Download TAR-RNA binding by HIV-1 Tat protein is

2886–2890 Nucleic Acids Research, 1998, Vol. 26, No. 12
 1998 Oxford University Press
TAR-RNA binding by HIV-1 Tat protein is selectively
inhibited by its L-enantiomer
Anna Garbesi, François Hamy1,*, Mauro Maffini+, Geneviève Albrecht1 and
Thomas Klimkait§
Consiglio Nazionale delle Ricerche, I. Co. C.E.A., Bologna, Italy and 1Novartis Pharma Research,
Department of Oncology, K-125 3.09, CH-4002 Basle, Switzerland
Received March 17, 1998; Revised and Accepted April 28, 1998
ABSTRACT
An oligoribonucleotide, corresponding to the Tat-interactive top half of the HIV-1 TAR RNA stem–loop, was
synthesized in both the natural D- and the enantiomeric
L-configurations. The affinity of Tat for the two RNAs,
assessed by competition binding experiments, was
found to be identical and is reduced 10-fold for both,
upon replacement of the critical bulge residue U23 with
cytidine. It is suggested that this interaction of the
flexible Tat protein depends strongly upon the tertiary
structure of a binding pocket within TAR, but not upon
its handedness, and may be described by a ‘handin-mitten’ model.
INTRODUCTION
The HIV-1 Tat protein, required for efficient virus production,
stimulates the elongation efficiency of RNA polymerase II (1).
Moreover, Tat can be released from acutely infected cells as a
biologically active protein (2), which, as indicated by a substantial
body of evidence, contributes to the pathogenesis of AIDSassociated diseases (3) like Kaposi’s sarcoma, and is suggested to
participate in the induction of apoptosis even in uninfected
lymphocytes (4). Because of the pleiotropic role played by this
viral protein, any Tat ligand endowed with good affinity and
specificity could be valuable both as a research tool and as a
potential lead compound for therapeutical application.
The primary mechanism of Tat activation of HIV-1 replication is
mediated by its binding to a stem–loop region (TAR) present near
the 5′ terminus of all retroviral mRNAs (5,6). Details of the
stoichiometric binding of Tat to TAR have become available
which define a trinucleotide bulge within TAR together with
flanking base pairs to form a unique binding pocket (7–11). Given
the very tight association of the Tat–TAR complex (Kd of 1–3
nM), a TAR decoy, i.e. a short synthetic RNA, which features the
wild-type sequence of the Tat binding site, should have the
potential to function as an efficient and specific ligand for the viral
Tat protein. In fact, this strategy has been applied successfully to
inhibit HIV-1 replication in cells transduced with retroviral
vectors and overexpressing the TAR sequence (12,13). Since our
initial aim was to develop a Tat ligand that could be delivered to
cells in culture and bind both intracellular and secreted protein, a
native TAR decoy would have been unsuitable, given the well
known instability of the phosphodiester backbone in biological
media (14). The so called ‘mirror-image’ DNAs and RNAs are
probably the most nuclease-resistant oligonucleotide analogs
presently known (15). These compounds are also called enantioor L-nucleic acids, since they contain, in place of the natural
D-ribose, its enantiomer L-ribose. We (16) and others (17–22)
have already evaluated their potential as antisense and antigene
agents for the control of gene expression, but, with the exception
of highly biased sequences (20–22), they do not anneal to natural
nucleic acids (23). To our knowledge, a potential interaction of
L-DNAs and -RNAs with nucleic acid binding proteins has never
been reported, we tested the L-enantiomer of a shortened form of
the native TAR sequence for its affinity to the viral protein.
According to a generally accepted view (24), the structural
complementarity between natural nucleic acids and proteins,
i.e. D-nucleic acid forming complexes with L-proteins (as D–L),
would imply a highly reduced affinity of the diastereoisomeric
complexes L–L and D–D. Nevertheless, aside from this inference,
we took into account the following considerations to support our
working hypothesis: (i) the structural complementarity between
D-nucleic acids and L-proteins, an extension of the ‘key and lock’
analogy (25), coined by Emil Fisher in 1894 to illustrate enzyme
specificity, is considered particularly compelling when the
enzyme substrate is a chiral molecule. However, exceptions to the
enantiospecific behavior of enzymes have been described for the
phosphotransferases of carrot and human prostate acting on
uridine (26), and for HSV-1 TK and human dCK which
phosphorylate efficiently both enantiomers of thymidine and
2′-deoxycytidine, respectively (27,28). Moreover L-form nucleosides have been described as RT inhibitors and anti-HIV drugs
(29). These findings can be interpreted by Koshland’s ‘induced
fit’ theory of enzyme activity, which, while maintaining the
*To whom correspondence should be addressed. Tel: +41 61 6965093; Fax: +41 61 6967096; Email: [email protected]
Present addresses: +Pro. Bio. Sint., 20/22 Via Valverde, 21100 Varese, Italy and §Deptartment of Molecular Biology, Institute of Medical Microbiology,
University of Basle, 10 Petersplatz, CH-4003 Basle, Switzerland
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Fisher’s concept of shape complementarity of enzyme and
substrate, introduced the idea of enzyme flexibility, whereby the
mutual fitting of the interacting partners is described by the
‘hand-in-glove’ metaphor (30). (ii) The idea that natural nucleic
acids have generally higher affinities for L- than for D-proteins
had already been experimentally disproved in at least two cases,
in which L- or D-polypeptides were shown to bind nucleic acids
with equal affinities (31,32). Moreover, while the present study
was progressing, it was reported that the L- and D-enantiomers of
a 36 amino acid peptide, which contains the RNA-binding
domain of Tat, bind TAR RNA with very close affinities (33).
(iii) The interaction of the M13 ssDNA binding protein with
D-(dA)6 and L-(dA)6 is merely selective for the chirality of the
sugar-phosphate backbone (A.Garbesi, unpublished observations).
(iv) It has been shown that the RNA binding domain of HIV Tat
forms in solution an extended, unstructured loop (34). Upon
contact with the flexible TAR-RNA (35), both interacting
molecules assume specific structural rearrangements, which lead
to the observed tight complex. Hence, the high stability of the
Tat–TAR complex can be seen as the end point of a process of
mutual fitting and refolding, rather than as the result of a built-in
shape complementarity between the interacting partners. This
view, reminiscent of the ‘induced-fit’ model for enzyme–substrate
interactions, led us to the idea that the very flexible arginine-rich
Tat domain could recognize the ‘moldable’ contact site of TAR,
independently of its particular chiral sense.
2887
Figure 1. (a) Secondary structure of TAR33 RNA. The two terminal GC base
pairs at the bottom of the stem are included for enhanced stability. A bold ‘T’
at the 3′ terminus indicates that this residue for synthetic reasons is always in
D-form. Underlined bases are involved in the formation of a Tat-binding pocket.
An inactivating U→C mutation at position 23 is indicated. (b) Thermal
denaturation profile of both enantiomeric forms of TAR33.
for ∼30 min, allowed to cool slowly to room temperature, then
kept at 4C for a few hours, before starting the melting
experiments. The half-melting temperature was determined from
the first derivative of the denaturation curve.
CD experiments
MATERIALS AND METHODS
Oligoribonucleotides synthesis
All the oligonucleotides used in this work were synthesized by
solid phase on a Pharmacia Gene Assembler II plus using
standard phosphoramidite method at 0.2 and 1 µmol scales. The
D-amidites were purchased from ABI, while L-amidites were
prepared as follows. The L-nucleosides were obtained from
L-arabinose by epimerization to L-ribose, followed by conversion
in three steps to 1-O-acetyl-2,3,5-tri-O-benzoyl-β-ribufuranoside
(36), and reaction with in situ silylated N-protected bases (37).
After complete deprotection, the nucleosides were protected at
their exocyclic amino group with benzoyl for adenosine and
cytidine, and with isobutyryl for guanosine (38). The base-protected
nucleosides were finally converted in three steps to their
5′-O-terbutyldimethylsylyl-3′-O-(2-cyanoethyl-N,N′-diisopropyl)phosphoramidites following usual procedures for D-isomers
(39,40). Removal of the protecting groups was performed as
described by Sproat et al. (41). The crude oligonucleotides were
purified by anion exchange HPLC on a Waters 600E HPLC with
a DEAE 5PW column, at 30C, flow rate 1 ml/min and gradient:
0.00 min, 100% buffer A; 40 min, 60% buffer A (buffer A:
20 mmol LiClO4, 20 mmol NH4OAc in H2O/CH3CN 90:10;
buffer B: 600 mmol LiClO4, 20 mmol NH4OAc in H2O/CH3CN
90:10).
UV experiments
UV melting experiments were conducted on a Perkin-Elmer 554
spectrophotometer equipped with a MGW Lauda thermostat and
a MGW Lauda R40/2 digital thermometer. An electronic device
was used to generate a linear temperature gradient (0.6C/min).
The oligoribonucleotide was dissolved in a buffer containing 0.1 M
Tris–HCl, 0.1 M NaCl at pH 7.0; the solution was heated at 90C
Annealing of the oligoribonucleotides was performed as described
for UV melting experiments. CD spectra of the annealed
oligoribonucleotides were recorded with a Jasco J700A spectropolarimeter in 0.1 M NaCl and 0.1 M Tris–HCl buffer, pH 7.0, at
room temperature.
Gel-shift experiments
Recombinant Tat protein was prepared and binding assays were
carried out as previously described (9). 33mer D-TAR oligoribonucleotide was labeled using [γ-32P] (3000 Ci/mmol) (Amersham,
UK) and T4 Polynucleotide kinase (New England Biolabs,
Beverly, MA). Competition binding assays (25 µl) included 0.1%
Triton X-100, 50 mM Tris–HCl (pH 8.0), 20 mM KCl, 0.1 M DTT,
12.5 nM 32P-labelled 33mer D-TAR RNA (20 000 c.p.m.),
15–20 nM Tat protein and various concentrations of unlabelled
competitor RNA. Binding reactions were incubated on ice for
15 min, and non-denaturing PAGE was carried out at 4C (7.2%
gel, 20 × 20 cm) in 0.5× TB (TB = 44.5 mM Tris base, 44.5 mM
boric acid), 0.1% Triton X-100 for 1 h at 200 V. Gels were dried
and complexes detected by autoradiography. Relative competition
constants were calculated after densitometrically scanning the
resulting autoradiographs using a Biorad GS-700 imaging
densitometer.
RESULTS
Chemical synthesis and characterization of
TAR-oligoribonucleotides
All sequences were obtained by solid-phase synthesis using D- or
Due to the use, in all syntheses, of a
commercial support loaded with D-thymidine, two unpaired
nucleotides are present at the 3′ terminus. The penultimate residue
has the same chirality of the main sequence and was introduced
L-phosphoramidites.
2888 Nucleic Acids Research, 1998, Vol. 26, No. 12
a
Figure 2. CD (top) and absorption (bottom) spectra of equimolar solutions of
L- and D-TAR33
to avoid skeletal deformation in the bottom part of the stem of the
L-TAR RNAs. Sequence and characteristic features are depicted
in Figure 1a. The final sequences were characterized by UV
melting experiments and CD spectroscopy. The half-melting
temperatures, obtained from the corresponding thermal denaturation
profiles (Fig. 1b), were in both cases in the range of 71–72C. As
shown in Figure 2 for the wild-type sequences with optimal
intramolecular base-pairing (i.e. annealing as described in
Materials and Methods), the CD spectrum of a D-RNA is the
mirror image of that of the L-RNA at the same concentration
(i.e. having an identical UV spectrum), which demonstrates their
enantiomeric relationship.
Tat binds D- and L-TAR with the same apparent affinity
and discriminates mutants
As a model of the natural TAR sequence (59 residues), we used, as
described by others (6–10), a shorter synthetic oligoribonucleotide
(Fig. 1a), corresponding to the top half of the TAR RNA
stem–loop (TAR33). This shortened version of TAR, which
includes two unnatural GC basepairs at the terminus to increase
stability, has been shown previously to bind Tat protein with only
a slight reduction in apparent affinity (20 nM) when compared
with that of the full-length 59mer TAR RNA.
Figure 3a shows the results of a competition binding experiment
using a fixed concentration of Tat protein, 32P-labelled D-TAR33
RNA and increasing concentrations of unlabelled TAR-RNA
wild-type and U23→C mutant from both L- and D-series. As
suggested by a similar decrease in intensity of the retarded RNA
band, as densitometrically quantified in Figure 3b, both the L- and
D-forms of TAR33 wild-type RNA were equally efficient as
competitors for D-TAR–Tat complex formation. As a control for
Tat binding, a 30mer L-RNA, which carries a deletion of the
U-rich bulge (L-TAR∆U) and hence disrupts the Tat binding site, was
prepared. This RNA was unable to compete for Tat binding to
TAR33 RNA (not shown) up to concentrations of at least 500 nM.
This demonstrates clearly that both TAR RNAs from the L- and
D-series provide a suitable binding site for Tat. In order to study
the selective interaction between Tat and L-TAR RNA further, we
introduced a more subtle change into synthetic L- and D-RNAs.
b
Figure 3. (a) Competition gel shift for the Tat–L-TAR complex. Control lanes,
‘-Tat’ includes 15 nM of 32P-labeled D-TAR33 RNA; in the ‘0’ controls and
throughout the competition lanes, Tat is present at a constant concentration
(∼20 nM). In the adjacent lanes, cold competitor RNAs (as marked above the
autoradiographies) were co-incubated at indicated concentrations. (b) Competition
curves obtained by quantitative densitometric analysis of the gels shown in
(a). Shift intensity is plotted as a function of concentration of RNA competitor
as indicated.
Although we could demonstrate that Tat protein binds to
synthetic L-TAR with wild-type affinity, it seemed crucial to
confirm that the main contributor to this interaction was not just
an unselective protein–bulged-RNA binding. Therefore, we
performed competition binding experiments, this time comparing
L- and D-TAR as mutants, in which the uridine residue at position
23 was replaced by a cytidine residue (Fig. 1a). This point
mutation has been described to induce a significant reduction of
binding affinity (∼10-fold) (9). As expected, the mutant D-TARC23
showed an elevated D1/2 value, 10-fold greater than wild-type
D-TAR. The same difference in competition ability was observed
for the oligonucleotides of the L-series. We conclude that Tat does
not only recognize TAR RNA in both isomeric forms, but is also
able to discriminate the wild-type sequence and structure from
point-mutated bulge sequences in both the natural D- as well as the
non-natural L-RNA.
DISCUSSION
Very recently, Nolte et al. reported an elegant application of the
SELEX methodology, which led to the identification of L-oligo-
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nucleotides that interact with a nucleic acid binding peptide of
HIV Tat (42). This and other published examples have proven
that SELEX can be a powerful tool for the selection of nucleic
acid sequences having very high affinity for a variety of
biological molecules, including HIV-1 reverse transcriptase (43).
Nolte’s 38mer L-RNA, originally developed as a ligand for
L-arginine, is sequence-wise unrelated to TAR and binds to a short
peptide (12 residues) corresponding to the basic region of HIV-1
Tat protein with a Kd of 26 µM. In contrast, our competition
binding experiments demonstrate that the affinity of the mirrorimage (L-enantiomer) of wild-type TAR for the Tat protein is
1000-fold better, thereby matching the affinity of the D-sequence.
Moreover, our data show that in both cases the stability of the
complexes is significantly lowered upon changing the bulge
residue U23 of TAR RNA to cytidine, i.e. Tat is able to
discriminate a critical bulge mutation in both RNA enantiomers.
Thus, a central—and somewhat surprising—finding of our work
is that the selective binding of Tat to the TAR element is
unaffected by the chirality of the receptor RNA sequence. In
agreement with the principles of stereochemistry, which also
apply to chiral biopolymers (44), our experimental results show
that L- and D-TAR RNAs have the same secondary structure
(same thermal stability) and the same spatial folding but in
opposite direction (mirror-image CD spectra). This led to the
following inference: a similar thermal stability (same Tm values)
suggests an identical secondary structure (base pairing) of both
enantiomers. The mirror image of the CD-spectra indicates that
the spatial folding similarly is a mirror image. Thus, the logical
consequence of same sequence (primary structure), same base
pairing (secondary structure) and same, but mirror image, folding
(tertiary structure) of L- and D-TAR is that they engage in a
molecular interaction with the same intrinsic energy content.
In addition, at least the initial event in Tat–TAR recognition,
i.e. the fit of the Arg side-chain into the unique binding pocket
(formed by G26:C39, U23, and A22:U40) (35,45) and the
subsequent conformational RNA change (46), are not expected to
differ significantly from natural D-TAR to its mirror image
L-TAR. In our study, beyond this entropy-conserving step of
cation-π* van der Waals interaction, the peculiar observation that
the apparent affinity of the natural complex is retained was less
expected. Obviously, our results do not allow us to conclude that
every single electrostatic interaction between protein and RNA is
kept as such; nevertheless, the sum of binding energies is the same
for both enantiomers. This would imply a great ‘cost-neutral’
flexibility of the structure of Tat, since it can accommodate two
enantiomeric RNAs and still depend, for both, on the presence of
a unique binding pocket (discrimination of U23 mutants).
The findings reported here, combined with those recently
published by Huq et al. (33) on the high-affinity interaction of a
D-Tat-peptide with D-TAR-RNA, strongly support the view that
both partners in the Tat–TAR interaction are moldable. Thus,
rather than being modelled as a rigid key-lock, or even as a
hand-in-glove fitting, we suggest the Tat–TAR interaction to be
best represented as a ‘hand-in-mitten’, in which the thumb would
be the intrusion of an Arg side-chain in the binding-pocket of TAR,
with the rest of the hand (Tat) fitting, not in the exact same way, but
as well as, both right- or left-hand mitten (L- or D-TAR RNA).
Whatever the detailed structure of the Tat–L-TAR complex,
which could be further elucidated e.g. by NMR studies, the
present finding is, to the best of our knowledge, the first example
of a nucleic acid–protein interaction that depends strongly upon
2889
a specific tertiary structure of the binding pocket, but not upon its
particular chiral sense.
The apparent flexibility of the Tat structure invites the
speculation that it could be utilized by the viral protein to
alternatively act on other nucleic acid targets like cis-regulatory
DNA elements in the viral genome and/or of the genome of the
host. If such interactions were to be identified, they could shed
light on yet unexplained effects of Tat on the infected cell. Then,
beyond preventing the Tat–TAR trans-activation, the L-TAR
could be considered as a ‘multi-task’ decoy to divert the protein
from other potential function(s) and associated pathologies.
ACKNOWLEDGEMENT
A.G. dedicates this work to the memory of Prof. Antonino Fava.
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