From the Department of Cancer Immunology & AIDS,
Dana-Farber Cancer Institute and § Department of Medicine,
Harvard Medical School, Boston, Massachusetts 02115
Received for publication, August 13, 2002, and in revised form, October 24, 2002
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ABSTRACT |
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Human immunodeficiency virus, type 1 (HIV-1)
replication requires the interaction of Tat protein with the human
cyclinT1 (hCyclinT1) subunit of the positive transcription elongation
factor (P-TEFb) complex, which then cooperatively binds to
transactivation response element (TAR) RNA to transactivate
HIV transcription. In this report, a non-immune human single-chain
antibody (sFv) phage display library was used to isolate anti-hCyclinT1
sFvs that could disrupt hCyclinT1-Tat interactions. The N-terminal 272 residues of hCyclinT1, including the entire cyclin domains and the
Tat·TAR recognition motif (TRM), that fully support Tat
transactivation was used for panning, and of the five unique
anti-hCyclinT1 sFvs that were obtained, three bound to the cyclin box
domains and two bound to TRM. All sFvs could be expressed as
intrabodies at high levels in transiently transfected 293T and in
stable Jurkat and SupT1 transfectants and could specifically
co-immunoprecipitate co-expressed hCyclinT1 in 293T cells with varying
efficacy without disrupting hCyclinT1-Cdk9 interactions. In
addition, two sFv clones (3R6-1 and 2R6-21) that mapped to the cyclin
box domains markedly inhibited Tat-mediated transactivation in several
transiently transfected cell lines without inhibiting basal
transcription or inducing apoptosis. When HIV-1 challenge studies were
performed on stable 3R6-1-expressing Jurkat T cells, near complete
inhibition of viral replication was obtained at a low challenge dose,
and 74-88% inhibition to HIV-1 replication was achieved at a high
infection dose in SupT1 cells. These results provide proof-in-principle
that anti-hCyclinT1 intrabodies can be designed to block HIV-1
replication without causing cellular toxicity, and as a result, they
may be useful agents for "intracellular immunization"-based gene
therapy strategies for HIV-1 infection/AIDS.
The HIV1 transactivator
protein Tat is absolutely required for viral replication. In the
absence of Tat the transcription of HIV mRNAs can be initiated but
cannot be efficiently elongated to produce full-length viral RNA genome
(1). Tat interacts specifically with human cyclinT1 (hCyclinT1), a
regulatory partner of cyclin-dependent kinase 9 (Cdk9) in
the positive transcription elongation factor (P-TEFb) complex. Tat
binds cooperatively with hCyclinT1 to the transactivation response
element (TAR), a bulged RNA loop structure located at the 5'-end of
nascent viral RNA transcripts to recruit P-TEFb and promote HIV
transcription elongation. The assembly of the Tat·TAR·P-TEFb
complex to the HIV promoter activates the Cdk9 kinase
activities, which further autophosphorylates P-TEFb and
hyperphosphorylates the C-terminal domain of RNA polymerase II (2),
leading to the formation of processive elongation complexes that
synthesize full-length HIV viral mRNA. In addition to hCyclinT1 and
Cdk9 subunits, the nascent P-TEFb complex also contains hCyclinT2a, T2b, (3) and recently described 7SK small nuclear RNAs as well as
several other proteins of unknown identity (4, 5). Among these, only
Cdk9/hCyclinT1 heterodimer functions as both a general and an HIV-1
Tat-specific transcription factor (2, 6, 7, 8).
The transactivation mechanism is a particularly attractive target for
the development of new anti-retroviral drug therapies, because Tat is
required for viral gene expression not only during the exponential
growth of the virus, but also, critically, it is required during the
activation of the integrated proviral genomes that give rise to
drug-resistant strains of HIV-1 (9). With the recent advances in our
understanding of the molecular mechanisms of transactivation, it is
clear that one novel anti-retroviral strategy that would bypass the
ability of HIV-1 to adapt to traditional anti-viral therapies is to
develop agents that target hCyclinT1. This would be an attractive
target, if it was possible to selectively disrupt Tat-hCylinT1
interactions without disrupting the function of the P-TEFb complex,
which is required for basal cellular transcription. Indeed, although
hCyclinT1 contains 726 amino acids (see Fig. 1), the N-terminal 272 residues, including the entire cyclin domain (residues 1-250) and a
TAR recognition motif (TRM, residues 250-272 of hCyclinT1) fully
support the Tat transactivation of HIV-1 LTR (long terminal repeat)
(10), whereas the C-terminal 300 amino acids (residues 402-701) are
believed to mediate the coupling of general transcription elongation
and pre-mRNA splicing (11).
In previous studies we have demonstrated that an intrabody to HIV-1 Tat
could significantly inhibit HIV-1 replication in vitro (12,
13) indicating that the Tat transactivation pathway could be targeted
by intrabodies specific for the viral component of this pathway. Based
on these studies, we further hypothesized that, because the molecular
interaction between HIV-1 Tat and the N-terminal region of hCyclinT1 is
necessary for HIV-1 replication, the selective disruption of this
binding by anti-hCyclinT1 intrabodies could inhibit Tat-mediated
transactivation and HIV-1 replication without having a detrimental
effect on basal transcription. To test this hypothesis, we used the
N-terminal region of hCyclinT1 and a synthetic TRM peptide to screen a
very large non-immune human single-chain antibody (sFv) phage display
library to identify sFvs that could be engineered as anti-hCyclinT1
intrabodies. Of the five hCyclinT1-specific single chain antibody (sFv)
clones that were identified, one sFv (3R6-1) not only disrupted the
molecular interaction between Tat and hCyclinT1 in co-transfected human CD4+ T cell lines but also inhibited HIV-1 replication upon
viral challenge. Importantly, no disruption of basal cellular
transcription was observed. These results provide proof-in-principle
that anti-hCyclinT1 intrabodies can be designed to selectively block
Tat-mediated transactivation and HIV-1 replication. They may also
provide a promising new agent for the gene therapy of HIV-1 infection.
Antibodies--
Anti-M13 monoclonal antibody (mAb) were
obtained from Amersham Biosciences. Goat anti-human cyclinT1, rabbit
anti-human Cdk9, rabbit anti-HA, rabbit anti-His, and goat anti-HA
IgG-beads were purchased from Santa Cruz Biotechnology. Horseradish
peroxidase (HRP)-conjugated goat anti-mouse IgG and HRP-conjugated
mouse anti-goat IgG were purchased from Pierce (Rockford).
Construction of Expression Vectors--
Prokaryotic expression
vectors pGST-hT1-250 and pGST-hT1-300 for producing truncated
hCyclinT1-GST fusion proteins were kindly provided by Dr. Peterlin
(10). To express GST-full-length hCyclinT1 fusion protein, the
hCyclinT1 gene amplified by PCR with built-in BamHI and
EcoRI sites was inserted into pGEX-2T (Amersham
Biosciences), resulting in pGST-hT1-726. PGST-hT1-200 was created by
digesting pGST-hT1-726 with EcoRI and BsmI
followed by blunt-end ligation to remove amino acids 202-726 in
hCyclinT1 (see Fig. 3A). The eukaryotic expression vector
for hCyclinT1 (pCI-86) was provided by Dr. Jones (8). PCI-86(His)
vector for expressing C-terminally His-tagged hCyclinT1 was constructed
by replacing the original C-terminal BstBI/NotI
fragment with a similar PCR-modified fragment containing sequence
coding for six histidines.
For expressing soluble sFvs in bacterial cells, sFv coding sequences
were isolated from the pFarber phage display vector by NcoI
and NotI digestion and inserted into the pSyn1 vector in the
same orientation. The sFvs expressed in bacterial cells were C-terminally tagged with c-myc and His6 (Fig.
2A). For expressing sFv intrabodies in mammalian cells, the
sFv coding sequences were released from pFarber phage display vectors
by SfiI and NotI digestion and ligated into an
sFv expression cassette in psFv-C Purification of Bacterially Expressed GST and sFv Fusion
Proteins--
GST fusion proteins were purified using
glutathione-Sepharose 4B beads following the manufacturer's
instructions (Amersham Biosciences). Single colonies of transformed
XL1-blue bacteria were cultured at 37 °C overnight in 10 ml of 2xYT
medium containing 100 µg/ml ampicillin. The overnight culture was
used to inoculate 1 liter of fresh medium followed by shaking for 3-5
h until 0.3-0.5 A600 nm was achieved.
Subsequently, isopropyl-1-thio-
To purify His-tagged sFvs, bacterial cultures were processed as
described above. Supernatants of cell lysates were clarified through a
syringe filter. Proteins from 1 liter of culture were precipitated in
50% saturated ammonium sulfate and dissolved in 10 ml of PBS buffer
containing 10 mM imidazole before being loaded onto a
nickel-bound chelating Sepharose column (Amersham Biosciences). After
washing with 20-200 mM imidazole in PBS buffer, His
tag-bound proteins were eluted with PBS containing 300 mM imidazole.
Screening of Human sFv-phagemid Library--
Construction of the
pFarber phagemid display vector and characterization of a 15-billion
member non-immune human sFv phage display library will be described
elsewhere.2 Phage particles
displaying human sFv molecules from TG1 Escherichia coli host cells by VCS M13 helper phage and concentrated by
PEG/NaCl precipitation and diluted to 1012-13
plaque-forming units/ml before screening (14-16).
To identify sFv clones that specifically bind to epitopes in the
N-terminal region of hCyclinT1 that are involved in Tat
transactivation, the phage display library containing 1012
plaque-forming units was panned against affinity-purified GST-free hCyclinT1-300-coated Maxisorb immunotubes (20 µg/ml, Nalge Nunc International (15, 16)) for three successive rounds. After extensive
washing with 0.1% Tween-20/PBS, bound phages were eluted with 100 mM triethylamine for 30 and 60 min. Pooled samples were used for rescue and further panning. After the last round of selection, selected sFv phages were diluted and used to infect TG1 E. coli strain. Culture medium containing sFv phage particles rescued from individual colonies in 24-well plates were submitted for ELISA screening.
To identify TRM-specific sFvs, a biotin-labeled TRM peptide
(NRLKRIWNWRACEAAKKGGK-biotin) was synthesized for screening the library from 200 to 2 ng concentration. Peptide-bound phages
were trapped by streptavidin Immunobeads and eluted with 0.1 M HCl-glycine (pH 2.2) for 30 and 60 min. Individual clones
rescued from rounds 3-7 were further analyzed by ELISA screening.
Phage ELISA--
96-well microtiter plates were coated with
purified GST-hCyclinT1-726 protein (10 µg/ml) followed by incubation
with sFv phage particles (1010 plaque-forming units). The
binding activity of anti-hCyclinT1 sFv phages to the antigens was
determined by incubation with anti-M13 monoclonal antibodies and goat
anti-mouse IgG-HRP. Positive samples were further tested with
GST-coated plates to remove GST-specific sFv binders. VCS M13 helper
phage stocks with a similar number of virus particles were used as
negative control. SFv clones from peptide panning were screened on
streptavidin-coated microtiter plates followed by incubation with
biotin-TRM peptide and then with sFv phage particles. TRM-positive
binders were further tested with purified hCyclinT1 as described above.
Real-time Biacore (Biospecific Interaction Analysis)--
The
binding affinity and kinetic profiles of isolated anti-hCyclinT1 sFvs
to hCyclinT1 were analyzed by surface plasmon resonance on the Biacore
1000 apparatus (Amersham Biosciences). The flow cells of a CM5 sensor
chip was tested under three different pH conditions. The GST-free
hCyclinT1-300 could not be efficiently immobilized to the CM5 sensor
chips under neutral (5 mM maleate, pH 6.0-7.0) or acidic
pH conditions (10 mM acetate, pH 4.8); however, the sensor
chips were effectively coupled with GST-hCyclinT1-300 (20 µg/ml) at
pH 4.8 in the presence of 10 mM acetate. The level of
immobilization was 24 ng of protein/mm2 surface, equivalent
to 24,000 relative units/mm2 over background. Binding
kinetic parameters for individual sFv clones were measured with His
tag-purified sFv proteins at different concentrations. 2R6-21 and 3R6-1
were measured at 6.6, 33, 165, 330, and 660 nM, whereas
2R6-113 was measured at 330, 825, 1650, and 3300 nM.
Association (Kon) and dissociation
(Koff) constants of sFvs to hCyclinT1 were
calculated with Biacore evaluation software based on simulated
binding curves of actual measurements.
Establishment of sFv-intrabody Expressing Stable T Cell
Transfectants--
Human Jurkat and SupT1 cell lines (ARRRP-NIH) were
cultured in RPMI 1640 growth medium supplemented with 10% fetal calf
serum and 100 units of penicillin and streptomycin. To establish T cell transfectants that stably expressed anti-hCyclinT1 intrabodies for
HIV-1 challenge, Jurkat and SupT1 cells were split 48 h prior to
DNA transfection, washed in PBS, and then diluted to 3 × 106 cells/ml in RPMI 1640 growth medium. The diluted cells
(1 ml) were gently mixed with ScaI-linearized psFv-C HIV-1 Infection of T Lymphocytes--
Bulk populations of stably
transfected Jurkat and SupT cells in log phase of growth were harvested
and washed in PBS. Subsequently, 5 × 105 cells were
resuspended in 1 ml of growth medium containing 50 or 2500 TCID50 infection units of HIV-1 IIIB virus
(17). After 2-h incubation at 37 °C, infected cells were washed in
serum-free medium twice and were cultured in duplicate 6-well dishes
for 3 weeks. Culture medium (1 ml) was collected at intervals of 2-3 days and frozen at HIV p24 Antigen Assay--
The concentration of HIV-1 core
protein antigen in culture medium was measured by using a p24
immunoassay from Coulter Corp. (Westbrook, ME). The results were read
on a Dynatech MR5000 enzyme-linked immunosorbent assay (ELISA) plate
reader (Dynatech Laboratories Inc., Chantilly, VA). Medium samples were
first tested without dilution. Samples that gave optical density
readings of more than 1.0 were diluted at 1:10, 1:102,
1:103, 1:104, and 1:105 and were
re-assayed.
Identification of Human sFvs against hCyclinT1--
To
identify hCyclinT1-specific sFvs, the affinity-purified hCyclinT1
polypeptide (hCyclinT1-300) was used to coat immunotubes for screening
the phage display library. The bait protein contained the N-terminal
region 300 amino acids of hCyclinT1 that participates in Tat
transactivation (Fig. 1). After three
rounds of selection, individual colonies were tested by phage ELISA
against purified GST-hCyclinT1-300 and GST. Approximately 80%
colonies from the third round of panning were positive for hCyclinT1,
and only 1 of 200 colonies reacted to GST. DNA sequence analysis of 50 binders positive for hCyclinT1 identified one unique sFv clone (3R6-1). To identify more diversified anti-hCyclinT1 sFv clones, phages from the
second round of panning were used for ELISA screening. In addition to
3R6-1, two more sFv clones, 2R6-21 and 2R6-113, were identified. 3R6-1
and 2R6-21 clones predominated in the phage populations of the second
round of selection, whereas 2R6-113 represented a small proportion
(2%) of the positive binders.
The TRM motif of hCyclinT1 contains a cysteine residue (Cys-261)
that has been shown to be critical for Tat and hCyclinT1 interaction
(Fig. 1) (18). To identify TRM-specific sFvs, immunotubes were coated
with the synthetic 20-mer peptide and one clone (PS11) was isolated
after the second round of selection. When the same peptide was used for
liquid phase selection with streptavidin beads, one additional sFv
clone (S84) was isolated after seven rounds of panning. Table
I shows the comparison of deduced amino acid sequences of the five hCyclinT1-specific sFv clones. It is noteworthy that 3R6-1 and 2R6-21 share identical heavy chains, but have
13 amino acid differences in their light chains. These variations are
mainly located in FW1 (PCR primer annealing region) and FW4 (different
J Epitope Mapping of Anti-hCyclinT1 sFv Clones--
To
characterize the biochemical and binding properties of the
anti-hCyclinT1 sFvs, the sFv coding sequences were subcloned into the
pSyn1 bacterial expression vector to produce soluble 30-kDa sFv
proteins that contain C-terminal c-myc followed by a
His (x6) tag (Fig. 2A). Fig.
2B shows SDS-PAGE analysis of the sFv proteins purified from
XL1-blue E. coli bacteria using nickel-chelating Sepharose
columns. Purified GST fusion proteins containing different-length N-terminal hCyclinT1 amino acids or GST alone were used to coat microtiter plates to determine sFv binding epitopes on hCyclinT1 (Fig.
3A). To avoid nonspecific
binding activity, the sFv concentrations used in these studies were
first pre-determined by testing the serially diluted purified sFvs
against a fixed amount of GST-free hCyclinT1-300, and then the sFvs
were tested for binding at low concentrations above the end point of
their binding activity (refer to Fig. 3C). As shown in Fig.
3B, the five sFvs bound specifically to GST-hCyclinT1 fusion
proteins but not to GST. SFv 3R6-1, 2R6-21, and 2R6-113 bound strongly
to GST-T1-200, GST-T1-250, and GST-T1-300, indicating that the
epitopes for these sFvs were located within the cyclin box domains of
the N-terminal 200 amino acids. Although the sFv 3R6-1 and 2R6-21 have
identical VH chains and closely related light chains, there
was a 2-fold higher binding signal for 3R6-1 compared with 2R6-21 when
tested at 0.05 µg/ml sFv protein that reflects a difference in
binding affinities for the two sFvs under these assay conditions. PS11
displayed stronger binding activity to GST-T1-300 than to GST-T1-250
as expected, because the TRM peptide used for panning corresponded to
amino acids 250-266 of hCyclinT1. However, S84 showed only weak
binding activity to GST-T1-300 and similarly cross-reacted with
GST-T1-250 as well, indicating that a TRM-like epitope may be present
in residues 200-250 of hCyclinT1.
Determination of sFv Binding Affinities to hCyclinT1--
To
further characterize the binding profiles of the anti-hCyclinT1 sFvs,
microtiter plates were coated with purified GST-free hCyclinT1-300 (20 µg/ml, equivalent to 660 nM), and the purified sFvs were
tested for binding activity over the 0.033-1320 nM range (equivalent to 0.001-40 µg/ml). As shown in Fig. 3C, the
2R6-21 and 3R6-1 clones yielded the strongest binding activities to
purified hCyclinT1 with detectable binding at concentrations as low as 0.16 nM. Clones 2R6-113 and PS11 had similar binding
profiles, however, their binding end points to hCyclinT1 were about
3.33 nM and were much higher than those seen for clones
2R6-21 and 3R6-1. Clone S84 had the lowest end point binding activity
(82.5 nM) to the purified hCyclinT1. In addition, only the
binding of sFv S84 and PS11, but not sFv 3R6-1, 2R6-21, or 2R6-113, to
hCyclinT1-300 could be specifically blocked by the TRM peptide used
for their selection in an ELISA-based peptide-blocking assay. As
expected, a higher concentration of TRM peptide was required to compete for sFv PS11 compared with sFv S84 binding, which is most likely due to
the relatively higher binding affinity of sFv PS11 to hCyclinT1-300 (data not shown).
To more accurately characterize the binding affinities of these sFvs to
hCyclinT1, real-time Biacore analysis was performed and the association
(Kon) and dissociation
(Koff) constants for anti-hCyclinT1 sFv binding
to hCyclinT1-300 were experimentally determined. Table
II summarizes the results for the
real-time Biacore analyses. The binding affinity of sFv 2R6-21
(KD = 6.95 × 10 Purified Anti-hCyclinT1 sFvs Could Precipitate Human CyclinT1-GST
Fusion Proteins--
To demonstrate whether purified anti-hCyclinT1
sFvs could bind to soluble hCyclinT1, an immunoprecipitation assay was
conducted with purified His-tagged sFvs and GST-hT1-300 fusion
proteins. The sFv·hCyclinT1 complexes were co-immunoprecipitated by
anti-His mAb-conjugated microbeads. As shown in Fig.
4, sFv 3R6-1, 2R6-21, and 2R6-113
effectively bound to soluble GST-hT1-300. Compared with these sFvs,
S84 showed less efficiency in co-immunoprecipitation, which is
consistent with its lower binding affinity to hCyclinT1.
Anti-hCyclinT1 sFv Intrabodies Expressed in Mammalian Cells Bind to
hCyclinT1--
It has been previously reported that the addition of a
human immunoglobulin kappa chain constant region to an anti-HIV-1 Tat sFv significantly increases the ability of the anti-Tat intrabody to
inhibit Tat-mediated trans-activation and HIV-1 replication, presumably
by improving intracellular folding and increasing binding affinity due
to dimerization (12). Therefore, to characterize the biological
functions of anti-hCyclinT1 intrabodies in mammalian cells, the sFv
coding sequences were subcloned into a pcDNA-based expression
vector that directs the cytoplasmic expression of sFv-C
To determine whether the anti-hCyclinT1 sFv-C Inhibition of Tat-mediated Transactivation by Anti-hCyclinT1
Intrabodies--
The anti-hCyclinT1 intrabodies were next
co-transfected into HIV-1-susceptible T cells with the HIV-1 Tat
reporter gene system (pSV-tat and pHIV-1 LTR-luc) to evaluate their
ability to inhibit Tat-mediated transactivation. A Anti-hCyclinT1 Intrabodies Expressed in Human T Cell Lines Suppress
T-tropic HIV-1 Replication--
To evaluate whether the anti-hCyclinT1
intrabodies could inhibit HIV-1 replication in HIV-1-susceptible T
cells, the intrabody expression vectors were stably transfected into
Jurkat and SupT1 T cell lines. The expression of anti-hCyclinT1
sFv-C
HIV-1 Tat protein is initially expressed in the cytoplasm of infected
cells from spliced HIV-1 tat mRNA and subsequently translocated into the nuclei and nucleoli where it interacts with P-TEFb to increase
the efficiency of elongation of full-length HIV-1 genomic RNA. Because
this interaction occurs in nuclei and nucleoli of infected cells,
nuclear and nucleolar targeting vectors were constructed to evaluate
whether expression of the anti-hCyclinT1 intrabodies in the cytoplasm
or in the nuclear or nucleolar compartments would give rise to stronger
inhibition of HIV-1 replication.
Stable SupT1 transfectants that expressed anti-hCyclinT1 3R6-1 and
control A3H5 intrabodies containing the SV40 nuclear localization signal (NLS) or the HIV-1 Tat nucleolar localization signal (NOL) (20)
were additionally established and then infected with a high
concentration of HIV-1 IIIB (2500 units of
TCID50 per million cells). Marked syncytia formations were
seen with all SupT1 cells that expressed control intrabodies (A3H5,
A3H5-NLS, and A3H5-NOL) beginning day 3 post-infection. As the viral
replication progressed, cytopathic effects became more extensive and
ballooning fused cells that contained hundreds of nuclei were seen on
day 11 post-infection. In contrast, the cytopathic effects of HIV-1
IIIB on infected SupT1 cells that expressed the
anti-hCyclinT1 intrabody 3R6-1 and its NLS or NOL derivatives were much
less severe. For these cells, the onset of syncytia formation was
delayed until day 6 post-infection, and only small syncytium formation
was observed in 3R6-1-expressing cells, whereas SupT1 3R6-1-NLS cells
showed no syncytial formation and SupT1 3R6-1-NOL cells displayed only rare small fused cells. These results indicated that the expression of
anti-hCyclinT1 intrabodies 3R6-1 and its NLS- or NOL-containing derivatives significantly decreased the cytopathic effects of HIV-1
BIII on infected SupT1 cells (data not shown).
During the initial 4-day period post-infection, the p24 levels in the
culture medium showed no significant differences between SupT1 cells
that expressed different versions of A3H5 control and 3R6-1
intrabodies. However, p24 production in SupT1 cells that expressed
anti-hCyclinT1 intrabodies 3R6-1 and its NLS and NOL derivatives were
significantly reduced compared with their A3H5 counterparts (Fig. 8,
B-D). On day 11 post-infection the viral replication in
A3H5 transfectants progressed very rapidly and produced 4,545 ng/ml p24
antigen. Compared with this, the viral production in 3R6-1
transfectants was only 646 ng/ml, representing an 86% reduction in the
viral replication at the same time point (Fig. 8B). For the
SupT1 cells expressing NLS-containing intrabodies (Fig. 8C),
the p24 production in 3R6-1-NLS transfectants on day 11 post-infection
was 4,337 ng/ml, which represented only 12% of viral production in
A3H5-NLS control (35,080 ng/ml). As for SupT1 cells that expressed NOL
derivatives (Fig. 8D), on day 7 post-infection the viral
replication was well controlled in the 3R6-1 transfectants and yielded
11 ng/ml p24 antigen, representing 0.2% of the viral production in
control cells (4,464 ng/ml). On day 11 post-infection, compared with
A3H5-NOL-expressing cells (77,640 ng/ml), the peak p24 levels in
3R6-1-NOL cell supernatants (22,200 ng/ml) was reduced about 71%.
Interestingly, a cross comparison between SupT1 cells expressing 3R6-1
and its NLS or NOL derivatives showed that SupT1 transfectants that
expressed 3R6-1-NLS and -NOL intrabodies displayed much less severe
cytopathic effects compared with 3R6-1 transfectants. These results
demonstrate that the anti-hCyclinT1 intrabody 3R6-1 is a potent agent
that can significantly reduce HIV-1 cytopathic effects and dramatically
suppress HIV-1 replication in vitro.
The cytopathic effects of HIV-1 on CD4+ T lymphocytes
cause rapid and continuous destruction of CD4+ T helper
cells and lead to chronic, persistent infection and immune deficiency
(21-23). The molecular interaction between HIV-1 Tat protein and
hCyclinT1 in the P-TEFb complex is one of the most important pathways
that regulates HIV-1 replication. The presence of proviruses in resting
CD4+ T lymphocytes and the persistent release of infectious
viral particles by activation of these latently infected cells results in a life-long, latent viral infection. Several investigators have
demonstrated that the Tat-mediated transactivation pathway can be
disrupted by genetic "intracellular immunization"-based strategies
using anti-Tat intrabodies (24), antisense RNA and ribozyme-based
genetic inactivation (25), anti-tat/rev interference RNA (26), and
others (27). A major limitation of these approaches is the sequence
variations in the HIV-1 Tat proteins or tat gene among the
different virus isolates (28).
HCyclinT1 is a conserved cellular protein that is absolutely necessary
for HIV-1 Tat-mediated transactivation and HIV-1 replication but is
also a critical component of the cellular P-TEFb complex that is
required for normal cellular transcription elongation. Therefore, the
identification of human anti-hCyclinT1 intrabodies that specifically
disrupt the molecular interaction between Tat and hCyclinT1 but do not
interfere with its normal cellular function would be an ideal agent for
HIV-1 gene therapy against diverse viral isolates. Toward this end, we
employed a purified hCyclinT1-300 polypeptide containing the
N-terminal region of hCyclinT1 as bait for screening a human phage
display library and identified five distinct anti-hCyclinT1 sFvs. Three
of these sFvs bound to the cyclin box domains and two sFvs mapped to
the TRM motif. The hCyclinT1 N-terminal region, including the cyclin
domains and TRM motif, fully supports HIV-1 Tat-mediated
transactivation. Co-immunoprecipitation studies demonstrated that all
of these anti-hCyclinT1 intrabodies specifically bound to co-expressed
hCyclinT1 in 293T cells. SFv clones 3R6-1 and 2R6-21 that bound to the
cyclin box domain inhibited HIV-1 Tat-mediated transactivation in
co-transfected Jurkat and SupT1 cells without significant effects on
cellular function based on SFv clones 2R6-21 and 3R6-1 share the same VH but have
different VL chains. Their binding epitopes were mapped to
the cyclin box domains; however, it is not clear whether the two sFvs
bind to the same epitopes. The in vivo binding activity of
the 2R6-21 intrabody to the nascent hCyclinT1 was only slightly weaker
than 3R6-1; however, sFv 2R6-21 did not inhibit HIV-1 replication upon challenge in Jurkat T cells, although it did significantly suppress Tat-mediated transactivation in transfected Jurkat, Jurkat-Tat, and
SupT1 cells. It remains to be determined whether this lack of
inhibition of HIV-1 replication for sFv 2R6-21 is caused by its lower
in vivo binding affinity or its binding to a different epitope on hCyclinT1. It will be very interesting in future studies to
determine the exact binding sites for these sFvs to hCyclinT1. This may
shed further light on the one or more mechanisms by which these two
intrabodies differentially block Tat-mediated transactivation and HIV-1 replication.
The TRM motif lies at the extreme C terminus of the cyclin domain
(residues 250-265) and contains multiple residues that are critical to
the binding of the hCyclinT1·Tat complex to TAR RNA (18). The TRM
motif makes independent contact with Tat and TAR RNA, especially
residues 259 (arginine) and 261 (cysteine) that are important for
hCyclinT1-Tat interaction, which requires Zn2+ ions. In the
current study, efforts were made to identify TRM-specific sFvs using
the purified TRM-containing hCyclinT1-300 polypeptide for screening
the phage display library. Three sFv clones were found that bound
specifically for the cyclin box domains. However, no binders for the
TRM were identified suggesting that the TRM may be masked by
neighboring residues in the hCyclinT1-300 polypeptide that might
prevent phage sFvs from binding, even though this region has a
predicted high hydrophilicity index suggesting that it should be
exposed to the aqueous phase. To overcome this problem, we used a
biotinylated 20-amino acid polypeptide that contains the major part of
the TRM (amino acids 250-266) to screen the sFv-phage library and
identified two TRM-specific sFv clones. SFv S84 was identified using
streptavidin beads to capture the polypeptide-bound phage. This sFv had
very low binding affinity to denatured hCyclinT1-300 in the ELISA
assay, showed good binding activity to the nascent hCyclinT1, and
yielded mild inhibition to the HIV-1 Tat-mediated transactivation in
Jurkat and Jurkat-Tat T cells but did not inhibit HIV-1 replication
upon challenge. This may be due to its low binding affinity to
endogenous hCyclinT1. The other clone, PS11, was identified using TRM
peptide-coated immunotubes, and this sFv had a much higher binding
affinity to denatured hCyclinT1-300 than S84, however, it only had
weak binding activity to nascent hCyclinT1 and showed no inhibition to
Tat transactivation. Although the one or more reasons for lack of a
significant inhibitory effect of these two TRM-specific sFvs on
Tat-mediated transactivation or HIV-1 replication are not clear, it has
been previously demonstrated that synthetic TRM peptides failed to form
a specific complex with Tat and TAR, as did other fragments of
hCyclinT1 lacking small regions of the cyclin box domain (18). These
results suggest that maintenance of the natural conformation of the TRM
in hCyclinT1 is necessary for Tat·TAR recognition by hCyclinT1 and
this may also be important for isolating high affinity TRM-specific
sFvs. Strategies that will allow us to better expose the TRM motif in
hCyclinT1 and to simultaneously maintain its natural conformation will
be important in future attempts to obtain high affinity human sFvs to
completely epitope map that surface of hCyclinT1. These anti-hCyclinT1
sFvs can then be used for in vitro biochemical studies and
in vivo animal studies to better understand how to more
efficiently inhibit Tat transactivation and HIV-1 replication.
A priority for anti-HIV-1 gene therapy using intracellular
immunization-based strategies is to reconstruct the defective immune system with cells that will be genetically resistant to HIV-1 infection
and/or replication. Infusion of transduced autologous CD4+ T-cells
expressing anti-HIV-1 resistance genes is the most immediate way to
demonstrate proof-in-principle that the transduced cells have a
survival advantage in vivo in HIV-1-infected individuals. However, pluripotent hematopoietic CD34+ stem cells are the
best choice for the delivery of therapeutic constructs into humans long
term, because these cells are self-renewing and can differentiate into
multiple lineages of hematopoietic phenotypes, including HIV-1 targets
T lymphocytes, macrophages, and dendritic cells that will aid in
reconstituting severely damaged hematopoietic compartments. In
addition, transduced CD34+ stem cells can provide sustained
transgene expression in vivo for prolonged periods of time
(29). The SCID-hu mouse model has been recognized as a unique in
vivo system (30) to investigate HIV-1 pathogenesis (31, 32) and
latent infection (33, 34) and is useful for the pre-clinical evaluation
of the biological effects of anti-HIV-1 constructs on stem cell
differentiation (35-37). In the current study, we have successfully
isolated a high affinity anti-hCyclinT1 sFv clone (3R6-1) that can
specifically bind to its cellular target protein in vivo and
have a significant inhibitory effect on HIV-1 replication without
apparent toxicity. Our results indicate that the sFv 3R6-1 clone could
be very useful for HIV-1 gene therapy studies. Accordingly, it will be
important to assess the in vivo biological effects of 3R6-1
and other new anti-hCyclinT1 intrabodies on stem cell differentiation
and HIV-1 replication in this small animal model as a first step in our pre-clinical studies.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-HA (Fig. 5A).
-D-galactopyranoside was
added at 0.5 mM to induce the expression of GST fusion
proteins at 30 °C with vigorous shaking for 3-5 h. The bacterial
pellets were resuspended in 50 ml of cold PBS containing protease
inhibitor cocktails (Roche Molecular Biochemicals) and sonicated for 3 min in an ice bath. After centrifugation, the clear protein
supernatants were mixed with pre-washed glutathione-Sepharose beads for
30 min. The column bed was then washed in cold PBS, and GST fusion proteins were eluted with 10 mM reduced glutathione
solution and stored at 4 °C for ELISA assay or immunoprecipitation.
For the isolation of GST-free hCyclinT1-200, -250, and -300 polypeptides, 1 ml of thrombin/PBS was added to the Sepharose bed.
Cleaved hCyclinT1 polypeptides were collected by draining the column
and washing with PBS. Protein concentrations were determined by the
Bio-Rad protein assay.
-HA
(8 µg) and transferred to a sterile chamber followed by
electroporation. The transfected cells were cultured at 37 °C for
48 h to allow cells to recover, and, subsequently, the cells were
selected in growth medium containing G418 (1 mg/ml) for 3 weeks to
derive G418-resistant cells.
70 °C for p24 assay.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The molecular structure of the human cyclinT1
(hCyclinT1). The N-terminal 272 amino acids participate in the
HIV-1 Tat-mediated transactivation. The C-terminal region is predicted
to participate in basal and Tat-stimulated HIV transcription through
interaction with Tat-SF1 and recruitment of Tat-SF1-U snRNP
spliceosomes to RNA polymerase II (11).
1-
5,
helix motifs; TRM,
Tat·TAR recognition motif. The arrowhead indicates the
point mutation of a key amino acid (C261Y) in mouse cyclin T1 that
abolishes the molecular interaction between mouse cyclinT1 and HIV-1
Tat (10, 18). Asterisks highlight the sequence differences
of the TRM motif between mouse and human cyclinT1 proteins.
segments) of VL with one amino acid substitution in
CDR2 and two substitutions in CDR3 that might account for their differential binding affinities in vitro and in mammalian
cells described below. In addition, although PS11 and S84 were specific to the TRM motif, their amino acid sequences were very different.
Heavy and light chain regions
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Fig. 2.
Purification of anti-hCyclinT1 sFv proteins
from bacterial cells. A, prokaryotic expression vector
pSyn1. SFv inserts were isolated from the pFarber phage display vectors
by NcoI and NotI digestion and ligated into pSyn1
vectors cleaved with the same restriction enzymes. SFv proteins
expressed from transformed XL-1 Blue were C-terminally tagged with Myc
and six histidines to facilitate affinity purification on nickel-bound
chelating Sepharose column as described under "Experimental
Procedures." B, His tag-purified 30-kDa anti-hCyclinT1
sFvs were analyzed in a 10% SDS-PAGE gel and stained with Coomassie
Brilliant Blue. The amounts of each protein sample loaded are
indicated.
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Fig. 3.
Epitope mapping of anti-hCyclinT1 sFvs.
A, prokaryotic expression constructs used for producing
GST-hCyclinT1 fusion proteins that contained 200, 250, and 300 amino
acids of the hCyclinT1 N-terminal region. GST and GST fusion proteins
were purified using a glutathione-Sepharose 4B column. B,
epitope mapping by ELISA. Microtiter plates were coated with 10 µg/ml
GST and GST-hT1 fusion proteins overnight at pH 9.0. Duplicate wells
were incubated for 1 h at room temperature with varying
concentrations of purified His-tagged sFvs followed by incubation with
rabbit anti-His antibody (1:500) as primary antibody and horseradish
peroxidase (HRP)-labeled goat anti-rabbit IgG (1:25,000) as secondary
antibody. The optimal concentrations of sFvs used in this assay were
pre-determined and chosen to be above their lowest detectable level of
binding (end point binding activity) to avoid nonspecific reactions
that could occur due to the use of high concentrations of sFv proteins.
3R6-1 and 2R6-21: 0.05 µg/ml; 2R6-113 and PS11: 0.5 µg/ml; S84: 5 µg/ml. C, determination of binding properties for
anti-hCyclinT1 sFvs. Microtiter plates were coated with purified
GST-free hCyclinT1-300 polypeptide (20 µg/ml) overnight. Binding of
the different concentrations of purified His-tagged sFvs (0.033-1320
nM) to the plates was determined.
8 M)
was 1.5-fold higher than 3R6-1 (KD = 1.07 × 10
7 M) and 11-fold higher than 2R6-113
(KD = 7.94 × 10
7 M).
Because 2R6-21 and 3R6-1 share identical heavy chains, this result is
consistent with the known finding that the majority of binding energy
is often supplied by VH CDR3 and different VL chains can variably contribute to the overall energy of binding. Although S84 gave rise to positive binding signals at 82.5-660 nM concentrations in ELISA (Fig. 3C), it only
showed background binding to hCyclinT1 in the Biacore assay even at
much higher concentrations (330-3300 nM, data not shown),
indicating that the Biacore assay had lower sensitivity than the ELISA
assay. The binding parameters for PS11 were not measured using Biacore, however, the ELISA binding profile for PS11 is similar to that for sFv
2R6-113, suggesting that these two sFvs have similar binding affinities. Taken together, the binding affinity rank for these five
anti-hCyclinT1 sFvs was determined to be 2R6-21 > 3R6-1 > 2R6-113
PS11
S84.
Affinity binding constants for anti-hCyclin T1 sFVs as determined by
Biacore analysis
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Fig. 4.
Immunoprecipitation of human cyclinT1 by
purified anti-hCyclinT1 sFvs. GST-hT1-300 (20 µg) was incubated
with each His-tagged sFv (5 µg) in 500 µl of binding buffer (PBS
supplemented with 0.1% bovine serum albumin and 0.5 M
NaCl) for 2 h at room temperature followed by incubation overnight
at 4 °C with 20 µl of microbeads containing 40 µg of anti-His
mAbs. The microbeads were pre-blocked with 5% milk/binding buffer for
1 h at room temperature before being added to the binding
reaction. After serial washes, sFv/GST-hT1-300 immune complexes pulled
down by anti-His mAb-conjugated microbeads were analyzed on 10%
SDS-PAGE followed by detection for precipitated GST-hT1-300 by Western
blot using HRP-conjugated mouse anti-GST mAbs.
-HA fusion
proteins from the CMV promoter (Fig.
5A). As shown in Fig. 5B, the ~43-kDa anti-hCyclinT1 sFv-C
-HA-tagged fusion
proteins were all expressed at comparable levels in transfected 293T
cells except for PS11, which was expressed at a lower level.
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Fig. 5.
The expression of anti-hCyclinT1 intrabodies
in mammalian cells. A, eukaryotic vectors (psFv-C -HA) for
expressing anti-hCycinT1 intrabodies. SFv inserts from the pFarber
phage display vector were isolated by SfiI and
NotI digestion and ligated into the vector to create sFvs
that are fused to the human Ig
chain constant region to increase
intracellular folding and binding affinity (23). B,
anti-hCyclinT1 intrabody expression vectors were transfected into 293T
cells by using Qiagen Polyfect reagent and 24 h post-transfection,
cells were lysed with 1 ml of lysis buffer (20 mM Hepes,
pH.7.9, 0.5 M NaCl, 1% Nonidet P-40, 5 mM
EDTA, 1 mM dithiothreitol, and 1 mM
phenylmethylsulfonyl fluoride). The cell lysates (20 µl) were
analyzed on 10% SDS-PAGE followed by Western blot with rabbit anti-HA
primary antibody and HRP-conjugated goat anti-rabbit IgG secondary
antibody. The sFv fusion proteins are ~43 kDa.
-HA intrabodies
expressed in mammalian cells could bind to nascent cellular hCyclinT1,
co-transfection and co-immunoprecipitation experiments were performed.
An irrelevant sFv-C
-HA intrabody (A3H5) served as a negative
control. A full-length hCyclinT1-726 expression vector was modified to
produce a C-terminal His-tagged hCyclinT1 protein (hCyclinT1-His) for
these studies (8). Fig. 6A
shows that all five anti-hCyclinT1 intrabodies and the A3H5 negative control were expressed at similar levels in transfected 293T cells, although PS11 is again expressed at lower levels. In addition, the
hCyclinT1-His expression levels in the total cell lysates among the
co-transfected 293T cells were very similar. As shown in Fig.
6B, the anti-hCyclinT1 sFv-C
-HA intrabodies were also co-precipitated by goat anti-His IgG-conjugated agarose beads (Santa
Cruz Biotechnology, Santa Cruz, CA) under conditions in which about
98% of the hCyclinT1-His was precipitated (data not shown). HCyclinT1
heterodimerizes with its partner Cdk9 to form the P-TEFb complex, which
is associated with the 7SK snRNA (4). The precipitated immune
complexes also contained Cdk9 molecules, indicating that the P-TEFb
complex was present and, importantly, that the anti-hCyclinT1
intrabodies did not disrupt the heterodimerization between hCyclinT1
and Cdk9. Also shown in Fig. 6B is that different amounts of
anti-hCyclinT1 intrabodies were bound to co-expressed hCyclinT1-His and
that the negative control sFv A3H5 showed no interactions with
hCyclinT1-His, indicating that the co-immunoprecipitation was specific.
Based on the signal intensities of the bound anti-hCyclinT1 intrabodies
to hCyclinT1-His, the in vivo binding affinities of these
anti-hCyclinT1 sFvs ranked 3R6-1 > 2R6-21 > 2R6-113
S84 > PS11. For reasons that are not clear, in
co-immunoprecipitation studies with anti-HA antibodies we were unable
to effectively pull down the cellular components of the p-TEFb complex
(data not shown).
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Fig. 6.
Co-immunoprecipitation of nascent human
cyclinT1 by anti-hCyclinT1 intrabodies expressed in mammalian
cells. 293T cells were seeded overnight prior to DNA transfection.
Each psFv-C -HA construct (4 µg) and an equal amount of pCI-86(His)
that directed the expression of C-terminally His-tagged human cyclinT1
were co-transfected into 293T cells in 10-cm dishes by Polyfect reagent
(Qiagen). 24 h post-transfection, total cell lysate was made with
1 ml of lysis buffer in the presence of protease inhibitors.
A, detection of anti-hCyclinT1 intrabodies and human
cyclinT1 co-expressed in co-transfected 293T cells. Total cell lysates
(20 µl) from each transfection were analyzed by Western blot. Rabbit
anti-HA primary antibodies and HRP-labeled goat anti-rabbit IgG
secondary antibody were used to detect sFv-C
-HA intrabodies and goat
anti-His primary antibodies, and HRP-labeled mouse anti-goat IgG mAbs
were used to detect human cyclinT1 proteins, respectively.
B, co-immunoprecipitation of human cyclinT1-His and
anti-hCyclinT1 intrabodies with anti-His mAbs. Cell lysate (200 µl)
was mixed with 30 µl of mouse anti-His IgG-conjugated agarose beads
that were pre-blocked with 2% milk/lysis buffer for 30 min on a
rocking platform, and the mixtures were rocked overnight at 4 °C
followed by three washes with the lysis buffer and two washes with PBS.
The beads were then resuspended into 60 µl of 1× protein loading
buffer containing 100 mM dithiothreitol, boiled for 5 min,
and analyzed on 10% SDS-PAGE followed by Western blotting for
co-immunoprecipitation of hCyclinT1, sFv-C
-HA, and Cdk9 with
different antibodies against hCyclinT1 and sFv-K-HA as described in
A. The human Cdk9 was detected by rabbit anti-human Cdk9
primary antibody and HRP-labeled goat anti-rabbit secondary
antibody.
-galactosidase
expression vector driven by the SV40 promoter was used for normalizing
the transfection efficiency. Only the three of the anti-hCyclinT1
intrabodies with the strongest in vivo binding affinities
had inhibitory activity. When compared with the irrelevant negative
control A3H5, the sFv 3R6-1 and 2R6-21 intrabodies showed 80 and 60%
reduction in Tat-mediated transactivation in Jurkat T cells, whereas
sFv S84 displayed only 28% inhibition (Fig.
7A). In SupT1 cells, 3R6-1 and
2R6-21 showed 71 and 52% inhibition of transactivation but
transactivation was increased with S84 compared with the A3H5 control
(Fig. 7B). Stable Jurkat-Tat cells were also co-transfected
with the anti-hCyclinT1 sFvs and pHIV-1 LTR-luc (19). As shown in Fig.
7C, the anti-hCyclinT1 intrabodies 3R6-1, 2R6-21, and S84
yielded 73, 67, and 20% reduction in Tat transactivation compared with
the A3H5 control, a result consistent with the results obtained with
Jurkat and SupT1 cells (Fig. 7, B and C). The
anti-hCyclinT1 intrabodies 2R6-113 and PS11 showed no significant
inhibition to Tat activation with any of the three cell lines tested
(data not shown).
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Fig. 7.
Inhibition of HIV-1 Tat-mediated
transactivation in human T cell lines by anti-hCyclinT1
intrabodies. Human Jurkat T cells (A) and SupT1 T cells
(B) were transiently co-transfected with three
anti-hCyclinT1 psFv-C -HA intrabody constructs (3R6-1, 2R6-21, and
S84, 5 µg each) together with pSV-tat (25 ng) and pHIV-1-LTR-luc (25 ng) by using Qiagen SuperFect reagent. HIV-1 Tat expressed from pSV-tat
vector transactivated the reporter gene luciferase expression through
HIV-1 LTR. A pSV-
-gal vector (1 µg) was included in each
transfection experiment to normalize the transfection efficiency. The
irrelevant sFv A3H5 was used as a negative control. 24 h
post-transfection cell lysate was made with 1 ml of cell culture lysis
buffer and 50 µl of lysate was used to perform a luciferase assay
according to the manufacturer's instruction (Promega). The luciferase
activity for the negative control sFv A3H5 construct was set to 100%,
and the percentage of luciferase activities for each anti-hCyclinT1
intrabody construct was calculated accordingly. C, the
transfection was conducted similarly except that Jurkat Tat cells
stably express HIV-1 Tat proteins (19), therefore, the pSV-tat vector
was omitted.
-HA intrabodies 3R6-1, 2R6-21, and S84 as well as the negative
control A3H5 showed no toxic effects on Jurkat- and SupT1-stable
transfectants (data not shown). The bulk populations of stable Jurkat
transfectants were infected with HIV-1IIIB (50 units of
TCID50 per million cells) and cultured for 3 weeks. The
control A3H5 and S84 transfectants showed extensive large syncytia
formation on days 14-19 post-infection. The 2R6-21 transfectants also
displayed syncytia formation but to a lesser extent, indicating that
anti-hCyclinT1 2R6-21 and S84 did not protect infected Jurkat cells
from cytocidal effects. The absence of inhibition of HIV-1 replication
was not caused by the loss of 2R6-21 or S84 intrabody expression based
on Western blot analysis (data not shown). In contrast, 3R6-1
transfectants showed no syncytia formation and were morphologically
normal during the 21-day challenge period (data not shown). The results
of p24 antigen assays for the HIV-1 challenge experiments with 3R6-1 and control A3H5 are shown in Fig.
8A. Virus production from A3H5 transfectants was detected first on day 8 post-infection (182 pg/ml) and rapidly reached to the peak level of 347 ng/ml on day 21 post-infection. However, the viral replication in 3R6-1
transfectants was significantly inhibited and was below a detectable
level on day 11. Subsequently, the viral replication remained markedly inhibited on day 21 post-infection (11 ng/ml, p24), the latest day
post-infection that was examined, which represented ~97% reduction in the viral production compared with A3H5 control cells. When 3R6-1,
2R6-21, S84, or A3H5 control transfectants were infected with a 50-fold
higher dose of HIV-1 IIIB (2500 TCID50 per
million cells), massive syncytia formation, severe cytopathic effects, and cell death were seen on day 7 post-infection indicating that the
infection doses were too high to identify any differences in viral
infection between the 3R6-1 and other anti-hCyclinT1 and control
intrabody-expressing transfectants.
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Fig. 8.
In vitro HIV-1 challenge of Jurkat
and SupT1 cells stably expressing anti-hCyclinT1 intrabodies.
Jurkat or SupT1 transfectants were split 48 h before challenge.
Cells were washed in PBS and resuspended into 1 ml of viral dilution
medium containing T-tropic HIV-1 BIII virus and incubated
at 37 °C for 2 h. Subsequently, the cells were washed and
resuspended into 6 ml of growth medium supplemented with 10% fetal
calf serum and 100 units of penicillin and streptomycin. Each set of
infected cells was cultured in two wells in six-well plates. The medium
was harvested at intervals of 2-3 days and stored at 70 °C for
p24 assays. The infected cells were split simultaneously by 1:2
dilution. In A, Jurkat T cells were infected with HIV-1
IIIB at 50 units of TCID50. In B-D,
SupT1 cells were infected with 2500 units of TCID50.
A3H5, negative control intrabody; 3R6-1,
anti-hCyclinT1 intrabody. nls (sFv-C
-NLS-HA) and
nol (sFv-C
-NOL-HA) represent intrabodies containing
nuclear localization and nucleolar localization signals,
respectively.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-galactosidase activity. The expression
of these sFvs did not promote apoptosis of Jurkat and SupT1-stable
transfectants and did not disrupt hCyclinT1-Cdk9 interaction indicating
that the P-TEFb functions were not affected. Upon HIV-1 challenge of stably transfected Jurkat and SupT1 cells, the 3R6-1 intrabodies that
displayed the highest binding affinity to nascent hCyclinT1 not only
significantly reduced the cytopathic effects of HIV-1 BIII
but also markedly suppressed viral replication in infected Jurkat T
cells at a lower challenge dose and achieved 71-88% inhibition to
HIV-1 replication even at higher infection doses in SupT1 cells. These
results provide a proof of concept that targeting Tat-hCyclinT1 interaction by anti-hCyclinT1 intrabodies is an effective approach for
HIV-1 gene therapy that specifically disrupts the molecular interaction
between HIV-1 Tat and hCyclinT1.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. Peterlin, who kindly provided us with GST-hT1 expression vectors, Dr. Kathy Jones, who kindly provided hCyclinT1 pCI-84 vector, and the National Institutes of Health AIDS Research and Reference Reagent Program that provided Jurkat and SupT1 cell lines and HIV-1 virus strains.
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FOOTNOTES |
---|
* This work was supported by a National Research Service Award and Center for AIDS Research Development Award (to J. B.), by Grants 5R01-RR14447 and 2R01-AI28785 from the National Institutes of Health (to W. A. M.), and by a joint Dana-Farber Cancer Institute-Beth-Israel Deaconess Medical Center and Children's Hospital Center for AIDS Research grant. W. Marasco has a financial interest in Abgenix.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Cancer Immunology and AIDS, Dana-Farber Cancer Institute, JFB# 824, 44 Binney St., Boston, MA 02115. Tel.: 617-632-2153; Fax: 617-632-3113; E-mail: Wayne_Marasco@DFCI.Harvard.Edu.
Published, JBC Papers in Press, October 24, 2002, DOI 10.1074/jbc.M208297200
2 S. Mehta, Y. Wang, A. St. Clair Tallarico, and W. A. Marasco, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are: HIV, human immunodeficiency virus; hCyclinT1, human cyclinT1; Cdk9, cyclin-dependent kinase 9; P-TEFb, positive transcription elongation factor complex; TAR, transactivation response element; TRM, Tat·TAR recognition motif; LTR, long terminal repeat; sFv, single-chain antibody; mAb, monoclonal antibody; HA, hemagglutinin; HRP, horseradish peroxidase; GST, glutathione S-transferase; PBS, phosphate-buffered saline; ELISA, enzyme-linked immunosorbent assay; CMV, cytomegalovirus; NLS, nuclear localization signal; NOL, nucleolar localization signal; snRNA, small nuclear RNA; TCID50, 50% tissue culture infectious dose.
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REFERENCES |
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