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Journal of Virology, January 1999, p. 214-224, Vol. 73, No. 1
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Transactivation of a Ribosomal Gene by Simian Virus 40 Large-T
Antigen Requires at Least Three Activities of the Protein
Jane F.
Cavender,1,*
Christine
Mummert,2 and
Mary
Judith
Tevethia2
Department of Biology, Elizabethtown College,
Elizabethtown, Pennsylvania 17022,1 and
Department of Microbiology and Immunology, Pennsylvania
State University College of Medicine, Hershey, Pennsylvania
170332
Received 30 January 1998/Accepted 18 September 1998
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ABSTRACT |
Simian virus 40 large-T antigen transactivates the ribosomal genes
which are transcribed by RNA polymerase (pol I), as well as genes that
are dependent on either pol II or pol III. This report identifies
regions and activities of T antigen that are required to transactivate
a pol I-dependent rat ribosomal gene promoter. By using the rat
ribosomal gene (rDNA) promoter linked to a chloramphenicol
acetyltransferase gene, we show that at least three separable T-antigen
regions are necessary to achieve wild-type levels of transactivation of
rDNA in transiently transfected monkey cells. One activity depends on
the region of T antigen shared with small-t antigen (T/t common
region). A second activity maps to amino acids 109 to 626 and is highly
sensitive to mutational inactivation. Complementation analyses suggest
that at least one activity in this region is independent of
and must be in cis with the activity within the T/t common
region. In addition, a functional nuclear localization signal is
required for maximal T-antigen-mediated transactivation of rat rDNA.
The three activities work in concert to override cellular
species-specific controls and transactivate the rat ribosomal gene
promoter. Finally, we provide evidence that although the tumor
suppressor protein Rb has been shown to repress a pol I-dependent
promoter, transactivation of the rat rDNA promoter does not depend on T
antigen's ability to bind the tumor suppressor product Rb.
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INTRODUCTION |
Expression of the simian virus
40 (SV40) large-T antigen (or, for simplicity, T antigen) is sufficient
to initiate and maintain transformation of cells in culture
and tumorigenesis in experimental animals (see references 21
and 40 for recent reviews). Regions and activities of
the multifunctional protein that are involved in specific growth
property changes that accompany transformation have been defined
(40). The aggressive growth that characterizes transformed
and tumor cells places increased demands on protein synthesis. In
support of this contention, current evidence indicates that increased
protein synthesis, brought about by overexpression of translation
initiation factors, can result in transformation (for a review, see
reference 30). Increased expression of ribosomal genes might provide a second mode of increasing protein
synthesis. One consequence of T-antigen expression is
transactivation of ribosomal genes (36, 53, 55, 68). The
relationship of this activity to transformation remains to be
determined. Genetic analysis to identify the regions of T antigen
required for transactivation of the ribosomal genes in vivo is a first
step in correlating this capability with the oncogenic activities of
the protein.
The ribosomal genes are transcribed by RNA polymerase I (pol I)
in a species-specific manner with the aid of at least two transcription factors, the upstream binding factor (UBF) and the species selectivity factor SL1. The rat ribosomal gene (rDNA) contains
a core promoter element (CPE) located between nucleotides (nt) 
31 and
+6 (6, 20, 27, 36, 69, 70), an upstream promoter element
(UPE), whose exact location varies slightly between species (nt 50 to
186) (27, 50), an enhancer (nt 2357 to 2183)
(16), and terminator sequences (29). UBF binds to
specific sequences in both the UPE and the CPE (2, 42) and
stimulates pol I-mediated transcription. SL1 has low DNA-binding
affinity unless it is accompanied by UBF (2). SL1 is needed
for efficient transcription of the ribosomal genes and is responsible
for conferring the species-specific nature of the transcription
(3, 25, 35).
Human SL1 is a complex composed of the TATA-binding protein (TBP; 38 kDa) and three TATA-associated factors, TAFI48 (48 kDa), TAFI63 (63 kDa), and TAFI110 (110 kDa)
(12, 18). TAFI48 and TBP efficiently bind to
UBF, whereas TAFI110 and TAFI63 contact the
promoter directly (1). Thus, during transcription, UBF and
pol I interact with cis elements in the promoter, whereas SL1 associates with these proteins to form the active initiation complex (1, 12).
T antigen is a promiscuous transcriptional transactivator. It
transactivates the ribosomal genes, which are transcribed by pol I
(36, 53, 54), as well as genes that are dependent on either
pol II or pol III. T antigen's ability to transactivate pol II- and
III-dependent promoters (38, 47, 63) and the regions of the
protein involved have been investigated in detail (4, 26, 31, 56,
72). However, less is known concerning the T-antigen activities
required for transactivation of pol I-dependent promoters. In HeLa cell
extracts, purified T antigen increases in vitro transcription from a
human ribosomal promoter (36). Recently, Zhai et al.
(71) showed that T antigen binds to the SL1 complex through
interactions with TAFI48, TAFI110, and TBP and
that the T antigen-SL1 association is crucial to activation of the
ribosomal gene promoter. They showed further that T-antigen amino acids
1 to 436 were sufficient to bind SL1 and that amino acids 1 to 538 were
sufficient to stimulate pol I-mediated transcription of the human
ribosomal gene in HeLa cell extracts.
Early investigations into T antigen's ability to override the species
specific nature of ribosomal transcription in vivo assessed reactivation silent rDNA promoters within mouse-human hybrid cell lines
(53, 55). These assays defined the T-antigen region necessary for the reactivation of a heterologous rRNA gene as amino
acids 1 to 509 (54).
The role of specific T-antigen activities in pol I-dependent
transactivation remains to be determined. It was shown recently that
the retinoblastoma gene product, Rb, plays a role in the regulation of
ribosomal transcription (7, 67). In vitro Rb binds to UBF
(7) and inhibits its ability to bind to the UPE. This
interaction represses transcription from the ribosomal promoter (67). It is not known whether T-antigen binding to Rb is
involved in activation of ribosomal genes.
The study reported here focused on the ability of T antigen to
transcriptionally activate the rat ribosomal gene promoter when
transiently transfected into monkey cells and investigated the specific
regions and activities of T antigen necessary for this heterologous
ribosomal gene transactivation. The results indicate that at least
three activities cooperate to transactivate the rat ribosomal gene and
that Rb binding is not essential.
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MATERIALS AND METHODS |
Plasmids.
The rat ribosomal promoter was cloned into plasmid
pU3RIIICAT (51) as follows. Plasmid pDJ3 (16),
used as the source of the rat ribosomal promoter, contains the rat
ribosomal promoter within 2.16 kb of external transcribed sequence. The
promoter was released from pDJ3 by first digesting the plasmid with
KpnI. The KpnI end was made blunt by excessive
treatment with the Klenow fragment of DNA polymerase, and an
XhoI linker was added. The resulting DNA was digested with
XhoI and HindIII, and the released fragment
containing nt 527 to +124 was gel purified. Plasmid pU3RIIICAT was
digested with SalI and HindIII to release the
human immunodeficiency virus long terminal repeat (LTR), and the rDNA promoter was inserted in its place to generate the reporter plasmid prDCAT. Loss of the SalI site provided evidence of
insertion. The rat promoter cloned upstream of the chloramphenicol
acetyltransferase (CAT) gene contains the CPE and UPE (167 to +124),
encompassing the region protected by rat UBF binding (69),
and 360 bp of far-upstream sequence.
Plasmid pPVU0 (33) contains the SV40 enhancer, promoter,
origin, and early region coding sequences from the PvuII (nt
272) to BamHI (nt 2533) sites cloned into pBR328 at the
corresponding sites. pPVU0 produces functional large-T and small-t
antigens. Plasmid Wt-2 was generated by cloning the entire SV40 genome
into the EcoRI site of pBR322. Plasmid pdl2005 contains the
genome of the mutant dl2005, which has a 230-bp deletion
within the large-T intron and does not produce detectable quantities of
small-t antigen (49).
Plasmid pdl536 was generously provided by L. Sompayrac. The pdl536
(
52) construct contains the genome of the deletion mutant
dl536 cloned into the
BamHI site of pBR322. The
dl536 deletion
removes the splice acceptor site for the
large-T-antigen and small-t-antigen
mRNAs. Thus, pdl536 encodes an
authentic small-t antigen but no
large-T
antigen.
Plasmids
dl1265,
dl1066,
dl2465,
dl1061,
dl2433, and
dl1263 contain the
deletion mutant genomes cloned into pBR322. The mutants
and their
properties have been described previously (
62,
65).
They
encode T antigens containing amino acids 1 to 699, 1 to 670,
1 to 626, and 1 to 590 and T antigen missing amino acids 586 to
589 and 663 to
674, respectively. Plasmid K1 (
33) encodes a
T antigen
(T-Glu107Lys) with the amino acid substitution Glu107Lys.
Plasmid D10
(
33) encodes a T antigen (T-Lys128Thr) with the
amino acid
substitution
Lys128Thr.
Plasmids
dl105-108 (
dl2441) (
73),
dl127-250 (S11-S24) (
33),
dl252-300,
dl301-350,
dl336-484,
dl347-370,
dl357-370,
dl351-400,
dl382-400,
dl400,
dl401-436,
dl401-450,
dl434-444,
dl451-465,
dl451-532,
dl501, and
dl501-550 (
34) have been
described previously.
They contain the early coding regions of the
mutants in a pBR328
plasmid backbone. They express T antigens missing
the amino acids
indicated by the plasmid
names.
The SV40 nuclear localization signal (NLS; amino acids 126 to 132)
(
32) was inserted into D10, and also into
dl127-250,
between amino acids 650 and 651 in the following
steps. Initially,
annealed complementary oligonucleotides containing
T-antigen codons
126 to 132 bounded by
EcoRI-compatible ends
of appropriate length
to conserve the reading frame were inserted at
the
EcoRI site
between codons 650 and 651 of plasmid
pPVU0RI650 (
34). Then
the small DNA fragment generated by
digesting the resulting plasmid,
pPVU0RI650NLS, with
PvuII
and
BamHI was purified from an agarose
gel following
electrophoresis and was ligated to the large
PvuII-plus-
BamHI
digest fragment of
dl127-250 to produce
dl127-250NLS650. The NLS
was
inserted into plasmid D10 by the equivalent fragment exchange
between
dl127-250NLS650 and plasmid D10. In a separate construction,
the SV40 NLS was inserted between codons 126 and 251 of
dl127-250
by using annealed oligonucleotides as described
for the generation
of
pPVU0RI650NLS.
Plasmid CAV83-708 was constructed as follows. First, the SV40 early
coding region and promoter/enhancer contained between
the
KpnI and
BamHI sites was cloned into the vector
p-Select (Promega).
Then the
EcoRI site in the vector was
converted to a blunt end
to remove the enzyme recognition sequence.
Oligonucleotide-directed
mutagenesis was performed on the resulting
plasmid to insert an
EcoRI linker between codons 82 and 83. The resulting plasmid was
digested with
EcoRI and
SalI. The fragment released from the vector
contained the
early-region sequences encoding T-antigen amino
acids 83 to 708 and
the poly(A) site. The released fragment was
cloned between the
corresponding sites of the modified (
8)
vector pBluescript
SK
+ (Stratagene) and subsequently was transferred into the
modified
expression vector as described previously (
8). The
final construct
encodes a fusion protein containing the first seven
amino acids
of the
-galactosidase
-peptide followed by 31 amino
acids encoded
by the synthetic polylinker in pBluescript SK, two amino
acids
encoded by the
EcoRI linker, and T-antigen amino acids
83 to 708
under control of the cytomegalovirus transcriptional
promoter/enhancer.
CAT assays.
Two and a half micrograms of prDCAT reporter, 5 µg of salmon sperm (carrier) DNA, and 5 µg of wild-type or mutant
T-antigen plasmid or an additional 5 µg of carrier DNA were
transfected into TC7 cells by the DEAE-dextran-chloroquine procedure
essentially as described elsewhere (8). For complementation
assays, the carrier DNA was replaced by 5 µg of DNA of the
complementing mutant. Specifically, 6 × 105 cells
were seeded into 60-cm2 tissue culture dishes 1 day prior
to transfection. For each DNA sample, two to four replicate plates were
seeded in Dulbecco's modified Eagle's medium supplemented with 100 µg of streptomycin per ml, 100 µg of kanamycin per ml, 100 U of
penicillin per ml, 0.03% glutamine, 0.15%
Na2HCO3, 25 mM HEPES, and 10% fetal bovine serum (DMEM10×2+HEPES). For each transfection, DNA was added to 750 µl of Tris-buffered saline (TBS; pH 7.4). Then 250 µl of a 2-mg/ml stock solution of DEAE-dextran was added to give a final DEAE-dextran concentration of 500 µg/ml. Cell monolayers were rinsed
once with TBS. One-half of each DNA mixture then was placed onto each
of two cultures, and the dishes were rocked for 20 min at room
temperature. TBS (5 ml) was added to each plate, aspirated, and
replaced with 4.5 ml DMEM10×2+HEPES containing 100 µM chloroquine phosphate. The cultures were incubated at 37°C for precisely 3.5 h. The medium then was replaced with DMEM10×2+HEPES, and the cells were incubated at 37°C for an additional 48 h.
Proteins were extracted from the transfected cells, and the extracts
were processed for CAT assays essentially as described
previously
(
48). Briefly, medium was removed from the cells,
and the
monolayers were washed once with 4 ml of TBS. Monolayers
were
incubated in an additional 4 ml of TBS for 5 min. Following
removal of
the TBS, 0.5 ml of 0.25 M Tris-Cl (pH 8.0) was added,
and the
monolayers were incubated at room temperature for an additional
5 min.
Cell monolayers were scraped into the buffer, and the cell
suspensions
were placed into microcentrifuge tubes. Cells were
lysed by three
cycles of freezing and thawing, and endogenous
acetylase activity was
inactivated by heating at 65°C for 10 min.
Lysates were cleared by
microcentrifugation at 14,000 rpm for
10 min at 4°C. Then 70 µl of
each extract was assayed for CAT
activity after addition of
n-butyryl coenzyme A (0.2 mg/ml), 100
µCi of
[
14C]chloramphenicol, and 0.25 M Tris-Cl (pH 8.0) to a
final volume
of 125 µl. The samples were incubated at 37°C for 16 to 20 h.
The butyrated product was isolated by xylene extraction.
Specifically,
350 µl of mixed xylenes was added to each reaction; the
tubes
were vortexed for 40 s and centrifuged for 5 min at
14,000 rpm.
Next, 300 µl of the upper layer containing the
butyrated product
was transferred to a microcentrifuge tube containing
125 µl of
0.25 M Tris-Cl (pH 8.0), vortexed for 20 s, and
centrifuged for
5 min at 14,000 rpm. Two hundred microliters of the
butyrated
product was transferred to a glass scintillation vial, and 5 ml
of aqueous fluor was added. The samples were counted for 1 min
in a
Beckman scintillation counter. The means of the counts per
minute of
replicate samples were calculated. The level of transactivation
is
expressed as the mean percentage of wild-type activity in the
same
experiment; the percent standard error of the mean was
calculated.
Antibodies.
To detect T antigen and various mutant T
antigens, monoclonal antibodies PAb901 and PAb419 were used. PAb901
recognizes a denaturation-resistant epitope located between amino acids
684 and 698 (57). PAb419 is directed towards the N-terminal
82 amino acids of large-T and small-t antigens, and it recognizes a
denaturation-resistant epitope (28).
Immunoblot analysis.
Immunoblot analysis was performed
essentially as described by Cavender et al. (8). TC7 cells
were seeded into T150 tissue culture flasks. Cells were transfected by
the DEAE-dextran-chloroquine method with 40 µg of plasmid DNA.
Proteins were extracted 48 h posttransfection. Equal protein
amounts were used in immunoprecipitation reactions. Detection of the
specific proteins was accomplished by using protein A-Sepharose
conjugated with horseradish peroxidase obtained from Amersham.
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RESULTS |
Transactivation of the rat ribosomal promoter by wild-type large-T
antigen.
Figure 1A shows the results
of representative experiments assessing the ability of large-T and
small-t antigens to transactivate the rat ribosomal promoter in
transiently transfected monkey cells. The basal level of CAT activity
produced from the rat ribosomal promoter reporter plasmid was not
significantly different from that in extracts prepared from cells
transfected with carrier DNA only, confirming the species-specific
nature of the rDNA promoter. Since there was no measurable activity
from the rat ribosomal promoter in the monkey cells, fold activation by
T antigen could not be measured. Therefore, transactivation is
expressed as percentage of wild-type activity in the same experiment.
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FIG. 1.
Transactivation of the rat ribosomal promoter by
wild-type large-T antigens and the accumulated levels of T antigens in
transiently transfected cells. (A) Transactivation of the rat ribosomal
promoter-CAT construct was assayed by determining the amount of
butyrated chloramphenicol produced by extracts prepared from cells
transfected with the reporter construct only or in conjunction with
plasmids that produce wild-type large-T antigen only, small-t antigen
only, or both large-T and small-t antigens (pPVU0 or Wt-2), as
described in Materials and Methods. Each construct or construct
combination was transfected in duplicate or quadruplicate. Replicate
assays were performed on extracts from each transfection. Means were
determined from the four or eight replicate samples. The level of
transactivation is expressed as the mean percentage of wild-type (wt)
activity in the same experiment. Each error bar represents the percent
standard error of the mean; the absence of an error bar indicates that
the error was less than 2%. The entire experiment was performed four
times. The pattern of results was consistent; results of one
representative experiment are shown. (B) Accumulated levels of T
antigen produced from cells transiently transfected with pPVU0, Wt-2,
or a T-antigen-only (T1-708) construct. Immunoblot analysis of
transfected cell protein extracts was performed as described in
Materials and Methods with monoclonal antibody PAb901, which recognizes
an epitope in the C terminus of T antigen.
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Initially, three large-T-antigen expression plasmids were tested.
In the experiments described below, the majority of mutant
T antigens
used were cloned in or derived from two plasmid sources.
Wt-2 and
dl2005 contain SV40 genetic information in a pBR322
background.
In pPVU0, the SV40 information is contained in the pBR328
background.
All three plasmids showed a high level of transactivation.
As
shown in Fig.
1A,
3A, and
4, cells transfected with prDCAT and
the
large-T antigen-expressing plasmid
dl2005 or plasmid Wt-2,
which expresses large-T and small-t antigens, produced a higher
level of CAT activity than cells cotransfected with plasmid pPVU0.
pPVU0, like Wt-2, produces both large-T and small-t antigens.
Immunoblot analysis of cells transfected with the plasmids is
shown in
Fig.
1B. In all cases, T antigen accumulated to approximately
the same
level. Therefore, the reason for the differences in levels
of
transactivation is not clear. The majority of mutant T-antigen
constructs examined in subsequent transactivation experiments
were
derived from pPVU0. Therefore, in all experiments the levels
of
transactivation were compared with results for pPVU0. In addition,
in
those cases involving mutant T-antigen constructs in a pBR322
background, the level of transactivation achieved by Wt-2 was
included. Small-t antigen did not contribute to the
transactivation
by wild-type large-T antigen, as the percentages
of transactivation
conferred by Wt-2 and
dl2005 were
similar. Thus, as shown previously
by microinjection (
53,
55) and in vitro transcription assays
(
36), large-T
antigen alone was sufficient to transactivate
the rat rDNA promoter in
transient transfection
assays.
Transactivation capacity of C-terminally truncated T antigens.
The C-terminal region of T antigen is dispensable for multiple
functions of the protein (10, 45, 62, 65, 66). To determine
the extent to which the C-terminal sequences of T antigen are necessary
for transactivation of the rat ribosomal promoter in monkey cells, each
of a series of deletion mutants was cotransfected with the reporter
plasmid and tested three to six times. The pattern of transactivation
by the mutants was consistent. Figure 2A
shows the results of a representative experiment. T antigen missing amino acids 700 to 708 (T1-699), 671 to 708 (T1-670), 663 to 674 (Tdl663-674), or 627-708 (T1-626) transactivated the rat
ribosomal promoter to wild-type levels. However, transactivation was
abrogated by deleting amino acids 591 to 708 (T1-590) or 587 to 589 (Tdl586-590). Immunoblot analysis of cells transiently
transfected with the mutant T-antigen constructs is shown in Fig. 2B.
T1-699 and Tdl663-674 accumulated approximately to the level
of T1-708. The levels of the T1-670, T1-626, and Tdl586-590
were readily detected but reduced; the T1-591 protein did not
accumulate to a detectable level. Therefore, a T antigen consisting
of amino acids 1 to 626 was sufficient to transactivate the
rat ribosomal promoter to wild-type levels, and wild-type levels
of protein are not required to achieve full transactivation
potential. In addition, removal of amino acids 586 to 590 abrogated transactivation, suggesting a requirement for integrity
of that portion of T antigen. The results obtained with T1-590 also
confirmed that small-t antigen was not sufficient for transactivation
of the ribosomal promoter.
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FIG. 2.
Transactivation by C-terminally truncated T antigens and
the accumulated levels of mutant T antigens in transiently transfected
cells. (A) The ability of each mutant T antigen to transactivate the
rat ribosomal promoter was tested three to six times as described for
Fig. 1A. The pattern of results was consistent; results of a
representative experiment are shown. Each error bar represents the
percent standard error of the mean; the absence of an error bar
indicates that the error was less than 2%. (B) Accumulated levels of T
antigen produced from cells transiently transfected with Wt-2,
dl1265 (T1-699), dl1066 (T1-670),
dl1263 (Tdl663-674), dl2465 (T1-626),
dl1061 (T1-590), and dl2433
(Tdl586-590) constructs. Immunoblot analysis of transfected
cell protein extracts was performed as described in Materials and
Methods with monoclonal antibodies PAb901, which recognizes an epitope
in the C terminus of T antigen, and PAb419, which recognizes an epitope
in the T/t common region.
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Transactivation by T antigens containing internal or N-terminal
deletions.
To further define the T-antigen segments that are
sufficient to transactivate the rat ribosomal promoter in vivo, mutants with deletions spanning amino acids 127 to 550 were examined. Figure
3A shows that the T antigen missing amino
acids 105 to 108 transactivated to a wild-type level. However, T
antigens bearing internal deletions of amino acids 127 to 250, 252 to
300, 301 to 350, 336 to 484, 347 to 370, 357 to 370, 351 to 400, 382 to 400, 400, 401 to 436, 401 to 450, 434 to 444, 451 to 532, 451 to 465, 501, or 501 to 550 failed to transactivate the rat ribosomal promoter.
Even deletion of the single amino acid at position 400 or 501 yielded
no transactivational activity. Figure 3B shows that the mutant T
antigens accumulated to approximately wild-type levels or greater with
the exception of Tdl336-494. However, T antigens with
smaller deletions within the region encompassed by amino acids 336 to
494 accumulated to the wild-type level. These results suggest that
transactivation of the rat ribosomal gene promoter by T antigen
requires integrity of the T-antigen region between amino acids 108 and
550.
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FIG. 3.
Transactivation by T antigens containing internal or
N-terminal deletions and the accumulated levels of mutant T antigens in
transiently transfected cells. (A) The ability of each mutant T antigen
to transactivate the rat ribosomal promoter was determined as described
for Fig. 1A. Each mutant was tested two to six times. The pattern of
results was consistent; the results of one representative experiment
are shown. Each error bar represents the percent standard error of the
mean; the absence of an error bar indicates that the error was less
than 2%. (B) Accumulated levels of T antigen produced from cells
transiently transfected with each mutant T-antigen-producing construct.
Immunoblot analysis of transfected cell protein extracts was performed
as described in Materials and Methods with monoclonal antibody PAb901,
which recognizes an epitope in the C terminus of T antigen.
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To determine whether N-terminal amino acids were needed, mutant
T83-708, which is missing amino acids 1 to 82, the region
shared by
large-T and small-t antigens (the T/t common region),
was examined. The
results presented in Fig.
4 showed that
removal
of this T/t common region substantially reduced
transactivation.
Immunoblot analysis (Fig.
5) indicated that T83-708 accumulated
to
a level lower than the wild-type level but greater than those
for other
mutants that retained full transactivation potential
(Fig.
2B). These
results suggested that alterations in the N terminus
as well as in the
region from amino acids 108 to 550 of T antigen
abrogate
transactivation.
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FIG. 4.
Complementation of an N-terminally truncated T antigen.
The ability of the N-terminally truncated T antigen T83-708 was tested
for its ability to transactivate the rat ribosomal promoter either
alone and in combination with Tdl400 or small-t antigen.
Each T antigen or combination was tested at least six times. The
pattern of results was consistent; results of a representative
experiment for each complementation are shown. Each error bar
represents the percent standard error of the mean; the absence of an
error bar indicates that the error was less than 2%.
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FIG. 5.
Immunoblot analysis of mutant T antigens used in
complementation experiments and the effect of cotransfection on protein
accumulation. Immunoblot analysis of transfected cell protein extracts
was performed as described in Materials and Methods with monoclonal
antibody PAb901, which recognizes an epitope in the C terminus of T
antigen.
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Complementation assays.
The observation that interrupting
multiple regions of T antigen disrupted transactivation suggested two
possibilities. Multiple independent activities may cooperate to provide
full transactivating potential, or transactivation may depend on a
specific conformation of T antigen dictated by several regions of the
protein. Complementation assays were performed to distinguish between
these alternatives. Initially, complementation between the small
internal deletion (dl400) and T83-708 was examined. The
results in Fig. 4 showed that neither mutant T antigen alone
transactivated the rat ribosomal promoter substantially; however,
wild-type levels of transactivation were achieved in complementation
assays between Tdl400 and T83-708. Figure 5 shows the
relative levels of the mutant T antigens when transfected singly or in
combination. In cells expressing both T83-708 and Tdl400,
the level of T83-708 was unchanged. However, the level of
Tdl400 was lower than the level in cells transfected with
Tdl400 alone. This reduction may result from promoter
competition between the strong cytomegalovirus promoter driving
expression of T83-708 and the SV40 promoter driving expression of
Tdl400. Nonetheless, the level of each protein was
sufficient for complementation to the wild-type level. This
complementation suggested that at least two T-antigen activities were
required for transactivation and that one or both could operate in
trans. One of these activities requires integrity of the T/t
common region. However, cotransfection of the N-terminally deleted T
antigen, T83-708, and the small-t-antigen-expressing plasmid did not
substantially increase the level of transactivation (Fig. 4). Thus, the
T/t common region could not act in trans to restore
wild-type levels of transactivation, suggesting that the N-terminal
activity either required large T-antigen-specific sequences beyond
amino acid 82 or required another region of T antigen to be supplied in
cis.
If the N-terminal activity extended beyond amino acid 82 and could be
supplied in
trans, then the C-terminal limit of the
region
might be determined by examining the ability of T83-708
to complement T
antigens containing additional internal deletions.
The results of such
an analysis appear in Fig.
6. In no case
was
complementation to a wild-type level achieved. In this and a second
experiment (data not shown), slight apparent increases in
transactivation
were observed when T83-708 was cotransfected with
T
dl127-250,
T
dl351-400, T
dl401-436,
or T
dl501-550 and the reporter plasmid;
nonetheless, in contrast to complementation between T83-708 and
T
dl400, they did not approach the wild-type level. Because
coexpression
of T83-708 and T
dl400 resulted in reduced
levels of T
dl400, it
was important to examine the protein
levels of the complementation
pairs (Fig.
7). The protein levels of mutant T
antigens T
dl301-350,
T
dl351-400,
T
dl401-436, T
dl451-532, and T
dl501-550
were reduced
substantially in cotransfected cells (Fig.
7); lighter
exposure
of the immunoblot (not shown) revealed a decreased level of
T
dl252-300.
Since the minimal amount of T antigen needed for
complementation
is not known, it was not possible to define the limit
of the activity
marked by the deletion of amino acids 1 to 82. The
protein level
of T
dl127-250, however, was not diminished in
cells cotransfected
with the T83-708 plasmid. Thus, it was
possible to conclude that
removal of amino acids 127 to 250 prevented
complementation. These
results suggested three possibilities.
The deletion could be part
of the N-terminal activity compromised in
T83-708. Alternatively,
the deletion could impinge on the activity
marked by
dl400 or
could identify a third activity which
must be in
cis with the
N terminus.
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FIG. 6.
Complementation between internally deleted T antigens
and T83-708 or Tdl400. The abilities of the T antigens with
internal deletions to transactivate the rat ribosomal promoter either
alone and in combination with T83-708 or Tdl400 were
examined. Each T antigen or combination was tested twice. The pattern
of results was consistent; results of a representative experiment for
each complementation are shown. Each error bar represents the percent
standard error of the mean; the absence of an error bar indicates that
the error was less than 2%.
|
|
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FIG. 7.
Accumulated T-antigen levels in cells coexpressing
T83-708 and other mutant T antigens. Immunoblot analysis of transfected
cell protein extracts was performed as described in Materials and
Methods with monoclonal antibody PAb901, which recognizes an epitope in
the C terminus of T antigen.
|
|
To further explore whether the T83-708 T-antigen segment contains
two complementing activities, one or both of which operate
in
cis with the N terminus of T antigen, we tested
mutants with
internal deletions for the ability to complement mutant
dl400.
Mutant
dl400 would contain the putative
N-terminal activity in
cis with the region containing amino
acids 127 to 250 or 252 to
300, for instance. Similarly, T antigens
with internal deletions
(except T
dl401-450) would contain
the N-terminal activity in
cis with the activity marked by
dl400. The results in Fig.
6 showed
that
dl400
could not cooperate with any of the internal deletion
mutants tested to
transactivate the rat ribosomal promoter. Immunoblot
analysis (not
shown) showed no evidence of substantial reduction
in T-antigen
accumulation in cells coexpressing mutant T antigens
under
transcriptional control of the SV40 promoter. Therefore,
although
T83-708 complements
dl400,
trans-complementing
activities
between amino acids 127 and 550 of T antigen were not
identified.
The results did not distinguish between the possibilities
that
transactivation requires the structural integrity of that large
region of T antigen or that more than two activities of the protein
are
required in
cis in order to complement T83-708.
Involvement of specific T-antigen functions in
transactivation.
Two activities located in the N terminus of T
antigen, Rb binding and nuclear localization, were examined for
involvement in transactivation of the rat ribosomal gene
promoter. The amino acid deletion or substitution in the
conserved LXCXE (amino acids 103 to 107) motif
(43) of T antigen disables binding of the Rb family
(Rb/p107/p130) of proteins (14). The mutant T antigens T-Glu107Lys and Tdl105-108 were examined for protein
accumulation and the ability to transactivate the ribosomal gene
promoter. Both proteins accumulated to wild-type protein levels (Fig.
8 and 3B) and transactivated (Fig.
9 and 3A) to wild-type levels, indicating
that Rb/p107/p130 binding was not required.
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FIG. 8.
Immunoblot analysis of mutant T antigens with or without
an NLS or Rb-binding capability in single transfections and
cotransfections with T83-708. Immunoblot analysis of transfected cell
protein extracts was performed as described in Materials and Methods
with monoclonal antibody PAb901, which recognizes an epitope in the C
terminus of T antigen.
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|
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FIG. 9.
Involvement of specific T-antigen functions in
transactivation of the ribosomal promoter. The impacts of Rb binding
and nuclear localization were investigated. Each mutant was tested at
least twice for the ability to transactivate the rat ribosomal
promoter; results of representative experiments are shown. The graph is
divided into four units for ease of comparing related functions.
T-Glu107Lys does not bind the Rb family (Rb/p107/p130) of proteins.
Tdl127-250NLS250 and Tdl127-250NLS650 are T
antigens missing amino acids 127 to 250 and containing the SV40 NLS
immediately preceding amino acid 250 and between amino acids 650 and
651, respectively. T-Lys128ThrNLS contains the SV40 NLS between amino
acids 650 and 651. Each error bar represents the percent standard
error of the mean; the absence of an error bar indicates that the error
was less than 2%.
|
|
The mutant T antigen T
dl127-250 accumulates in the
cytoplasm. Its inability to transactivate suggested three
possibilities:
that nuclear localization is essential; that the amino
acids comprising
the NLS (amino acids 126 to 132) are part of a larger
region containing
a required activity; and that nuclear localization is
inconsequential,
but amino acids 133 to 250 are required. To
distinguish among
these possibilities, an NLS was introduced into
T
dl127-250 between
amino acids 126 and 250 or between amino
acids 650 and 651. The
location adjacent to amino acid 126 was used in
order to place
the NLS close to its natural position in the protein.
The location
between amino acids 650 and 651 was chosen on the basis of
two
observations. We showed previously that extraneous sequences could
be inserted at that position without compromising protein accumulation
or any of the T-antigen functions tested (
22,
61). In
addition,
we show here that removal of amino acids 626 to 708 did not
compromise
transactivation of the rat ribosomal promoter (Fig.
2A).
Both
mutant T antigens containing the NLS accumulated in the nucleus
in
immunofluorescence assays (data not shown) and accumulated
to greater
than wild-type levels in transiently transfected cells
(Fig.
8). The
results in Fig.
9 showed that neither T antigen
transactivated,
suggesting that disruption of the region from
amino acids 127 to 250 abrogates transactivation even when the
protein accumulates in the
nucleus.
To investigate whether amino acids that constitute the NLS are required
only to direct the protein to the nucleus or are part
of a region
directly associated with transactivation, we examined
the ability of
the mutant T-Lys128Thr to transactivate the rat
ribosomal gene
promoter. The Lys128Thr mutation inactivates transport
to the nucleus,
resulting in cytoplasmic accumulation of the protein.
As
shown in Fig.
9, T-Lys128Thr did not transactivate the rat
ribosomal promoter. To determine whether nuclear localization
was the
only activity disrupted by the Lys128Thr mutation, the
NLS was
introduced between amino acids 650 and 651; the resulting
T antigen was
shown to accumulate in the nucleus (data not shown)
and to wild-type
levels (Fig.
5 and
8) in transiently transfected
cells. Introduction of
the NLS into T-Lys128Thr did not restore
a wild-type level of
transactivation in transiently transfected
(Fig.
9). It appeared,
therefore, that nuclear localization affected
transactivation of the
rat ribosomal gene promoter. However, the
amino acid substitution
at position 128 significantly diminished
the ability of T antigen to
transactivate.
We next determined whether the Lys128Thr mutation could be complemented
by T83-708 and the impact of nuclear location on that
complementation.
The results are shown in Fig.
9; corresponding
T-antigen levels are
shown in Fig.
5 and
8. T-Lys128ThrNLS650
and T83-708 complemented
to produce a nearly wild-type level of
transactivation, whereas
coexpression of T-Lys128Thr and T83-708
did not. Coexpression of
T-Lys128ThrNLS650 and T83-708 resulted
in an increased level
of T83-708 relative to the amount of protein
that accumulates in cells
transfected with the T83-708-expressing
plasmid (Fig.
5 and
8). The
reason for this increase is not known.
Nonetheless, it is unlikely that
this increase in the level of
T83-708 is sufficient to explain the near
wild-levels of transactivation.
A lighter exposure of the immunoblot in
Fig.
7 (not shown) revealed
a similar increase in T83-708 when cells
were coexpressing T
dl252-300,
yet complementation was not
observed. These results indicated
a requirement for nuclear
accumulation to achieve maximal transactivation
of the rat ribosomal
gene promoter in complementation
assays.
Since mutation of amino acid 128 abrogated transactivation independent
of its effect on nuclear localization, it was appropriate
to determine
whether the activities marked by the alterations
at amino acids 400 and
128 were separable and operated in
trans.
Therefore, we
performed complementation assays between the two
mutants. The results
appear in Fig.
9; corresponding protein levels
appear in Fig.
5 and
8.
T-Lys128-Thr and T
dl400 did not complement.
Introduction of
an NLS into T-Lys128Thr did not alter the result.
Thus, the activities
marked by the
dl400 and Lys128Thr mutations
either
constitute distal elements of a compound function or represent
independent activities that operate only in
cis.
| |
DISCUSSION |
The genetic analysis presented here showed that a T antigen
containing amino acids 1 to 626 was sufficient to transactivate the rat
ribosomal promoter linked to a CAT reporter gene in transiently transfected monkey cells. These results are consistent with other reports of T antigen's capacity to transactivate pol I-dependent promoters. Previously, Soprano et al. (54) demonstrated
reactivation of silent human ribosomal genes by microinjecting cloned
T-antigen segments into mouse-human hybrid cell lines. They found that
a mutant T antigen containing amino acids 1 to 509 (T1-509) was sufficient for reactivation. More recently, using in vitro
transcription assays, Zhai et al. (71) showed that a
T-antigen segment consisting of amino acids 1 to 538 (T1-538) was
sufficient to transactivate a human ribosomal gene promoter in HeLa
cell extracts. Each of these investigations indicated that C-terminal
regions of T antigen are not needed to transactivate pol I-dependent
promoters. The reasons for the difference observed in the T-antigen
segment required may relate to the methodologies used (microinjection,
transfection, in vitro transcription) or the steady-state level of the
mutant proteins maintained in the various systems. Finally, these
studies may indicate that different activities of T antigen are
required to transactivate a rat ribosomal gene in monkey cells compared to the human genes in a mouse-human hybrid cell line or in vitro.
The genetic analysis presented here indicated that at least three
T-antigen regions or activities were needed to transactivate the rat
ribosomal promoter in vivo. One of these relied on the integrity of
amino acids spanning the length of the T polypeptide from
amino acids 109 to 626. The requirement for an extensive region of T
antigen in vivo is consistent with results obtained by others using in
vitro transcription assays. Zhai et al. (71) showed that a T
antigen containing amino acids 1 to 538 was sufficient to stimulate
transcription of a human rDNA gene in HeLa cell extracts and that
deletion of amino acids 436 to 538 from T antigen sharply diminished
transcriptional stimulation. They showed further that T1-538 contained
two regions essential for transactivation. An N-terminal T antigen
segment extending to amino acid 436 was sufficient to bind the SL1
components TBP and the polymerase-specific TAFs that are required for
efficient transcription of pol I-dependent promoters and that confer
the species-specific nature of transcription. However, the additional
amino acids between positions 436 and 538 were needed for
transactivation. The data presented here examined the consequence in
vivo of deletions within the T1-538 region and indicated that the
T-antigen region extending from amino acids 109 to 550 is
highly sensitive to mutational inactivation of its ability to
transactivate the rat ribosomal gene promoter. Even small deletions
within that region such as deletion of amino acid 400 or 501 abolished T antigen's capacity to transactivate. These findings
suggested that additional functions in the region that is sufficient
for SL1 binding are needed to transactivate pol I-dependent promoters,
that SL1 binding requires integrity of the entire region, or that even
small alterations in amino acid sequence distort this region of T
antigen globally or lead to rapid degradation of the protein.
Immunoblot analyses of the mutant T antigens (Fig. 2B, 3B, and 8)
revealed that less than wild-type protein levels were sufficient to
transactivate, and rarely could the lack of transactivation be directly
related to a reduced level of the mutant T antigen. It is
unlikely, therefore, that differences in protein accumulation can
account for failure of mutant T antigens with deletions between amino
acids 109 and 550 to transactivate the ribosomal gene promoter.
As summarized in Fig. 10, each of the T
antigens with internal deletions of amino acids 127 to 350, 451 to 532, 400, or 501, as well as T-Lys128Thr, retained the ability to bind p53
and to immortalize primary cells. Integrity of activities in both the N
and C termini of T antigen are required for immortalization (13,
58), and separate regions within the C-terminal half of the
protein are required for binding the tumor suppressor p53 in vivo
(34). Thus, retention of these activities indicates that the
proteins are not distorted globally. Nonetheless, they all failed to
transactivate the ribosomal promoter. It seems likely, therefore, that
maintaining integrity of at least the regions containing amino acids
127 to 350, 451 to 532, 400, 501, and Lys128Thr is essential for
transactivation. The T antigens containing deletions within the regions
defined by amino acids 351 to 450 and 532 to 626 lose simultaneously
the abilities to bind the tumor suppressor p53 and to immortalize
primary mouse cells (34). The loss of multiple T-antigen
activities may indicate substantial distortion of at least the
C-terminal half of the protein. Thus, on the basis of deletion mutant
analysis, the involvement of the regions 351 to 450 and 532 to 626 in
transactivation of pol I-dependent promoters in vivo is less certain.
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FIG. 10.
Diagrammatic representation of mutant T antigens used
in this study. The names of the plasmids encoding the mutant T antigens
are given on the left. The corresponding T antigens are represented by
lines in which the deleted amino acids are represented by gaps. Single
amino acid substitutions and deletions of three or fewer amino acids
are represented by vertical lines at the positions of their occurrence.
Insertions of amino acids are represented by an inverted triangle at
the position of the insertion. The deleted amino acids, substitutions,
and insertions are indicated above the sites of alteration. Mutant T
antigen sequences cloned in a pBR322 background are underlined; mutant
T-antigen sequences cloned in a pBR328 background are not underlined;
mutant T antigens that complement one another are designated by an
asterisk or dot. Numbers in superscript correspond to literature
citations.
|
|
The finding that T83-708 could not transactivate the rat ribosomal gene
promoter indicated that a second region, the extreme N-terminal portion
of T antigen, was required. Complementation analyses were
performed to determine whether the activity marked by the deletion of
amino acids 1 to 82 was separable from that marked by internal
deletions. T83-708 complemented Tdl400. Thus, deletion of
amino acids 1 to 82 and deletion of amino acid 400 alter different
T-antigen activities that can operate in trans to increase
transcription of a pol I-dependent promoter. To determine if the
C-terminal limit of the activity marked by deletion of amino acids 1 to
82 extends further into the protein, attempts were made to complement
T83-708 with additional T antigens containing internal deletions. This
analysis was compromised by decreased levels of mutant T antigens in
cells coexpressing T83-708. Nonetheless, the finding that deletion of
amino acids 105 to 108 did not diminish transactivation of the rat
ribosomal gene promoter indicated that amino acids 1 to 82 is not part
of a contiguous transactivation function of T antigen extending beyond
amino acid 104.
The ability of T antigen to localize to the nucleus also affected
activation of the rat ribosomal gene promoter. Mutant T antigens
with alterations that prevent nuclear localization failed to
transactivate alone or in combination with T83-708. Addition of an NLS
to T-Lys128Thr at an alternate site did not result in transactivation
to wild-type levels, indicating that this mutation marks a
required activity independent of nuclear localization. The
observation that T-Lys128ThrNLS complemented the N-terminally truncated
protein T83-708 indicated that the activity marked by the substitution
at residue 128 is independent of the activity lost when the first 82 amino acids of T antigen are deleted. In addition, the results showed
that accumulation of T antigen in the nucleus was essential for
wild-type levels of transactivation.
The finding that T-Lys128ThrNLS would not complement dl400
suggested two possibilities. Either T-Lys128ThrNLS and
Tdl400 must be present on the same molecule to
cooperate functionally or they mark a single activity that depends
on integrity of a substantial portion of T antigen. The finding
that T antigens with internal deletions other than dl400
were devoid of transactivating function supports the latter contention.
The capacity of T83-708 and dl400 and of T83-708 and
T-Lys128ThrNLS to complement constitutes intracistronic
complementation. Intracistronic complementation could occur
if transactivation operated through the independent action of
mutant T-antigen monomers. Alternatively, oligomerization of T antigens
with alterations in separate regions might be required to
form an active protein structure. Although oligomerization is not
needed for transactivation of pol II-dependent promoters
(31), it is not known whether transactivation of pol
I-dependent promoters depends on oligomerization of T antigen.
T-antigen regions between amino acids 114 and 152 and the C-terminal
region up to 669 are necessary and sufficient for oligomerization
(41), yet a T-antigen fragment extending only to amino acid
538 transactivates the human ribosomal gene promoter in vitro
(71). The close correspondence between the T-antigen regions
required for in vitro and in vivo transcription of pol I-dependent
promoters suggests that oligomerization would not be needed. If
oligomerization were required, it clearly would not be sufficient.
T83-708 has been shown to oligomerize in a fashion identical to
that of wild-type T antigen (37), indicating that it should
be able to form oligomers with mutant T antigen that retain
oligomerization domains. With the exception of T125-250 and its
derivatives containing a translocated NLS, and T-Lys128Thr and its
NLS-containing derivative, all of the mutant T antigens tested contain
the T-antigen regions that are sufficient for oligomerization, yet complementation to wild-type levels was observed only in the two
cases noted.
It was shown recently that Rb represses transcription of ribosomal
genes by binding to UBF and disrupting initiation complexes (7,
67). Thus, it seemed likely that T antigen might transactivate ribosomal genes by binding to Rb and preventing its association with
UBF. This, however, does not appear to be the case. T-Glu107Lys, in
which T-Rb binding is disrupted, and Tdl105-108, which bears a deletion within the Rb-binding site, both transactivated to wild-type levels. Thus, T-antigen binding to Rb is not the mechanism used to transactivate the rat ribosomal gene.
The relationship between activities of the SV40 early proteins that
participate in transactivation of pol I-dependent genes and those
reported to contribute to transactivation of pol II-dependent genes is
difficult to decipher. Large-T antigen transactivates a variety of pol
II-dependent promoters containing different upstream activation
sequences (23, 46). The requirement for specific activities
of T antigen in transcriptional activation of pol II-dependent genes
varies with the specific promoters utilized. For instance, transactivation of the Rous sarcoma virus (RSV) LTR and SV40 late promoters by T antigen does not require integrity of the
Rb-binding site (72), whereas Rb binding appears to
participate in transactivation of the E2A promoter (39).
Nonetheless, some comparisons can be made. Like transactivation of the
RSV and SV40 late promoters, transactivation of the rat rDNA did not
require an intact T-antigen Rb-binding region. In addition,
efficient nuclear localization was not essential for transactivation of
the RSV or SV40 late promoter (72). However, nuclear
localization substantially affected transactivation of the rat
ribosomal gene promoter. The N-terminal 121-amino-acid segment of T
antigen is sufficient to transactivate certain pol II-dependent
promoters in transient transfection assays (56), and
multiple insertions and internal deletions in T antigen are tolerated
with little or no substantial loss of transactivating activity
(72). In contrast, we show here that with the exception of
amino acids 105 to 108, internal deletions within T antigen abrogated
transactivation of the rat ribosomal gene promoter. Similarly,
the role of small-t antigen in transactivation depends on the
specific promoter utilized. Small-t antigen transactivates the pol
II-dependent adenovirus E2A and SV40 early promoters but not the RSV or
SV40 late promoter. Small-t antigen did not independently transactivate
the rat ribosomal gene promoter to a measurable extent (Fig. 1A and
2A), nor could it supply the transactivating activity missing from
T83-708. Thus, the activities of large-T antigen that are reported to
be sufficient to transactivate a pol II-dependent promoter are not
sufficient to transactivate a rat pol I-dependent promoter in a
transient transfection assay in monkey cells.
Four conclusions can be drawn from the results presented here. First,
activation of the rat ribosomal gene in monkey cells depends on an
N-terminal activity of T antigen that can be supplied in
trans by large-T but not small-t antigen. Second,
accumulation of T antigen in the nucleus is required for maximal
transactivation of the rat rDNA promoter. Third, one or more activities
encompassed by amino acids 109 to 626 are required. The results do not
distinguish between the possibilities that multiple activities within
the region from amino acids 109 to 626 must be in cis, as is
the case for activities involved in replication of the viral
genome (11, 19), or that a single activity determined
by a specific conformation of the region is involved. Finally, the
capacity to transactivate ribosomal genes did not correlate with the
capacity of T antigen to immortalize primary cells or to bind either
the Rb or p53 tumor suppressor.
| |
ACKNOWLEDGMENTS |
This work was supported by Public Health Service grant CA67303
(J.F.C.) and (in part) CA24694 (M.J.T.) from the National Cancer Institute of the National Institutes of Health.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biology, Elizabethtown College, One Alpha Dr., PA 17022-2298. Phone: (717) 361-1448. Fax: (717) 361-1176. E-mail:
cavender{at}acad.etown.edu.
| |
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Journal of Virology, January 1999, p. 214-224, Vol. 73, No. 1
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
