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Journal of Virology, October 1999, p. 8890-8897, Vol. 73, No. 10
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Assessment of Bovine Leukemia Virus Transcripts
In Vivo
Joel
Rovnak* and
James W.
Casey
Department of Microbiology and Immunology,
College of Veterinary Medicine, Cornell University, Ithaca, New
York 14853
Received 16 February 1999/Accepted 9 July 1999
 |
ABSTRACT |
Reverse transcriptase PCR (RT-PCR) consistently detected bovine
leukemia virus transcripts in fresh cells, and competitive RT-PCR
enumerated these transcripts. The detection of transcripts in limited
numbers of tumor cells indicated that expression occurs in a minority
of cells. The data suggest that individual cells contain hundreds of
copies of the tax/rex transcript in vivo.
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TEXT |
Bovine leukemia virus (BLV), a
B-cell lymphotropic retrovirus of the BLV-human T-cell leukemia virus
(HTLV) genus, causes a persistent B-lymphocytosis (PL) and/or a
neoplastic proliferation of B lymphocytes in a minority of infected
animals after prolonged infection (reviewed in reference
28). Virus expression in vivo is highly restricted
throughout disease progression and in leukosis. This has made it
difficult to identify a discrete mechanism by which BLV can mediate
leukemogenesis. Amplification by reverse transcriptase PCR (RT-PCR) has
detected BLV mRNAs in freshly isolated peripheral blood mononuclear
cells (PBMC) and tumor cells of infected animals (1, 18, 19, 23,
38). Overt virus expression occurs upon cultivation of these
cells in vitro.
The genome of BLV, like that of HTLV type 1 (HTLV-1), contains a
region, X, which encodes proteins that regulate virus expression, have
the capacity to mediate host gene expression, and have been implicated
in cellular transformation (11, 12, 26, 40, 49, 51). The
expression of the open reading frames of the X region is dependent upon
spliced transcripts (Fig. 1). pp18 Rex, a
transregulator of mRNA processing, and p34 Tax, a transcriptional transactivator, are encoded by overlapping reading frames in the 3'
proximal portion of the X region from a doubly spliced transcript, tax/rex (39, 41, 55), which has been detected in
vivo (1, 18, 19, 23, 38). A singly spliced version of the
tax/rex transcript, alt, has the second exon
excluded and has also been detected in vivo (19). This
transcript is capable of encoding a truncated Rex protein (Trex; Fig.
1) which migrates at 14 to 16 kDa (1, 19, 42, 55). The
alt transcript is similar to a singly spliced transcript of
HTLV-1, which produces a truncated Rex protein, p21 Rex. This splicing
pattern is common to all members of the BLV-HTLV genus (34).
Several spliced transcripts with the capacity to express 5' proximal
portions of the X region have been identified (1, 7) (Fig.
1). These transcripts are present at low levels in productively
infected cells. Their significance is indicated by their conservation
in members of the BLV-HTLV genus and the reduction of in vivo
replicative capacity of viruses with mutations in this region (7,
10, 27, 52, 54).
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FIG. 1.
Representation of the BLV genome, open reading frames,
transcripts, and the products of translation of spliced transcripts.
The locations of primers are indicated on the genomic RNA, and their
identifiers are underlined. Vertical dashed lines align common termini
of transcripts, exons, and reading frames. Splice donor and acceptor
sites are numbered according to the proviral sequence described by
Sagata et al. (42). Names and splice sites of alternate
transcripts and their predicted protein products are from references
19 (alt), 7
(tof/rof), and 1 (RIII,
305/7018, GIV, and 502/7157).
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A sensitive quantitative competitive (QC) RT-PCR technique utilizes a
size- and sequence-matched RNA competitor in order to allow high PCR
cycle numbers without loss of quantitation (31). RT-PCR
products of competitor constructs with a modified restriction endonuclease sequence are distinguished by restriction digestion. In
addition to the quantitation of viral transcripts in a pool of cells,
mRNAs can be detected by RT-PCR in limited numbers of cells to assess
their distribution in the population (14). BLV expression
has been detected, rarely, in individual PBMC by in situ hybridization
(30) and RT-PCR on sorted cells (18) and in fresh
lymphoid tissues and lymphomas by immunohistochemistry (20).
We sought to quantitate BLV X region transcripts in the PBMC of animals
with PL (PL animals) and cells from malignant lymphomas (ML) and to
assess the distribution of these transcripts in ML cells. QC RT-PCR was
utilized to measure tax/rex and alt transcripts, and direct RT-PCR amplification of RNA from limited numbers of cells
was used to enumerate cells with these transcripts.
Detection of BLV X region transcripts.
Primers 5'B1 and 3'B2
(Table 1; Fig. 1) are capable of
amplifying all reported variations in splicing of BLV X region
transcripts (1, 7, 19). Figure
2 shows the products of RT-PCR of 50 ng
of RNA with this primer pair after 45 cycles of amplification with a
63°C annealing temperature. All RNAs were treated with DNase I,
selected by chromatography on oligo(dT) cellulose, and quantitated by
spectrophotometry. Southern blotted amplicons were hybridized with an
end-labeled oligonucleotide probe, X (Table 1; Fig. 1), which is also
common to known BLV transcripts and is internal to the primer
sequences. PBMC and tumor cells were collected from naturally infected
animals. Samples included RNAs from the PBMC of one BLV-negative
animal, six BLV-positive, hematologically normal animals (non-PL
animals), and 12 PL animals and ML cells from 18 animals. Five-nanogram
quantities of RNAs from BLV-negative BL3 cells (ATTC CRL 8037),
BLV-positive BL3* cells (18) and NBC-13 cells
(16), and NBC-13 cells treated with 10 nM
phorbol-12-myristate-13-acetate (PMA; Sigma) were also assayed. The
BL3* and NBC-13 cell lines were previously single cell cloned by
endpoint dilution. Mock cDNA preparations from NBC-13 RNA (RT
) served
as a negative control.
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FIG. 2.
Amplification of X region transcripts. Fifty-nanogram
quantities of DNase I-treated, poly(A)+ RNA preparations
were amplified by RT-PCR with BLV primers 5'B1 and 3'B2 (top panel) or
with actin primers 5'ACT1 and 3'ACT2 (middle panel). The bottom panel
shows the results of hybridization of BLV amplicons with X probe.
Samples include PBMC from a BLV-seronegative animal (BLV), PBMC from
seropositive, non-PL animals (BLV+), PBMC from seropositive, PL animals
(PL), and ML cells. Five-nanogram quantities of control
poly(A)+ RNA preparations were used (BL3, BL3*, NBC-13, and
NBC-13 plus PMA). The numbers over the bottom panel indicate samples
which were subsequently subjected to QC RT-PCR. The molecular weight
(MW) lane contained a 50-bp ladder. The amplified products of
tax/rex and alt transcripts are 414 and 191 bp,
respectively.
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Products corresponding to the
tax/rex and
alt
transcripts (414 and 191 bp, respectively) were detected in most, but
not all,
samples tested (Fig.
2).
tax/rex was detected in
five of six samples
from non-PL animals, and
alt was
detected in four of six. In PL
samples,
tax/rex was detected
in all samples, and
alt was detected
in 11 of 12. All of the
tumor samples contained BLV transcripts,
but one contained
alt and not
tax/rex, and 12 of 18 contained
alt. Additional RT-PCR products, with sizes corresponding to
previously
detected BLV transcripts as well as to unidentified
transcripts,
were observed to varying degrees in PL and ML
samples.
The detection of
tax/rex and
alt transcripts in
fresh cells has been observed previously (
1,
18,
19,
23,
38). One
tumor sample was negative for
tax/rex, and
although it was positive
for
alt, this example further
weakens the case for viral protein
function in the maintenance of the
transformed state. At this
stage of disease the cell genome is marked
by chromosomal abnormalities
(
43), which could account for
this phenotype. BLV transcripts
were not detected in cells from all
BLV-positive, non-PL animals,
but few of the PBMC from these animals
are infected (
9,
32);
the inability to detect BLV
transcripts in all samples at this
stage of disease could be due to
limitations of the
assay.
BLV transcription commonly occurs upon in vitro cultivation of infected
cells. To address the possible onset of transcription
ex vivo, the
following conditions were used during the collection
of tumors: (i)
tumors were excised within 10 min of the death
of the animal; (ii)
tumor masses were immediately frozen on dry
ice or cut with scissors in
phosphate-buffered saline on ice to
release cells into suspension, and
(iii) 500 µg of actinomycin
D per ml was included in cell suspension
buffers, in selected
samples, to block transcription. No differences
were observed
for such preparations, in either the detection or
relative levels
of the BLV transcripts, compared to identical samples
prepared
at ambient temperatures and without actinomycin D (data not
shown).
Figure
2 also shows the products of RT-PCR of the same
poly(A)
+ RNA preparations with primers 5'ACT1 and 3'ACT2
(Table
1) for
22 cycles in separate reactions. These primers yield a
437-bp
amplicon from bovine
-actin mRNA. Amplification of serial
twofold
RNA dilutions showed that 22 cycles provided a linear response
of amplicon quantity to input RNA in these reactions (data not
shown).
This allowed an evaluation of the RNAs, quantitated by
spectrophotometry, in the context of RT-PCR.
QC RT-PCR of BLV transcripts.
Analysis of the levels of viral
gene transcription in vivo focused on the tax/rex and
alt transcripts. Clones of these transcripts were prepared
in pBluescript SK() (Stratagene) by RT-PCR of RNA from persistently
infected fetal lamb kidney cells (FLK-BLV [48]) with
primers 5'C1 and 3'TR2Hind (Table 1; Fig. 1). 3'TR2Hind introduces two
point mutations, which alter a native BglI site to a
HindIII site. Competitor RNAs transcribed from these
clones were quantitated by spectrophotometry, and their sizes were
confirmed by gel electrophoresis. Competitor dilutions were included in RT-PCRs of 5 ng of DNase I-treated, poly(A)+, cellular
RNAs. Specific amplification of either the tax/rex or
alt transcript was achieved with the common 3' primer 3'TR2 and a 5' primer in the second exon of tax/rex, 5'TR1, or a
5' primer which crosses the alt splice junction, 5'ALT
(Table 1; Fig. 1), and 40 cycles of amplification with 63°C
annealing. Twenty-microliter aliquots of QC RT-PCR products were
digested with 5 U of HindIII or BglI for
2 h at 37°C and subjected to electrophoresis. Southern blots
were hybridized with the end-labeled oligonucleotide probe X. The
quantity of native transcripts was determined to be equal to the
quantity of competitor when the products of BglI and
HindIII digestion, 211 and 206 bp, respectively, were equivalent.
Figure
3 shows an example of quantitation
of 5 ng of NBC-13 poly(A)
+ RNA, where the
tax/rex transcript quantity was assessed at 5
fg of
competitor. Samples were first tested against fivefold competitor
dilutions in order to define a range of appropriate twofold dilutions.
Results are presented in Table
2 as
femtogram equivalents of
competitor RNA per nanogram of cellular RNA
and corresponding
estimates of copies of mRNA per cell. These estimates
were based
upon an approximation of 500 fg of total mRNA per cell (from
typical
lymphocyte yields of poly[A]
+ RNA). Molecules of
individual transcripts or competitor RNAs
per femtogram were calculated
by their lengths (
tax/rex mRNA,
1,800 bases;
tax/rex competitor RNA, 398 bases;
alt mRNA,
1,600
bases;
alt competitor RNA, 176 bases).
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FIG. 3.
Quantitation of tax/rex transcript in NBC-13
cells. BglI- or HindIII-digested products of
QC RT-PCR were subjected to electrophoresis and Southern analysis with
the X probe. The input of cellular RNA, in nanograms of
poly(A)+ RNA, and competitor RNA, in femtograms of
competitor, and the presence of RT in reaction mixtures are shown above
the autoradiographs. An arrow indicates the relative equivalence point.
The molecular weight (mw) lane contains a 50-bp ladder with an intense
band at 350 bp. BglI digestion yields a 211-bp fragment from
tax/rex transcript amplicons, and HindIII
digestion yields a 206-bp band from tax/rex competitor RNA
amplicons.
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A total of 18 samples, indicated by numbers in Fig.
2, were quantitated
(Table
2). Samples included PBMC from five PL animals,
10 tumor
samples, and the BLV-positive cell lines BL3*, NBC-13,
and PMA-treated
NBC-13. Quantities of the
tax/rex transcript were
consistent
in PL PBMC at approximately one to two copies per cell.
In tumor cells,
tax/rex levels varied, from approximately 0.2
to 5 copies
per cell. Levels of the
alt transcript were significantly
lower, with copy numbers in the range of 0.02 to 0.1 per cell.
Most of
the samples contained less than one copy of the
alt
transcript
per cell, and three tumor samples were negative for
alt, but one
tumor contained approximately five copies per
cell. Quantities
of transcripts lower than a single copy per cell
suggest that
these transcripts are present only in a subpopulation of
the
cells.
Levels of the
tax/rex transcript in the NBC-13 cell line
were comparable to those observed in tumor cells. This cell line
can be
stimulated with PMA to induce BLV expression (
24), and
such
stimulation, here, increased
tax/rex levels in these cells
approximately 50-fold to levels similar to those observed in
productively
infected BL3* cells. NBC-13 and BL3* cells had
approximately 250
copies of the
alt transcript per cell, and
the levels of the
alt transcript were not significantly
enhanced by treatment of NBC-13
cells with
PMA.
RT-PCR of limited numbers of cells.
The detection of
transcripts in limited numbers of tumor cells in a direct lysis
procedure allowed the evaluation of the distribution of the
tax/rex and alt transcripts in this cell
population. Selected tumors were 3 to 5 cm in diameter, and gross
tissue was completely replaced by neoplastic cells. Suspensions of
cells were derived from the interior of the tumor mass by cutting
fresh, decapsulated tissues with scissors in phosphate-buffered saline
supplemented with actinomycin D. Cells were diluted to appropriate
concentrations to yield fixed cell numbers in 1-µl aliquots and
distributed into wells of a 96-well plate on ice. Cell numbers were
visually confirmed. The cells were lysed by the addition of 9 µl of
0.025% Nonidet P-40 (Sigma), 2 mM dithiothreitol, and 1 U of RNasin
per µl, incubated on ice for 20 min, and frozen at 80°C. Lysates
were thawed on ice, supplemented with 2 µl of 25 mM Tris-HCl (pH
8.3), 37.5 mM KCl, 1.5 mM MgCl2, and 1 U of DNase I per
µl, and incubated for 15 min at room temperature. Three microliters
of 1 mM EDTA was added, and the lysates were heated to 65°C for 10 min and chilled on ice. After the addition of 10 µl of buffer
containing 125 mM Tris-HCl (pH 8.3), 187.5 mM KCl, 7.5 mM
MgCl2, a 62.5 mM concentration of each deoxynucleoside
triphosphate, 5 mM dithiothreitol, 2.5 U of RNasin per µl, 5 µM
primer 3'B2, 0.25 µM primer 3'ACT2 (Table 1; Fig. 1), and 10 U of RT
(Superscript II; Life Technologies), samples were incubated for 1 h at 45°C followed by 10 min at 70°C. Negative controls received
the same mix prior to the addition of RT. cDNA preparations were
overlaid with a PCR mix containing 2 µM primer 5'B1 and 0.1 µM
primer 5'ACT1 (Table 1) and amplified for 45 cycles with a 63°C
annealing temperature. One microliter of a 1:100 dilution of the
product of this PCR was reamplified with nested primer pairs for either
tax/rex (5'TR1 and 3'TR2; 2 µM), alt (5'ALT and
3'TR2; 2 µM), or -actin (5'ACTN1 and 3'ACTN2; 0.1 µM;
Table 1) for 35 cycles, and 20 µl was subjected to electrophoresis and Southern analysis with the oligonucleotide probe X or ACT (Table
1).
Figure
4 shows an example of the products
from
tax/rex amplification of serial twofold cell dilutions.
Only three of five
preparations of each dilution received RT. The
number of cells
required to yield a consistent PCR signal in three of
three samples
with RT was interpreted as containing, at least, a single
cell
with
tax/rex transcripts. This cutoff provides a
conservative
estimation of the numbers of positive cells within the
population.
Signals obtained in fewer than three of three dilutions
suggested
that individual or few cells contained sufficient transcripts
for detection. This assay detected
-
actin in single tumor
cells.
Results for the five tumors tested ranged from 1 cell in 30 (3.3%;
sample shown in Fig.
4) to 1 in 250 (0.4%), with an average of
1 in 125 cells or 0.8%. In similar assays the
tax/rex
transcript
was limited to approximately 5% of NBC-13 cells and present
in
single BL3* cells (Fig.
4). The
alt transcript was rarely
detected
in fewer than 100 tumor cells (not shown) but was readily
detected
in single NBC-13 or BL3* cells (Fig.
4).
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FIG. 4.
RT-PCR of limited numbers of BLV-infected cells. Twofold
serial dilutions of cells were lysed and subjected to RT-PCR for BLV or
-actin transcripts. The actual numbers of cells in dilutions under
50 were confirmed visually and varied by no more than three cells. (A)
Five samples at each dilution of ML cells were tested for
tax/rex transcripts, two without and three with RT enzyme.
The products of nested primers, 5'TR1 and 3'TR2, are 241 bp. (B)
Samples were the same as in panel A but were amplified with nested
-actin primers 5'ACTN1 and 3'ACTN2, which yield a 165-bp
product. (C) RT-PCR of lysates from serial twofold dilutions of NBC-13
cells with tax/rex-specific primers 5'TR1 and 3'TR2 (241 bp)
or alt specific primers 5'ALT and 3'TR2 (89 bp). (D)
Amplification of two BL3* cells as described for panel C. Southern
blots were hybridized with X probe or actin probe (Table 1).
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The possibility that the positive cells present in the tumor mass do
not belong to the neoplastic pool cannot be excluded.
Such tumors do
contain low levels of infiltrating T lymphocytes
as determined by
immunohistology (
6), but these cells have
not been found to
be significant targets of BLV infection (
32)
and are
unlikely to be responsible for the signal observed in
the tumor cell
preparations.
Further characterization of NBC-13 cells.
BLV expression in
NBC-13 cells is restricted and can be induced with PMA (24).
Analysis of NBC-13 DNA revealed the presence of a full-length provirus
and one with a deletion of 4,620 bp between positions 2217 and 6837 (data not shown). The deletion precludes the expression of
tax/rex, but not singly spliced alt, from this provirus.
Immunofluorescence assays of acetone/methanol-fixed preparations of
NBC-13 cells with monoclonal anti-BLV p24 (4'G9) demonstrated
the
presence of a small number of virus-expressing cells (Fig.
5A). Flow cytometric analysis of 10,000 methanol-fixed NBC-13
cells labelled with anti-p24 demonstrated that
0.5% of this clonal
cell line expressed BLV antigen (Fig.
5C).
Treatment of these
cells with PMA stimulated the expression of p24 in
20% of cells
after 24 h (Fig.
5B and C). Analysis regions
included less than
0.04% of antibody controls.
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FIG. 5.
Indirect immunofluorescence staining of NBC-13 cells.
(A) Photomicrograph of a rare positive cell, stained with a monoclonal
antibody for BLV p24 and goat anti-mouse immunoglobulin G-fluorescein
isothiocyanate, prior to PMA treatment (magnification, ×600; Evans
blue counterstain). (B) NBC-13 cells 24 h after treatment with 10 nM PMA (magnification, ×600). (C) Flow cytometric analysis of NBC-13
cell preparations as described for panels A and B stained with a
control monoclonal antibody (Ab) or with anti-p24.
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The
alt transcript is persistently expressed in NBC-13
cells. The Rex amino terminus, deleted with the second exon in the
singly spliced
alt transcript, includes an arginine-rich
region
implicated in RNA binding and nuclear localization. The HTLV-1
protein homolog p21X has been identified in tumor cells and transformed
cell lines (
3,
35). p21X is localized in the cytoplasm, and
its HTLV-2 homolog has been demonstrated to act as a dominant-negative
inhibitor of Rex function (
8,
29). Considering that the Rex
proteins of BLV and HTLV-1 are functionally interchangeable
(
15),
the BLV truncated Rex protein could also act as an
inhibitor of
Rex function. Such a mechanism could explain the
restricted BLV
expression observed in NBC-13 cells, and the ability to
manipulate
this expression experimentally offers an in vitro model for
the
control of BLV
expression.
The work presented here addresses the long-standing paradox of
pathogenesis in BLV and HTLV-1: the paucity of expression of
virus.
While much effort has been expended to define the oncogenicity
of
proteins encoded by these viruses, evidence for significant
levels of
virus expression during disease progression is lacking.
While it
remains clear that BLV expression in vivo is limited,
the results
demonstrate the continued presence of X region transcripts
in
significant numbers of cells. The
tax/rex transcript was
present
at levels approximating 0.2 to 5 copies per cell in PL PBMC and
ML cells, and detection of this transcript in limited numbers
of tumor
cells indicated expression in a minimum of 0.8% of cells.
In light of
the estimates of copy numbers, the results suggest
that these cells
actually contained hundreds of copies of the
tax/rex
transcript and could have contained functional levels
of
tax/rex protein products. Limited virus expression was also
observed in NBC-13 cells, and these results demonstrate that variation
in BLV expression exists in cloned cell populations. This selective
viral gene expression was further demonstrated in NBC-13 cells
by
immunofluorescence. The mechanism whereby viral gene transcription
is
restricted to a subpopulation of clonal cells is unknown. A
possible
distinction of these cells is their position in the cell
cycle. BLV
expression has been shown to be cell cycle dependent
in FLK-BLV cells
(
45) and associated with the induction of cell
division by
PMA in NBC-13 cells (
24). BLV transcription could
also be
associated with
apoptosis.
A product of translation of the
tax/rex transcript, p34 Tax,
has the demonstrated capacity to affect cell function. It facilitates
the activation of cyclic AMP response element-binding protein
(CREB) to
transactivate viral gene transcription (
4,
13,
26,
51,
53).
Tax has immortalization potential in cell culture
(
50,
51)
and activates the transcription of cellular genes
via CREB
(
26). The HTLV-1 Tax protein, in addition to the
transactivation
of viral gene transcription via CREB, can activate or
repress
the transcription of cellular genes involved in the control of
cell proliferation (
2,
17,
21,
46), DNA repair
(
22),
tumor suppression (
5), and apoptosis
(
47), induce cell division
by interacting directly with
proteins, such as p53 (
36,
37),
p16
INK4a
(
44), and cyclin D (
33), and disrupt the mitotic
checkpoint
by binding to human mitotic arrest-defective protein 1 (
25).
The loss of this checkpoint allows the premature onset
of anaphase
and the development of karyotypic abnormalities similar to
those
observed in malignancy. If the Tax protein of BLV is capable of
functions similar to that of HTLV-1, then it has the capacity
to
promote proliferation and perturb host chromosomal integrity.
The
evidence presented here shows that the
tax/rex gene of BLV
is transcribed throughout infection in significant quantities
in a
minority of infected cells. The interrelationship between
selective
viral gene expression and BLV disease progression remains
enigmatic.
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ACKNOWLEDGMENTS |
This work was supported in part by USDA grant 93-37204-9213. J.R.
was supported by USDA National Needs Graduate Fellowship 92-38420-7366.
We thank Richard A. Reyes, Gary L. Cockerell, and Thomas J. Divers for
the generous provision of samples and Craig Schultz and the USDA staff
at the Taylor Packing Co. for support in the rapid collection of tumor
material. NBC-13 cells were provided by Jorge F. Ferrer, and 4'G9
anti-p24 monoclonal antibody was provided by Daniel Portetelle.
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FOOTNOTES |
*
Corresponding author. Present address: Department of
Molecular Biosciences, 7047 Haworth Hall, University of Kansas,
Lawrence, KS 66045-2106. Phone: (785) 864-4022. Fax: (785) 864-5294. E-mail: jr38{at}cornell.edu.
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REFERENCES |
| 1.
|
Alexandersen, S.,
S. Carpenter,
J. Christensen,
T. Storgaard,
B. Viuff,
Y. Wannemuehler,
J. Belousov, and J. A. Roth.
1993.
Identification of alternatively spliced mRNAs encoding potential new regulatory proteins in cattle infected with bovine leukemia virus.
J. Virol.
67:39-52[Abstract/Free Full Text].
|
| 2.
|
Béraud, C.,
S.-C. Sun,
P. Ganchi,
D. W. Ballard, and W. C. Greene.
1994.
Human T-cell leukemia virus type I Tax associates with and is negatively regulated by the NF- B2 p100 gene product: implications for viral latency.
Mol. Cell. Biol.
14:1374-1382[Abstract/Free Full Text].
|
| 3.
|
Bhat, N. K.,
Y. Adachi,
K. P. Samuel, and D. Derse.
1993.
HTLV-1 gene expression by defective proviruses in an infected T-cell line.
Virology
196:15-24[Medline].
|
| 4.
|
Boros, I. M.,
F. Tie, and C.-Z. Giam.
1995.
Interaction of bovine leukemia virus transactivator Tax with bZip proteins.
Virology
214:207-214[Medline].
|
| 5.
|
Brauweiler, A.,
J. E. Garrus,
J. C. Reed, and J. K. Nyborg.
1997.
Repression of bax gene expression by the HTLV-I Tax protein: implications for suppression of apoptosis in virally infected cells.
Virology
231:135-140[Medline].
|
| 6.
|
Chiba, T.,
T. Ajito, and K. Okada.
1994.
Phenotype analysis of lymphocytes present in different stages of neoplasia induced by bovine leukemia virus.
Leukemia
8:S206-S210.
|
| 7.
|
Ciminale, V.,
G. N. Pavlakis,
D. Derse,
C. P. Cunningham, and B. K. Felber.
1992.
Complex splicing in the human T-cell leukemia virus (HTLV) family of retroviruses: novel mRNAs and proteins produced by HTLV type I.
J. Virol.
66:1737-1745[Abstract/Free Full Text].
|
| 8.
|
Ciminale, V.,
L. Zotti,
D. M. D'Agostino, and L. Chieco-Bianchi.
1997.
Inhibition of human T-cell leukemia virus type 2 Rex function by truncated forms of Rex encoded in alternatively spliced mRNAs.
J. Virol.
71:2810-2818[Abstract].
|
| 9.
|
Cockerell, G. L., and J. Rovnak.
1988.
The correlation between the direct and indirect detection of bovine leukemia virus infection in cattle.
Leuk. Res.
12:465-469[Medline].
|
| 10.
|
Cockerell, G. L.,
J. Rovnak,
P. L. Green, and I. S. Y. Chen.
1996.
A deletion in the proximal untranslated pX region of human T-cell leukemia virus type II decreases viral replication but not infectivity in vivo.
Blood
87:1030-1035[Abstract/Free Full Text].
|
| 11.
|
Derse, D.
1987.
Bovine leukemia virus transcription is controlled by a virus-encoded trans-acting factor and by cis-acting response elements.
J. Virol.
61:2462-2471[Abstract/Free Full Text].
|
| 12.
|
Derse, D.
1988.
trans-acting regulation of bovine leukemia virus mRNA processing.
J. Virol.
62:1115-1119[Abstract/Free Full Text].
|
| 13.
|
Derse, D., and J. W. Casey.
1986.
Two elements in the bovine leukemia virus long terminal repeat that regulate gene expression.
Science
231:1437-1440[Abstract/Free Full Text].
|
| 14.
|
Edmands, S.,
J. Kirk,
A. Lee, and J. Radich.
1994.
Rapid RT-PCR amplification from limited cell numbers.
PCR Methods Appl.
3:317-319[Medline].
|
| 15.
|
Felber, B. K.,
D. Derse,
A. Athanassopoulos,
M. Campbell, and G. N. Pavlakis.
1989.
Cross-activation of the Rex proteins of HTLV-I and BLV and of the Rev protein of HIV-1 and nonreciprocal interactions with their RNA responsive elements.
New Biol.
1:318-328[Medline].
|
| 16.
|
Ferrer, J. F.,
N. D. Stock, and P. Lin.
1971.
Detection of replicating C-type viruses in continuous cell cultures established from cows with leukemia: effect of the culture medium.
J. Natl. Cancer Inst.
47:613-621.
|
| 17.
|
Fujii, M.,
P. Sassone-Corsi, and I. M. Verma.
1988.
c-fos promoter trans-activation by the tax1 protein of human T-cell leukemia virus type 1.
Proc. Natl. Acad. Sci. USA
85:8526-8530[Abstract/Free Full Text].
|
| 18.
|
Gaynor, E. M.,
M. L. Mirsky, and H. A. Lewin.
1996.
Use of flow cytometry and RT-PCR for detecting gene expression by single cells.
BioTechniques
21:286-291[Medline].
|
| 19.
|
Haas, L.,
T. Divers, and J. W. Casey.
1992.
Bovine leukemia virus gene expression in vivo.
J. Virol.
66:6223-6225[Abstract/Free Full Text].
|
| 20.
|
Heeny, J. L.,
P. J. S. Valli,
R. M. Jacobs, and V. E. O. Valli.
1992.
Evidence for bovine leukemia virus infection of peripheral blood monocytes and limited antigen expression in bovine lymphoid tissue.
Lab. Investig.
66:608-617[Medline].
|
| 21.
|
Hirai, H.,
T. Suzuki,
J. Fujisawa,
J. Inoue, and M. Yoshida.
1994.
Tax protein of human T-cell leukemia virus type I binds to the ankyrin motifs of inhibitory factor kappa B and induces nuclear translocation of transcription factor NF-kappaB proteins for transcriptional activation.
Proc. Natl. Acad. Sci. USA
91:3584-3588[Abstract/Free Full Text].
|
| 22.
|
Jeang, K.-T.,
S. G. Widen,
O. J. Semmes, and S. H. Wilson.
1990.
HTLV-I transactivator protein, Tax, is a trans-repressor of the human beta-polymerase gene.
Science
247:1082-1084[Abstract/Free Full Text].
|
| 23.
|
Jensen, W. A.,
J. Rovnak, and G. L. Cockerell.
1991.
In vivo transcription of the bovine leukemia virus tax/rex region in normal and neoplastic lymphocytes of cattle and sheep.
J. Virol.
65:2484-2490[Abstract/Free Full Text].
|
| 24.
|
Jensen, W. A.,
B. J. Wicks-Beard, and G. L. Cockerell.
1992.
Inhibition of protein kinase C results in decreased expression of bovine leukemia virus.
J. Virol.
66:4427-4433[Abstract/Free Full Text].
|
| 25.
|
Jin, D.-Y.,
F. Spencer, and K.-T. Jeang.
1998.
Human T-cell leukemia virus type 1 oncoprotein Tax targets the human mitotic checkpoint protein MAD1.
Cell
93:81-91[Medline].
|
| 26.
|
Katoh, I.,
Y. Yoshinaka, and Y. Ikawa.
1989.
Bovine leukemia virus trans-activator p34Tax activates heterologous promoters with a common sequence known as a cAMP-responsive element or the binding site of a cellular transcription factor ATF.
EMBO J.
8:497-503[Medline].
|
| 27.
|
Kerkhofs, P.,
H. Heremans,
A. Burny,
R. Kettman, and L. Willems.
1998.
In vitro and in vivo oncogenic potential of bovine leukemia virus G4 protein.
J. Virol.
72:2554-2559[Abstract/Free Full Text].
|
| 28.
|
Kettmann, R.,
A. Burny,
I. Callebaut,
L. Droogmans,
M. Mammericks,
L. Willems, and D. Portetelle.
1994.
Bovine leukemia virus, vol. 3.
Plenum Press, New York, N.Y.
|
| 29.
|
Kubota, S.,
M. Hatanaka, and R. J. Pomerantz.
1996.
Nucleo-cytoplasmic redistribution of the HTLV-I rex protein: alterations by coexpression of the HTLV-I p21x protein.
Virology
220:502-507[Medline].
|
| 30.
|
Lagarias, D. M., and K. Radke.
1989.
Transcriptional activation of bovine leukemia virus in blood cells from experimentally infected, asymptomatic sheep with latent infections.
J. Virol.
63:2099-2107[Abstract/Free Full Text].
|
| 31.
|
McCulloch, R. K.,
C. S. Choong, and D. M. Hurley.
1995.
An evaluation of competitor type and size for use in the determination of mRNA by competitive PCR.
PCR Methods Appl.
4:219-226[Medline].
|
| 32.
|
Mirsky, M. L.,
C. A. Olmstead,
Y. Da, and H. A. Lewin.
1996.
The prevalence of proviral bovine leukemia virus in peripheral blood mononuclear cells at two subclinical stages of infection.
J. Virol.
70:2178-2183[Abstract].
|
| 33.
|
Neuveut, C.,
K. G. Low,
F. Maldarelli,
I. Schmitt,
F. Majone,
R. Grassmann, and K.-T. Jeang.
1998.
Human T-cell leukemia virus type 1 Tax and cell cycle progression: role of cyclin D-cdk and p110Rb.
Mol. Cell. Biol.
18:3620-3632[Abstract/Free Full Text].
|
| 34.
|
Orita, O.,
S. Sato,
Y. Aono,
N. Minoura,
T. Yamashita,
Y. Hinuma, and H. Igarashi.
1993.
Identification of novel singly spliced pX mRNA transcripts common to all human T-cell leukemia virus type 1-related retroviruses.
Virus Genes
7:197-204[Medline].
|
| 35.
|
Orita, S.,
H. Kobayashi,
Y. Aono,
A. Saiga,
M. Maeda, and H. Igarashi.
1993.
p21X mRNA is expressed as a singly spliced transcript from defective provirus genomes having a partial deletion of the pol-env region in human T-cell leukemia virus type 1-infected cells.
Nucleic Acids Res.
21:3799-3807[Abstract/Free Full Text].
|
| 36.
|
Pise-Masison, C. A.,
M. Radonovich,
K. Sakaguchi,
E. Appella, and J. N. Brady.
1998.
Phosphorylation of p53: a novel pathway for p53 inactivation in human T-cell lymphotropic virus type 1-transformed cells.
J. Virol.
72:6348-6355[Abstract/Free Full Text].
|
| 37.
|
Reid, R. L.,
P. F. Lindholm,
A. Mireskandari,
J. Dittmer, and J. N. Brady.
1993.
Stabilization of wildtype p53 in human T lymphocytes transformed by HTLV-1.
Oncogene
8:3029-3036[Medline].
|
| 38.
|
Reyes, R. A., and G. L. Cockerell.
1996.
Unintegrated bovine leukemia virus DNA: association with viral expression.
J. Virol.
70:4961-4965[Abstract/Free Full Text].
|
| 39.
|
Rice, N. R.,
R. M. Stephens,
D. Couez,
J. Deschamps,
R. Kettmann,
A. Burny, and R. Gilden.
1984.
The nucleotide sequence of the env and post-env region of bovine leukemia virus.
Virology
138:82-93[Medline].
|
| 40.
|
Rosen, C. A.,
J. Sodroski,
R. Kettmann,
A. Burny, and W. Haseltine.
1984.
Trans activation of the bovine leukemia virus long terminal repeat in BLV-infected cells.
Science
227:320-322.
|
| 41.
|
Sagata, N.,
J. Tsuzuku-Kawamura,
M. Nagayoshi-Aida,
F. Shimizu,
K. I. Imagawa, and Y. Ikawa.
1985.
Identification of some biochemical properties of the major XBL gene product of bovine leukemia virus.
Proc. Natl. Acad. Sci. USA
82:7879-7883[Abstract/Free Full Text].
|
| 42.
|
Sagata, N.,
T. Yasunaga,
J. Tsuzuku-Kawamura,
K. Ohishi,
Y. Ogawa, and Y. Ikawa.
1985.
Complete nucleotide sequence of the genome of bovine leukemia virus: its evolutionary relationship to other retroviruses.
Proc. Natl. Acad. Sci. USA
82:677-681[Abstract/Free Full Text].
|
| 43.
|
Schnurr, M. W.,
R. F. Carter,
I. D. Dube,
V. E. Valli, and R. M. Jacobs.
1994.
Nonrandom chromosomal abnormalities in bovine lymphoma.
Leuk. Res.
18:91-99[Medline].
|
| 44.
|
Suzuki, T.,
S. Kitao,
H. Matsushime, and M. Yoshida.
1996.
HTLV-1 tax protein interacts with cyclin-dependent kinase inhibitor p16INK4A and counteracts its inhibitory activity towards CDK4.
EMBO J.
15:1607-1614[Medline].
|
| 45.
|
Takashima, I., and C. Olson.
1980.
Effect of mitogens and anti-bovine leukosis serums on DNA synthesis of lymphocytes from cattle.
Eur. J. Cancer
16:639-645.
|
| 46.
|
Uittenbogaard, M.,
A. P. Armstrong,
A. Chiaramello, and J. K. Nyborg.
1994.
HTLV-I Tax protein represses gene expression through the bHLH family of transcription factors.
J. Biol. Chem.
269:22466-22469[Abstract/Free Full Text].
|
| 47.
|
Uittenbogaard, M.,
H. A. Giebler,
D. Reisman, and J. K. Nyborg.
1995.
Transcriptional repression of p53 by human T-cell leukemia virus type 1 tax protein.
J. Biol. Chem.
270:28503-28506[Abstract/Free Full Text].
|
| 48.
|
Van Der Maaten, M. J., and J. M. Miller.
1976.
Replication of bovine leukemia virus in monolayer cell cultures.
Bibl. Haematol.
43:360-362.
|
| 49.
|
Willems, L.,
A. Gegonne,
G. Chen,
A. Burny,
R. Kettmann, and J. Ghysdael.
1987.
The bovine leukemia virus p34 is a transactivator protein.
EMBO J.
6:3385-3389[Medline].
|
| 50.
|
Willems, L.,
C. Grimonpont,
H. Heremans,
N. Rebeyrotte,
G. Chen,
D. Portetelle,
A. Burny, and R. Kettmann.
1992.
Mutations in the bovine leukemia virus tax protein can abrogate the long terminal repeat-directed transactivating activity without concomitant loss of transforming potential.
Proc. Natl. Acad. Sci. USA
89:3957-3961[Abstract/Free Full Text].
|
| 51.
|
Willems, L.,
H. Heremans,
G. Chen,
D. Portetelle,
A. Billiau,
A. Burny, and R. Kettmann.
1990.
Cooperation between bovine leukemia virus transactivator protein and Ha-ras oncogene in cellular transformation.
EMBO J.
9:1577-1581[Medline].
|
| 52.
|
Willems, L.,
P. Kerkhofs,
F. Dequiedt,
D. Portetelle,
M. Mammerickx,
A. Burny, and R. Kettmann.
1994.
Attenuation of bovine leukemia virus by deletion of R3 and G4 open reading frames.
Proc. Natl. Acad. Sci. USA
91:11532-11536[Abstract/Free Full Text].
|
| 53.
|
Willems, L.,
R. Kettmann,
G. Chen,
D. Portetelle,
A. Burny, and D. Derse.
1992.
A cyclic AMP-responsive DNA-binding protein (CREB2) is a cellular transactivator of the bovine leukemia virus long terminal repeat.
J. Virol.
66:766-772[Abstract/Free Full Text].
|
| 54.
|
Willems, L.,
R. Kettmann,
F. Dequiedt,
D. Portetelle,
V. Vonèche,
I. Cornil,
P. Kerkhofs,
A. Burny, and M. Mammerickx.
1993.
In vivo infection of sheep by bovine leukemia virus mutants.
J. Virol.
67:4078-4085[Abstract/Free Full Text].
|
| 55.
|
Yoshinaka, Y., and S. Oroszian.
1985.
Bovine leukemia virus post-envelope gene coded protein: evidence for expression in natural infection.
Biochem. Biophys. Res. Commun.
131:347-354[Medline].
|
Journal of Virology, October 1999, p. 8890-8897, Vol. 73, No. 10
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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