Previous Article | Next Article 
Journal of Virology, July 2000, p. 6659-6668, Vol. 74, No. 14
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Novel Tat-Encoding Bicistronic Human
Immunodeficiency Virus Type 1-Based Gene Transfer Vectors for
High-Level Transgene Expression
Narasimhachar
Srinivasakumar* and
Friedrich
Schuening
Division of Hematology-Oncology, Department
of Medicine, Vanderbilt University, Nashville, Tennessee
Received 22 October 1999/Accepted 14 April 2000
 |
ABSTRACT |
We describe bicistronic single-exon Tat (72-amino-acid Tat
[Tat72])- and full-length Tat (Tat86)-encoding gene transfer vectors based on human immunodeficiency virus type 1 (HIV-1). We created versions of these vectors that were rendered Rev independent by using
the constitutive transport element (CTE) from Mason-Pfizer monkey virus
(MPMV). Tat72-encoding vectors performed better than Tat86-expressing
vectors in gene transfer experiments. CTE-containing vectors, produced
in a Rev-independent packaging system, had gene transfer efficiencies
nearly equivalent to those produced using a combination RNA transport
(CTE and Rev-Rev response element)-based packaging system. The
Tat72-encoding vectors could be efficiently transduced into a variety
of cell types, showed higher levels of transgene expression than
vectors with the simian cytomegalovirus immediate-early or the simian
virus 40 early promoter, and provide an alternative to HIV-1 vectors
with internal promoters.
| |
TEXT |
Lentiviruses can transduce
terminally differentiated and growth-arrested cells (45, 46)
and are therefore being developed for gene therapy purposes. Most human
immunodeficiency virus type 1 (HIV-1)-based gene transfer vectors
express marker genes under control of internal promoters. The HIV-1
long terminal repeat (LTR) in the presence of Tat is one of strongest
promoters known. The Tat protein of HIV specifically binds to the
transactivation response (TAR) RNA element at the 5' end of nascent
viral transcript to not only increase initiation of RNA synthesis from
the viral LTR promoter (44) (a minor effect) but also
enhance the efficiency of elongation or processivity of RNA polymerase
II (a pronounced effect) (21, 25, 33-36). This allows
accumulation of abundant full-length HIV messages in the infected
cells. The full-length message and its spliced products are necessary
for the synthesis of viral structural and regulatory proteins.
For situations that demand high levels of transgene expression, it
would be advantageous to design HIV vectors that express transgenes
under control of the viral LTR. Another potential problem of using
heterologous internal promoters in retroviral vectors is the
possibility of promoter interference between the internal promoter and
the viral LTR, although the evidence for this thus far has been scanty
for HIV vectors. Thus, using the viral LTR as a promoter may overcome
any potential conflict between the LTR and internal promoters. An
alternative approach to overcome promoter competition is by
inactivation of the viral promoter by introducing promoter-debilitating
mutations in the 3' U3 region of the gene transfer vector. This results
in the inactivation of viral LTR promoter during the process of
infection. Several groups have developed such self-inactivating
lentivirus vectors that give titers equivalent to vectors without these
mutations (51, 74).
The HIV-1 Tat protein is encoded in two exons. The full-length protein
is between 86 and 130 amino acids long, depending on the strain of HIV
(33). Generally, gene transfer vectors derived from HIV-1
that express Tat use both coding exons coupled with a deletion within
env (which is an intron for tat mRNA) (1, 42, 52, 56, 66). This results in the retention of a considerable portion of the HIV-1 sequence in the middle of the genome, a situation that increases the possibility of generating replication-competent HIV-1 by homologous recombination. It was previously demonstrated that
the transactivation function of Tat resides almost entirely in the
first coding exon (23, 53, 64), although sequences in the
second coding exon can improve transactivation in certain cell types
(68). There is also a report that indicates that Tat has
another novel function, i.e., in the efficient reverse transcription of
HIV-1 RNA as well. This function of Tat is also carried out by the
protein encoded in the first-exon of tat (27, 30). It should thus be possible to construct a
single-exon-Tat-producing HIV-1-based gene transfer vector.
Description of gene transfer vectors.
We created four
Tat-expressing HIV-1 gene transfer vectors (Fig.
1) in which the Tat
coding sequences were positioned downstream of an internal ribosome
entry site (IRES) of encephalomyocarditis virus (EMCV) to allow
expression of Tat when used in a bicistronic configuration. The vector
pN-IT72 encodes a single-exon Tat (72-amino-acid Tat [Tat72]), while
pN-IT86 encodes a full-length Tat (86-amino-acid Tat [Tat86]). We
created versions of these plasmids containing the constitutive
transport element (CTE) derived from Mason-Pfizer monkey virus
(8) (MPMV) (pN-IT72CTE and pN-IT86CTE). In MPMV, the CTE
performs a function that is analogous to that of Rev-Rev response
element (RRE) of lentiviruses, which is the nucleocytoplasmic transport
of unspliced or partially spliced viral mRNAs (20). The CTE
can completely substitute for Rev-RRE function not only in subgenomic
HIV-1 structural protein expression vectors but also in the context of
the HIV-1 provirus itself. This is evidenced by the fact that a
replication-competent HIV-1 that lacks functional Rev and RRE can be
made by utilizing this RNA element in the proviral sequence (8,
73). Likewise, Rev-independent HIV-1-based gene transfer vectors
that contain this RNA element have been described (63, 64).
View larger version (24K):
[in this window]
[in a new window]
|
FIG. 1.
Schematic representation of HIV-1 packaging constructs
(A), provirus (B), and gene transfer vectors (C). The HIV-1 packaging
constructs and gene transfer vectors were derivatives of the molecular
clone pNL4-3 (GenBank accession no. M19921). For all packaging
constructs, viral proteins were expressed under control of the sCMV
immediate-early promoter-enhancer elements, and RNA transport was
regulated using the MPMV CTE and poly(A) signal. The sCMV
promoter-enhancer corresponds to bp 681 to 1349 of the IE94 gene
(GenBank accession no. M16019), and the CTE and poly(A) region
corresponds to bp 8007 to 8557 of MPMV (GenBank accession no. M12349).
In pgpirin and pgp3virin, Rev and Nef were expressed using IRES derived
from Harvey murine sarcoma virus (HaMSV) and EMCV, respectively. The
HaMSV IRES-Rev and EMCV IRES-Nef were positioned upstream of MPMV
CTE-poly(A). The viral accessory and regulatory proteins expressed by
the constructs are tabulated at the right. Restriction enzyme sites and
their nucleotide positions in pNL4-3 pertinent for the creation of the
packaging plasmids are indicated above the provirus. The packaging
constructs contain a deletion within the encapsidation signal (  )
and have been described previously (64). The gene transfer
vectors were derived from pTR167 (61). They contain a
deletion of HIV coding sequences between the proximal NsiI
site in gag to the distal NsiI site in
env. Restriction enzyme sites and their nucleotide positions
in pNL4-3 that were used for the creation of the gene transfer vectors
are indicated below the provirus. A frameshift mutation was introduced
into the gag open reading frame between codons 9 and 10 by
inserting an A residue (64). All transgene expression
cassettes were positioned downstream of the 3' splice acceptor site of
Tat and Rev between the BamHI in the second coding exon of
rev and XhoI site in nef. Tat72 or
Tat86 sequences were amplified by PCR from pGEM-NL4-3 and pCMVtat,
respectively, and cloned into a workshop vector to assemble the EMCV
IRES-Tat cassettes, which were then introduced between the indicated
restriction enzyme sites of the modified pTR167 to obtain pN-IT72 and
pN-IT86 or between the BamHI and SalI sites of
pTR167-CTE (64), to obtain pN-IT72CTE and pN-IT86CTE. CTE
(GenBank accession no. M12349; bp 8007 to 8240)-containing
Tat-expressing vectors do not have the frameshift mutation in
gag. pN-sCMV contains the sCMV promoter (bp 681 to 1349 of
the IE94 gene; GenBank accession no. M16019), whereas pN-SV contains
the SV40 early promoter (bp 5175 to 5243 and 1 to 272; GenBank
accession no. J02400). The promoters in both vectors are situated
between the indicated restriction enzyme sites of the modified pTR167.
Multiple cloning sites have been introduced downstream of these
promoters to allow insertion of any foreign or marker gene of interest.
Marker genes (luciferase or enhanced GFP) were introduced upstream of
the IRES-Tat cassette between the unique BamHI and
NotI restriction enzyme sites or downstream of the sCMV or
SV40 promoter between unique BamHI and XhoI
sites. Details of plasmid construction will be provided on request.
5'ss, 5' splice acceptor site.
|
|
The Tat-encoding HIV-1 vectors were engineered to contain unique
restriction enzyme sites to allow introduction of any gene
of interest
upstream of the IRES but situated downstream of the
3' splice acceptor
site of
tat and
rev to enable expression of
the transgene off the spliced message. We also created HIV-1 vectors
that express marker genes under the control of internal promoters
(Fig.
1). In pN-sCMV, the marker gene is expressed under the control
of the
simian cytomegalovirus (sCMV) immediate-early promoter-enhancer
elements; in pN-SV, the marker is expressed under control of the
simian
virus 40 (SV40) virus early
promoter.
Transactivation of HIV-1 LTR by Tat-encoding vectors.
The
Tat-encoding vectors were first tested for the production of functional
Tat protein by cotransfecting 293 cells with each of the vectors
together with an HIV LTR-luciferase reporter (pLTR-luc). If Tat is
produced, it should lead to the transactivation of the HIV-1 LTR,
resulting in increased luciferase activity. As controls, 293 cells were
transfected with pLTR-Luc alone (for basal promoter activity) or
pLTR-Luc and a plasmid (pCMVtat) (63) that encodes Tat86
under control of the sCMV immediate-early promoter as a positive
control. Mock-transfected cells were used as negative controls.
Parallel transfections with each gene transfer vector received pCMVtat.
To normalize for transfection efficiency, all transfections also
received pCMV
-gal, which expresses -galactosidase under control
of the sCMV immediate-early promoter.
Cells lysates were prepared 48 h posttransfection and assayed for
luciferase and
-galactosidase activities using commercially
available reagents. Luciferase activity, normalized to
-galactosidase
activity, for each vector is shown in Fig.
2A. The data indicate
that all vectors
expressed a functional Tat protein. While there
was some variation in
the transactivation levels produced by the
different vectors, there was
no clear indication for perturbation
of transactivation function by the
presence or absence of the
CTE. Cotransfection with pCMVtat increased
luciferase activity
with all Tat-encoding vectors. This indicated that
either the
vectors were producing suboptimal levels of Tat or the
reporter
plasmid (pLTR-luc) was present in relative excess. A titration
experiment using decreasing amounts of pLTR-luc with constant
amount of
Tat72-encoding vector (pN-IT72) confirmed that the reporter
construct
appeared to be in excess (data not shown).
View larger version (46K):
[in this window]
[in a new window]
|
FIG. 2.
Transactivation of HIV-1 LTR by tat
expression gene transfer vectors. 293 cells were transfected with 1 µg of each vector together with either 2 µg of pLTR-luc and 1 µg
of pCMV-gal (A) or 2 µg of each bicistronic vector with 1 µg of
pCMV-gal (B) by the CaPO4 transfection method
(64). Parallel transfections also received 1 µg of pCMVtat
(64). pLTR-luc contains the 3' HIV-1 LTR of pNL4-3, inserted
within the multiple cloning site of pBluescript II SK(+). The firefly
luciferase gene is positioned downstream of the HIV-1 LTR. Cell lysates
were prepared 48 h posttransfection and assayed for luciferase and
-galactosidase activities using commercially available kits
according to the recommended protocols (Pharmingen, San Diego, Calif.,
for luciferase and Clontech, Palo Alto, Calif., for -galactosidase).
Error bars correspond to 1 standard deviation and were derived from
duplicate experiments.
|
|
Additional support for the latter hypothesis was provided in
experiments carried out using bicistronic vectors in which the
marker
gene was expressed within the gene transfer vector itself
along with
Tat72 or Tat86. In this situation, the Tat protein
would upregulate
expression from its own promoter, which would
also lead to increased
marker gene activity. To test this hypothesis,
we introduced the
luciferase gene upstream of the IRES in each
of the Tat-encoding
vectors (Fig.
1) to create pN-LIT72, pN-LIT86,
pN-LIT72CTE, and
pN-LIT86CTE. The bicistronic vectors were transfected
into 293 cells
together with pCMV-
-gal to normalize for transfection
efficiency.
Parallel transfections received pCMVtat. Cell lysates
were harvested
and assayed for luciferase and
-galactosidase
activities as
described earlier. The data (Fig.
2B) indicate that
high levels of
luciferase activity were observed with all bicistronic
constructs but
to various levels. Cotransfection with pCMVtat
enhanced luciferase
activity either marginally (pN-LIT86 and pN-LIT72CTE)
or not at all
(pN-LIT72 and pN-LIT86CTE). This suggested that
some of the vectors
were producing adequate amounts of Tat whereas
others were producing
less than saturating amounts of Tat. The
CTE-containing vectors yielded
lower luciferase activity than
the vectors lacking CTE. This difference
was statistically significant
(
P 
0.05). A previous
report showed that the two-exon Tat provides,
depending on cell type,
approximately two-fold-higher transcriptional
activation in comparison
to single-exon Tat (
68). In contrast
to this, other reports
(
23,
53,
64) have not found any such
difference between the
activities of Tat72 and Tat86. The results
of our experiments are in
agreement with those of the latter
studies.
Gene transfer efficiency of Tat72- and Tat86-encoding vectors.
Next, we wished to determine the efficiency of gene transfer by each of
the luciferase-encoding bicistronic tat expression vectors.
To produce virus stocks, each vector was cotransfected into 293 cells
with a helper plasmid, pgpirin or pgp (Fig. 1), that produced viral
packaging proteins and a vesicular stomatitis virus G envelope
glycoprotein (VSV-G)-expressing plasmid (pMD.G). The packaging plasmid,
pgpirin, is a polycistronic construct that expresses HIV-1 Gag, Pol,
Rev, and Nef. Rev and Nef were expressed via the Harvey murine sarcoma
virus IRES and EMCV IRES, respectively. The other construct, pgp,
expresses only HIV Gag and Pol. Both constructs express viral proteins
using the sCMV immediate-early promoter, with RNA transport being
regulated by the MPMV CTE and poly(A). These packaging plasmids have
been described previously (64). Virus stocks, produced with
each of the vectors, were then used for infection of 293 (adenovirus-transformed human embryonic kidney cell line) and D17
(canine osteosarcoma) cells. Cell lysates were prepared 48 h
after transduction and assayed for luciferase activity
(expressed as relative light units [RLU]). Since results were similar
for both D17 and 293 targets, only the results of experiments with D17
cells are shown in Fig. 3.
View larger version (38K):
[in this window]
[in a new window]
|
FIG. 3.
(A) Gene transfer into D17 cells by
luciferase-expressing HIV-1 vectors encoding Tat72 or Tat86 and either
containing or lacking CTE. Virus stocks were produced separately for
each indicated vector by transfecting 293 cells with 7.5 µg of each
vector, 3.75 µg of indicated packaging plasmid, and 0.2 µg of
pMD.G. Parallel transfections received 1 µg of pCMVtat. Supernatants
were harvested 72 h posttransfection, cleared of debris by
centrifugation at 2,500 rpm (1,430 × g) for 15 min at
4°C, and used for infection of D17 cells. For determining luciferase
activity, transduced cell lysates were prepared 72 h
postinfection, and an aliquot, after appropriate dilution, was assayed
for luciferase activity using a commercial kit as described in the
legend to Fig. 2. The luciferase activity, normalized to p24 levels, is
shown. (B) Gene transfer into 293T by GFP-expressing vectors encoding
Tat72 or Tat86 and either containing or lacking CTE. Virus stocks were
produced separately for each indicated GFP-expressing vector by
cotransfecting 293T cells with 3.75 µg of pgp3virin or pgp3 v, 0.5 µg of pMD.G, and 7.5 µg of each vector. Supernatants were harvested
as for panel A and used for infection of 293T cells. The cells were
harvested 72 h postinfection, fixed with 5% paraformaldehyde, and
analyzed by flow cytometry, as described for Table 2, to obtain the
number of GFC per milliliter. The GFP titers (as GFC per milliliter)
were normalized to p24 levels (which ranged between 10 and 22 ng/ml for
the different vectors) in the supernatants used for infection. The
number above each bar is the average titer obtained for the
corresponding vector in units of 100,000. Thus, the titer of pN-GIT72
was 2.9 × 105 GFC/ml. The amount of p24 in the
supernatant was quantitated using a commercial kit (Cellular Products
or Zeptometrix Corp., Buffalo, N.Y.) (64). Error bars
correspond to 1 standard deviation and were derived from duplicate
experiments.
|
|
Virus stocks for pN-LIT72 and pN-LIT86 were produced using pgpirin as
the packaging plasmid. Both vectors are expected to
be Rev dependent
since they contain RRE but no CTE. The results
of gene transfer
experiments with pN-LIT72 and pN-LIT86 in D17
cells indicate that the
Tat72-encoding bicistronic vector was
transduced at two- to
threefold-higher efficiency than the Tat86-expressing
vector. In
contrast to pN-LIT72 and pN-LIT86, the CTE-containing
vectors,
pN-LIT72CTE and pN-LIT86CTE, are expected to be Rev independent
since they contain the MPMV CTE (
63,
64). We therefore
wanted
to test pN-LIT72CTE and pN-LIT86CTE in a packaging system devoid
of Rev. To produce virus stocks, pN-LIT72CTE or pN-LIT86CTE was
cotransfected with the helper plasmid pgp, which does not code
for
either Rev or Nef. To allow comparison with virus stocks produced
with
pgpirin, which expresses Nef and Rev, a CMV-Nef-encoding
plasmid,
pCMVnef (
63), was included during virus stock
production.
This ensured that the only difference between packaging of
pN-LIT72
or pN-LIGT86 by pgpirin and pN-LIT72CTE or pN-LIT86CTE was the
absence of Rev in the latter case. Virus stocks produced with
the
Rev-independent vector (pN-LIT72CTE) in a packaging system
lacking Rev
had approximately 70% of the gene transfer efficiency
of the
vector stock produced with pN-LIT72 using pgpirin (Fig.
3). Again,
pN-LIT72CTE gave two- to threefold-higher gene transfer
efficiencies than pN-LIT86CTE.
Tat has been shown previously to be required for efficient reverse
transcription in addition to its role in transactivation
of the HIV-1
LTR (
27). To confirm that levels of Tat produced
by the
vectors were adequate to allow maximal production of vector
RNA for
packaging, and also test if coexpression of full-length
Tat affected
gene transfer, virus stocks were produced for each
vector with and
without pCMVtat. Gene transfer efficiencies for
all vectors were
similar both in the presence and absence of a
full-length-Tat-expressing plasmid (Fig.
3). This demonstrated
that
adequate amounts of Tat were being produced by the vectors
and that the
single-exon Tat was sufficient for gene delivery
into D17 or 293 cells.
These results were consistent with our
previous results, which also
showed that Tat72 was adequate for
gene transfer into growing and
growth-arrested target cells (
64).
In our previously
reported experiments Tat72 was produced by the
packaging plasmid,
whereas in the experiments presented here,
Tat is encoded by the HIV-1
gene transfer
vector.
It was not clear from the preceding experiments if the lower levels of
luciferase activity observed in transduced cells with
the Tat86 vectors
was due to lower levels of gene expression,
to lower titers, or to a
combination of the two. To address this
question and to confirm and
extend the results using an independent
marker gene, we tested
bicistronic
tat expression vectors expressing
the enhanced
version of green fluorescent protein (GFP). These
vectors were packaged
using the packaging plasmid pgp3v or pgp3virin
(Fig.
1). The packaging
plasmid pgp3v encodes HIV-1 Gag, Pol,
Vif, Vpr, Vpu, and Tat72, while
pgp3virin encodes Rev and Nef
in addition to those proteins present in
pgp3v (Fig.
1). These
plasmids yield higher titers than vector stocks
produced with
pgpirin or pgp (
64). The pgp3v construct does
not encode for
Rev and can therefore be used for packaging gene
transfer vectors
that are Rev independent (i.e., those containing the
CTE), while
pgp3virin, which encodes both Rev and Nef, would be
suitable for
packaging Rev-dependent vectors (i.e., those containing
RRE but
no CTE). The four GFP-encoding vectors, pN-GIT72, pN-GIT86,
pN-GIT72CTE,
and pN-GIT86CTE, were used to produce separate virus
stocks using
either pgp3v or pgp3virin. The vectors were pseudotyped
with VSV-G.
A Nef-expressing plasmid was included for production of
virus
stocks with pgp3v to allow comparison with pgp3virin. Each of
the
vector stocks was used for infection of 293T cells. Transduced
cells
were harvested 3 days postinfection, fixed with 5% paraformaldehyde,
and analyzed by flow cytometry. The titers, as deduced from the
number
of green fluorescent cells (GFC) per ml, normalized to
p24 levels, are
depicted in Fig.
3B. Consistent with the results
for the
luciferase-encoding vectors, Tat72-expressing vectors
(pN-GIT72 and
pN-GIT72CTE) gave approximately two- to threefold-higher
titers than
the Tat86 vectors (pN-GIT86 and pN-GIT86CTE). Rev-dependent
Tat72 and
Tat86 vectors (pN-GIT72 and pN-GIT86) packaged with
pgp3virin had
titers of 1.6 × 10
4 and 0.7 × 10
4
GFC/ng of p24, respectively. In contrast, the same vectors packaged
in
a system lacking Rev gave, as anticipated, 16.5- and 14.9-fold-lower
titers, respectively. This result is in accordance with our previous
observations (
63,
64) showing the requirement for Rev for
vectors that contain the RRE but no CTE. Also consistent with
our
previous observations (
63,
64) was the finding that
CTE-containing
vectors (pN-GIT72CTE and pN-GIT86CTE) could be
efficiently packaged
and transduced into target cells by the pgp3v
packaging plasmid.
Thus, pN-GIT72CTE and pN-GIT86CTE, packaged in a
system lacking
Rev, gave titers of 1.5 × 10
4 and
0.6 × 10
4 GFC/ng of p24, respectively, which were
essentially identical
to titers of vectors lacking the CTE (pN-GIT72
and pN-GIT86) and
produced in a Rev-containing packaging system. These
same vectors
(pN-GIT72CTE and pN-GIT86CTE) could also be efficiently
packaged
with pgp3virin and interestingly, provided 1.4- to
2-fold-higher
titers than corresponding vectors without CTE
packaged with the
same packaging plasmid. These titers were also higher
than the
titers obtained with the same vectors packaged in a
Rev-independent
system using pgp3v. Statisfical analysis indicated that
the differences
in titers between the CTE-containing vectors and the
non-CTE-containing
vectors when packaged with pgp3virin were not
significant (
P >
0.05).
In a separate experiment, levels of GFP expressed by Tat72 and Tat86
vectors in transduced target cells were determined by
measuring the
geometric mean of fluorescent intensity (GMFI).
D17 cells transduced
with pN-GIT72 had a GMFI of 268 ± 40 (mean
± standard
deviation), while pN-GIT86-transduced cells had a GMFI
of 328 ± 24. Cells transduced with the CTE-containing vector,
pN-GIT72CTE, had a
GMFI of 221 ± 4.45, while those transduced
with pN-GIT86CTE had a
GMFI of 259 ± 13.2. Thus, Tat72-encoding
vectors produced similar
levels of GFP expression in transduced
D17 cells as Tat86-expressing
vectors.
Comparison of bicistronic Tat-encoding vector with vectors that
express transgenes under control of internal promoters.
Next, we
wished to compare the HIV-1 LTR as a promoter with the sCMV and SV40
promoters for transgene expression in a variety of cell lines. To do
this we used two types of reporter genes. One series (pN-LIT72,
pN-sCMVluc, and pN-SVluc) had the firefly luciferase gene, and a
corresponding set (pN-GIT72, pN-sCMVGFP, and pN-SVGFP) contained the
GFP gene under control of the HIV-1 LTR and the sCMV and SV40
promoters, respectively. Virus stocks were produced as described above,
using pgp3virin to provide helper function. All vectors were
pseudotyped with VSV-G. We first compared virus stocks produced using
pN-LIT72, pN-sCMVluc (previously referred to as pN-FS-sCMVluc
[64]), and pN-SVluc. Virus stocks of each of these
vectors were used to transduce D17, 293, HeLa, and 3T3 cell lines.
Transduced cell lysates were harvested 48 h postinfection and
assayed for luciferase activity. The results of this experiment are
shown in Table 1. D17 and 293 cells
transduced with pN-LIT72 showed higher luciferase activity than cells
transduced with either pN-sCMVluc or pN-SVluc. In HeLa targets,
transduction with pN-sCMVluc resulted in higher luciferase activity
than transduction with pN-SVluc or pN-LIT72. Low levels of luciferase
activity were noted with all vectors in 3T3 cells.
View this table:
[in this window]
[in a new window]
|
TABLE 1.
Transduction of various cell lines by HIV-1 vectors
encoding luciferase under the control of different promoters
|
|
The differences between the vectors could be either due to differences
in titers or due to differences in the strength of
each of the
promoters in the various cell lines. To address this
more directly, we
used vectors with GFP as the reporter. GFP would
allow us to detect and
quantitate gene expression at a single-cell
level. Again, virus stocks
were prepared with each of these vectors
and used for transduction of
the various cell lines, some of which
were also used as targets for
luciferase-encoding vectors. Transduced
cells were harvested 72 h
postinfection by trypsinization, washed
and fixed with 4%
paraformaldehyde, and analyzed by flow cytometry.
The results are shown
in Table
2. In all cases, less than 10%
of cells were transduced, judging from proportion of cells that
were
positive for GFP (data not shown), suggesting that most cells
harbored
only one virus genome. By enumerating the number of transduced
cells
per well for each virus stock, we were able to estimate
the number of
transducing units per milliliter for each vector.
View this table:
[in this window]
[in a new window]
|
TABLE 2.
Transduction of various cell lines by HIV-1 vectors
encoding GFP under the control of different promoters
|
|
Flow cytometry also allowed us to obtain the GMFI of transduced cells
for each vector (Fig.
4). Representative
flow diagrams
obtained with three cell lines are shown in Fig.
4. In
D17, 293,
and THP-1 (human acute monocytic leukemia cell line) cells,
pN-GIT72
achieved 4- to 104-fold-higher GFP expression than vectors
with
sCMV or SV40 promoters (as deduced from GMFI in transduced cells
[Table
2]). In HeLa cells, although pN-sCMVGFP produced an overall
lower GMFI than pN-GIT72, there was considerable overlap in expression
levels of the two vectors (Fig.
4), with the peaks exhibiting
similar
GMFIs. In contrast, the LTR proved less efficient in 3T3
cells. All of
the three promoters tested provided quite low GFP
expression in 3T3
cells and in fact were difficult to visualize
by fluorescence
microscopy, but they could be distinguished by
flow cytometry from
untransduced cells, using a second detector
for measuring emission
between 564 and 604 nm. This allowed gating
of untransduced cells
exhibiting autofluorescence from cells that
were transduced with the
HIV vector and therefore expressing GFP
(data not shown).
View larger version (47K):
[in this window]
[in a new window]
|
FIG. 4.
Flow cytometric analysis of 293, HeLa, and 3T3 cells
transduced with pN-GIT72, pN-sCMVGFP, or pN-SVGFP. Virus stocks were
prepared as described in the text and in the legend to Fig. 3. One
half-milliliter of each virus stock was used for infection of indicated
cell lines. The cells were harvested approximately 60 h
postinfection, fixed, and analyzed by flow cytometry as described for
Table 2. The horizontal axis shows GFP expression in logarithmic scale;
the vertical axis shows cell number. The gate used for analysis (M1) is
shown for each cell line. The GMFIs of the positive peaks are
indicated.
|
|
The titers of the GFP-encoding vectors were lower in THP-1 cells than
the other cell lines tested. This is probably due to
the higher
background or autofluorescence in these cells, which
in turn interfered
with the accurate enumeration of GFP-expressing
cells by flow
cytometry.
Thus, the results were comparable for both luciferase and GFP vectors
in all cell lines tested except HeLa. In HeLa cells,
the pN-sCMVluc
vector showed higher luciferase activity than the
others (Table
2),
whereas pN-GIT72-transduced cells showed levels
of GFP expression
marginally higher than those in cells transduced
by pN-sCMVGFP (Fig.
4;
Table
2). We have no explanation for this
discrepancy. Nevertheless,
these results demonstrated that the
Tat-encoding bicistronic vectors
were in general more efficient
than the vectors that expressed the
transgene under the control
of either the sCMV or SV40 internal
promoter in many cell lines,
but not in those cell lines where the
HIV-1 LTR has been shown
to be weak (e.g., mouse cells) (
13,
43). Since most published
work with HIV vectors has used the
human CMV immediate-early promoter
to drive transgene expression, we
also tested a vector that expressed
GFP under control of this promoter.
The CMV promoter for this
vector was derived from pcDNA3 (Invitrogen
Corp., San Diego, Calif.).
This vector achieved somewhat lower levels
of gene expression
(GMFI of 19.9 ± 0.04) than the sCMV
promoter-containing vector
in 293 T cells (GMFI of 29.1 ± 0.2).
Another puzzling observation was that both the sCMV and SV40 promoters,
as part of the HIV-1 vector, seemed to work very poorly
in 3T3 cells.
The same promoters gave higher levels of gene expression
in the context
of murine leukemia virus (MLV)-based vectors in
3T3 cells (data not
shown). This would imply that the HIV-1 vector
backbone can negatively
affect transgene expression under control
of heterologous internal
promoters in some cell
types.
Test for pseudotransduction by VSV-G-pseudotyped vectors.
Several recent studies have shown the disconcerting possibility of
pseudotransduction of marker genes by VSV-G-pseudotyped vectors
(10, 22, 47). Pseudotransduction is a type of
protein or DNA delivery in the absence of bona fide infection. The
marker protein or plasmid DNA is incorporated into the virion or
pseudovirion. Upon binding and fusion of the virus particles with
target cell plasma membrane, the protein or DNA is delivered to the
cytoplasm. If enough protein or DNA is delivered, one may wrongly
conclude that the target cells were expressing the protein as a result of authentic retrovirus infection process. Pseudotransduction has been
observed with highly concentrated preparations of VSV-G-pseudotyped vectors. To rule out pseudotransduction, we did the following experiment.
We prepared virus stocks of pN-GIT72 pseudotyped with either VSV-G or
amphotropic MLV envelope glycoprotein. The amphotropic
envelope-pseudotyped virus would serve as a negative control since
pseudotransduction has been observed only with VSV-G-pseudotyped
virus.
The virus stocks were concentrated approximately 30-fold
using
Centricon Plus-20 centrifugal concentrators (Millipore Corp.,
Bedford,
Mass.). To eliminate the possibility of plasmid DNA carryover
from
transfected cells, virus stocks were treated with RNase-free
DNase
(Promega, Madison, Wis.) and then used for infection of
D17 target
cells. Preliminary studies showed that DNase treatment
did not
adversely affect titer (data not shown). Infections were
carried out
with cells that were pretreated with 10 or 20 mM azidothymidine
(AZT;
Sigma, St. Louis, Mo.). AZT was present through out infection
and up to
the time of harvest. The treatment of target cells with
AZT should
allow the virus particles to bind and fuse with the
plasma membrane but
interfere with the reverse transcription of
incoming virus RNA and
thereby abort the infection process. Under
these circumstances,
pseudotransduction by protein or DNA delivery
is still possible, but no
true transduction can occur. Transduced
cells were harvested 48 h
after infection, fixed with 4% paraformaldehyde,
and analyzed by flow
cytometry. The results of this experiment
are shown in Table
3. Amphotropic envelope-pseudotyped virus
had a titer of 6.5 × 10
6 transducing units/ml, while
the VSV-G-pseudotyped virus had a
titer of 1.2 × 10
7
transducing units/ml on D17 cells. Treatment of cells with either
10 or
20 mM AZT decreased transduction of VSV-G-pseudotyped virus
by 93 and
97% and that of amphotropic envelope pseudotyped virus
by 96 and 98%,
respectively, compared to gene transfer into untreated
cells. This
indicated that pseudotransduction was occurring at
very low levels, if
at all. The difference between our study and
those described previously
(
10,
22,
47) may be related to
the method of concentration
of virus particles, the marker gene
used within the gene transfer
vector, the cell line used for production
of virus stocks, or the
amount of VSV-G used for production of
virus stocks. It is also
possible that concentration by ultracentrifugation
may result in
copurification of marker protein and/or plasmid
DNA with the virus
particles resulting in pseudotransduction of
target cells, whereas
ultrafiltration may not have these drawbacks.
View this table:
[in this window]
[in a new window]
|
TABLE 3.
Effect of AZT on gene transfer into D17 cells by
amphotropic MLV envelope or VSV-G-pseudotyped HIV vectors
|
|
In this report, we describe novel Tat-encoding HIV-1-based gene
transfer vectors that allow high levels of transgene expression
using
the HIV-1 LTR promoter. Vectors that expressed Tat72 were
more
efficient than those encoding Tat86. There are several possible
explanations for why Tat86-encoding vectors give lower gene transfer
efficiencies than Tat72-encoding vectors. One possible explanation
is
that inserting a cDNA encoding the complete Tat coding sequence
downstream of the second exon of
tat will result in
duplication
of sequences of a second coding exon on either side of the
marker-IRES
cassette (Fig.
1). Since retroviruses can delete sequences
between
repeats during reverse transcription (
16,
37), this
process
could have resulted in the elimination of the marker-IRES
sequence
during the infection process, thus accounting for the lower
gene
transfer efficiency with the full-length Tat-expressing vectors.
We are currently conducting experiments to confirm this hypothesis.
Other possibilities include interference in the RNA secondary
structures between the Tat86 sequences and IRES RNA or unknown
function
of the Tat86 sequences not present in
Tat72.
Finally, the results demonstrated that the CTE-containing vector could
transduce genes nearly as efficiently as the vector
without the CTE.
This Rev-independent system may prove useful
for expressing high levels
of transdominant Rev in HIV-1 susceptible
targets. An additional
benefit of the RNA produced from this vector
is that it contains the
TAR element and RRE, both of which can
act as decoys in the event of
infection of the intracellularly
immune cells by wild-type virus
(
15). The CTE-containing vector
would also be useful to
define the requirement for envelope sequences
(RRE) in HIV-1-based gene
transfer vectors. The vectors described
here still retain 1.7 kb of
envelope sequence that contains the
RRE and the
tat/rev
splice acceptor site. In the CTE-containing
vectors, it should be
possible to eliminate much of this envelope
sequence and thereby render
the vectors safer by removal of unnecessary
sequences and also increase
its capacity to accommodate larger
transgenes.
Our results using CTE in the packaging system are at odds with those
obtained by Kim et al. (
42) and Gasmi et al. (
24)
but consistent with previously published results of our group.
In the
study of Kim et al., the gene transfer vector was based
on Rev and RRE
and the vector also coded for Rev, while the packaging
plasmid
contained the CTE. In the study by Gasmi and coauthors,
again the
packaging plasmid contained the CTE and the gene transfer
vector
contained the RRE, but Rev was expressed using a separate
plasmid. Kim
et al. found that the Rev- and RRE-based packaging
system resulted in
nearly 100-fold-higher titers than the system
that used the CTE, while
Gasmi and coworkers found a difference
of about 10-fold between the two
packaging constructs. One possible
explanation that has been offered
previously is that the placement
or length of CTE used might have
influenced titers of vectors
containing this element (
42).
There are several significant
differences between our study and the
above-mentioned ones. We
have used the CTE not only in the packaging
plasmids but also
in the gene transfer vector to create a truly
Rev-independent
system. Both of the above-mentioned studies required
the coexpression
of Rev to allow export of RRE-containing and thereby
Rev-dependent
vector RNAs. This is similar to the combination packaging
system
(using CTE for expression of packaging plasmid and RRE-Rev for
expression of gene transfer vector) we have described in this
study and
in a previous one (
64). Unlike the studies of Kim
et al.
(
42) and Gasmi et al. (
24), we have not compared
a
purely RRE-Rev-based packaging system directly with the CTE-based
packaging system. However, in an earlier study (
64), we
found
that the combination packaging system gave gene transfer
efficiencies
comparable to those of an RRE-Rev-based packaging system.
We are
now attempting to reconcile these differences among
studies.
Tat-encoding vectors may have some unique advantages in overcoming two
important hurdles to the implementation of gene therapy.
The first is
the gradual loss of transgene expression in the transduced
target cells
(
26,
31,
39,
50,
55,
60). This effect
has been ascribed to
chromatin remodeling by methylation of DNA
(
26,
31,
32) or
due to deacetylation of histones (
11,
12). Tat has been
shown to recruit histone acetyltransferases
to the viral promoter
(
7,
40,
48,
72). This may keep
the chromatin in an open
configuration and allow prolonged transgene
expression. Even if
transgene expression diminishes over time,
it may be possible to
reverse this silencing by using inhibitors
of histone deacetylases such
as trichostatin A or sodium butyrate
or its analogs such as
phenylbutyrate (
11,
40). It remains
to be demonstrated that
the Tat-encoding vectors can indeed allow
long-term transgene
expression.
The second hurdle for gene therapy is immune-mediated elimination of
transduced cells expressing a protein recognized as foreign
by the
host. A biological property of Tat that may be useful in
this context,
is the ability of Tat to downregulate the expression
of major
histocompatibility complex (MHC) class I and class II
genes (
9,
28,
29,
38,
69), although there are other
reports which seem to
indicate that Tat possesses no such activity
(
49,
68). If
Tat can indeed down modulate MHC expression,
this may prevent
immune-mediated elimination of transduced target
cells by decreasing
antigen presentation in the transduced cells.
This property is,
however, principally localized to the second
coding exon of Tat
(
29). Therefore, one will have to use a
full-length-Tat-encoding
vector to prevent immune responses to
transduced cells. Thus,
Tat-encoding vectors may provide answers to the
central problems
currently besetting gene
therapy.
On the other hand, it is also possible that Tat (particularly Tat72)
may engender an immune response to the transduced cells
leading to
their elimination. Moreover, Tat, which is known to
be secreted from
infected cells and act in a paracrine manner
on neighboring cells, may
produce other untoward consequences
(
14,
53,
54,
57-59,
65,
67,
70). One concern is the
observation that Tat may be involved in
the induction of Kaposi's
sarcoma (
2-5,
17-19).
Notwithstanding its effect on MHC expression,
Tat may also produce
other effects on the immune system. For example,
Tat has been shown to
induce interleukins 2 and 8 in peripheral
blood lymphocytes and T-cell
lines when used in conjunction with
antibodies to CD3 and CD28
(
53). These activities can have unforeseen
consequences in
vivo. Both of the above-mentioned activities of
Tat (immune modulation
and induction of Kaposi's sarcoma) most
likely require that Tat be
secreted from one cell and then act
on neighboring cells. Although Tat
can traverse through the plasma
membrane via signals present
in the first coding exon of Tat (
41,
62), such activities as
immune modulation and induction of Kaposi's
sarcoma may require that
Tat bind to the integrin receptors on
the cell membrane via the RGD
sequence present in the second coding
exon (
6,
53,
71).
While the single-exon-Tat-encoding vector,
which lacks the RGD
sequence, may allow high levels of transgene
expression without the
possible untoward consequences envisaged
with full-length Tat, it is
not clear which of the Tat-encoding
vectors will eventually prove
useful for gene therapy. Clearly,
the vectors need to be tested in
suitable animal models to establish
their safety and efficacy.
Conversely, these vectors may prove
useful for illuminating the role of
Tat in the pathogenesis of
AIDS in in vivo animal
models.
| |
ACKNOWLEDGMENTS |
This study was supported by grants from the National Institute of
Diabetes and Digestive and Kidney Diseases, National Institutes of
Health, to N.S. (DK53929) and F.S. (DK48265).
We thank Brian Klahn for expert technical assistance, Cristina Cueto
for help with plasmid construction, Michail Zaboikin for providing
IRES-containing constructs and the 3T3 cell line, Kathleen Schell and
Janet Lewis of the flow cytometry facility at the University of
Wisconsin
Madison and David McFarland of the HHMI Flow Cytometry
Facility at Vanderbilt University for help with running and analyzing
samples, Kendra Tutsch and staff of the Analytical Laboratory for the
use of the spectrophotometer and ELISA readers, Chinnasamy Jagannath
for the THP-1 cell line, Antonito Panganiban for the D17 cell line,
Didier Trono for pMD.G, David Camerini for pCDM8-luc, and David Rekosh
and Marie-Lou Hammarskjöld for generously sharing numerous
plasmid constructs, including ones with CTE. The following reagents
were obtained through the AIDS Research and Reference Reagent Program,
Division of AIDS, NIAID, NIH: 293 from Andrew Rice,
HeLa-CD4-LTR--gal from Michael Emerman, and pSV-A-MLV-Env from
Nathaniel Landau.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: 547 MRBII, 2220 Pierce Ave., Division of Hematology-Oncology, Department of Medicine, Vanderbilt University, Nashville, TN 37232-6305. Phone: (615) 936-2134. Fax: (615) 936-3853. E-mail:
narasimhachar.srinivasakumar{at}mcmail.vanderbilt.edu.
| |
REFERENCES |
| 1.
|
Akkina, R. K.,
R. M. Walton,
M. L. Chen,
Q. X. Li,
V. Planelles, and I. S. Chen.
1996.
High-efficiency gene transfer into CD34+ cells with a human immunodeficiency virus type 1-based retroviral vector pseudotyped with vesicular stomatitis virus envelope glycoprotein G.
J. Virol.
70:2581-2585[Abstract].
|
| 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.
|
Albini, A.,
R. Benelli,
M. Presta,
M. Rusnati,
M. Ziche,
A. Rubartelli,
G. Paglialunga,
F. Bussolino, and D. Noonan.
1996.
HIV-tat protein is a heparin-binding angiogenic growth factor.
Oncogene
12:289-297[Medline].
|
| 4.
|
Albini, A.,
G. Fontanini,
L. Masiello,
C. Tacchetti,
D. Bigini,
P. Luzzi,
D. M. Noonan, and W. G. Stetler-Stevenson.
1994.
Angiogenic potential in vivo by Kaposi's sarcoma cell-free supernatants and HIV-1 tat product: inhibition of KS-like lesions by tissue inhibitor of metalloproteinase-2.
AIDS
8:1237-1244[Medline].
|
| 5.
|
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].
|
| 6.
|
Barillari, G.,
C. Sgadari,
C. Palladino,
R. Gendelman,
A. Caputo,
C. B. Morris,
B. C. Nair,
P. Markham,
A. Nel,
M. Stürzl, and B. Ensoli.
1999.
Inflammatory cytokines synergize with the HIV-1 Tat protein to promote angiogenesis and Kaposi's sarcoma via induction of basic fibroblast growth factor and the alpha v beta 3 integrin.
J. Immunol.
163:1929-1935[Abstract/Free Full Text].
|
| 7.
|
Benkirane, M.,
R. F. Chun,
H. Xiao,
V. V. Ogryzko,
B. H. Howard,
Y. Nakatani, and K. T. Jeang.
1998.
Activation of integrated provirus requires histone acetyltransferase. p300 and P/CAF are coactivators for HIV-1 Tat.
J. Biol. Chem.
273:24898-24905[Abstract/Free Full Text].
|
| 8.
|
Bray, M.,
S. Prasad,
J. W. Dubay,
E. Hunter,
K. T. Jeang,
D. Rekosh, and M. L. Hammarskjold.
1994.
A small element from the Mason-Pfizer monkey virus genome makes human immunodeficiency virus type 1 expression and replication Rev-independent.
Proc. Natl. Acad. Sci. USA
91:1256-1260[Abstract/Free Full Text].
|
| 9.
|
Brown, J. A.,
T. K. Howcroft, and D. S. Singer.
1998.
HIV Tat protein requirements for transactivation and repression of transcription are separable.
J. Acquir. Immune Defic. Syndr. Hum. Retrovirol.
17:9-16[Medline].
|
| 10.
|
Case, S. S.,
M. A. Price,
C. T. Jordan,
X. J. Yu,
L. Wang,
G. Bauer,
D. L. Haas,
D. Xu,
R. Stripecke,
L. Naldini,
D. B. Kohn, and G. M. Crooks.
1999.
Stable transduction of quiescent CD34(+)CD38( ) human hematopoietic cells by HIV-1-based lentiviral vectors.
Proc. Natl. Acad. Sci. USA
96:2988-2993[Abstract/Free Full Text].
|
| 11.
|
Chen, W. Y.,
E. C. Bailey,
S. L. McCune,
J. Y. Dong, and T. M. Townes.
1997.
Reactivation of silenced, virally transduced genes by inhibitors of histone deacetylase.
Proc. Natl. Acad. Sci. USA
94:5798-5803[Abstract/Free Full Text].
|
| 12.
|
Chen, W. Y., and T. M. Townes.
2000.
Molecular mechanism for silencing virally transduced genes involves histone deacetylation and chromatin condensation.
Proc. Natl. Acad. Sci. USA
97:377-382[Abstract/Free Full Text].
|
| 13.
|
Chesebro, B.,
K. Wehrly, and W. Maury.
1990.
Differential expression in human and mouse cells of human immunodeficiency virus pseudotyped by murine retroviruses.
J. Virol.
64:4553-4557[Abstract/Free Full Text].
|
| 14.
|
Chirmule, N.,
S. Than,
S. A. Khan, and S. Pahwa.
1995.
Human immunodeficiency virus Tat induces functional unresponsiveness in T cells.
J. Virol.
69:492-498[Abstract].
|
| 15.
|
Corbeau, P., and F. Wong-Staal.
1998.
Anti-HIV effects of HIV vectors.
Virology
243:268-274[CrossRef][Medline].
|
| 16.
|
Delviks, K. A.,
W. S. Hu, and V. K. Pathak.
1997.
Psi-vectors: murine leukemia virus-based self-inactivating and self-activating retroviral vectors.
J. Virol.
71:6218-6224[Abstract].
|
| 17.
|
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].
|
| 18.
|
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].
|
| 19.
|
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].
|
| 20.
|
Ernst, R. K.,
M. Bray,
D. Rekosh, and M. L. Hammarskjold.
1997.
A structured retroviral RNA element that mediates nucleocytoplasmic export of intron-containing RNA.
Mol. Cell. Biol.
17:135-144[Abstract].
|
| 21.
|
Feinberg, M. B.,
D. Baltimore, and A. D. Frankel.
1991.
The role of Tat in the human immunodeficiency virus life cycle indicates a primary effect on transcriptional elongation.
Proc. Natl. Acad. Sci. USA
88:4045-4049[Abstract/Free Full Text].
|
| 22.
|
Gallardo, H. F.,
C. Tan,
D. Ory, and M. Sadelain.
1997.
Recombinant retroviruses pseudotyped with the vesicular stomatitis virus G glycoprotein mediate both stable gene transfer and pseudotransduction in human peripheral blood lymphocytes.
Blood
90:952-957[Abstract/Free Full Text].
|
| 23.
|
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].
|
| 24.
|
Gasmi, M.,
J. Glynn,
M. J. Jin,
D. J. Jolly,
J. K. Yee, and S. T. Chen.
1999.
Requirements for efficient production and transduction of human immunodeficiency virus type 1-based vectors.
J. Virol.
73:1828-1834[Abstract/Free Full Text].
|
| 25.
|
Gaynor, R. B.
1995.
Regulation of HIV-1 gene expression by the transactivator protein Tat.
Curr. Top. Microbiol. Immunol.
193:51-77[Medline].
|
| 26.
|
Harbers, K.,
A. Schnieke,
H. Stuhlmann,
D. Jahner, and R. Jaenisch.
1981.
DNA methylation and gene expression: endogenous retroviral genome becomes infectious after molecular cloning.
Proc. Natl. Acad. Sci. USA
78:7609-7613[Abstract/Free Full Text].
|
| 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.
|
Howcroft, T. K.,
L. A. Palmer,
J. Brown,
B. Rellahan,
F. Kashanchi,
J. N. Brady, and D. S. Singer.
1995.
HIV Tat represses transcription through Sp1-like elements in the basal promoter.
Immunity
3:127-138[CrossRef][Medline].
|
| 29.
|
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].
|
| 30.
|
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].
|
| 31.
|
Jaenisch, R.,
K. Harbers,
D. Jahner,
C. Stewart, and H. Stuhlmann.
1982.
DNA methylation, retroviruses, and embryogenesis.
J. Cell. Biochem.
20:331-336[CrossRef][Medline].
|
| 32.
|
Jahner, D.,
H. Stuhlmann,
C. L. Stewart,
K. Harbers,
J. Lohler,
I. Simon, and R. Jaenisch.
1982.
De novo methylation and expression of retroviral genomes during mouse embryogenesis.
Nature
298:623-628[CrossRef][Medline].
|
| 33.
|
Jeang, K. T.,
Y. Chang,
B. Berkhout,
M. L. Hammarskjold, and D. Rekosh.
1991.
Regulation of HIV expression: mechanisms of action of Tat and Rev.
AIDS
5(Suppl. 2):S3-S14.
|
| 34.
|
Jeang, K. T.,
H. Xiao, and E. A. Rich.
1999.
Multifaceted activities of the HIV-1 transactivator of transcription, Tat.
J. Biol. Chem.
274:28837-28840[Free Full Text].
|
| 35.
|
Jones, K. A.
1997.
Taking a new TAK on tat transactivation.
Genes Dev.
11:2593-2599[Free Full Text].
|
| 36.
|
Jones, K. A., and B. M. Peterlin.
1994.
Control of RNA initiation and elongation at the HIV-1 promoter.
Annu. Rev. Biochem.
63:717-743[CrossRef][Medline].
|
| 37.
|
Julias, J. G.,
D. Hash, and V. K. Pathak.
1995.
E vectors: development of novel self-inactivating and self-activating retroviral vectors for safer gene therapy.
J. Virol.
69:6839-6846[Abstract].
|
| 38.
|
Kanazawa, S.,
T. Okamoto, and B. M. Peterlin.
2000.
Tat competes with CIITA for the binding to P-TEFb and blocks the expression of MHC class II genes in HIV infection.
Immunity
12:61-70[CrossRef][Medline].
|
| 39.
|
Kaptein, L. C.,
M. Breuer,
D. Valerio, and V. W. van Beusechem.
1998.
Expression pattern of CD2 locus control region containing retroviral vectors in hemopoietic cells in vitro and in vivo.
Gene Ther.
5:320-330[CrossRef][Medline].
|
| 40.
|
Kiernan, R. E.,
C. Vanhulle,
L. Schiltz,
E. Adam,
H. Xiao,
F. Maudoux,
C. Calomme,
A. Burny,
Y. Nakatani,
K. T. Jeang,
M. Benkirane, and C. Van Lint.
1999.
HIV-1 tat transcriptional activity is regulated by acetylation.
EMBO J.
18:6106-6118[CrossRef][Medline].
|
| 41.
|
Kim, D. T.,
D. J. Mitchell,
D. G. Brockstedt,
L. Fong,
G. P. Nolan,
C. G. Fathman,
E. G. Engleman, and J. B. Rothbard.
1997.
Introduction of soluble proteins into the MHC class I pathway by conjugation to an HIV tat peptide.
J. Immunol.
159:1666-1668[Abstract].
|
| 42.
|
Kim, V. N.,
K. Mitrophanous,
S. M. Kingsman, and A. J. Kingsman.
1998.
Minimal requirement for a lentivirus vector based on human immunodeficiency virus type 1.
J. Virol.
72:811-816[Abstract/Free Full Text].
|
| 43.
|
Kwak, Y. T.,
D. Ivanov,
J. Guo,
E. Nee, and R. B. Gaynor.
1999.
Role of the human and murine cyclin T proteins in regulating HIV-1 tat-activation.
J. Mol. Biol.
288:57-69[CrossRef][Medline].
|
| 44.
|
Laspia, M. F.,
A. P. Rice, and M. B. Mathews.
1989.
HIV-1 Tat protein increases transcriptional initiation and stabilizes elongation.
Cell
59:283-292[CrossRef][Medline].
|
| 45.
|
Lewis, P.,
M. Hensel, and M. Emerman.
1992.
Human immunodeficiency virus infection of cells arrested in the cell cycle.
EMBO J.
11:3053-3058[Medline].
|
| 46.
|
Lewis, P. F., and M. Emerman.
1994.
Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus.
J. Virol.
68:510-516[Abstract/Free Full Text].
|
| 47.
|
Liu, M. L.,
B. L. Winther, and M. A. Kay.
1996.
Pseudotransduction of hepatocytes by using concentrated pseudotyped vesicular stomatitis virus G glycoprotein (VSV-G)-Moloney murine leukemia virus-derived retrovirus vectors: comparison of VSV-G and amphotropic vectors for hepatic gene transfer.
J. Virol.
70:2497-2502[Abstract].
|
| 48.
|
Marzio, G.,
M. Tyagi,
M. I. Gutierrez, and M. Giacca.
1998.
HIV-1 tat transactivator recruits p300 and CREB-binding protein histone acetyltransferases to the viral promoter.
Proc. Natl. Acad. Sci. USA
95:13519-13524[Abstract/Free Full Text].
|
| 49.
|
Matsui, M.,
R. J. Warburton,
P. C. Cogswell,
A. S. Baldwin, Jr., and J. A. Frelinger.
1996.
Effects of HIV-1 Tat on expression of HLA class I molecules.
J. Acquir. Immune Defic. Syndr. Hum. Retrovirol.
11:233-240[Medline].
|
| 50.
|
McCune, S. L., and T. M. Townes.
1994.
Retroviral vector sequences inhibit human beta-globin gene expression in transgenic mice.
Nucleic Acids Res.
22:4477-4481[Abstract/Free Full Text].
|
| 51.
|
Miyoshi, H.,
U. Blomer,
M. Takahashi,
F. H. Gage, and I. M. Verma.
1998.
Development of a self-inactivating lentivirus vector.
J. Virol.
72:8150-8157[Abstract/Free Full Text].
|
| 52.
|
Mochizuki, H.,
J. P. Schwartz,
K. Tanaka,
R. O. Brady, and J. Reiser.
1998.
High-titer human immunodeficiency virus type 1-based vector systems for gene delivery into nondividing cells.
J. Virol.
72:8873-8883[Abstract/Free Full Text].
|
| 53.
|
Ott, M.,
S. Emiliani,
C. Van Lint,
G. Herbein,
J. Lovett,
N. Chirmule,
T. McCloskey,
S. Pahwa, and E. Verdin.
1997.
Immune hyperactivation of HIV-1-infected T cells mediated by Tat and the CD28 pathway.
Science
275:1481-1485[Abstract/Free Full Text].
|
| 54.
|
Ott, M.,
J. L. Lovett,
L. Mueller, and E. Verdin.
1998.
Superinduction of IL-8 in T cells by HIV-1 Tat protein is mediated through NF-kappaB factors.
J. Immunol.
160:2872-2880[Abstract/Free Full Text].
|
| 55.
|
Palmer, T. D.,
G. J. Rosman,
W. R. Osborne, and A. D. Miller.
1991.
Genetically modified skin fibroblasts persist long after transplantation but gradually inactivate introduced genes.
Proc. Natl. Acad. Sci. USA
88:1330-1334[Abstract/Free Full Text].
|
| 56.
|
Parolin, P.,
B. Taddeo,
G. Palu, and J. Sodroski.
1996.
Use of cis- and trans-acting regulatory sequences to improve expression of human immunodeficiency virus vectors in human lymphocytes.
Virology
222:415-422[CrossRef][Medline].
|
| 57.
|
Puri, R. K.,
P. Leland, and B. B. Aggarwal.
1995.
Constitutive expression of human immunodeficiency virus type 1 tat gene inhibits interleukin 2 and interleukin 2 receptor expression in a human CD4+ T lymphoid (H9) cell line.
AIDS Res. Hum. Retroviruses
11:31-40[Medline].
|
| 58.
|
Rautonen, N.,
J. Rautonen,
N. L. Martin, and D. W. Wara.
1994.
HIV-1 Tat induces cytokine synthesis by uninfected mononuclear cells.
AIDS
8:1504-1506[Medline].
|
| 59.
|
Reinhold, D.,
S. Wrenger,
U. Bank,
F. Buhling,
T. Hoffmann,
K. Neubert,
M. Kraft,
R. Frank, and S. Ansorge.
1996.
CD26 mediates the action of HIV-1 Tat protein on DNA synthesis and cytokine production in U937 cells.
Immunobiology
195:119-128[Medline].
|
| 60.
|
Richards, C. A., and B. E. Huber.
1993.
Generation of a transgenic model for retrovirus-mediated gene therapy for hepatocellular carcinoma is thwarted by the lack of transgene expression.
Hum. Gene Ther.
4:143-150[Medline].
|
| 61.
|
Rizvi, T. A., and A. T. Panganiban.
1993.
Simian immunodeficiency virus RNA is efficiently encapsidated by human immunodeficiency virus type 1 particles.
J. Virol.
67:2681-2688[Abstract/Free Full Text].
|
| 62.
|
Schwarze, S. R.,
A. Ho,
A. Vocero-Akbani, and S. F. Dowdy.
1999.
In vivo protein transduction: delivery of a biologically active protein into the mouse.
Science
285:1569-1572[Abstract/Free Full Text].
|
| 63.
|
Srinivasakumar, N.,
N. Chazal,
C. Helga-Maria,
S. Prasad,
M. L. Hammarskjold, and D. Rekosh.
1997.
The effect of viral regulatory protein expression on gene delivery by human immunodeficiency virus type 1 vectors produced in stable packaging cell lines.
J. Virol.
71:5841-5848[Abstract].
|
| 64.
|
Srinivasakumar, N., and F. Schuening.
1999.
A lentivirus packaging system based on alternative RNA transport mechanisms to express helper and gene transfer vector RNAs and its use to study the requirement of accessory proteins for particle formation and gene delivery.
J. Virol.
73:9589-9598[Abstract/Free Full Text].
|
| 65.
|
Subramanyam, M.,
W. G. Gutheil,
W. W. Bachovchin, and B. T. Huber.
1993.
Mechanism of HIV-1 Tat induced inhibition of antigen-specific T cell responsiveness.
J. Immunol.
150:2544-2553[Abstract].
|
| 66.
|
Sutton, R. E.,
H. T. Wu,
R. Rigg,
E. Bohnlein, and P. O. Brown.
1998.
Human immunodeficiency virus type 1 vectors efficiently transduce human hematopoietic stem cells.
J. Virol.
72:5781-5788[Abstract/Free Full Text].
|
| 67.
|
Vacca, A.,
M. Farina,
M. Maroder,
E. Alesse,
I. Screpanti,
L. Frati, and A. Gulino.
1994.
Human immunodeficiency virus type-1 tat enhances interleukin-2 promoter activity through synergism with phorbol ester and calcium-mediated activation of the NF-AT cis-regulatory motif.
Biochem. Biophys. Res. Commun.
205:467-474[CrossRef][Medline].
|
| 68.
|
Verhoef, K.,
M. Bauer,
A. Meyerhans, and B. Berkhout.
1998.
On the role of the second coding exon of the HIV-1 Tat protein in virus replication and MHC class I downregulation.
AIDS Res. Hum. Retroviruses
14:1553-1559[Medline].
|
| 69.
|
Weissman, J. D.,
J. A. Brown,
T. K. Howcroft,
J. Hwang,
A. Chawla,
P. A. Roche,
L. Schiltz,
Y. Nakatani, and D. S. Singer.
1998.
HIV-1 tat binds TAFII250 and represses TAFII250-dependent transcription of major histocompatibility class I genes.
Proc. Natl. Acad. Sci. USA
95:11601-11606[Abstract/Free Full Text].
|
| 70.
|
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].
|
| 71.
|
Wu, Z.,
U. Cavallaro,
P. C. Marchisio,
M. R. Soria, and J. A. Maier.
1998.
Fibronectin modulates the effects of HIV-1 Tat on the growth of murine Kaposi's sarcoma-like cells through the down-regulation of tyrosine phosphorylation.
Am. J. Pathol.
152:1599-1605[Abstract].
|
| 72.
|
Yamamoto, T., and M. Horikoshi.
1997.
Novel substrate specificity of the histone acetyltransferase activity of HIV-1-Tat interactive protein Tip60.
J. Biol. Chem.
272:30595-30598[Abstract/Free Full Text].
|
| 73.
|
Zolotukhin, A. S.,
A. Valentin,
G. N. Pavlakis, and B. K. Felber.
1994.
Continuous propagation of RRE() and Rev()RRE() human immunodeficiency virus type 1 molecular clones containing a cis-acting element of simian retrovirus type 1 in human peripheral blood lymphocytes.
J. Virol.
68:7944-7952[Abstract/Free Full Text].
|
| 74.
|
Zufferey, R.,
T. Dull,
R. J. Mandel,
A. Bukovsky,
D. Quiroz,
L. Naldini, and D. Trono.
1998.
Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery.
J. Virol.
72:9873-9880[Abstract/Free Full Text].
|
Journal of Virology, July 2000, p. 6659-6668, Vol. 74, No. 14
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Schuening, F. G., Srinivasakumar, N., Zaboikin, M. M., Zaboikina, T. N.
(2005). HIV-1 Vector Mediated Expression of Human B-Domain Deleted FVIII in Primary Canine Marrow Mononuclear Cells.. ASH ANNUAL MEETING ABSTRACTS
106: 5524-5524
[Abstract]
-
Srinivasakumar, N., Zaboikin, M., Zaboikina, T., Schuening, F.
(2002). Evaluation of Tat-Encoding Bicistronic Human Immunodeficiency Virus Type 1 Gene Transfer Vectors in Primary Canine Bone Marrow Mononuclear Cells. J. Virol.
76: 7334-7342
[Abstract]
[Full Text]
Top
Abstract
Text
