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Journal of Virology, June 1999, p. 5191-5195, Vol. 73, No. 6
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Potent Inhibition of Human Immunodeficiency Virus
Type 1 (HIV-1) Gene Expression and Virus Production by an HIV-2 Tat
Activation-Response RNA Decoy
Catherine M.
Browning,1
Laurence
Cagnon,2
Paul D.
Good,3
John
Rossi,2
David R.
Engelke,3 and
David M.
Markovitz4,*
Department of Microbiology and
Immunology,1 Department of Biological
Chemistry,3 and Department of
Internal Medicine,4 University of Michigan, Ann
Arbor, Michigan 48109, and Department of Molecular Biology,
Beckman Research Institute of the City of Hope, Duarte, California
910102
Received 2 November 1998/Accepted 28 February 1999
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ABSTRACT |
Tat activation-response region (TAR) decoys have been developed for
use in gene therapy for people infected with human immunodeficiency virus type 1 (HIV-1). When a TAR RNA decoy is overexpressed, it will
bind Tat, thus leaving less of this crucial protein to bind to and
activate the natural transcriptional promoter of HIV-1. Previous TAR
decoy constructs have used HIV-1 TAR. However, recent epidemiological
and biological data began to suggest that the TAR region from the human
immunodeficiency virus type 2 (HIV-2) may suppress HIV-1 transcription
and hence replication. We created a vector which overexpresses TAR-2
under the control of the human U6 small nuclear RNA gene promoter and
here show that the U6-TAR-2 decoy construct potently inhibits both
HIV-2 and HIV-1 gene expression. Further, this decoy construct is able
to markedly suppress HIV-1 replication. Thus, we have directly proven
that TAR-2 can suppress HIV-1 replication and suggest that the HIV-2
TAR decoy may prove useful for combating HIV-1 infection.
| |
TEXT |
AIDS is caused by infection with
either human immunodeficiency virus type 1 (HIV-1) or HIV-2. The two
types of HIV are related in origin but differ in rates of transmission
and disease progression (reviewed in reference 31).
Despite the most aggressive pharmaceutical treatment of HIV-infected
patients, HIV remains in the immune system, and long-term therapy is
necessary (18, 42). Thus, the use of gene therapy to combat
HIV pathogenesis is an attractive concept. For this reason, much work
has been dedicated to finding genetic constructs that would render the
cells of the immune system resistant to HIV replication (reviewed in
references 22 and 27).
Gene therapy approaches to treating HIV-1 infection have focused on
inhibiting the regulation of viral gene expression by the viral
proteins Rev and Tat. Rev and Tat are key regulators of HIV gene
expression and are required for effective replication (6, 14-16,
19, 29). Both Tat and Rev proteins mediate their regulatory
effects through RNA targets on the HIV transcripts: the Tat
activation-response region (TAR) and the Rev-response element. The
function of these two key viral regulatory proteins has been blocked by
the expression of dominant negative mutant proteins (9, 41).
However, though these dominant negative mutant proteins demonstrate
promise for suppressing Tat and Rev function in vivo, expression of
foreign proteins or peptides in host cells has several drawbacks from a
gene therapy perspective (discussed in reference
27). The Tat and Rev proteins can also be
functionally inactivated by sequestering them from the HIV RNA through
the overexpression of the respective RNA-response elements in the cell,
to act as molecular decoys (reviewed in reference
32). Tat-mediated transactivation of HIV-1 gene
expression has been targeted by using expression constructs that
constitutively express the TAR region of HIV-1 (TAR-1) to function as a
molecular decoy. Early decoy experiments with constructs expressing
TAR-1 (26, 28, 30) or circular TAR-1 RNA decoys
(7) were reasonably effective against HIV-1. The mechanism
of action of such TAR-1 decoys was found to be consistent with
sequestration of Tat (8).
The current TAR RNA decoy approaches to decrease Tat activity have all
focused on TAR-1, which is structurally divergent from the TAR region
of HIV-2 (TAR-2) (35). Likewise, the HIV-1 and HIV-2 Tat
transactivating proteins (Tat-1 and Tat-2, respectively) exhibit
functional differences (reviewed in references 10
and 21). Although Tat-1 and Tat-2 show considerable
similarity, and Tat-1 can transactivate HIV-2 gene expression through
the TAR-2 element, Tat-2 is unable to effectively interact with TAR-1 to transactivate the HIV-1 promoter (reviewed in reference
10). This nonreciprocal activity of the Tat-TAR
interactions, while perhaps due to a more stringent RNA site
requirement for the Tat-2 protein than for the Tat-1 protein
(12), may also suggest that TAR-2 is a more versatile
structure for functional interactions with Tat proteins than is TAR-1.
Further, there is in vitro binding (20, 35) and in vivo
functional (17) data to support interaction between at least
two of the hairpin structures of TAR-2 with Tat, suggesting an avidity
of interaction that would increase the effectiveness of a TAR-2 decoy
relative to that of the current TAR-1-based systems.
Consistent with the above observations, preliminary findings suggest
that TAR-2 may be capable of inhibiting Tat-1 in vivo. For example,
results of epidemiological studies in Senegal suggested that infection
with HIV-2 provides protection against subsequent infection with HIV-1
(though findings from Gambian cohort studies have recently disputed
this effect [4, 38, 39]). The trans inhibition of HIV-1 by HIV-2 was also investigated in vitro, and it was
suggested, although not directly demonstrated, that HIV-2 suppresses
the activity of the HIV-1 long terminal repeat due to differences in
the TAR RNAs of HIV-1 and HIV-2 (34). This trans
inhibition was also examined by another group, and the results suggested that inhibition may occur by competition between the viral
RNAs for Tat and cellular factors (5). Further, HIV-2 infection has been shown to decrease HIV-1 replication in primary human
monocytes (3), further suggesting that trans
inhibition does indeed occur in vivo.
The above findings led us to believe that a decoy construct expressing
TAR-2 could be a potent inhibitor of HIV-1 replication. To test this,
we cloned the coding sequence for the entire TAR-2 superstructure into
an expression vector demonstrated to express high levels of structured
RNA molecules, synthesized by RNA polymerase III from the human U6
small nuclear RNA promoter, and to target their delivery to the cell
nucleus (23). The targeting signals serve to concentrate the
transcripts in the nucleus and thus decrease the chance of any toxic
effects of the RNA on cytoplasmic functions such as translation. Since
the transcripts are not translated, the construct generates no
potentially immunogenic proteins and should therefore not elicit an
adverse immune response. Here we show that the resulting TAR-2 decoy
construct was highly effective in decreasing HIV gene expression and
suppressing HIV-1 replication. This inhibition was of greater potency
than that of other constructs that we tested, thus directly
demonstrating the ability of TAR-2 to interfere with HIV-1 replication
and suggesting that TAR-2 is a highly effective decoy molecule.
TAR-2 decoy potently inhibits HIV gene expression.
To test the
ability of the TAR-2 decoy to inhibit transactivation of the HIV-2
promoter by Tat-2, we generated plasmid pTZU6+19 TAR-2. The DNA coding
sequence for the entire TAR-2 region (from +1 to +160) was amplified by
PCR from the HIV-2 CAT (chloramphenicol acetyltransferase) plasmid
(15), using primers which introduced restriction sites for
SalI and XbaI on the 5' and 3' ends,
respectively. The TAR-2 coding region was then cloned into the pTZU6+19
RNA polymerase III expression vector to generate pTZU6+19 TAR-2 (Fig. 1; reference 23). This
construct was then tested for its effectiveness in inhibiting
transactivation by Tat in cotransfection experiments using an HIV-2
promoter-driven reporter plasmid (15). The Tat-2 expression
construct (37) was used at the concentration which gave
effective transactivation in our previous studies (10). The
TAR-2 decoy plasmids strongly inhibited Tat-2 transactivation of the
parental HIV-2 promoter in a dose-dependent manner (Fig. 2). Promoter activity in the presence of
Tat-2 was reduced to basal levels in the presence of 2 µg of the
decoy vector.
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FIG. 1.
The pTZU6 TAR-2 decoy expression plasmid. The expression
cassette that uses the RNA polymerase III promoter for U6 RNA has been
described elsewhere (23). The DNA coding sequence for the
entire TAR-2 element was cloned downstream of the +19 site of the
expression plasmid. The integrity of the insert was confirmed by DNA
sequencing.
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FIG. 2.
The TAR-2 decoy inhibits the response of the HIV-2
promoter to Tat-2. Jurkat T cells were transfected with 5 µg of HIV-2
CAT and 0.5 µg of the Tat-2 expression vector, plus 0, 0.5, 1, or 2 µg of the TAR-2 decoy construct pTZU6+19 TAR-2 and 5, 4.5, 4, or 3 µg of the empty vector, respectively. Percent inhibition was
calculated in comparison to 5 µg of HIV-2 CAT cotransfected with 0.5 µg of Tat-2 expression vector, no decoy vector, and 5 µg of the
empty pTZU6+19 vector. CAT assays were normalized for protein
concentration and performed as described elsewhere (10). The
data presented are compiled from three separate experiments.
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The TAR-2 decoy was also able to potently inhibit transactivation of
the HIV-1 promoter by Tat-1 (Fig.
3).
Inhibition to near
basal levels again occurred in the presence of 2 µg of the pTZU6+19
TAR-2 vector (Fig.
3). Such potent inhibition of
Tat transactivation
suggested that the pTZU6+19 TAR-2 decoy would be
effective at
lowering virus production during infection; therefore, it
was
then tested in cotransfection experiments with HIV-1 infectious
clones.
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FIG. 3.
The TAR-2 decoy construct inhibits HIV-1 promoter
activity in Jurkat T cells. Transfections were performed as for
Fig. 2 except that HIV-1 CAT was substituted for HIV-2 CAT and an
expression vector for Tat-1 (10) was used. The data
presented are compiled from three separate experiments.
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The TAR-2 decoy construct is a potent inhibitor of HIV-1
replication.
The TAR-2 decoy construct was tested for efficacy in
inhibiting HIV-1 replication in the 293 human kidney cell line, which is a readily transfectable and highly productive host for HIV-1. The
293 cells were transfected by using the Lipofectamine reagent (GibcoBRL), according to the manufacturer's protocol, with 2 µg of
decoy or carrier plasmid DNA, 200 ng of the HIV-1 infectious clone
pNL4-3 (1) (acquired from the AIDS Repository), and 6 µl
of Lipofectamine reagent per well. The transfections were performed in
triplicate, and clarified supernatants were harvested 40 h later.
The TAR-2 decoy showed potent inhibition of HIV-1 replication, as
indicated by decreased p24 antigen production (kit from SAIC Frederick)
(Fig. 4A). Compared to several other
recently developed decoy RNA expression constructs which effectively
sequester the HIV-1 Rev protein (23), the TAR-2 decoy showed
superior inhibition of HIV-1 replication (Fig. 4A). Further, the TAR-2
decoy inhibited HIV-1 in a dose-dependent manner, with HIV-1 production
falling below detectable levels when 20 µg of the TAR-2 decoy was
used (Fig. 4B). Such inhibition is particularly impressive, as the replication of HIV-1 in 293 cells is generally more refractory to
inhibitors than is replication in T cells and monocytic cells (36).
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FIG. 4.
(A) The TAR-2 decoy construct is a potent inhibitor of
HIV-1 replication in 293 cells compared to other U6-based anti-HIV
constructs. The RBE constructs express Rev-binding decoy RNAs. The
human embryonal kidney cell line 293 was transfected with 200 ng of the
pNL4-3 HIV-1 infectious clone and 10 µg of the plasmids indicated.
The cell supernatants were harvested 40 h posttransfection and assayed
for p24 antigen. The data are from triplicate samples, and the standard
deviations were used to determine the error bars. (B) The TAR-2 decoy
inhibits HIV-1 replication in a dose-dependent manner. Transfections
were performed as for panel A except that 5, 10, and 20 µg of the
TAR-2 decoy construct were used where indicated.
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The TAR-2 decoy was next assessed in cotransfection experiments using
the more physiologically relevant U937 monocytic cell
line with a
different HIV-1 infectious clone, pHXB3 (
24). The
inhibition
of HIV-1 production, as measured by reverse transcriptase
(RT) activity
(
2), was dose dependent and occurred during the
entire time
course of the infection (Fig.
5). Ten
micrograms of
the decoy construct reduced the RT activity to almost
baseline
levels throughout the course of the experiment. Thus, the
TAR-2
decoy is able to potently inhibit the replication of at least
two
different clones of HIV-1. This effect is seen in the 293
cell line,
which has been shown to be an extremely high producer
of HIV-1 in
previous experiments (
11), and in the more physiologically
relevant monocytic cells.
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FIG. 5.
The TAR-2 decoy construct is a potent inhibitor of HIV-1
replication in U937 monocytic cells. Monocytic cells were cotransfected
with 1 µg of the pHXB3 HIV-1 infectious clone DNA and 0, 1, 2.5, 5, or 10 µg of the TAR-2 decoy vector and 10, 9, 7.5, 5, or 0 µg of
the empty pTZU6+19 vector, respectively, to normalize for DNA content.
The cells were plated out in duplicate wells, and cellular supernatants
were harvested every 2 days for assessment of RT activity. Peak RT
activity is displayed in arbitrary units.
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Discussion.
The vector for expressing the decoy presented in
these studies has several benefits over other current anti-HIV gene
therapy constructs. The RNA polymerase III promoter for U6 RNA is a
highly productive promoter capable of generating complex RNA structures with high integrity, as the natural role of RNA polymerase III in cells
is to transcribe tRNA and other highly structured RNA molecules.
Further, the presence of U6 upstream flanking sequences allows for the
TAR-2 decoy molecules to be targeted to the cell nucleus, where they
can more effectively compete for nuclear transcription factors and the
viral transactivator protein Tat. In addition, the terminal hairpin
serves to protect the TAR RNA from degradation by cellular RNases
(23).
Our experiments show that the use of TAR-2, rather than TAR-1, as the
RNA decoy for Tat and cellular cofactors may prove a
significant
advance in the design of gene therapy constructs for
HIV. This finding
is compatible with very recent epidemiological
data (
38,
39), molecular experiments (
5,
34), and cellular
assays (
3) which had begun to suggest that TAR-2 may be an
effective decoy construct. Further, we have shown that Tat-1 is
able to
interact functionally with TAR-2 even when the RNA contains
substantial
mutations in primary sequence and structure (
10),
suggesting
that the interaction between the Tat-1 protein and
TAR-2 RNA is very
robust. The studies presented here demonstrate
that the TAR-2 decoy is,
indeed, a highly potent inhibitor of
HIV-1 gene expression and
replication. In direct comparisons,
it appears to be at least as
effective as other vectors used to
inhibit HIV-1 replication and may
prove superior (Fig.
4A and
reference
36).
The potency of TAR-2 RNA as a molecular decoy for inhibition of HIV
replication could stem from its multiple sites for interaction
with Tat
and/or its ability to sequester cellular factors. The
importance of
cellular factors for the transactivation of HIV
by Tat, particularly
cyclin-dependent kinases and their associated
cyclins (
13,
25,
33,
40,
43,
44), has been clearly
demonstrated (reviewed in
references
10 and
21). Multiple
binding sites for human cellular proteins have been identified
on the
TAR RNA structure (discussed in reference
10). The
TAR-2
decoy could potentially function by competing with the viral
nascent
transcript for association with any of these cellular factors
in addition to the viral protein Tat. The TAR-2 superstructure
possesses three separate loop regions and may therefore more
effectively
compete with the single stem-loop structured TAR-1 for
loop-binding
cellular factors. Lack of interaction with cellular
cofactors
for Tat might also explain why a minimal TAR-1 decoy,
expressed
from the same polymerase III promoter construct as our TAR-2
decoy,
did not inhibit HIV-1 replication (
23).
In this study, we have shown that expression of TAR-2 RNA in
trans can inhibit HIV-1 replication. The pTZU6 TAR-2 decoy
expression
construct generated in this study is a very potent inhibitor
of
Tat activation of both the HIV-1 and HIV-2 promoters in both
monocytic
and T-cell lines, which represent the key cellular hosts
during
the course of HIV-1 infection. Further, we have shown that the
TAR-2 decoy is highly effective at blocking HIV-1 replication
in at
least two human cell lines. These data demonstrate that
use of the
TAR-2 decoy is a promising new stratagem in the effort
to control HIV
pathogenesis by gene therapy approaches. Further
studies are under way
to determine the scope and duration of HIV
inhibition by this construct
and assess its performance in combination
with other gene therapy
modalities.
| |
ACKNOWLEDGMENTS |
We thank Michael Imperiale, David Friedman, Victor DiRita, and
Clive Woffendin for helpful discussions and training.
This work was supported by grants AI-36685 and HL-57885 to D.M.M. from
the National Institutes of Health. C.M.B. was supported by fellowships
from the Cellular Biotechnology Training Program (5 T32 GM08353) and
the Cancer Biology Training Program (T32 CA09676) of the University of
Michigan. L.C. and J.R. were supported by grant NIH AI38592 from the
National Institutes of Health.
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FOOTNOTES |
*
Corresponding author. Mailing address: 5220 MSRB III,
University of Michigan Medical Center, 1150 W. Medical Center Dr., Ann Arbor, MI 48109-0640. Phone: (734) 647-1786. Fax: (734) 764-0101. E-mail: DMARKOV{at}UMICH.EDU.
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Journal of Virology, June 1999, p. 5191-5195, Vol. 73, No. 6
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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