in
both late gene expression and cell cycle-independent replication leads
us to speculate that these functions of the E1B 55-kDa protein may be linked.
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INTRODUCTION |
The early region 1B (E1B) of
adenovirus type 5 (Ad5) encodes overlapping functions essential for
virus mediated cellular transformation in cooperation with E1A by
suppressing p53-mediated apoptosis or a G1 growth arrest
(52, 62). The E1A proteins are potent transactivators that
relieve cellular growth suppression and induce quiescent cells to enter
S phase by binding members of the retinoblastoma protein family and
transcription factors such as p300 (reviewed in reference
17). This action of E1A results in the accumulation of p53. p53 is a cellular growth suppressor that acts as a
G1 checkpoint control (reviewed in references
37 and 74). In response to viral
challenge, p53 may induce a G1 growth arrest by inducing
genes such as the cyclin-dependent kinase inhibitor p21/WAF1/Cip1 gene
(16, 69) or apoptosis by inducing genes such as
bax1 (43). Either response by p53 is expected to
severely hinder Ad replication and transformation (38).
The E1B 55-kDa and 19-kDa proteins are required to fully transform
cells (4, 19, 23, 56, 64). The E1B 19-kDa protein is a
functional homologue of the proto-oncogene-encoded Bcl-2 and prevents
apoptosis by similar mechanisms (12, 52). The E1B 55-kDa
proteins of Ad5 and Ad12 have been hypothesized to permit E1A-induced
DNA synthesis and transactivation by preventing a p53-mediated
G1 growth arrest (51, 60, 61). The 55-kDa protein complexes with the amino-terminal end of p53 and inhibits its
activity as a transcription factor (33, 72, 73). This inhibition of p53-mediated transactivation by the large E1B protein is
required for transformation by both the weakly oncogenic group C and
highly oncogenic group A Ads (33, 72, 75, 76). E1B 55-kDa
protein-mediated inactivation of p53 has been hypothesized to be
required for viral replication in the lytic infection (7); however, this has not been demonstrated.
The Ad E4orf6 protein was recently shown to bind p53 and block
transcriptional activation mediated by p53 (14, 46).
Subsequently, the E4orf6 protein was shown to cooperate with the E1A
and E1B proteins to transform baby rat kidney cells (46), to
convert the nontumorigenic 293 human cell line (24) into a
tumorigenic cell line in nude mice, and to block p53-dependent
apoptosis (44). In both transformed and productively
infected cells, coexpression of the E1B 55-kDa and the E4orf6 proteins
decreased the stability of p53 (44, 46, 50). These findings
suggest that the E4orf6 protein may encode some overlapping and
redundant functions with the E1B 55-kDa protein with regard to transformation.
At late times in the lytic infection, the E1B 55-kDa protein
facilitates the transport of viral late mRNA while inhibiting the
transport of most cellular mRNA in association with the E4orf6 protein
(3, 8, 26, 36, 49). Ad mutants that fail to express the E1B
55-kDa protein are defective for expression of late viral proteins and
replicate poorly. The functional interaction between the E1B 55-kDa and
E4orf6 proteins may be mediated by primate-specific cellular factors
(22, 47). The transport of several cellular messages,
including the heat shock protein 70, 
-tubulin, and
interferon-inducible Mx-A and 6-16 mRNAs, requires the E1B 55-kDa
protein late in Ad infection. This effect correlates with activation of
their transcription during the late phase (45, 71). In
addition to selectively blocking transport of most cellular mRNA, the
E1B 55-kDa protein further modulates host cell shutoff by inhibiting
host protein synthesis by mechanisms unrelated to the inhibition of
mRNA transport (2). We have recently demonstrated that the
E1B 55-kDa protein functions in promoting Ad replication independently
of the cell cycle. E1B 55-kDa mutant Ads produce virus most efficiently
when cells are infected during S phase and are restricted from
replication in cells infected during G1 (20).
These findings suggest that the E1B 55-kDa protein plays a role in
deregulating the cell cycle to the advantage of the lytic infection.
Perhaps this function represents a link between the functions of the
E1B 55-kDa protein in the lytic infection and transformation.
Because the E1B 55-kDa protein inhibits the function of p53, it has
been hypothesized that an E1B 55-kDa mutant Ad can replicate only in
cells lacking a functional p53. Evidence supporting this hypothesis
includes the finding that the E1B 55-kDa mutant Ad dl1520
failed to lyse U2OS cells which contain wild-type p53. Furthermore, the
E1B 55-kDa mutant virus induced more severe cytopathic effect in the
RKO human colon cancer cell line transfected with a dominant negative
p53 gene than in the parental cell line containing a wild-type p53 gene
(7). However, p53 null tumor cells were not more susceptible
to lysis by the E1B 55-kDa mutant virus than some cancer cells
containing wild-type p53 (28). Furthermore, the relative
ability of the E1B 55-kDa mutant virus to suppress tumor growth
compared to the wild-type Ad was unaffected by the status of p53
(7). By contrast, Ridgway et al. (54) suggest that the interaction between p53 and the E1B 55-kDa protein is necessary for efficient Ad replication and that neither the wild-type nor E1B 55-kDa mutant virus replicated in p53-mutant cell lines. These
authors reported that the wild-type Ad failed to efficiently shut off
-actin synthesis, synthesize viral DNA, and induce cytopathic effect
in the p53 mutant cell lines T98G and 143B. They suggest that p53 may
mediate the interaction between the E1B 55-kDa and E4orf6 proteins in
the lytic infection (54). However, Rubenwolf et al.
(55) demonstrated that p53 is not required for
coimmunoprecipitation of the E1B 55-kDa and E4orf6 proteins although
the two proteins have been shown to bind p53 independently (14,
55, 57).
The work presented here demonstrates that the inability of the E1B
mutant virus to replicate efficiently and produce virus in all infected
cells is not strictly due to the failure to abrogate p53 function.
Among a variety of cell lines analyzed, the capacity of the E1B 55-kDa
mutant virus to replicate, synthesize viral DNA, and produce virus in
all infected cells did not correlate with the status of p53. However,
the ability of the E1B 55-kDa mutant virus to replicate differed among
the cell types studied. Furthermore, the ability of the E1B 55-kDa
mutant virus to induce cell killing correlated with permissivity to
virus growth and not the status of p53. In a cell line expressing a
temperature-sensitive p53 allele, active p53 only moderately affected
the replication of the E1B 55-kDa mutant virus. The defect in E1B
55-kDa mutant virus replication due to reduced temperature was as much
as 10-fold greater than the defect due to p53 function. Indeed, the E1B
55-kDa mutant virus produced equivalent yields of virus and synthesized equivalent levels of late viral proteins compared to the wild-type virus in cells maintained at 39°C but not at 32°C. The cell cycle restriction of the E1B 55-kDa mutant virus was also partially overcome
at 39°C, and progeny virus were produced in a larger fraction of
infected cells. By contrast, the apparent cell cycle restriction was
exacerbated at 32°C. Since both the cell cycle restriction and the
defect in late gene expression exhibited a cold-sensitive phenotype, we
speculate that the functions of the E1B 55-kDa protein in promoting
mRNA transport and cell cycle-independent viral replication may be linked.
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MATERIALS AND METHODS |
Cell culture.
Cell culture media, cell culture supplements,
and serum were obtained from Life Technologies (Gibco/BRL,
Gaithersburg, Md.) through the Tissue Culture Core Laboratory of the
Comprehensive Cancer Center of Wake Forest University. HeLa (ATCC CCL
2; American Type Culture Collection, Rockville, Md.), A549 (ATCC CCL
185), and 293 (ATCC CRL 1573) cells were maintained as monolayers in Dulbecco's modified Eagle's minimal essential medium (DMEM)
supplemented with 10% newborn calf serum (CS), 100 U of penicillin per
ml, and 100 µg of streptomycin per ml. Saos-2 cells were a generous gift of Jerry Zambetti (St. Jude Children's Research Hospital, Memphis, Tenn.) and were maintained as monolayers in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U of penicillin per ml, and 100 µg of streptomycin per ml. NCI-H460 (ATCC HTB 117) and NCI-H358 (ATCC
CTL 5807) cells were maintained in antibiotic-free RPMI 1640 supplemented with 10% FBS and essential amino acids. U2OS cells (ATCC
HTB 96) were maintained in McCoy's 5A medium supplemented with 10%
FBS, 100 U of penicillin per ml, and 100 µg of streptomycin per ml.
H1299 cells and H1299-p53 cells were a generous gift of Nancy
Raab-Traub (University of North Carolina, Chapel Hill) and were
maintained in DMEM supplemented with 10% FBS, 100 U of penicillin per
ml, and 100 µg of streptomycin per ml. The H1299-p53 cells were
maintained at 39°C with 0.5 mg of Geneticin (Life Science
Technologies, Gibco/BRL, Gaithersburg, Md.) per ml. p53 in H1299-p53
cells is in the wild-type conformation when cells are shifted to
32°C. Cells were maintained in subconfluent adherent cultures in a
5% CO2 atmosphere at the appropriate temperature by
passage twice weekly. Cells were preserved in liquid nitrogen in 93%
FBS-7% dimethyl sulfoxide.
Synchronization of the HeLa cell cycle was achieved by a combination of
mitotic detachment and hydroxyurea block as described previously
(20). HeLa cells were passaged 1:5 into 75-cm2
flasks for synchronization 12 to 16 h prior to the mitotic
detachment. All but 5 ml of growth medium was removed, and the flasks
were tapped sharply six times on each side. The detached cells were resuspended in DMEM supplemented with 10% CS and 2 mM hydroxyurea (Sigma, St. Louis, Mo.), replated at 2 × 105 cells
per ml, and incubated at 37°C in a 5% CO2 atmosphere.
After 1 h, the medium and nonadherent cells were replaced with
fresh DMEM supplemented with 10% CS and 2 mM hydroxyurea and incubated for 12 h at 37°C in a 5% CO2 atmosphere. At the
completion of the incubation with hydroxyurea, the cells were washed
once with warm phosphate-buffered saline (PBS; 137 mM NaCl, 3 mM KCl,
1.76 mM KH2PO4, 10 mM
Na2HPO4), and the last wash was replaced with normal growth medium to release the G1/S block.
Viruses.
The phenotypically wild-type Ad5 parent virus,
dl309, used in these studies lacks a portion of the region
encoding the E3 region that has been shown to be dispensable for growth
in tissue culture (30). The E1B mutant virus
dl338 contains a 524-bp deletion in the 55-kDa
protein-coding region (49). Mutant virus dl1520D contains an 827-bp deletion in the region encoding the 55-kDa protein
in combination with a stop codon to ensure that a truncated 55-kDa
product cannot be expressed (4). The E1B 19-kDa mutant virus, dl337, contains a 146-bp deletion in the E1B 19-kDa
protein-coding region between nucleotide sequence positions 1770 and
1916 (48).
The propagation of these viruses has been described elsewhere
(
30). In brief, virus stocks were prepared by infecting 293
cells at a multiplicity of infection (MOI) of 1. Virus was harvested
4 days postinfection from a concentrated freeze-thaw lysate by
sequential
centrifugation in discontinuous and equilibrium cesium
chloride
gradients (
31). The gradient-purified virus was supplemented
with 5 volumes of 12 mM HEPES (pH 7.4)-120 mM NaCl-0.1 mg of bovine
serum albumin (fraction V; Life Technologies Inc.) per ml-50%
glycerol (Fisher Scientific, Pittsburgh, Pa.) and stored at
20°C.
The titer of each virus stock was determined by plaque assays
on 293 cells (
31).
For infection with Ad, cells were passaged 16 to 24 h prior to
infection to a density of 2 × 10
5 cells per ml. Cells
were washed once with PBS, and the final
wash was replaced with virus
(3 to 20 PFU per cell) in Ad infection
medium (PBS supplemented with
0.2 mM CaCl
2, 0.2 mM MgCl
2, 2% CS,
100 U of
penicillin per ml, and 100 µg of streptomycin per ml).
The virus was
added at one-fourth the normal culture volume, and
the cells were
gently rocked for 60 to 90 min at 37°C. The virus
suspension was then
replaced with normal growth medium, and the
infected cells were
returned to 37°C. For temperature-sensitive
and cold-sensitive
experiments, cells were infected at 39 or 32°C.
Infectivity.
The infectivities of HeLa, U2OS, Saos-2, C33A,
and A549 cells were determined by infecting each cell line with
dl309, dl338, or dl1520 at MOIs of 1, 10, and 30 PFU per cell. The virus titers were determined by plaque
assays on 293 cells (24). At 14 h postinfection,
infected monolayers of cells were harvested with trypsin to achieve a
single-cell suspension. Cells were fixed in 4% formaldehyde for 20 min, permeabilized with 0.1% Triton X-100 for 4 min, and secondarily
fixed in cold methanol for 5 min. The Ad E2A 72-kDa DNA binding protein
was stained with the E2A 72-kDa protein-specific monoclonal antibody
(clone B6-8) (53) used as hybridoma cell medium diluted 1:2
in Tris-buffered saline (137 mM NaCl, 3 mM KCl, 25 mM Tris-Cl [pH
8.0], 1.5 mM MgCl2, 0.5% bovine serum albumin, 0.1%
glycine, 0.05% Tween 20, 0.02% sodium azide). The primary antibody
was detected with fluorescein isothiocyanate (FITC)-conjugated goat
antibodies specific for mouse immunoglobulin G (Jackson ImmunoResearch,
West Grove, Pa.). Cells were resuspended in PBS, filtered through a
nylon mesh, and passed through a 27.5-gauge needle to achieve a
single-cell suspension. Cells stained with the E2A 72-kDa
protein-specific antibody were counted by fluorescence-activated cell
sorting (FACS) using a Coulter Epics XL flow cytometer (Coulter Corp.,
Miami, Fla.) with an argon laser as the excitement source. In each
case, 40,000 events were measured. H1299, H460, and H358 cell
infectivity was determined similarly except that cells were fixed and
stained as a monolayer. Cells staining with the E2A 72-kDa
protein-specific antibody were identified by epifluorescence with a
Leitz Dialux 20 EB microscope. Approximately 200 cells were evaluated
for each experiment.
Electron microscopy.
Cells were fixed for transmission
electron microscopy with 2.5% glutaraldehyde (Polysciences,
Warrington, Pa.) in PBS-1.5 mM MgCl2 or in 0.1 M sodium
cacodylate at 20 to 30 h postinfection. The fixed cell pellet was
postfixed with osmium tetroxide in cacodylate buffer and dehydrated in
a graded series of alcohol. Specimens were infiltrated with Spurr's
resin-propylene oxide and cut into approximately 100-nm-thick sections
with a diamond knife. Sections were collected on copper grids, stained
with uranyl acetate and lead citrate, and analyzed at 80 keV with a
Philips 400 transmission electron microscope. Specimens were embedded
and sectioned by MicroMed, the Electron Microscopy Core Laboratory of
the Comprehensive Cancer Center of Wake Forest University.
Flow cytometry.
HeLa cells were harvested by treatment with
trypsin and fixed in 70% ethanol for 1 h to overnight. The
ethanol was removed, and the cells were resuspended to approximately
106 cells per ml in propidium iodide buffer (100 mM NaCl,
36 mM sodium citrate, 50 µg of propidium iodide per ml, 0.6% Nonidet
P-40) supplemented with 0.04 mg of RNase (Sigma) per ml. The cells were filtered through nylon mesh and passed through a 27.5-gauge needle to
achieve a single-cell suspension. The DNA content of individual cells
was measured by FACS using a Coulter Epics XL flow cytometer (Coulter
Corp.) with an argon laser as the excitement source (488 nm). The
emitted light was analyzed for forward and 90°C scatter, pulse width
(to discriminate doublets), and red fluorescence (>630 nm) of
propidium iodide to determine the DNA content per nucleus; 40,000 events were measured in each analysis. The resulting data were acquired
in list mode for discriminatory analysis such as the use of standard
gating procedures to define distinct populations of cells, doublets,
and debris. All flow cytometric analyses were conducted by the Steroid
Receptor Laboratory in cooperation with the Hematology Flow Cytometry
Laboratory of North Carolina Baptist Hospital.
Plaque assays for viral yields.
Detailed methods for Ad
plaque assays have been described elsewhere (31). In brief,
virus was harvested from cells in culture medium 48 to 72 h
postinfection by multiple cycles of freezing and thawing. The cell
lysates were clarified by centrifugation and serially diluted in
infection medium for infection of 293 cells for plaque assays. After
incubation with dilute virus for 1 h, the infected cells were
overlaid with 0.7% SeaKem ME agarose (FMC, Rockland, Maine) in DMEM
containing 0.75% sodium bicarbonate and 4% CS. The cells were fed
with additional agar overlays every third day for 7 days. The plaques
were visualized by staining with 0.01% neutral red in an agarose
overlay on the seventh day.
DNA slot blotting.
The DNA slot blotting procedure has been
described in detail previously (1, 32). Briefly, total
cellular DNA was isolated from infected HeLa, A549, U2OS, and C33A
cells. Cells were collected, pelleted, and resuspended in 10 mM Tris
(pH 8.0). An equal volume of lysis buffer (400 mM Tris [pH 8.0], 100 mM EDTA [pH 8.0], 1% sodium dodecyl sulfate [SDS], 200 µg of
proteinase K per ml) was added, and the cells were kept at 50°C for
1 h. DNA was extracted with phenol-chloroform, precipitated, and
quantified by spectrophotometry (A260).
Equivalent amounts of total cellular DNA was blotted onto Nytran nylon
membranes (Schleicher & Schuell, Keene, N.H.) by using a manifold
device (Life Technologies) and vacuum. The immobilized DNA was
denatured, neutralized, and cross-linked to the matrix with UV light
(Stratalinker; Stratagene, La Jolla, Calif.). The DNA was then
hybridized with an excess of [-32P]dATP-labeled (ICN,
Costa Mesa, Calif.) DNA probe generated by random-primed synthesis of
wild-type Ad DNA (1). Hybridized probe was quantified with
the use of a Molecular Dynamics (Sunnyvale, Calif.) PhosphorImager and
ImageQuant analysis software.
Late viral protein synthesis.
Late viral protein synthesis
was analyzed by pulse-labeling infected H1299 cells for 1 h with
0.1 mCi of 35S-labeled amino acids (Trans35S
label; ICN Biochemicals) per ml in cysteine- and methionine-free DMEM
supplemented with 2% FBS at 32 or 64 h postinfection. Cells were
then scraped and resuspended and lysed in 2× protein sample buffer
(2% SDS, 125 mM Tris [pH 6.8], 20% glycerol, 5%
-mercaptoethanol, 100 mM dithiothreitol, 0.01% bromophenol blue).
Protein from 1 × 105 to 2 × 105
cell equivalents was separated by SDS-polyacrylamide gel
electrophoresis (PAGE) on an 8% polyacrylamide gel (36:1
acrylamide/N,N'-methylenebisacrylamide ratio;
Polysciences). Polyacrylamide gels were fixed in 15% glacial acetic
acid (Fisher Scientific)-7.5% methanol. Proteins were quantified with
the use of a Molecular Dynamics PhosphorImager and ImageQuant analysis
software. Ad late proteins were identified by reference to virion
standards that were synthesized in the presence of
14C-labeled mixed amino acids (Amersham, Arlington Heights,
Ill.) and gradient purified from 293 cells infected with the wild-type Ad dl309 as described elsewhere (31).
Cell killing assays.
Virus-induced cell killing was measured
in a time course fashion by using calcein uptake and ethidium exclusion
(LIVE/DEAD assay; Molecular Probes, Eugene, Oreg.). Synchronized HeLa
cells or asynchronous population of A549, C33A, U2OS, or Saos-2 cells were plated in six-well dishes at 5 × 104 cells per
ml. Cells were mock infected or infected with dl309 or
dl1520 at 3 and 10 PFU per cell. At 24, 48, 72, 96, 120, 168, 192, and 240 h after infection, cells were trypsinized and
collected with the nonadherent cells from the media. Cells were washed
once with PBS, exposed as living cells to 0.1 ml of 4 µM ethidium
homodimer and 2 µM calcein AM in PBS for 40 min, and then diluted
with 0.4 ml of PBS, Calcein AM is converted from a nonfluorescent
cell-permeant to an intensely fluorescent nonpermeant compound by
ubiquitous intracellular esterase activity in live cells. Ethidium
homodimer enters cells with damaged membranes, binds nucleic acids, and fluoresces bright red in dead cells but is excluded from live cells.
The fraction of live and dead cells was measured by FACS using a
Coulter Epics XL flow cytometer (Coulter Corp.) with an argon laser as
the excitement source (488 nm) and standard gating procedures. All flow
cytometric analyses were conducted by the Hematology Flow Cytometry
Laboratory of North Carolina Baptist Hospital.
Apoptosis assay.
HeLa cells synchronized to S phase or
G1 were infected with dl309, dl1520,
or dl337 at 10 PFU per cell or were mock infected. At
24 h postinfection, phosphatidylserine exposed on the surface of
cells was visualized with annexin V-FITC conjugate (3 µg/ml) in
binding buffer (10 mM HEPES [pH 7.4], 150 mM NaCl, 5 mM KCl, 1 mM
MgCl2, 1.8 mM CaCl2; Trevigen, Gaithersburg,
Md.). The annexin V-FITC reagent was generously provided by Greg Kucera
(Wake Forest University School of Medicine, Winston-Salem, N.C.). Cells
were washed in binding buffer and fixed in 4% formaldehyde with 1.8 mM
CaCl2. Cell nuclei were visualized by staining with
4,6-diamidino-2-phenylindole (DAPI; Sigma), a nonspecific DNA binding
stain. Cells were examined by epifluorescence with a Leitz Dialux 20 EB microscope.
Computer-assisted graphics.
Radioactive samples were
visualized with the use of a Molecular Dynamics PhosphorImager and
ImageQuant analysis software. Radioactive images were obtained as
16-bit gray scale images and then converted to 8 bit gray scale images.
The chemiluminescent images were recorded on film, which was scanned at
300 dots/in. with a Hewlett-Packard scanner fitted with a transparency
adapter into an 8-bit gray scale image. The density ranges were
adjusted for printing without further contrast or image enhancement.
The digitized images were imported at 300 dots/in. into the graphic software Canvas (Deneba Software, Miami, Fla.) operating on a Macintosh
microcomputer to create the final figures.
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RESULTS |
Replication of the E1B 55-kDa mutant virus is not correlated with
the status of p53.
Levels of replication of the phenotypically
wild-type Ad dl309 and two E1B 55-kDa mutant viruses,
dl338 and dl1520, were compared among cell lines
of known p53 status. Both mutant viruses contain large deletions within
the gene encoding the E1B 55-kDa protein. However, dl1520
contains a termination codon at the second position to preclude
expression of any E1B 55-kDa-related protein (4), whereas
the deletion present in the dl338 genome could allow
expression of a truncated (17-kDa) protein. The eight cell lines
analyzed included HeLa cells, three p53 wild-type cell lines, and four p53 null or mutant cell lines. HeLa cells have a wild-type p53 sequence
but are functionally mutant due to the presence of the human
papillomavirus E6 protein (59, 66). However, during an Ad
infection, active p53 accumulates in infected HeLa cells (10,
25). Therefore, in Ad-infected HeLa cells, p53 might be expected
to inhibit virus replication in the absence of the E1B 55-kDa protein
(10). Table 1 summarizes the
status of p53 in each of the other cell lines tested, which include
three p53 wild-type cell lines, A549 (35), H460 (9,
63), and U2OS (13), and four p53-deficient cell lines,
C33A (11, 58), H358 (9, 63), H1299
(42), and Saos-2 (39).
The infectivity of Ad varied significantly among the cell lines
analyzed, as illustrated by the results shown in Fig.
1. For
this experiment, HeLa, C33A, or
U2OS cells were infected with
approximately 1 PFU of
dl1520
per cell (the virus titer was determined
on 293 cells). Cells were
fixed and labeled by indirect immunofluorescence
for a representative
early gene product, the E2A 72-kDa protein.
Cells expressing the E2A
72-kDa protein were quantified by flow
cytometry using mock-infected
cells as the negative control. Typically,
infectivity in HeLa cells
reflected the titer measured with 293
cells. In this experiment, we
measured 89% infected HeLa cells.
The C33A cells appeared to infect
more readily, and more than
95% of these cells were infected by 1 PFU
per cell. By contrast,
only 20% of the U2OS cells were infected by the
same amount of
virus. We measured similar results by indirect
immunofluorescence
microscopy of infected U2OS cells even after
allowing the infection
to continue for 3 days.
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FIG. 1.
Infectivity determined after infection with a constant
amount of virus varies significantly between different cell lines. The
cell lines indicated in each panel were infected with approximately 1 PFU of dl1520 (as measured by titer on 293 cells) per cell.
At 15 h postinfection, cells were stained for the E2A 72-kDa
protein by indirect immunofluorescence, and the fraction of infected
cells was determined by FACS. The abscissa indicates the relative
number of cells counted at the fluorescence intensity indicated on the
ordinate. The filled curves were derived from uninfected cells; the
nonfilled curves were obtained from infected cells. The percentage of
positive cells indicated in each panel was determined by fitting the
data to two Gaussian curves.
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The amount of virus required to infected 80 to 90% of the cells was
determined for each cell line by the method illustrated
in Fig.
1. As
defined by titer on 293 cells, HeLa, A549, and C33A
cells required 1 to
3 PFU per cell to infect 80 to 90% of the
cells. H1299, H358, and H460
cells required 3-fold more virus
whereas Saos-2 and U2OS cells required
10-fold more virus to infect
an equivalent number of cells. Each cell
line was infected at
an MOI sufficient to infect at least 80% of the
cells. At 48 to
72 h postinfection, the cells were harvested and
virus yields
were measured by titer on 293 cells. The results of these
measurements,
summarized in Fig.
2,
suggest that the status of p53 does not
predict the ability of the cell
line to support growth of the
E1B 55-kDa mutant virus.
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FIG. 2.
The inability of the E1B 55-kDa mutant virus to
replicate is not mediated by p53. Monolayer cultures of HeLa cells, p53
wild-type cell lines, or p53 mutant cell lines were infected with the
wild-type virus dl309 or the E1B 55-kDa mutant virus
dl338 or dl1520 at an MOI sufficient to infect
>80% of the cells (3 to 30 PFU/cell). Cells were lysed at 48 h
postinfection, and virus yields were measured by plaque assay on 293 cells. The yields shown, expressed as percentages of virus produced
relative to the wild-type virus, are averages of at least three
independent experiments; the range of values is indicated by the
brackets.
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As we and others have reported previously, HeLa cells did not permit
efficient replication of the E1B 55-kDa mutant viruses
(
20).
The E1B 55-kDa mutant viruses
dl338 and
dl1520
grew to
averages of 7 and 2%, respectively, of the wild-type virus
yield
in HeLa cells (Fig.
2A). Two of the three wild-type p53 cell
lines
restricted growth of the mutant viruses. In H460 and U2OS cells,
the mutant virus produced approximately 8 and 10% of the virus
obtained from an infection with wild-type virus (Fig.
2B). However,
the
E1B 55-kDa mutant virus grew to an average of 30 to 50% of
the
wild-type yield in A549 cells (Fig.
2B). In some experiments,
the yield
of the mutant virus was the same as the yield of the
wild-type virus
from A549 cells. Some of the p53 mutant cell lines
also restricted
growth of the E1B 55-kDa mutant viruses (Fig.
2C). Saos-2 and H1299
cells restricted growth of the mutant viruses
to an averages of 14 and
8% of the level produced by the wild-type
virus, respectively. H358
cells exhibited lesser restriction,
producing an average of 20% of the
wild-type virus yield, with
a maximum value of 38% recorded in one of
eight independent experiments.
Finally, the p53 mutant C33A cell line
was least restrictive to
the E1B 55-kDa mutant viruses, producing
between 40 and 65% of
the yield obtained from a wild-type virus
infection. In summary,
the E1B 55-kDa mutant viruses grew to the
highest titer in cell
lines containing a mutant p53 (C33A) and a
wild-type p53 (A549)
gene. It should be noted that human embryonic
kidney cells, which
would have wild-type p53, have also been reported
to be permissive
for growth of E1B 55-kDa mutant Ads (
6).
These results suggest
that although levels of replication of the E1B
55-kDa mutant viruses
varied among cell types, these differences may
not be mediated
by
p53.
E1B 55-kDa mutant Ads synthesize viral DNA to levels equivalent to
those of the wild-type virus in a permissive and a restrictive cell
line irrespective of the status of p53 in the infected cell.
Based
on the known function of p53, it is reasonable to expect that p53
could inhibit the replication of Ad in the absence of the E1B
55-kDa protein by inducing a G1 growth arrest and
inhibiting viral DNA synthesis. This effect may not be readily apparent
in the virus yields measured in Fig. 2; therefore, viral DNA synthesis was measured in two p53-wild-type cell lines, A549 and U2OS, and in one
p53 mutant cell line, C33A. Of the two wild-type p53 cell lines chosen,
A549 cells were permissive for E1B 55-kDa mutant virus replication and
U2OS cells were restrictive for E1B 55-kDa mutant virus replication
(Fig. 2). Hybridization analysis of total DNA isolated from cells
infected with wild-type or E1B 55-kDa mutant Ad demonstrated that cells
infected with either E1B 55-kDa mutant virus (dl338 or
dl1520) synthesized viral DNA to levels equivalent to that
of the same cells infected with the wild-type virus (Fig.
3). We previously reported that HeLa
cells infected with the E1B 55-kDa mutant virus dl338 also
synthesized wild-type levels of viral DNA (20). Among these
four cell lines, neither the permissivity toward replication of the E1B
55-kDa mutant virus nor p53 status affected viral DNA synthesis. These
results are consistent with the suggestion that the crucial role for
the E1B 55-kDa protein in viral replication occurs after the onset of viral DNA synthesis during the late phase of virus replication.
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FIG. 3.
E1B 55-kDa mutant virus-infected p53 wild-type (p53-wt)
and p53 mutant cells synthesize viral DNA to the same level as the
wild-type virus-infected cells. A549, U2OS, and C33A cell lines were
mock infected or infected with the wild-type virus dl309 or
the E1B 55-kDa mutant virus dl338 or dl1520 at an
MOI sufficient to infect >80% of the cells (10 to 30 PFU/cell). Total
DNA was isolated from equal numbers of Ad-infected cells at 20 h
postinfection. Total DNA in the amount indicated below each lane was
transferred to a nylon membrane, denatured, and hybridized with
radioactive Ad-specific DNA probes generated by random-primed
synthesis. Hybridized probe was quantified with a PhosphorImager, and
mock-infected background was subtracted. Identical amounts of viral DNA
were measured in each cell line infected with the E1B 55-kDa mutant
viruses and the wild-type virus.
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Cells that are permissive for the growth of the E1B 55-kDa mutant
virus produce progeny in a greater fraction of infected cells.
The
cell cycle-restricted phenotype of the E1B 55-kDa mutant virus was
first identified in HeLa cells by using electron microscopy (20). In infected populations of HeLa cells, only 20% of
the E1B 55-kDa mutant Ad-infected cells produced progeny virus, whereas nearly all cells infected with the wild-type Ad produced progeny virus.
The p53 wild-type and p53 null or mutant cell lines infected with the
wild-type or E1B 55-kDa mutant virus were examined by electron
microscopy at 32 h postinfection to determine the fraction of
infected cells producing E1B 55-kDa mutant virus progeny. The MOI was
adjusted for each cell line to infect greater than 80% of the cells.
Because the mutant virus-infected cells synthesized wild-type levels of
viral DNA, the infected cells were readily identified by the presence
of viral inclusions in the cell nucleus. The results of these
experiments are summarized in Table 2.
In all five cell lines analyzed, the wild-type virus produced progeny
virus in at least 90% of the infected cells. By contrast,
cell lines
that restricted replication of the E1B 55-kDa mutant
virus, H1299,
Saos-2, and U2OS, contained progeny mutant virus
in 28% or less of the
infected cells. This value is comparable
to the value of 22% reported
for asynchronously infected HeLa
cells (
20). The two cell
lines that best supported replication
of the E1B 55-kDa mutant virus,
A549 and C33A, contained progeny
virus in a greater fraction of cells
compared to the restrictive
cell lines. An average of 70% of the A549
and 90% of C33A cells
infected with
dl1520 contained
progeny virus. These results suggest
that replication of the E1B 55-kDa
mutant virus is not subjected
to a cell cycle growth restriction in
A549 and C33A cells. Furthermore,
because the E1B 55-kDa mutant virus
produced progeny in 70% or
more of the infected p53 wild-type cells
(A549) as well as the
p53 mutant cell line (C33A), it seems likely the
cell cycle growth
restriction is not strictly mediated by
p53.
Cell killing by the E1B 55-kDa mutant virus correlates with
permissivity to mutant virus replication and not the status of p53 in
the infected cell.
It was suggested that dl1520
(ONYX-015) selectively kills cells that lack normal p53 function
(7) although dl1520 was found to also kill some
tumor cells of wild-type p53 status (28). Therefore, the
relationship between p53, replication of an E1B 55-kDa mutant virus,
and virus-induced cell killing was analyzed in two p53 wild-type cell
lines (A549 and U2OS) and two p53 mutant cell lines (C33A and Saos-2).
As demonstrated by the results in Fig. 2, A549 and C33A cells were
permissive for growth of an E1B 55-kDa mutant virus whereas U2OS and
Saos-2 cells were restrictive for the growth of the E1B 55-kDa mutant
virus. For these experiments, replicate cultures of cells were mock
infected or infected with the wild-type virus dl309 or the
E1B 55-kDa mutant virus dl1520 at 3 and 10 PFU per cell for
A549 and C33A cells and at 9 and 30 PFU per cell for U2OS and Saos-2
cells. These multiplicities correspond to 3 and 10 infectious units per
cell for the respective cell lines. For 11 days after infection, cells
were harvested at the times indicated and the percentage of live cells
and dead cells was determined by a flow cytometric assay. The results
of these experiments suggest that cells that permitted growth of the
E1B 55-kDa mutant Ad were susceptible to killing by the E1B mutant
virus, irrespective of the status of p53.
The wild-type Ad
dl309 killed more than 50% of the cells by
day 6 or 7 in all cell lines examined (Fig.
4). By 9 days postinfection,
95% of the
cells were killed by the wild-type virus for each cell
line except
Saos-2 cells. By contrast, the E1B 55-kDa mutant virus
completely lysed
only A549 and C33A cell cultures. The onset of
E1B 55-kDa
mutant-induced cell killing was slightly delayed in
these cell lines
compared to the wild-type virus; the E1B 55-kDa
mutant virus induced
complete lysis by 11 days postinfection,
in contrast to the wild-type
virus, which required 9 days. U2OS
and Saos-2 cells were resistant to
lysis by the E1B 55-kDa mutant
virus. By 11 days postinfection, fewer
than 20% of the U2OS and
Saos-2 cells in the E1B mutant-infected
population had been killed.
Cell killing did not demonstrate a striking
multiplicity effect
over the range examined since cells infected at the
lower multiplicities
(3 or 9 PFU per cell) were lysed nearly as
efficiently as cells
infected at the higher multiplicities (10 or 30 PFU per cell).
The cells most susceptible to killing by the E1B 55-kDa
mutant
virus were those that produced the highest yields of virus as
measured in Fig.
2.
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FIG. 4.
Cell killing induced by the E1B 55-kDa mutant virus
correlates with permissivity to viral replication, not the status of
p53. U2OS, Saos-2, A549, and C33A cells cultured in six-well plates
were mock infected or infected with either the wild-type virus
dl309 or the E1B 55-kDa mutant virus dl1520 at 3 and 10 infectious units per cell. During a time course after infection,
replicate dishes of cells were harvested and the percentage of live
cells was determined by flow cytometry using the LIVE/DEAD assay. Live
cells with active esterases converted the nonfluorescent calcein AM to
a brightly fluorescing molecule (green). Dead cells were stained with
ethidium homodimer (red), which was excluded from living cells.
Typically 40,000 cells were analyzed. The data shown represent a single
experiment performed in duplicate. The top two graphs represent data
from wild-type p53 (p53+) cell lines (U2OS and A549); the
bottom two graphs represent data from a p53-mutant (p53 )
cell lines (Saos-2 and C33A); the two graphs on the left represent data
from infection of relatively non permissive cell lines (U2OS and
Saos-2); the two graphs on the right represent data from infection of
relatively permissive cell lines (A549 and C33A).
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S-phase cells are more susceptible than G1 cells to
cell killing by the E1B 55-kDa mutant virus.
We previously showed
that HeLa cells infected during S phase were permissive for E1B 55-kDa
mutant virus replication whereas HeLa cells infected during
G1 phase restricted E1B mutant virus replication. S-phase
cells infected with the E1B 55-kDa mutant virus also exhibited more
severe cytopathic effect than G1-infected cells
(20). Therefore, the possibility that HeLa cells infected during S phase were more susceptible to virus-induced cell killing than
HeLa cells infected during G1 phase was tested. Replicate cultures of synchronized HeLa cells were infected during S phase or
G1 with the wild-type (dl309) or E1B 55-kDa
mutant virus (dl1520) at 3 and 10 PFU per cell or were mock
infected. As before, cells were harvested at the indicated times, and
the fraction of living cells was determined. The wild-type virus killed
both cells infected at the onset of S phase and during G1
with almost equal efficiencies (Fig. 5).
The wild-type virus induced complete cell lysis by 11 days
postinfection in cells infected during S phase or G1;
however, killing was slightly delayed in cells infected during
G1 phase of the cell cycle. Unlike the wild-type virus, the
E1B 55-kDa mutant virus failed to kill cells infected in early
G1 until day 11, at which time a maximum of 50% of cell
death was measured (Fig. 5B). By contrast, S-phase-infected cells were
killed by the mutant virus as soon as 4 days postinfection. By 11 days
postinfection, only 20% of the cells infected during S phase with the
E1B 55-kDa mutant virus were still living (Fig. 5A). These data further
support the idea that cells permissive for growth of the mutant virus are most susceptible to killing by the virus.
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FIG. 5.
HeLa cells infected during S phase are more susceptible
than cells infected during G1 to cell killing by the E1B
55-kDa mutant virus. HeLa cells were synchronized and cultured in
six-well plates and then mock infected or infected with the wild-type
virus dl309 or the E1B 55-kDa mutant virus dl1520
at MOIs of 3 and 10 at the onset of S phase or G1. During a
time course after infection, replicate dishes of cells were harvested
and the percentage of live cells was determined by flow cytometry using
the LIVE/DEAD assay as described in Materials and Methods. The data
shown are from a single experiment and are representative of two
independent experiments each performed in duplicate.
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To determine if the mechanism of cell killing differed between cells
infected with the wild-type virus and the E1B 55-kDa
mutant virus,
synchronized and infected cells were labeled with
annexin V 24 h
postinfection (Table
3). Annexin V is a
calcium-dependent
phospholipid-binding protein with high affinity for
phosphatidylserine.
Phosphatidylserine is normally restricted to the
inner surface
of the plasma membrane. However, early in the apoptotic
pathway,
the asymmetry of the plasma membrane is lost and
phosphatidylserine
is externalized (reviewed in reference
65). The E1B 19-kDa mutant
virus
dl337
induces apoptosis in infected cells and was used as
a positive control
(
12,
48,
52). Only 2 to 3% of mock-infected
S-phase or
G
1 HeLa cells stained with annexin V. By contrast,
71% of
the HeLa cells infected during S phase and 90% of the HeLa
cells
infected during G
1 with
dl337 underwent
apoptosis. The reason
for increased apoptosis in G
1-phase
cultures infected with
dl337
is not known. The fraction of
apoptotic cells in cultures infected
with
dl309 or
dl1520 was similar to or only slightly greater than
that in
mock-infected cultures. Furthermore, S-phase HeLa cells
infected with
the E1B 55-kDa mutant virus were not more likely
to undergo apoptosis
than cells infected during G
1. These results
suggest that
although S-phase HeLa cells are more susceptible
to killing by the E1B
55-kDa mutant virus, the increased cell
killing is not due to increased
apoptosis.
p53 has a modest effect on growth of the E1B 55-kDa mutant viruses
in a cell line expressing a temperature-sensitive p53 allele.
A
limitation to using cell lines of defined p53 status to test the role
of p53 in virus growth is that other cell-specific changes that may
affect virus growth are not controlled between the cell lines. To
circumvent this limitation, growth of the E1B 55-kDa mutant viruses was
analyzed in H1299 cells that express a temperature-sensitive p53 allele
(18). The parental H1299 human lung carcinoma cell line
contains a homozygous deletion of the p53 gene (42) and
therefore allows analysis of ectopic p53 function without interference
from endogenous p53 protein. H1299 cells were stably transfected with
the temperature-sensitive mouse p53 allele
tsVal135 to create the H1299-p53 cell line
(18). The p53 protein expressed in H1299-p53 cells is
largely mutant at 39°C but is found predominantly in the wild-type
tetramer conformation at 32°C. Accumulation of active p53 leads to
the induction of the cell cycle inhibitor p21/WAF-1. p21 inhibits
cyclin-dependent kinase complex formation and can therefore induce a
G1 growth arrest (15, 16, 27, 69, 70). To
determine the point at which p53 became functional after the shift to
32°C, the induction of p21 was analyzed in a time course fashion
following the shift from 39 to 32°C by Western blotting (Fig.
6).
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FIG. 6.
p21 expression is induced in H1299-p53 cells by 12 h after the shift to the permissive temperature. H1299-p53 cells and
parental H1299 cells were shifted from 39°C (p53 mutant) to 32°C
(p53 wild-type). Equivalent numbers of cells were lysed at the time
indicated above each lane. Total cellular protein from 2 × 105 cells was separated by SDS-PAGE and then
electrophoretically transferred to nitrocellulose. p21 expression was
analyzed by standard Western blotting methods. The p21/WAF-1 protein
present in cellular lysates was visualized with the monoclonal antibody
clone EA10 (Santa Cruz Biotechnology, Inc., Santa Cruz, Calif.).
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Expression of p21 was readily apparent within 12 h after the shift
of H1299-p53 cells to 32°C (Fig.
6). p21 levels continued
to increase
through 32 h following the shift to the lower temperature.
As
expected, p21 was not induced in the parental H1299 cell line
at
32°C. Cells expressing the temperature-sensitive p53 allele
stopped
dividing after being shifted to 32°C for 24 h (
18,
41).
H1299-p53 cells shifted to 32°C were also examined for
changes
in cell cycle distribution (i.e., G
1 arrest) by
FACS. Growth of
the parental H1299 cell line at 32°C did not
appreciably alter
the cell cycle distribution. After 12 h at
32°C, the cell cycle
distribution of the H1299-p53 cells was the same
as for cells
maintained at 39°C. However, by 32 h after the
shift to the lower
temperature, the percentage of cells in S phase had
decreased
from 45 to 13%, with a concomitant increase in the fraction
of
cells in G
2/M phase (data not shown). This alteration in
cell
cycle distribution due to prolonged expression of p53 at 32°C
is
consistent with the findings of Michalovitz et al. (
41).
These data indicate that p53 adopts the wild-type conformation
within
12 h of the shift to the lower temperature. However, p53
function
has not appreciably altered the fraction of cells in
S phase after
12 h at 32°C. A decrease in the fraction of cells
in S phase
would be expected to affect the growth of the E1B 55-kDa
mutant
(
20).
Portions of both H1299 and H1299-p53 cells were shifted from 39 to
32°C for 12 h and then infected with the wild-type virus
(
dl309) or an E1B 55-kDa mutant virus (
dl338 or
dl1520) at an
MOI of 9 PFU per cell (Fig.
7). Virus yields were determined by
titer
on 293 cells at 48 h postinfection for cells at 39°C and
96 h postinfection for cells at 32°C. The wild-type virus produced
equivalent yields of virus in both cell lines irrespective of
the
status of p53. Strikingly, the E1B 55-kDa mutant viruses produced
wild-type virus yields in both the parental and H1299-p53 cell
lines
when cells were maintained at 39°C. However, growth of the
E1B 55-kDa
mutant Ads was restricted in the parental H1299 cells
at 32°C.
Approximately 30-fold (
dl338) and 16-fold
(
dl1520) less
virus was produced in cells maintained at
32°C than in those maintained
at 39°C. Wild-type p53 modestly
diminished the growth of the E1B
55-kDa mutant virus in H1299-p53 cells
at 32°C, reducing virus
yields 3.5-fold (
dl338) and
3.3-fold (
dl1520) beyond that of reduced
temperature alone.
Thus, p53 played a significant, albeit minimal,
role in inhibiting
growth of the E1B 55-kDa mutant Ad in H1299-p53
cells. By contrast, the
growth defect of the E1B 55-kDa mutant
viruses in these experiments was
primarily due to reduced temperature
and was no longer evident at the
elevated temperature.
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FIG. 7.
p53 only modestly affects replication of the E1B 55-kDa
mutant virus in H1299-p53 cells. Portions of H1299-p53 and parental
H1299 cells were shifted to 32°C or maintained at 39°C. Twelve
hours later, cells maintained at 39°C or shifted to 32°C were
infected with either the wild-type virus dl309 or the E1B
55-kDa mutant viruses dl338 and dl1520 at an MOI
of 9 PFU per cell. Virus yields were measured by plaque assay on 293 cells, using lysates harvested at 48 h postinfection for cells
infected at 39°C and 4 days postinfection for cells infected at
32°C. Virus yields are expressed as average PFU per cell derived from
two independent experiments performed in duplicate; error bars indicate
standard errors of the means.
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The cell cycle restriction of the E1B 55-kDa mutant virus may be
overcome at the elevated temperature.
The ability of the E1B
55-kDa mutant virus to replicate to near wild-type virus yields in
H1299 cells at 39°C may reflect the absence of a cell cycle
restriction at the elevated temperature. This possibility was examined
by using electron microscopy to measure the fraction of virus-producing
cells at 39 and 32°C. H1299 cells were maintained at 39 or 32°C and
infected with the wild-type virus dl309 or the E1B 55-kDa
mutant virus dl1520. Cells were fixed and processed for
transmission electron microscopy at 24 h postinfection for cells
maintained at 39°C and 48 h postinfection for cells maintained
at 32°C. Over 200 infected cells were scored for the presence of
progeny virus in each of two independent experiments, and the results
are summarized below.
Virus production in H1299 cells infected with the wild-type virus was
not affected by temperature. At both temperatures, 92%
of the infected
cells contained progeny wild-type virus. The apparent
cell cycle
restriction to E1B 55-kDa mutant virus replication
in H1299 cells may
have been partially overcome at 39°C. The E1B
55-kDa mutant virus
produced progeny in 73% ± 7% of the infected
cells that were
maintained and infected at 39°C. Similar results
were obtained for
HeLa cells grown and infected at elevated temperature.
When HeLa cells
were maintained and infected at 39°C, the E1B
55-kDa mutant virus
produced progeny in approximately 60% of the
infected cells,
suggesting that the cell cycle restriction to
E1B 55-kDa mutant virus
replication in HeLa cells can also be
partially overcome by elevated
temperature.
In contrast to H1299 cells infected at the elevated temperature, H1299
cells maintained and infected at 32°C with the E1B
55-kDa mutant
virus contained progeny virus in only 12% ± 3% of
the infected
cells. Because 28% of the H1299 cells maintained
and infected at
37°C contained E1B 55-kDa mutant virus progeny
(Table
2), it seems
likely that the cell cycle restriction is
more severe at the lower
temperature.
The cold-sensitive growth phenotype of the E1B 55-kDa mutant Ad in
H1299 cells appears to be linked to the defect in late gene
expression.
The E1B 55-kDa mutant viruses hr6 and
hr13 were reported to be cold-sensitive for growth
(29). Yields of hr6 and hr13 were reduced 213- and 75-fold, respectively, in HeLa cells maintained at
32.5°C compared to 38.5°C. Furthermore, the virus-mediated mRNA
transport defect of the E1B 55-kDa mutant virus was shown to be more
severe at 32°C than at 37 or 39°C (36, 68). Thus, it
seems likely that the cold-sensitive phenotype of E1B 55-kDa mutant Ad
growth in H1299 cells is related to the defects in mRNA transport or
late gene expression.
Late protein synthesis was analyzed in H1299 cells maintained at 32 or
39°C for 24 h and infected with the wild-type (
dl309)
or the E1B 55-kDa mutant (
dl1520) Ad. Cells were
pulse-labeled
with
35S-amino acids at 32 h
postinfection for cells maintained at 39°C
and 64 h
postinfection for cells maintained at 32°C. Cells maintained
and
infected at 39°C with the E1B 55-kDa mutant virus
dl1520
synthesized
late viral proteins to levels equivalent to or exceeding
that
of the wild-type virus (Fig.
8;
compare lanes 1 and 3). However,
the E1B 55-kDa mutant Ad was defective
for late viral gene expression
compared to the wild-type virus when
cells were maintained and
infected at 32°C (compare lanes 2 and 4).
This defect in late
gene expression in H1299 cells at 32°C is similar
to that observed
in HeLa cells at 37°C (
21,
68). H1299
cells grown at 32°C
and infected with either the wild-type or E1B
55-kDa mutant virus
synthesized reduced levels of late viral proteins
compared to
cells grown at 39°C. The reason for this effect of
reduced temperature
is not known, although it may reflect lower rates
of protein synthesis
at 32°C.
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FIG. 8.
H1299 cells infected with the E1B 55-kDa mutant virus
synthesize wild-type levels of late viral proteins when infected and
maintained at 39°C. Parental H1299 cells were shifted from 37 to 39 or 32°C for 24 h and then mock infected or infected with either
the wild-type virus dl309 or the E1B 55-kDa mutant virus
dl1520 at an MOI of 30. Cells were labeled with
35S-labeled amino acids for 1 h at 32 h
postinfection for cells maintained at 39°C or at 64 h
postinfection for cells maintained at 32°C. Proteins from
105 cells (per lane) were separated by SDS-PAGE. Five viral
late proteins were visualized and quantified with a PhosphorImager. The
positions of migration and masses (in kilodaltons) of molecular weight
standards are indicated on the right. The positions of five Ad late
proteins were determined by using Ad virion standards labeled with
14C-amino acids; these proteins are identified on the left.
Assoc, associated.
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The amount of radioactivity in the five late viral proteins identified
in Fig.
8 was measured with a PhosphorImager in two
independent
experiments. The amount of radioactivity present in
each of these
proteins synthesized in the E1B 55-kDa mutant virus-infected
cells is
expressed as a percentage of the corresponding protein
synthesized in
the wild-type virus-infected cell. These results,
summarized in Table
4, confirm that the E1B 55-kDa mutant
virus
directed the synthesis of wild-type levels of protein at 39°C.
By contrast,
dl1520-infected cells synthesized late viral
proteins
to an average of 44% of the level synthesized in wild-type
virus-infected
cells at 32°C. This findings also reveals that virus
production
is not accurately reflected by the amount of late viral
protein
synthesized; the 2-fold average reduction in late viral protein
synthesis measured in the mutant virus-infected cells is accompanied
by
a 16- to 30-fold reduction in virus yield.
| |
DISCUSSION |
In this work, we demonstrated that the inability of E1B 55-kDa
mutant Ads to replicate efficiently is not due to a failure to abrogate
the function of p53. Replication of the E1B 55-kDa mutant viruses
(dl338 and dl1520) was not correlated with the status of p53 in the seven cell lines examined in this study (Fig. 2).
Furthermore, viral DNA synthesis (Fig. 3), cell cycle-independent replication (Table 3), and virus-induced cell killing (Fig. 4) in cells
infected with the E1B 55-kDa mutant virus did not correlate with the
status of p53. Rather, cell killing by the E1B 55-kDa mutant virus
correlated with permissivity to viral replication (Fig. 4 and 5).
Yields of the E1B 55-kDa mutant viruses were reduced only modestly in
an H1299-p53 cell line expressing a temperature-sensitive allele of p53
in the wild-type conformation (Fig. 7). The defect in E1B 55-kDa mutant
virus replication resulting from reduced temperature was as much as
10-fold greater than that due to p53. Thus, E1B 55-kDa mutant virus
replication is restricted by mechanisms independent of p53. In addition
to replication, the defect in late viral gene expression (Fig. 8 and
Table 4) and the cell cycle restriction (see Results) of the E1B 55-kDa
mutant virus exhibited a cold-sensitive phenotype in H1299 cells. At
elevated temperatures, the E1B 55-kDa mutant virus synthesized
wild-type levels of viral late proteins and replicated independently of the cell cycle. Taken together, these results suggest that the ability
of the E1B 55-kDa protein to promote late gene expression may
contribute to cell cycle-independent replication of Ad.
The E1B 55-kDa protein has been hypothesized to contribute to both
lytic infection and transformation by preventing p53-mediated G1 growth arrest or apoptosis. This response by p53 to
viral challenge would be expected to severely hinder the ability of the
virus to transform cells, synthesize viral DNA, and establish a lytic infection in the absence of the E1B 55-kDa protein (12, 38, 67). Furthermore, p53 could potentially inhibit cell
cycle-independent Ad replication in the absence of the E1B 55-kDa
protein. Recently, Bischoff et al. (7) have suggested that
the E1B 55-kDa mutant virus (dl1520) selectively replicates
in and kills p53-deficient cells, suggesting that the interaction
between p53 and the E1B 55-kDa protein is essential to the lytic
infection. In accordance with the results of Fig. 2 and 4, they found
that U2OS cells (wild-type p53) were resistant to E1B mutant
virus-induced cell killing and defective for E1B mutant virus
replication whereas C33A cells (p53 mutant) were susceptible to E1B
mutant-induced cell killing and permissive for replication of the E1B
mutant virus. However, contrary to the hypothesis offered by Bischoff
et al. (7), A549 cells which express wild-type p53 were
permissive for E1B 55-kDa mutant virus replication and cell killing
(Fig. 2 and 4). In addition, Bernards et al. (6) reported
that human embryonic kidney cells, which contain wild-type p53,
produced near-wild-type yields of the E1B 55-kDa mutant virus.
Furthermore, a wild-type p53 status did not affect viral DNA synthesis
in E1B 55-kDa mutant-infected cells (Fig. 3). However, Bischoff et al.
(7) reported that the RKO human colon cancer cell line
transfected to express a dominant negative p53 gene exhibited more
severe cytopathic effects when infected with ONYX-015 compared to the
parental RKO cell line with normal p53 function. Alternatively, Ridgway
et al. (54) reported that wild-type Ads failed to replicate
efficiently in p53 mutant cells lines because of a requirement of the
E1B 55-kDa protein-p53 complex for viral growth and the shutoff of host
protein synthesis. The results of these investigators are inconsistent with the work presented here in that we do not find a correlation between viral replication and the status of p53. The E1B 55-kDa mutant
virus most efficiently replicated in and lysed p53 mutant C33A cells
(Fig. 2 and 4). Replication of the E1B 55-kDa mutant viruses relative
to the wild-type virus differed among various cell types. These
differences could not be attributed to the status of p53.
The E1B 55-kDa mutant virus ONYX-015 (dl1520) has been shown
to selectively kill cancerous cell lines while sparing normal cells and
to induce regression of human tumor xenografts in nude mice
(28). The application of oncolytic Ad vectors for cancer therapy through the ability of Ad to induce tumor regression by lytic
replication also has been demonstrated by others (77). However, clinical applications of replication-competent virus vectors
require selectivity for their efficacy. Bischoff et al. (7)
analyzed the ability of the wild-type and E1B 55-kDa mutant viruses to
induce regression of p53 null human tumor xenografts transplanted into
nude mice. In both p53 null and p53 wild-type tumors, the wild-type
virus was better able to retard tumor growth than the E1B 55-kDa mutant
virus. The ability of the E1B 55-kDa mutant virus to suppress tumor
growth compared to that of the wild-type virus was unaffected by the
status of p53 (7). Furthermore, cancer cell lines that were
mutant for p53 did not exhibit significantly greater susceptibility to
killing by the E1B 55-kDa mutant virus compared to cells with a
functional p53 (28). Therefore, the failure of the E1B
55-kDa mutant virus to replicate and lyse infected cells is not
strictly dictated by the status of p53 in the infected cell.
We found that cell killing induced by the E1B 55-kDa mutant virus
correlated with permissivity to viral replication and not the status of
p53 (Fig. 4). In the absence of the E1B 55-kDa protein, our work has
demonstrated that HeLa cells infected during S phase are more
permissive than HeLa cells infected during G1 for
replication of E1B 55-kDa mutant viruses (20). We report
here that S-phase HeLa cells are more susceptible than G1
HeLa cells to killing by the E1B 55-kDa mutant virus (Fig. 5). Perhaps
this host range restriction of the E1B 55-kDa mutant virus could be
used to effectively target those tumors that contain a significant
fraction of cells in S phase (5, 34, 40) for virus-induced
killing. Because selective killing of S-phase cells did not depend on
increased apoptosis (Table 3), E1B 55-kDa mutant viruses may be
appropriate oncolytic vectors for the treatment of tumor cells that
have lost the capacity to initiate an apoptotic pathway. We propose
that selectivity to replication of an E1B 55-kDa mutant virus is linked to the cell cycle. The molecular basis for selective replication of
cell cycle-restricted Ads continues to be investigated.
The p53 null cell lines (H358, Saos-2, U2OS, and H1299) were defective
for E1B 55-kDa mutant virus replication (Fig. 2) and produced virus in
only a fraction (
28%) of the infected cells (Table 2). This defect
in replication resembles the cell cycle-restricted phenotype previously
described for HeLa cells (20). By contrast, A549 (p53
wild-type) and C33A (p53 mutant) cells produced virus in 70 and 90%,
respectively, of the E1B 55-kDa mutant-infected cells, as determined by
electron microscopy. Therefore, the cell cycle-mediated growth
restriction is diminished or absent in these permissive cells. Perhaps
these cell lines can be used to elucidate the nature of the cell cycle restriction.
The effect of p53 on E1B 55-kDa mutant virus replication was examined
in H1299 cells stably expressing a temperature-sensitive p53 allele
(18). This approach was chosen to minimize unknown differences between cell lines other than the status of p53 that may
affect replication of an E1B 55-kDa mutant virus. Growth of the E1B
55-kDa mutant virus was substantially (16- to 30-fold) less than
wild-type virus growth at the lower temperature in the parental H1299
(p53 null) cell line (Fig. 7). By contrast, the presence of wild-type
p53 at the lower temperature modestly (3.3- to 3.5-fold) reduced
replication of the E1B 55-kDa mutant beyond the effect of temperature
alone. These results demonstrate that p53 affects E1B 55-kDa mutant
virus replication. However, the defect in replication of E1B 55-kDa
mutant virus resulting from reduced temperature was as much as 10-fold
greater than that from p53 alone. Therefore, the E1B 55-kDa protein may
interact with cellular regulatory factors in addition to p53 to permit
virus replication in the wild-type Ad infection.
Interestingly, the E1B 55-kDa mutant virus produced near-wild-type
yields of virus in H1299 or H1299-p53 cells maintained and infected at
39°C, as previously reported for HeLa cells (29). At the
higher temperature, the defect in late gene expression (Fig. 8 and
Table 4) and the cell cycle restriction for replication (see Results)
of the E1B 55-kDa mutant virus were no longer evident. H1299 cells
maintained and infected at 39°C synthesized wild-type levels of viral
late proteins and produced virus in 73% of the infected cells. These
results resemble those obtained after infection of HeLa cells during S
phase (20). HeLa cells infected during S phase produced
greater virus yields and produced virus in up to 75% of the infected
cells, compared to 25% of randomly cycling cells. By contrast, the
cell cycle restriction was exacerbated when H1299 cells were maintained
and infected at 32°C (see Results) or when HeLa cells were infected
during G1 phase (20). Under each of these
conditions, E1B 55-kDa mutant virus was produced in no more than 12%
of the infected cells. Furthermore, H1299 cells infected with the E1B
55-kDa mutant virus produced lower virus yields (Fig. 7) and
synthesized reduced levels of late viral proteins at 32°C (Fig. 8 and
Table 4). The defect in mRNA transport and, therefore, late viral gene
expression by the E1B 55-kDa mutant virus was previously shown to be
exacerbated at reduced temperatures in HeLa cells (36, 68).
Both the cell cycle restriction and the defect in late viral gene
expression of the E1B 55-kDa mutant virus were exacerbated by reduced
temperatures and ameliorated by elevated temperatures. Therefore, it is
conceivable that a cellular activity at 39°C that compensates for the
loss of the E1B 55-kDa protein is the same activity in S phase that
permits efficient replication of the E1B 55-kDa mutant virus. For
example, a cellular factor that promotes virus replication may be made
available or cellular factors that hinder virus replication may be
absent when cells are maintained and infected at 39°C or infected
during S phase. Such factors may participate in virus-mediated
mRNA transport and cell cycle-independent viral replication.
We would speculate that the proposed cellular factor(s)
interacts with the E1B 55-kDa-E4orf6 protein complex in Ad infection
(22, 47) or, alternatively, depends on the E1B-E4 complex
for efficient expression late in Ad infection.
| |
ACKNOWLEDGMENTS |
This work was supported in part by Public Health Service grant
AI35589 from the National Institute of Allergy and Infectious Disease
to D.A.O. and grant CA12197 from the National Cancer Institute to the
Comprehensive Cancer Center of Wake Forest University. Tissue culture
reagents and services were provided by the Tissue Culture Core
Laboratory of the Comprehensive Cancer Center of Wake Forest
University, supported in part by NIH grant CA12197.
We gratefully acknowledge Tom Shenk (Princeton University) for the
dl309, dl337, and dl338 viruses, Arnie
Berk (UCLA) for the dl1520D virus, and Arnie Levine
(Princeton University) for the B6-8 hybridoma cell line. We also thank
Nancy Raab-Traub and Katherine Fries (University of North Carolina,
Chapel Hill) for the H1299 and H1299-p53 cells and Gerry Zambetti (St.
Jude Children's Research Hospital) for the Saos-2 cells. The annexin
V-FITC reagent was the generous gift of Greg Kucera (Wake Forest
University School of Medicine, Winston-Salem, N.C.). We also thank
Natalie Walker for assistance with the FACS analysis.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology and Immunology, Wake Forest University School of Medicine, Bowman Gray Campus, Winston-Salem, NC 27157-1064. Phone: (336) 716-9332. Fax: (336) 716-9928. E-mail: ornelles{at}wfubmc.edu.
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Journal of Virology, December 1998, p. 9479-9490, Vol. 72, No. 12
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
