Previous Article | Next Article 
Journal of Virology, March 2000, p. 2703-2713, Vol. 74, No. 6
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Jembrana Disease Virus Tat Can Regulate Human Immunodeficiency
Virus (HIV) Long Terminal Repeat-Directed Gene Expression and Can
Substitute for HIV Tat in Viral Replication
Hexin
Chen,1
Jun
He,1
Steven
Fong,2
Graham
Wilcox,3 and
Charles
Wood1,*
School of Biological Sciences, University of
Nebraska
Lincoln, Lincoln, Nebraska 685881;
Division of Veterinary and Biomedical Sciences, Murdoch
University, Murdoch, WA 6150, Australia3; and
Division of Toxicology, U.S. Army Medical Research Institute
for Infectious Diseases, Fort Detrick, Frederick, Maryland
21702-50112
Received 3 August 1999/Accepted 8 December 1999
 |
ABSTRACT |
Jembrana disease virus (JDV) is a bovine lentivirus genetically
similar to bovine immunodeficiency virus; it causes an acute and
sometimes fatal disease in infected animals. This virus carries a very
potent Tat that can strongly activate not only its own long terminal
repeat (LTR) but also the human immunodeficiency virus (HIV) LTR. In
contrast, HIV Tat cannot reciprocally activate the JDV LTR (H. Chen,
G. E. Wilcox, G. Kertayadnya, and C. Wood, J. Virol.
73:658-666, 1999). This indicates that in transactivation JDV Tat may
utilize a mechanism similar to but not the same as that of the HIV Tat.
To further study the similarity of JDV and HIV tat in
transactivation, we first tested the responses of a series of HIV LTR
mutants to the JDV Tat. Cross-transactivation of HIV LTR by JDV Tat was
impaired by mutations that disrupted the HIV type 1 transactivation
response element (TAR) RNA stem-loop structure. Our results
demonstrated that JDV Tat, like HIV Tat, transactivated the HIV LTR at
least partially in a TAR-dependent manner. However, the sequence in the
loop region of TAR was not as critical for the function of JDV Tat as
it was for HIV Tat. The competitive inhibition of Tat-induced
transactivation by the truncated JDV or HIV Tat, which consisted only
of the activation domain, suggested that similar cellular factors were
involved in both JDV and HIV Tat-induced transactivation. Based on the one-round transfection assay with HIV tat mutant
proviruses, the cotransfected JDV tat plasmid can
functionally complement the HIV tat defect. To further
characterize the effect of JDV Tat on HIV, a stable chimeric HIV
carrying the JDV tat gene was generated. This chimeric HIV
replicated in a T-cell line, C8166, and in peripheral blood mononuclear
cells, which suggested that JDV Tat can functionally substitute for HIV
Tat. Further characterization of this chimeric virus will help to
elucidate how JDV Tat functions and to explain the differences between
HIV and JDV Tat transactivation.
| |
INTRODUCTION |
Lentiviruses are a group of
retroviruses usually associated with slowly developing diseases with a
number of different clinical manifestations of infection in different
virus-host systems (13, 14). For example, human
immunodeficiency virus (HIV) causes immunodeficiency and ultimately
leads to the death of infected patients several years after infection
(14). In contrast, a recently characterized bovine
lentivirus, Jembrana disease virus (JDV), causes an acute disease in
infected animals after a short incubation period (7, 60,
61). It is probable that an understanding of the differences in
molecular mechanisms involved in the pathogenesis of different
lentiviruses will provide important insights into methods of
controlling lentivirus-induced diseases such as AIDS.
The genomes of all lentiviruses contain three major structural genes,
gag, pol, and env, as well as several
accessory and regulatory genes flanked by the two long terminal repeats
(LTRs) (14). In HIV, at least six accessory and regulatory
genes, tat, rev, nef, vif,
vpr, and vpu, have been identified previously
(14). Studies of these accessory genes have suggested that
most of them are involved in viral replication and pathogenesis
(19, 33, 39, 42, 44, 46, 57). The HIV tat gene
has been extensively studied and is an essential determinant of viral
replication and the pathogenesis of infection (3, 4, 12, 16, 18,
31, 58, 62). Tat is a potent transactivator for HIV gene
expression (3, 4) and has also been shown to modulate the
expression of cellular genes, such as those for major
histocompatibility complex class I (30), tumor necrosis
factor alpha (8, 48), interleukin-2 (IL-2) (59),
IL-6 (53), and several extracellular matrix proteins
(54). The Tat protein is released from HIV type 1 (HIV-1)-infected cells and can be detected in sera of HIV-1-infected individuals (24). Extracellular Tat may be involved in
suppression of the host immune response and cellular disorders
associated with AIDS pathology (2, 23). Tat can synergize
with cellular basic fibroblast growth factor and may be involved in the
induction of Kaposi's sarcoma lesions (6, 22, 23). In
addition, Tat may participate in the induction of apoptosis in
lymphocytes and contribute to the depletion of the CD4+ T
cells in AIDS patients (40, 43, 58). The role of Tat in HIV
pathogenesis may not be limited to its transactivation function
(31). Tat may also play a role in viral replication. Indeed,
Tat has recently been shown to be required for efficient HIV-1 reverse
transcription (27, 28, 29). Thus, there is ample evidence to
suggest that the HIV Tat is a pleiotropic protein, but how Tat is
involved in HIV pathogenesis is still an enigma. Further understanding
of the molecular basis for how Tat plays a role in viral replication
and viral pathogenesis would be of fundamental importance to our
understanding of lentiviruses.
JDV is a recently identified lentivirus (9, 10, 61) related
to bovine immunodeficiency virus (BIV), but in contrast to BIV, it
causes an acute disease atypical of most lentivirus infections (7,
60, 61). JDV infection of cattle can lead to death of the animal
within 1 to 2 weeks (60, 61). The virus replicates to
extremely high titers in infected animals, and since Tat plays an
important role in viral replication, we hypothesized that JDV encodes a
potent transactivator, Tat, responsible for its robust replication
activity in vivo. In previous studies, we have shown that JDV encodes a
Tat that can strongly activate not only the JDV LTR but also the LTRs
of other lentiviruses, including the HIV LTR (11). Other
than BIV Tat, JDV Tat is the only other nonprimate viral Tat to
effectively transactivate the HIV LTR (41), which implies
that similar mechanisms may be shared between HIV and the bovine
lentivirus Tat proteins. It also suggests that bovine lentiviruses may
have a close evolutionary relationship with primate lentiviruses, at
least in terms of transactivation mechanism, and may be used as a model
to further dissect the molecular mechanism of Tat transactivation and
its role in the pathogenesis of lentivirus infections.
To further study the similarities and differences between the JDV and
the HIV Tat, we investigated the transactivation of the HIV LTR by the
JDV Tat. As expected, JDV Tat transactivated HIV LTR at least partially
in a TAR-dependent manner, and this may involve similar mechanisms and
common cellular factors in the two systems. Moreover, we demonstrated
that JDV Tat functionally substituted for the HIV Tat when the JDV
tat gene was introduced into the HIV genome. Our study
provides additional evidence that the JDV Tat is a potent
transactivator and indicates that further characterization of the
molecular mechanism involved in transactivation by JDV Tat is warranted.
| |
MATERIALS AND METHODS |
Plasmids.
Several HIV and JDV LTR promoter constructs were
used in this study. A series of HIV LTR mutants with deletions and
mutations in the TAR region of the LTR, TARCAT and Mod1CAT to Mod7CAT,
were used. The clone TARCAT, used as a positive control, contained the
HIV 5' LTR region (nucleotide [nt] 
121 to +82, encompassing the
intact TAR sequence) cloned in front of the indicator chloramphenicol acetyltransferase (CAT). Additional mutant HIV LTR promoter constructs used were derivatives of TARCAT; these were used previously to test
their responsiveness to Tat transactivation (25). A pHIV-CAT clone that contained the intact HIV U3R region was described previously (35). The constructs pHTLV-CAT and pCMV-Tax were a kind gift from Fatah Kashanchi (37). The mutant HIV Tat construct
p
Cys1-4 used was described previously (26). The JDV
promoter construct, pJU3R-CAT, was constructed as previously described
(14); this clone was generated by insertion of the CAT gene
into a site 3' of the U3R region of the JDV LTR.
Various HIV and JDV tat expression constructs were generated
using the expression plasmid pUC-RSV. The pUC-RSV plasmid was generated
by inserting the Rous sarcoma virus LTR and the bovine growth hormone
polyadenylation signal sequence from pRc/RSV (Invitrogen, Carlsbad,
Calif.) into pUC18. The HIV or the JDV tat exon 1 was PCR
amplified and inserted into vector pUC-RSV to generate pUC-Htat and
pUC-Jtat, both of which encoded the transactivation-competent Tat
peptides. The truncated HIV tat (corresponding to amino
acids 1 to 50) and the truncated JDV tat sequences
(corresponding to amino acids 1 to 67) were cloned into pUC-RSV to
generate plasmids pUC-H50 and pUC-J67. These two plasmids encoded
truncated peptides which had lost their transactivation abilities. Two
tat mutant proviral clones, pMtat() and pMtat30, were
obtained from the AIDS Research and References Program National
Institute of Allergy and Infectious Diseases [NIAID], National
Institutes of Health, Bethesda, Md.): pMtat() contains a termination
codon (TGA) in place of the ATG initiation codon and is unable to
synthesize Tat (49); pMtat30, which carries a Cys
Gly
substitution within the cysteine-rich region, produces a missense Tat
(50).
The HIV infectious proviral clone pNL43 was obtained from the AIDS
Repository (NIAID) (
1). This proviral clone was used
to
generate other modified HIV proviral clones. The
tat gene
mutant
pNL-tat
m proviral plasmid was generated by
introducing a stop
codon into the
tat gene of pNL43 using a
PCR-based mutagenesis
method (
5). This mutation was verified
by
EcoRI restriction
enzyme digestion, since a new
EcoRI site was generated in the
mutated
tat gene
(
TGG AAG CAT mutated to
TGA ATT CAT).
Using this plasmid, an
NdeI restriction site was introduced
into the ATG site of the
nef gene and was then used to
construct
another modified proviral clone that carried the JDV
tat gene.
The plasmid pNL-Jtat contained the JDV
tat gene, in which the
NdeI/
XhoI
fragment of the
nef gene of pNL-tat
m was
replaced by
the JDV
tat gene exon 1 sequences. In addition,
a random stretch
of sequence (32 bp) was introduced into the
nef gene of pNL-tat
m to generate pNL-nef, which
was used as the negative control in
the various experiments. As a
control, the plasmid Tat(
)1ex,
which contains the HIV
tat
in place of the
nef gene, was constructed
using a similar
strategy (
45), and was kindly provided by K.
T. Jeang
(NIAID).
Transfection, virus stock, and CAT ELISA.
CV-1, HeLa, and
293T cells were maintained in Dulbecco's modified Eagle's medium with
10% fetal calf serum. As described previously (11), 2 × 105 cells were seeded into 60-mm-diameter dishes 24 h prior to transfection. The cells were transfected with 1 to 2 µg of
CsCl-purified plasmid DNA using Lipofectamine (GIBCO BRL, Grand Island,
N.Y.). At 48 h after transfection, the cells were harvested and
lysed, and the amount of protein in the lysate was determined using a
bicinchoninic acid protein assay kit (Pierce, Rockford, Ill.). For
cells transfected with the promoter CAT construct plasmids, the level
of CAT expression was determined by a commercial CAT enzyme-linked
immunosorbent assay (ELISA) (Boehringer Mannheim, Indianapolis, Ind.)
according to the protocols described by the manufacturer.
Stocks of infectious virus were produced by transfection of cells with
proviral DNA. The medium from the transfected cells
was collected
48 h posttransfection, the cells and cellular debris
were removed
by centrifugation, and the supernatant was used as
a source of
infectious virus. The titer of virus was measured
by the p24 assay
according to protocols described by the manufacturer
(NEN Life Sciences
Products, Boston, Mass.).
Viral infection and p24 assays.
Human peripheral blood
mononuclear cells (PBMC) were maintained in RPMI 1640 with 10% fetal
bovine serum and IL-2 (2 U/ml) (Pharmacia, Piscataway, N.J.) and
stimulated with phytohemagglutinin (at a final concentration of 250 ng/ml) for 1 to 3 days prior to infection. For each infection, 2 × 106 PBMC in a 0.5-ml volume were incubated with virus at
37°C for 2 h. The virus-containing medium was then decanted and
replaced with fresh RPMI medium containing IL-2 (2 U/ml). The infected cultures were sampled every 6 to 7 days, and the amount of virus produced was measured by p24 assay.
Sequence analysis.
The alignment of Tat amino acid sequences
was performed using the Clustal W program in the GCG package (Madison,
Wis.). Phylogenetic reconstruction trees were generated using the
Evolution program in the GCG package. The Distance program (with
Kimura's formula) was used to generate a pairwise matrix of the
evolutionary distance of the amino acid sequences. Phylogenetic trees
were constructed from the same distance matrices with the Grow-tree
program (neighbor-joining algorithm or unweighted pair group method
with averages [UPGMA]).
| |
RESULTS |
Transactivation of HIV LTR by JDV Tat is TAR dependent.
Our
previous study demonstrated that JDV Tat was a potent transactivator
which could significantly increase the LTR-directed gene expression of
different animal and primate lentiviruses, including HIV (11,
14). We found that the JDV Tat not only stimulated its own LTR
significantly but also stimulated HIV LTR-directed CAT expression to
high levels equivalent to those obtained with HIV Tat (11).
The activation of JDV LTR by JDV Tat was mediated by TAR-like elements
located in the LTR, and the loop region in the TAR seemed to be less
critical for JDV-mediated transactivation.
To further investigate the activation of HIV LTR by JDV Tat and whether
similar mechanisms of transactivation were involved,
a series of HIV
TAR and LTR mutants were studied. These constructs
were generated
previously (
25) and consisted of either deletions
or
mutations in the stem, the bulge, or the loop region of the
HIV TAR.
These mutants are summarized in Fig.
1A
and were designated
TARCAT, Mod1CAT, Mod2CAT, Mod3CAT, Mod4CAT,
Mod5CAT, Mod6CAT,
and Mod7CAT. Each plasmid construct was cotransfected
into CV-1
cells with the JDV
tat expression plasmid,
pUC-Jtat, and tested
for its ability to be activated by the JDV Tat
(Fig.
1B). TARCAT
containing the intact TAR sequence was used as the
wild-type LTR.
Mod7CAT, which contained the minimum HIV TAR required
for HIV
Tat transactivation, showed wild-type levels of transactivation
in the presence of either JDV or HIV Tat. The responsiveness of
the
remaining HIV LTR mutants to transactivation by HIV Tat and
JDV Tat was
severely impaired and varied. The mutants Mod1CAT,
Mod2CAT, Mod4CAT,
and Mod6CAT, with mutations in the TAR stem
and bulge region, were
activated by JDV Tat about 60 to 80% less
than was the wild-type HIV
LTR. However, we consistently observed
that these mutant promoters
still responded better to the JDV
Tat than to the HIV Tat, even though
the activities were much
lower than that with the wild-type LTR. The
Mod3CAT mutant, which
had a 2-base substitution in the loop region,
retained most of
its responsiveness to JDV Tat, but not to HIV Tat.
Mod5CAT, which
had a 5-base insertion in the loop region, was impaired
in its
ability to be activated although it has a relatively higher
basal
level of promoter activity. The data demonstrated that the JDV
Tat transactivated the HIV LTR at least partially in a TAR-dependent
manner, and sequences in the loop region of HIV LTR seemed to
be less
important. The data further support our earlier results
that showed
that activation of the JDV LTR by JDV Tat did not
involve the loop
sequence of the TAR region (
11).
View larger version (41K):
[in this window]
[in a new window]
|
FIG. 1.
Effects of HIV TAR mutations on the transactivation of
HIV LTR by both JDV Tat and HIV Tat. (A) Schematic presentation of the
HIV LTR and mutant TAR constructs which were described in detail
previously (25). TARCAT represents the wild-type LTR. All
other modifications are as follows: Mod1CAT, 4-base deletion (GAUC) in
the bulge region; Mod2CAT, 5-base insertion (CGAUC) in the bulge
region; Mod3CAT, 2-base substitution (GG mutated to UU) in the loop
region; Mod4CAT, 4-base insertion (CUCG) between the bulge and the
upper stem-loop region; Mod5CAT, 5-base insertion (AAAAA) in the loop
region; Mod6CAT, 35-base deletion, including the bulge and the
downstream sequences; Mod7CAT, 8-base substitution (UUCCCGGG) in the
lower stem-loop region. The shaded letters represent the deleted
residues, and the boldface letters represent the substituted residues.
(B) A 0.05-µg amount of LTR mutants was cotransfected with 0.5 µg
of either pUC-Jtat or pUC-Htat. At 48 h posttransfection, cell
lysates were collected and subjected to bicinchoninic acid assay. By
using the same amount of total cellular protein, the expressed CAT
protein in the transfected cell lysate was measured with the CAT ELISA
kit as described in Materials and Methods. The relative CAT
concentrations (conc.) shown in the figure represent the results from
at least three independent experiments. The number above each bar
represents fold activation for each LTR, and the transactivation fold
was obtained by dividing the amount of CAT protein in the presence of
Tat protein by the amount of CAT in the absence of Tat
(11).
|
|
JDV and HIV Tat activation of the LTR can be competitively
inhibited by coexpression of the HIV or JDV Tat activation domain.
HIV Tat has been shown to transactivate the LTR by recruiting the
cyclin T-CDK9 complex to the TAR region which then phosphorylates the
C-terminal domain of RNA polymerase II (15, 32, 34, 47, 56).
In comparing the JDV Tat sequence with HIV Tat, we found that the JDV
Tat contained a very conserved cysteine-rich domain and a core domain,
which are both involved in the HIV Tat binding of cellular factors.
Therefore, it is likely that JDV Tat transactivates HIV LTR by a
similar mechanism and might involve similar cellular factors.
We constructed two truncated
tat plasmids, pUC-J67, which
encoded a JDV Tat peptide, JTat67 (amino acids 1 to 67), and pUC-H50,
which encoded an HIV Tat activation domain, HTat50 (amino acids
1 to
50). We first examined the ability of the expressed truncated
Tat
peptide JTat67 and HTat50 to inhibit the transactivation function
of
HIV Tat and the effect of intact JDV Tat on the HIV LTR (Fig.
2A and
B). These truncated proteins lost their
abilities to transactivate
HIV LTR and JDV LTR and did not inhibit the
basal level of LTR
expression (data not shown). We then cotransfected
CV-1 cells
with the target plasmid pHIV-CAT, effector plasmid pUC-Htat
or
pUC-Jtat, and varying concentrations of the competitor plasmid
pUC-H50 or pUC-J67 (Fig.
2A and B); the total amount of DNA in
each
transfection was normalized by cotransfection of the parent
vector
pUC18. As shown in Fig.
2A and B, JTat67 and HTat50 efficiently
inhibited transactivation of the HIV LTR by both HIV Tat and JDV
Tat,
in a dose-dependent manner. A 20-fold excess of the competitor
plasmid
pUC-J67 or pUC-H50 inhibited HIV Tat activation of the
HIV LTR by about
65% (Fig.
2A), whereas a 10-fold excess of pUC-J67
inhibited HIV Tat
transactivation of the HIV LTR by about 40%
and a 10-fold excess of
pUC-H50 resulted in a 50% decrease in
activity (Fig.
2A). However,
when only a 2.5-fold excess of competitor
was added, the JDV peptide
JTat67 was ineffective while the HIV
peptide inhibited activation about
30%. The inhibition of HIV
LTR activation by JDV Tat was similar (Fig.
2B): a 16-fold excess
of either pUC-J67 or pUC-H50 caused significant
inhibition of
HIV LTR activation, 40% inhibition with pUC-J67 and 50%
inhibition
with pUC-H50; lower concentrations of pUC-H50 seemed to
cause
greater inhibition (Fig.
2B).
View larger version (34K):
[in this window]
[in a new window]
|
FIG. 2.
Competitive inhibition of Tat-induced transactivation by
the truncated Tat proteins. (A) CV-1 cells were cotransfected with 0.05 µg of pHIV-CAT, 0.1 µg of HIV tat plasmid pUC-Htat, and
the indicated amount of the competitors (pUC-J67 expresses the
truncated JDV Tat protein and pUC-H50 expresses the truncated HIV Tat
protein). The indicated values represent the percentages of the amount
of CAT in the presence of competitor relative to that in the absence of
competitor (defined as 100%). (B) CV-1 cells were cotransfected with
0.05 µg of pHIV-CAT plasmid, 0.25 µg of JDV tat plasmid
pUC-Jtat, and the indicated amounts of competitors. (C) CV-1 cells were
cotransfected with 0.5 µg of pJU3R-CAT plasmid, 0.25 µg of JDV
tat plasmid pUC-Jtat, and the indicated amounts of
competitors. (D) CV-1 cells were cotransfected with 0.15 µg of
pHTLV-CAT plasmid, 0.1 µg of pCMV-Tax plasmid, and the indicated
amounts of competitors. The total amount of DNA used in each
transfection was equalized with a control plasmid. Control in the
figure represents no competitor addition.
|
|
We next examined the effect of the truncated Tat proteins on
transactivation of the JDV LTR by JDV Tat (Fig.
2C). Cotransfection
of
CV-1 cells with pJU3R-CAT, pUC-Jtat, and varying concentrations
of
competitor plasmids (either pUC-H50 or pUC-J67) inhibited JDV
Tat
transactivation to levels similar to those observed with the
HIV LTR. A
10-fold excess of either of the Tat inhibitors HTat50
and JTat67
inhibited activation by about 50%. At lower concentrations
of the
inhibitors, the HIV Tat truncated protein also seemed to
be more
effective than the JDV truncated Tat protein. These results
demonstrated that both truncated JDV and HIV Tat may inhibit
transactivation
by the intact Tat, possibly by competing for common
cellular factors
used by both JDV and HIV
Tat.
To confirm that the inhibition by the JDV and HIV Tat activation
domains was specific to Tat-mediated activation, we also
examined the
effects of these activation domains on the activation
of human T-cell
leukemia virus (HTLV) LTR by its cognate transactivator
Tax. The
presence of either HIV or JDV Tat transactivation domain
did not
dramatically affect the activation of the HTLV LTR by
Tax. However, a
slight inhibition by a fivefold or more excess
of HIV Tat
transactivation domain expression plasmid pUC-H50 and
a 10-fold or more
excess of JDV Tat transactivation domain expression
plasmid pUC-J67 was
observed. These results suggested that the
inhibition effects of the
JDV and HIV Tat activation domain were
Tat specific but may also have
involved some nonspecific transcription
factors (Fig.
2D).
JDV Tat can complement HIV tat() provirus
expression.
Tat is a potent and essential transcriptional
transactivator of HIV LTR, and HIV-1 proviruses mutated in
tat are nonviable (18, 49). However, this defect
can be complemented by coexpression of the HIV Tat in trans.
To determine whether JDV Tat can also complement the HIV
tat() provirus expression in trans, two
tat mutant HIV proviral plasmids, pMtat() and pMtat30,
were cotransfected into CV-1 cells with a JDV Tat expression plasmid.
Both tat mutants were demonstrated previously to be
defective due to the mutations at the initiation codon of the Tat
protein or at the cysteine-rich transactivation domain (49,
50). At 48 h posttransfection, supernatant was collected and
assayed for the expression of HIV by p24 analysis. It was determined
that transfection of the two tat mutant cDNA plasmids alone
into CV-1 cells did not produce detectable levels of HIV as measured by
p24 assays. However, in the presence of either the JDV tat
or the HIV tat plasmid, HIV p24 was detected (Fig.
3). An increase in the amount of the JDV or HIV Tat plasmid transfected resulted in higher levels of HIV production. Our results demonstrated that both tat HIV
mutants were complemented equally well by the presence of JDV or HIV
Tat. This complementation assay demonstrated that the JDV Tat could functionally complement the defect in the tat gene in the
HIV provirus to enable the replication of HIV in the proviral
DNA-transfected cell.
View larger version (18K):
[in this window]
[in a new window]
|
FIG. 3.
Complementation of the tat mutant HIV
proviruses by JDV Tat and HIV Tat. A 0.1-µg amount of tat
mutant HIV proviral DNA was cotransfected into CV-1 cells with varying
concentrations of the JDV and HIV tat plasmids, as
indicated. The culture supernatant was collected at 72 h
posttransfection, and the amount of HIV produced was determined by p24
ELISA. (A) Complementation of the HIV tat mutant clone
pMtat() by JDV Tat and HIV Tat. This mutant carries a termination
codon in place of the ATG (methionine) initiator codon in the Tat
coding region (49). (B) Complementation of the HIV
tat mutant clone pMtat30 by JDV and HIV Tat. This mutant has
a CysGly substitution within the Tat cysteine cluster and produces a
missense Tat (50).
|
|
Construction and characterization of chimeric HIV carrying the JDV
tat gene.
To further investigate whether JDV
tat could functionally substitute for the HIV tat
gene, we constructed a chimeric proviral clone by inserting the JDV
tat gene into HIV proviral backbone, pNL43 (Fig.
4). To avoid affecting the function of
other genes which overlap with the tat gene, we introduced a
stop codon into the tat region of the HIV proviral plasmid
pNL43 to inactivate tat, and an EcoRI site was
generated in the mutated tat region. The resulting plasmid,
pNL-tatm, with a tat mutation, otherwise isogenic to pNL43, was wild type in all other genes. A chimeric pNL-Jtat virus was also constructed, wherein a small fragment of
nef was replaced with cDNA coding for JDV Tat exon 1 but
still using the native nef ATG translation initiation site.
The pNL43 nef could be effectively replaced since such
nef mutations do not significantly affect the in vitro
replication and infectivity of the mutant HIV in T-cell lines
(45). As a positive control, a chimeric virus, Tat()1ex,
containing the HIV exon 1 tat, was constructed using a
similar strategy (45). A pNL-nef plasmid, in which random
sequences were inserted into the nef region of pNL-tatm to further inactivate nef, was used as
a negative control.
View larger version (23K):
[in this window]
[in a new window]
|
FIG. 4.
Schematic representation of the wild-type pNL43 and the
various modified virus genomes. The pNL-tatm was generated
by introducing a stop codon at amino acid 12 of the tat gene
to render it inactive. This clone was then used to construct the other
modified HIVs. pNL-Jtat express the JDV Tat exon 1 using the ATG of the
nef gene. pNL-nef contains a random sequence insertion in
the nef gene. Tat()1ex was used and described previously
(45).
|
|
We first characterized the ability of these modified proviral clones to
replicate and generate viruses in transfected cells.
When 1 µg of the
above plasmids was transfected into nonpermissive
cells such as CV-1,
HeLa, and 293T cells, the
tat mutant proviral
clones
pNL-tat
m and pNL-nef did not produce detectable levels
of
HIV in HeLa cells and produced much lower levels of HIV in
293T and
CV-1 cells than did wild-type virus pNL43 (Table
1).
In contrast, Tat(
)1ex and pNL-Jtat,
which contain the HIV
tat and JDV
tat,
respectively, produced wild-type levels of expression
of HIV in all the
tested cell lines.
To rule out the possibility that the failure to express virus in cells
transfected with pNL-tat
m or pNL-nef was due to other
unexpected defects besides the
tat mutation in the genome,
we
cotransfected these cells with
tat mutant proviral DNAs
and either
JDV or HIV Tat-expressing plasmids. For both
pNL-tat
m and pNL-nef,
in the presence of HIV or JDV
tat, the replication of the viruses
in the transfected cells
was restored and high levels of p24 were
detected in the culture
supernatant. Cotransfection of Tat-expressing
plasmids with Tat(
)1ex,
pNL-Jtat, or pNL43 proviral DNA only
slightly increased or did not
change the level of HIV expression.
Cotransfection of a mutant
HIV Tat-expressing plasmid, p
Cys1-4,
which carries four Cys
Ser
amino acid substitutions in the Cys-rich
region and has lost its
transactivation ability, failed to rescue
the
tat-defective
virus (Table
1).
To further determine whether the presence of JDV
tat in the
chimeric virus could fully complement the HIV
tat functions
and
enable the virus to productively infect CD4
+ T cells,
the viruses generated by transfection were used to infect
both primary
PBMC and a T-cell line, C8166 (
45). The amount
of virus
produced from the transfected 293T cells was determined
by p24 antigen
levels, a similar amount of virus from each transfection
experiment was
then used to infect both fresh PBMC and C8166 cells,
and the infected
cells were monitored for virus growth by p24
assay at various time
points after infection (Fig.
5A). The
control
chimeric virus Tat(
)1ex, containing the HIV
tat
gene, replicated
in the T-cell line as demonstrated previously
(
45). The NL-Jtat
virus, containing JDV
tat, also
replicated efficiently in this
T-cell line, and substantial amounts of
p24 were detected 14 days
after infection. Almost twice as much p24 was
detected in NL-Jtat-infected
cells as in those infected by the control
Tat(
)1ex virus. Our
results thus showed that the chimeric virus with
the HIV
tat replaced
by the JDV
tat was fully
competent in its ability to infect and
replicate in C8166 T cells. As
expected, the negative control
viruses, NL-tat
m and NL-nef,
did not replicate.
View larger version (22K):
[in this window]
[in a new window]
|
FIG. 5.
Growth and replication kinetics of the various
recombinant HIV-1 viruses in the C8166 T-cell line and PBMC. (A)
Different viral stocks generated by transfection of 293T cells were
used to infect the C8166 T-cell line. The 293T cells were transfected
with the recombinant proviral DNA using Lipofectamine. At 48 h
posttransfection, supernatants were collected, and the p24 levels were
determined. C8166 cells (2 × 105) were infected by 10 ng of the different viruses as determined by the p24 assay. The amount
of virus produced was monitored at the indicated time points by p24
analysis. The cross in the figure represents a p24 concentration of 103 ng/ml, which is beyond the y-axis scale. (B) PBMC (2 × 106) were infected with 10 ng of virus as determined by the
p24 assay, and the amount of virus produced was monitored at the
indicated time points as described above.
|
|
To further characterize the chimeric viruses, their ability to infect
and replicate in primary T cells was tested. It was
previously shown
that the HIV chimera with the HIV
nef gene replaced
by HIV
tat was unable to replicate in PBMC (
45),
suggesting
that either the HIV
tat exon 1 alone or the
destruction of the
HIV
nef affected the ability of HIV to
replicate in primary cells.
Indeed, when we tested our various chimeric
viruses in PBMC (Fig.
5B), the Tat(
)1ex virus containing the HIV
tat exon 1 did not
replicate in PBMC even though it
replicated in C8166 cells, similar
to results obtained previously with
this virus (
45). In contrast,
the chimeric NL-Jtat which
contained the JDV
tat infected and
replicated effectively in
the primary PBMC, even though the levels
seemed to be lower than those
of the wild-type virus NL43. It
produced lower levels of virus
expression initially, but by 14
days the levels seemed to have
increased and by 18 days were much
higher. Our results suggested that
the presence of JDV exon 1
tat complemented both HIV
tat and
nef to render the virus fully
replication
competent in both PBMC and T-cell lines. As expected,
both
tat mutant virus NL-tat
m and NL-nef failed to
replicate in
PBMC.
To rule out the possibility that infection of PBMC and C8166 cells by
chimeric virus NL-Jtat was due to the back mutation
of the HIV
tat mutation to wild type, the HIV
tat gene of
the
infected cells was analyzed by PCR and restriction analysis of
the
infected PBMC viral DNA (Fig.
6). Since
the mutated
tat gene
was engineered to have an
EcoRI site, the presence of the restriction
site would
indicate persistence of the mutation. Indeed, the amplified
tat gene product from the chimeric virus NL-Jtat-infected
cellular
DNA, but not the wild-type virus-infected cellular DNA, was
cut
completely by
EcoRI to generate a smaller
tat
gene fragment of
the expected size (Fig.
6A, compare lanes 1 and 4).
This result
suggested that the mutation in the
tat gene for
NL-Jtat persisted
and that reversion to the wild type was not
responsible for the
ability of this virus to replicate in PBMC. To
further confirm
that the JDV and HIV
tat gene insertion in
the
nef region had
not been deleted, the
nef
region of the viruses from the infected
PBMC was analyzed by PCR. As
was expected, the PCR-amplified fragments
from NL-Jtat and Tat(
)1ex
viruses were relatively larger than
the wild-type
nef
fragment, indicating that the
tat insert was
retained (Fig.
6B).
View larger version (41K):
[in this window]
[in a new window]
|
FIG. 6.
PCR analysis of the viruses generated from the infected
PBMC cultures. The figure shows PCR amplification of tat and
nef gene sequences from the infected PBMC cellular DNA. At
18 days after infection, PBMC cellular DNA was extracted from cells
infected with different viruses, and PCR amplification with the
appropriate primers was performed. (A) Verification of tat
mutation in the recombinant virus by EcoRI digestion. PCR
products of the tat region amplified from infected PBMC DNA
were purified from 1% agarose gels and then cut with EcoRI.
To avoid amplification of the inserted tat sequences in the
recombinant virus, the primers NL-tat1 (nt 5762 to 5781, 5' GTT TAT CCA
TTT CAG AAT TG 3') and NL-tat2 (nt 6428 to 6409, 5' CCA AAC ATT ATG TAC
CTC TG 3') used were located outside the tat sequences (nt
5830 to 6044) in the NL43 genome (GenBank accession no. M19921). Lane
1, NL43 virus tat DNA; lane 2, Tat()1ex virus
tat DNA; lane 3, NL-tatm virus tat
DNA; lane 4, NL-Jtat virus tat DNA. (B) PCR amplification of
nef sequences from the infected PBMC DNA to verify the
presence of the tat insert in the nef region of
the recombinant virus genome. PCR was performed with primers NL-nef1
(nt 8447 to 8465, 5' TCC ATT CGA TTA GTG AAC G3') and NL-nef2 (nt 8904 to 8886, 5' CTA CTT GTG ATT GCT CCA T3'). Lane 1, lambda/HindIII; lane 2, NL43; lane 3, Tat()1ex; lane
4, NL-tatm; lane 5, NL-nef, lane 6, NL-Jtat; lane 7, mock-infected control. wt, wild type.
|
|
Phylogenetic analysis of lentivirus Tat proteins.
The
cross-transactivation by JDV Tat of the HIV LTR suggested a close
evolutionary relationship between JDV and other primate lentiviruses.
To analyze the evolutionary genetic relationship between JDV and other
lentiviruses, a neighbor-joining phylogenetic tree based on the
published sequences of lentiviral Tat peptides which are competent in
transactivation was constructed (3, 11, 17, 20, 21, 36, 41, 51,
52, 55) (Fig. 7A). With the
constructed tree, two major subgroups of Tat were formed (primate
versus nonprimate lentivirus Tat proteins). Within the primate
lentivirus subgroup, the simian immunodeficiency virus Tat was more
closely related to HIV-2 Tat than to HIV-1 Tat. Within the nonprimate
lentivirus Tat group, the JDV and BIV Tat proteins were closely related
phylogenetically. Interestingly, the two bovine lentivirus Tat proteins
were more closely related to the primate lentivirus Tat proteins than
they were to the nonprimate lentivirus Tat proteins. Another
phylogenetic tree created by using UPGMA also showed a similar
phylogenetic relationship, and although no distinctive nonprimate
lentiviral Tat subgroup was formed with this method of analysis (Fig.
7B), JDV and BIV Tat proteins were within a subgroup which mainly
contained the primate lentivirus Tat proteins.
View larger version (11K):
[in this window]
[in a new window]
|
FIG. 7.
Phylogenetic analysis of the various lentivirus Tat
proteins. The Tat amino acid sequences were deduced from the
tat nucleotide sequences: JDV genome (GenBank accession no.
U21603), BIV (GenBank accession no. M32690), HIV-1 (GenBank locus
hivhxb2cg), HIV-2 (GenBank accession no. M38432), simian
immunodeficiency virus (SIV) (GenBank accession no. M19499), equine
infectious anemia virus (EIAV) (GenBank accession no. M30138), caprine
arthritis-encephalitis virus (CAEV) (GenBank accession no. M34092),
feline immunodeficiency virus (FIV) (GenBank accession no. M36968), and
visna virus tat sequence (accession no. J04359). The trees
were reconstructed using two separate programs as described in
Materials and Methods. Phylogenetic analysis used the Evolution program
in GCG. (A) Analysis using the neighbor-joining algorithm; (B) analysis
using the UPGMA algorithm.
|
|
| |
DISCUSSION |
A comparison of the amino acid sequences of the JDV and HIV Tat
showed a striking homology in the cysteine-rich and the core activation
domain, but very little similarity in the RNA binding basic domain
(11). Indeed, competitors containing the HIV-1 and JDV Tat
activation domain could inhibit the HIV and JDV Tat transactivation of
the HIV LTR or the JDV Tat transactivation of the JDV LTR. Since both
truncated proteins did not contain the basic RNA binding domain that is
responsible for the TAR binding, the inhibition was unlikely to be due
to competition for binding to TAR and more likely was due to the
effects of the transactivating domain itself. These observations
support the notion that the JDV and HIV Tat activation domains may
involve a similar transactivation mechanism and both may involve
interaction with cellular cofactors. The cross-inhibition of Tat
functions may be due to the sequestering of the cellular factors involved.
In this study, we have demonstrated that the transactivation effect of
JDV Tat on HIV LTR is at least partially TAR dependent. Mutations of
TAR in the HIV LTR, except for mutations in the loop region as
represented by mutant Mod3CAT, resulted in significant loss of
responsiveness to JDV Tat transactivation. However, differences in the
Tat of JDV and HIV were noted. First, as the HIV LTR mutant Mod3CAT was
responsive to transactivation by JDV Tat but not to that by HIV Tat,
this suggested that a sequence in the loop region of HIV TAR was not
critical for transactivation by JDV Tat but was essential for
transactivation by HIV Tat. Second, most HIV LTR mutants retained some
responsiveness to the JDV Tat even though they were not activated by
HIV Tat. These differences could be due to the JDV Tat transactivating
the HIV LTR using other mechanisms in addition to the TAR-dependent
pathway, or the JDV Tat-TAR interaction may be different from the HIV
Tat-TAR interaction. We favor the latter explanation, since our results
suggest that the loop sequence in the TAR is not critical for JDV
transactivation, which is different from that by HIV. Studies of HIV
have shown that the loop region is the binding site for cyclin T, and
the binding of cyclin T to the loop can significantly stabilize the HIV
Tat-TAR interaction (56). Recent data have convincingly
demonstrated that the binding of the HIV Tat activation domain to the
cyclin T1 subunit of the human TAK/P-TEFb transcription elongation
complex is a critical first step in TAR RNA recognition and
Tat-mediated transactivation (32). It is possible that JDV
Tat can directly interact with the TAR elements independent of cyclin T
complex binding, and thus the mutations of the cyclin target in the
loop region would not affect the transactivation function of the JDV
Tat. However, this does not exclude the possibility that the cyclin T
complex is still involved in the JDV Tat-induced transactivation of the HIV LTR. Further studies are necessary in order to decipher the role of
cyclin T in JDV Tat transactivation.
In addition to a transactivation function, Tat is essential for viral
replication and pathogenesis (12, 18, 45, 62). HIV Tat was
reported to activate quiescent T lymphocytes and induce apoptosis
(38, 40) and may contribute to the selective depletion of
CD4+ lymphocytes in vivo. Tat is also vital for viral
replication and infection; mutations in tat led to viruses
that were defective (3, 4). Relocation of tat in
the HIV genome or the substitution of HIV tat for the
nef gene still led to viruses that were defective; they were
able to replicate in T-cell lines but not in PBMC (45). Substitution for the HIV tat by alternative transactivators,
such as HTLV Tax and Vp16, which were cloned into HIV
tat() has resulted in viruses that replicate very poorly
in PBMC and other cell types (45). Our chimeric virus that
used JDV tat exon 1 is therefore unique, as JDV
tat exon 1 was able to substitute for HIV tat and enable successful replication in both a T-cell line and PBMC, even
though resulting virus replication was less than that with the
wild-type virus. Our results indicated that JDV Tat can functionally replace HIV Tat and suggest that JDV Tat should play a role in viral
replication and pathogenesis of infection similar to that of HIV Tat in
HIV. In addition, the JDV Tat could rescue a mutant HIV when it was
mutated not only in the tat but also in the nef gene, suggesting that JDV Tat can substitute for both HIV Tat and Nef
to enable the virus to replicate effectively in vitro. However, it is
possible that this HIV-JDV chimeric virus may be nonpathogenic and
attenuated in vivo. Further characterization of this virus and its
pathogenicity will be important not only for elucidation of the
functions of Tat but also for vaccine development if the HIV-JDV
chimeric virus is found to be attenuated.
| |
ACKNOWLEDGMENTS |
This study was supported in part by the University of Nebraska
Center of Biotechnology Area of Concentration grant to C.W.
We thank K. T. Jeang for kindly providing the plasmid Tat()1ex,
R. Gaynor for plasmid pCys1-4, and F. Kashanchi for plasmids pHTLV-CAT and pCMV-Tax. The plasmids pMtat() and pMtat30 were obtained through the AIDS Research and Reference Reagent Program from
Reza Sadaie. We also thank Malcolm Martin for providing the pNL43
plasmid through the AIDS Research and Reference Reagent Program.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: School of
Biological Sciences, University of NebraskaLincoln, E319 Beadle
Center, P.O. Box 880666, Lincoln, NE 68588-0666. Phone: (402) 472-4550. Fax: (402) 472-8722. E-mail: cwood1{at}unl.edu.
| |
REFERENCES |
| 1.
|
Adachi, A.,
S. Koenig,
H. E. Gendelman,
D. Daugherty,
S. Gattoni-Celli,
A. S. Fauci, and M. A. Martin.
1987.
Productive, persistent infection of human colorectal cell lines with human immunodeficiency virus.
J. Virol.
61:209-213[Abstract/Free Full Text].
|
| 2.
|
Albini, A.,
G. Barillari,
R. Benelli,
R. C. Gallo, and B. Ensoli.
1995.
Angiogenic properties of human immunodeficiency virus type 1 Tat protein.
Proc. Natl. Acad. Sci. USA
92:4838-4842[Abstract/Free Full Text].
|
| 3.
|
Arya, S. K.,
B. Beaver,
L. Jagodzinski,
B. Ensoli,
P. J. Kanki,
J. Albert,
E. M. Fenyo,
G. Biberfeld,
J. F. Zagury,
F. Laure,
M. Essex,
E. Norrby,
F. Wong-Staal, and R. C. Gallo.
1987.
New human and simian HIV-related retroviruses possess functional transactivator (tat) gene.
Nature
328:548-550[CrossRef][Medline].
|
| 4.
|
Arya, S. K.,
C. Guo,
S. F. Josephs, and F. Wong-Staal.
1985.
Trans-activator gene of human T-lymphotropic virus type III (HTLV-III).
Science
229:69-73[Abstract/Free Full Text].
|
| 5.
|
Ausubel, F. M.,
R. Brent,
R. E. Kingston,
D. D. Moore,
J. G. Seidman,
J. A. Smith, and K. Struhl (ed.).
1989.
Current protocols in molecular biology.
Greene Publishing Associates, New York, N.Y.
|
| 6.
|
Barillari, G.,
R. Gendelman,
R. C. Gallo, and B. Ensoli.
1993.
The Tat protein of human immunodeficiency virus type 1, a growth factor for AIDS Kaposi sarcoma and cytokine-activated vascular cells, induces adhesion of the same cell types by using integrin receptors recognizing the RGD amino acid sequence.
Proc. Natl. Acad. Sci. USA
90:7941-7945[Abstract/Free Full Text].
|
| 7.
|
Budiarso, I. T., and S. Hardjosworo.
1976.
Jembrana disease in Bali cattle.
Aust. Vet. J.
52:97.
|
| 8.
|
Buonaguro, L.,
G. Barillari,
H. K. Chang,
C. A. Bohan,
V. Kao,
R. Morgan,
R. C. Gallo, and B. Ensoli.
1992.
Effects of the human immunodeficiency virus type 1 Tat protein on the expression of inflammatory cytokines.
J. Virol.
66:7159-7167[Abstract/Free Full Text].
|
| 9.
|
Chadwick, B. J.,
R. J. Coelen,
L. M. Sammels,
G. Kertayadnya, and G. E. Wilcox.
1995.
Genomic sequence analysis identifies Jembrana disease virus as a new bovine lentivirus.
J. Gen. Virol.
76:189-192[Abstract/Free Full Text].
|
| 10.
|
Chadwick, B. J.,
R. J. Coelen,
G. E. Wilcox,
L. M. Sammels, and G. Kertayadnya.
1995.
Nucleotide sequence analysis of Jembrana disease virus: a bovine lentivirus associated with an acute disease syndrome.
J. Gen. Virol.
76:1637-1650[Abstract/Free Full Text].
|
| 11.
|
Chen, H.,
G. E. Wilcox,
G. Kertayadnya, and C. Wood.
1999.
Characterization of the Jembrana disease virus tat gene and the cis- and trans-regulatory elements in its long terminal repeats.
J. Virol.
73:658-666[Abstract/Free Full Text].
|
| 12.
|
Cheng-Mayer, C.,
T. Shioda, and J. A. Levy.
1991.
Host range, replicative, and cytopathic properties of human immunodeficiency virus type 1 are determined by very few amino acid changes in Tat and gp120.
J. Virol.
65:6931-6941[Abstract/Free Full Text].
|
| 13.
|
Clements, J. E., and M. C. Zink.
1996.
Molecular biology and pathogenesis of animal lentivirus infections.
Clin. Microbiol. Rev.
9:100-117[Abstract].
|
| 14.
|
Coffin, J. M.,
S. H. Hughes, and H. E. Varmus.
1997.
Retroviruses, p. 475-636.
Cold Spring Harbor Laboratory Press, Plainview, N.Y.
|
| 15.
|
Cullen, B. R.
1998.
HIV-1 auxiliary proteins: making connections in a dying cell.
Cell
93:685-692[CrossRef][Medline].
|
| 16.
|
Cullen, B. R.
1995.
Regulation of HIV gene expression.
AIDS
9:S19-32.
|
| 17.
|
Davis, J. L., and J. E. Clements.
1989.
Characterization of a cDNA clone encoding the visna virus transactivating protein.
Proc. Natl. Acad. Sci. USA
86:414-418[Abstract/Free Full Text].
|
| 18.
|
Dayton, A. I.,
J. G. Sodroski,
C. A. Rosen,
W. C. Goh, and W. A. Haseltine.
1986.
The trans-activator gene of the human T cell lymphotropic virus type III is required for replication.
Cell
44:941-947[CrossRef][Medline].
|
| 19.
|
Deacon, N. J.,
A. Tsykin,
A. Solomon,
K. Smith,
M. Ludford-Menting,
D. J. Hooker,
D. A. McPhee,
A. L. Greenway,
A. Ellett,
C. Chatfield,
V. A. Lawson,
S. Crowe,
D. Dowton, and J. Mills.
1995.
Genomic structure of an attenuated quasi species of HIV-1 from a blood transfusion donor and recipients.
Science
270:988-991[Abstract/Free Full Text].
|
| 20.
|
de Parseval, A., and J. H. Elder.
1999.
Demonstration that orf2 encodes the feline immunodeficiency virus transactivating (Tat) protein and characterization of a unique gene product with partial Rev activity.
J. Virol.
73:608-617[Abstract/Free Full Text].
|
| 21.
|
Dorn, P.,
L. DaSilva,
L. Martarano, and D. Derse.
1990.
Equine infectious anemia virus tat: insights into the structure, function, and evolution of lentivirus trans-activator proteins.
J. Virol.
64:1616-1624[Abstract/Free Full Text].
|
| 22.
|
Ensoli, B.,
R. Gendelman,
P. Markham,
V. Fiorelli,
S. Colombini,
M. Raffeld,
A. Cafaro,
H. K. Chang,
J. N. Brady, and R. C. Gallo.
1994.
Synergy between basic fibroblast growth factor and HIV-1 Tat protein in induction of Kaposi's sarcoma.
Nature
371:674-680[CrossRef][Medline].
|
| 23.
|
Ensoli, B.,
G. Barillari,
S. Z. Salahuddin,
R. C. Gallo, and F. Wong-Staal.
1990.
Tat protein of HIV-1 stimulates growth of cells derived from Kaposi's sarcoma lesions of AIDS patients.
Nature
345:84-86[CrossRef][Medline].
|
| 24.
|
Ensoli, B.,
L. Buonaguro,
G. Barillari,
V. Fiorelli,
R. Gendelman,
R. A. Morgan,
P. Wingfield, and R. C. Gallo.
1993.
Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation.
J. Virol.
67:277-287[Abstract/Free Full Text].
|
| 25.
|
Fong, S. E.,
P. Smanik,
T. Thais,
M. C. Smith, and S. R. Jaskunas.
1997.
Detection of specific human immunodeficiency virus type 1 Tat-TAR complexes in the presence of mild denaturing conditions.
J. Virol. Methods
66:91-101[CrossRef][Medline].
|
| 26.
|
Garcia, J. A.,
D. Harrich,
L. Pearson,
R. Mitsuyasu, and R. B. Gaynor.
1988.
Functional domains required for tat-induced transcriptional activation of the HIV-1 long terminal repeat.
EMBO J.
7:3143-3147[Medline].
|
| 27.
|
Harrich, D.,
C. Ulich,
L. F. Garcia-Martinez, and R. B. Gaynor.
1997.
Tat is required for efficient HIV-1 reverse transcription.
EMBO J.
16:1224-1235[CrossRef][Medline].
|
| 28.
|
Harrich, D.,
C. Ulich, and R. B. Gaynor.
1996.
A critical role for the TAR element in promoting efficient human immunodeficiency virus type 1 reverse transcription.
J. Virol.
70:4017-4027[Abstract].
|
| 29.
|
Harrich, D.,
G. Mavankal,
A. Mette-Snider, and R. B. Gaynor.
1995.
Human immunodeficiency virus type 1 TAR element revertant viruses define RNA structures required for efficient viral gene expression and replication.
J. Virol.
69:4906-4913[Abstract].
|
| 30.
|
Howcroft, T. K.,
K. Strebel,
M. A. Martin, and D. S. Singer.
1993.
Repression of MHC class I gene promoter activity by two-exon Tat of HIV.
Science
260:1320-1322[Abstract/Free Full Text].
|
| 31.
|
Huang, L. M.,
A. Joshi,
R. Willey,
J. Orenstein, and K. T. Jeang.
1994.
Human immunodeficiency viruses regulated by alternative trans-activators: genetic evidence for a novel non-transcriptional function of Tat in virion infectivity.
EMBO J.
13:2886-2896[Medline].
|
| 32.
|
Ivanov, D.,
Y. T. Kwak,
E. Nee,
J. Guo,
L. F. Garcia-Martinez, and R. B. Gaynor.
1999.
Cyclin T1 domains involved in complex formation with Tat and TAR RNA are critical for tat-activation.
J. Mol. Biol.
288:41-56[CrossRef][Medline].
|
| 33.
|
Iversen, A. K.,
E. G. Shpaer,
A. G. Rodrigo,
M. S. Hirsch,
B. D. Walker,
H. W. Sheppard,
T. C. Merigan, and J. I. Mullins.
1995.
Persistence of attenuated rev genes in a human immunodeficiency virus type 1-infected asymptomatic individual.
J. Virol.
69:5743-5753[Abstract].
|
| 34.
|
Jeang, K. T.
1998.
Tat, Tat-associated kinase, and transcription.
J. Biomed. Sci.
5:24-27[Medline].
|
| 35.
|
Jung, M., and C. Wood.
1991.
Activation of retroviral promoters by trans-acting factors.
Virology (Life Sci. Adv.)
10:77-88.
|
| 36.
|
Kalinski, H.,
P. Mashiah,
D. Rotem,
Y. Orzech,
L. Sherman,
T. Miki,
A. Yaniv,
A. Gazit, and S. R. Tronick.
1994.
Characterization of cDNA species encoding the Tat protein of caprine arthritis encephalitis virus.
Virology
204:828-834[CrossRef][Medline].
|
| 37.
|
Kashanchi, F.,
J. F. Duvall,
P. F. Lindholm,
M. F. Radonovich, and J. N. Brady.
1993.
Sequences downstream of the RNA initiation site regulate human T-cell lymphotropic virus type I basal gene expression.
J. Virol.
67:2894-2902[Abstract/Free Full Text].
|
| 38.
|
Katsikis, P. D.,
M. E. Garcia-Ojeda,
J. F. Torres-Roca,
D. R. Greenwald, and L. A. Herzenberg.
1997.
HIV type 1 Tat protein enhances activationbut not Fas (CD95)-induced peripheral blood T cell apoptosis in healthy individuals.
Int. Immunol.
9:835-841[Abstract/Free Full Text].
|
| 39.
|
Kirchhoff, F.,
P. J. Easterbrook,
N. Douglas,
M. Troop,
T. C. Greenough,
J. Weber,
S. Carl,
J. L. Sullivan, and R. S. Daniels.
1999.
Sequence variations in human immunodeficiency virus type 1 nef are associated with different stages of disease.
J. Virol.
73:5497-5508[Abstract/Free Full Text].
|
| 40.
|
Li, C. J.,
D. J. Friedman,
C. Wang,
V. Metelev, and A. B. Pardee.
1995.
Induction of apoptosis in uninfected lymphocytes by HIV-1 Tat protein.
Science
268:429-431[Abstract/Free Full Text].
|
| 41.
|
Liu, Z. Q.,
D. Sheridan, and C. Wood.
1992.
Identification and characterization of the bovine immunodeficiency-like virus tat gene.
J. Virol.
66:5137-5140[Abstract/Free Full Text].
|
| 42.
|
Mariani, R.,
F. Kirchhoff,
T. C. Greenough,
J. L. Sullivan,
R. C. Desrosiers, and J. Skowronski.
1996.
High frequency of defective nef alleles in a long-term survivor with nonprogressive human immunodeficiency virus type 1 infection.
J. Virol.
70:7752-7764[Abstract].
|
| 43.
|
McCloskey, T. W.,
M. Ott,
E. Tribble,
S. A. Khan,
S. Teichberg,
M. O. Paul,
S. Pahwa,
E. Verdin, and N. Chirmule.
1997.
Dual role of HIV Tat in regulation of apoptosis in T cells.
J. Immunol.
158:1014-1019[Abstract].
|
| 44.
|
Michael, N. L.,
G. Chang,
L. A. d'Arcy,
P. K. Ehrenberg,
R. Mariani,
M. P. Busch,
D. L. Birx, and D. H. Schwartz.
1995.
Defective accessory genes in a human immunodeficiency virus type 1-infected long-term survivor lacking recoverable virus.
J. Virol.
69:4228-4236[Abstract].
|
| 45.
|
Neuveut, C., and K. T. Jeang.
1996.
Recombinant human immunodeficiency virus type 1 genomes with tat unconstrained by overlapping reading frames reveal residues in Tat important for replication in tissue culture.
J. Virol.
70:5572-5581[Abstract/Free Full Text].
|
| 46.
|
Nowak, M. A.,
R. M. Anderson,
M. C. Boerlijst,
S. Bonhoeffer,
R. M. May, and A. J. McMichael.
1996.
HIV-1 evolution and disease progression.
Science
274:1008-1011[CrossRef][Medline].
|
| 47.
|
Parada, C. A., and R. G. Roeder.
1996.
Enhanced processivity of RNA polymerase II triggered by Tat-induced phosphorylation of its carboxyl-terminal domain.
Nature
384:375-378[CrossRef][Medline].
|
| 48.
|
Pocsik, E.,
M. Higuchi, and B. B. Aggarwal.
1992.
Down-modulation of cell surface expression of p80 form of the tumor necrosis factor receptor by human immunodeficiency virus-1 Tat gene.
Lymphokine Cytokine Res.
11:317-325[Medline].
|
| 49.
|
Sadaie, M. R.,
T. Benter, and F. Wong-Staal.
1988.
Site-directed mutagenesis of two trans-regulatory genes (tat-III, trs) of HIV-1.
Science
239:910-913[Abstract/Free Full Text].
|
| 50.
|
Sadaie, M. R.,
J. Rappaport,
T. Benter,
S. F. Josephs,
R. Willis, and F. Wong-Staal.
1988.
Missense mutations in an infectious human immunodeficiency viral genome: functional mapping of tat and identification of the rev splice acceptor.
Proc. Natl. Acad. Sci. USA
85:9224-9228[Abstract/Free Full Text].
|
| 51.
|
Sakuragi, J.,
S. Sakuragi,
S. Ueda, and A. Adachi.
1996.
Functional analysis of simian immunodeficiency virus SIVAGM long terminal repeat.
Virus Genes
12:21-25[CrossRef][Medline].
|
| 52.
|
Saltarelli, M. J.,
R. Schoborg,
S. L. Gdovin, and J. E. Clements.
1993.
The CAEV tat gene trans-activates the viral LTR and is necessary for efficient viral replication.
Virology
197:35-44[CrossRef][Medline].
|
| 53.
|
Scala, G.,
M. R. Ruocco,
C. Ambrosino,
M. Mallardo,
V. Giordano,
F. Baldassarre,
E. Dragonetti,
I. Quinto, and S. Venuta.
1994.
The expression of the interleukin 6 gene is induced by the human immunodeficiency virus 1 Tat protein.
J. Exp. Med.
179:961-971[Abstract/Free Full Text].
|
| 54.
|
Taylor, J. P.,
C. Cupp,
A. Diaz,
M. Chowdhury,
K. Khalili,
S. A. Jimenez, and S. Amini.
1992.
Activation of expression of genes coding for extracellular matrix proteins in Tat-producing glioblastoma cells.
Proc. Natl. Acad. Sci. USA
89:9617-9621[Abstract/Free Full Text].
|
| 55.
|
Viglianti, G. A., and J. I. Mullins.
1988.
Functional comparison of transactivation by simian immunodeficiency virus from rhesus macaques and human immunodeficiency virus type 1.
J. Virol.
62:4523-4532[Abstract/Free Full Text].
|
| 56.
|
Wei, P.,
M. E. Garber,
S. M. Fang,
W. H. Fischer, and K. A. Jones.
1998.
A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates its high-affinity, loop-specific binding to TAR RNA.
Cell
92:451-462[CrossRef][Medline].
|
| 57.
|
Weiss, R. A.
1993.
How does HIV cause AIDS?
Science
260:1273-1279[Abstract/Free Full Text].
|
| 58.
|
Westendorp, M. O.,
R. Frank,
C. Ochsenbauer,
K. Stricker,
J. Dhein,
H. Walczak,
K. M. Debatin, and P. H. Krammer.
1995.
Sensitization of T cells to CD95-mediated apoptosis by HIV-1 Tat and gp120.
Nature
375:497-500[CrossRef][Medline].
|
| 59.
|
Westendorp, M. O.,
M. Li-Weber,
R. W. Frank, and P. H. Krammer.
1994.
Human immunodeficiency virus type 1 Tat upregulates interleukin-2 secretion in activated T cells.
J. Virol.
68:4177-4185[Abstract/Free Full Text].
|
| 60.
|
Wilcox, G. E.,
B. J. Chadwick, and G. Kertayadnya.
1995.
Recent advances in the understanding of Jembrana disease.
Vet. Microbiol.
46:249-255[CrossRef][Medline].
|
| 61.
|
Wilcox, G. E.,
S. Soeharsono,
D. M. N. Dharma, and J. W. Copland.
1997.
Jembrana disease and the bovine lentiviruses.
ACIAR Proc.
75:10-75.
|
| 62.
|
Zagury, J. F.,
A. Sill,
W. Blattner,
A. Lachgar,
H. Le Buanec,
M. Richardson,
J. Rappaport,
H. Hendel,
B. Bizzini,
A. Gringeri,
M. Carcagno,
M. Criscuolo,
A. Burny,
R. C. Gallo, and D. Zagury.
1998.
Antibodies to the HIV-1 Tat protein correlated with nonprogression to AIDS: a rationale for the use of Tat toxoid as an HIV-1 vaccine.
J. Hum. Virol.
1:282-292[Medline].
|
Journal of Virology, March 2000, p. 2703-2713, Vol. 74, No. 6
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Xuan, C., Qiao, W., Gao, J., Liu, M., Zhang, X., Cao, Y., Chen, Q., Geng, Y., Zhou, J.
(2007). Regulation of Microtubule Assembly and Stability by the Transactivator of Transcription Protein of Jembrana Disease Virus. J. Biol. Chem.
282: 28800-28806
[Abstract]
[Full Text]
-
Calabro, V., Daugherty, M. D., Frankel, A. D.
(2005). A single intermolecular contact mediates intramolecular stabilization of both RNA and protein. Proc. Natl. Acad. Sci. USA
102: 6849-6854
[Abstract]
[Full Text]
-
Xie, B., Wainberg, M. A., Frankel, A. D.
(2003). Replication of Human Immunodeficiency Viruses Engineered with Heterologous Tat-Transactivation Response Element Interactions. J. Virol.
77: 1984-1991
[Abstract]
[Full Text]
Top
Abstract
Introduction
