(Received for publication, August 22, 1994; and in revised form, November 9, 1994)
From the
An oligonucleotide (I100-15) composed of only
deoxyguanosine and thymidine was able to inhibit human immunodeficiency
virus type-1 (HIV-1) in culture assay systems. I100-15 did not
block virus entry into cells but did reduce viral-specific transcripts.
As assessed by NMR and polyacrylamide gel methods, I100-15
appears to form a structure in which two stacked guanosine tetrads are
connected by three two-base long loops. Structure/activity experiments
indicated that formation of intramolecular guanosine tetrads was
necessary to achieve maximum antiviral activity. The single
deoxyguanosine nucleotide present in each loop was found to be
extremely important for the overall antiviral activity. The toxicity of
I100-15 was determined to be well above the 50% effective dose
(ED) in culture which yielded a high therapeutic index
(>100). The addition of a cholesterol moiety to the 3` terminus of
I100-15 (I100-23) reduced the ED
value to less
than 50 nM (from 0.12 µM for I100-15) and
increased the duration of viral suppression to greater than 21 days (versus 7-10 days for I100-15) after removal of
the drug from infected cell cultures. The favorable therapeutic index
of such molecules coupled with the prolonged suppression of HIV-1,
suggest that such compounds further warrant investigation as potential
therapeutic agents.
Oligonucleotides such as antisense molecules have been reported
to inhibit specific viral gene expression and hence decrease viral
production in culture assay systems. Originally, the central dogma
relating to antisense oligonucleotides was that they inhibit viruses by
interfering with the translation process via RNA:DNA duplex structure
formation by promoting RNase H activity and therefore represented a
``rational'' approach to drug design(1) . Recent
reports, however, indicate that a variety of possible mechanisms exist
by which oligonucleotides inhibit viral infections. For example,
oligodeoxycytidine, with a phosphorothioate (PT) ()internucleoside linkage (poly(SdC)), inhibited HIV-1
replication in culture(2, 3) . One potential mechanism
for this antiviral activity, competitive inhibition of HIV-1 reverse
transcriptase, was postulated by Marshall et al.(2) .
Poly(SdC) was also reported to inhibit avian myeloblastosis virus
reverse transcriptase, Pol I (Klenow fragment), and human polymerase
(2, 4) ,
, and
(4) . Gao et
al.(5) reported that PT-containing oligonucleotides
inhibited both human DNA polymerases and RNase H. Matsukura et al.(3) , using poly(SdC), showed that the inhibition of HIV-1
in culture was dependent on the length of the oligonucleotide. When
Marshall and Caruthers (6) reported the use of
phosphorodithioate antisense oligonucleotides against HIV-1 reverse
transcriptase in vitro, their random sequence control
oligonucleotides were very similar in both affinity (K
) for HIV-1 reverse transcriptase and
the median inhibitory dose (ID
).
Reports on alternative
mechanisms of action for antisense compounds against HIV-1 followed
earlier studies by Gao et al.(4, 7, 8) in which poly(SdC) was used to inhibit herpes simplex virus type 2. In this series
of experiments a number of different antiviral mechanisms of action
were determined for poly(SdC), including adsorption blocking and
inhibition of herpes simplex virus DNA polymerase(7) . The
largest contribution to the antiviral effects described was due to
blockage of adsorption and/or penetration of the virus into the cell.
Stein et al.(9) characterized the interaction of
poly(SdC) with the v3 loop of HIV-1 gp120 and determined that
poly(SdC)
specifically interacted with the positively
charged v3 loop with an equilibrium dissociation (K
) constant of poly(SdC) for rgp120 of
approximately 5
10
M. Stein et
al.(9) postulated that the specific interaction of
poly(dC) with the HIV-1 v3 loop may be a mechanism by which an
oligonucleotide could inhibit HIV-1 in vivo. More recently
Wyatt et al.(10) reported an oligonucleotide
containing a PT backbone and composed of only deoxyguanosine (G) and
thymidine (T), which folded into an intermolecular tetrad, bound to the
v3 loop of HIV-1. The G-quartet structure and PT backbone of this
molecule (T
G
T
) were reported to be
essential for preventing cell-to-cell and virus-to-cell spread of
infection(10) .
In this study we report that the G/T
oligonucleotide (GTO) I100-15, containing a natural
phosphodiester (PD) backbone, does not block virus adsorption but
rather may function by inhibiting viral-specific transcription. Our
data indicate that in tissue culture assays I100-15 inhibits
acute HIV-1 infection of SUP T1 cells with a 50% effective dose
(ED) in the submicromolar range. In addition, suppression
of HIV-1 is observed for greater then 21 days (depending on the
oligonucleotide modification used) after removal of the drug from the
infected cell culture. We provide evidence to show that this anti-HIV-1
compound folds upon itself to form a structure that is stabilized by
intramolecular G-tetrads, and therefore represents a new class of
potent antiviral oligonucleotide compound. In addition, when paired
with the data of Wyatt et al.(10) it appears that
tetrad-forming oligonucleotides may intervene in the progression of
HIV-1 infection by at least two distinct mechanisms.
For NMR measurements, subsequent to
purification by denaturing anion-exchange chromatography in base (10
mM LiOH, 0.2-0.7 M NaCl), oligonucleotide
purity was confirmed by denaturing gel electrophoresis (7 M urea, 65 °C). For NMR, the oligonucleotide was desalted and
transferred into 20 mM LiCl adjusted to pH 6.0, in order to
minimize tetrad formation. Oligonucleotide strand concentration was
held constant at 2.7 mM. H NMR at 500 mHz was
measured in H
0, employing a Redfield pulse sequence to
saturate the water resonance, as described previously(20) .
Extracted DNA (0.1
µg) was analyzed using a polymerase chain reaction (PCR) primer set
that amplifies a 200-bp portion of the viral genome spanning the repeat
element (R) into the gag gene. The primers used were
5`-ggctaactagggaacccactg-3` and 5`-cctgctgcgagagagctcctctgg-3`. In the
same reaction mixture a PCR primer set that amplifies a 220-bp region
of the human -actin gene was used. The
-actin primers used
were 5`-aaagacctgtacgccaacacagtgctgtctgg-3` (nucleotide position 1196)
and 5`-cgtcatactcctgcttgctgatccacatctgc-3` (nucleotide position
1415)(22) . The PCR reactions were performed as described by
Zack et al.(23) .
Total RNA (1.0 µg) extracted
from HIV-1 infected cells was analyzed by reverse transcriptas-PCR. In
this assay an antisense primer (5`-gcctattctgctatgtcgacaccc-3`
nucleotide position 5818) was used with Moloney murine leukemia virus
reverse transcriptase and extracted mRNA to synthesize a viral specific
cDNA strand as described previously (24) . The cDNA template
was mixed with the antisense primer and a sense primer
(5`-gtgtgcccgtctgttgtgtgactctggtaac-3` nucleotide position 588) and
then amplified using PCR. The positioning of the primer pairs would
preferentially amplify spliced viral mRNAs. The predicted size for the
amplified product is 214 bp. -Actin primers were used as internal
controls in this experiment.
The GTOs were purified after
synthesis using anion exchange high performance liquid chromatography.
Using this procedure an oligonucleotide is purified in the presence of
sodium ions. Monovalent cations are known to encourage self-associated
structures for G-rich molecules, all of which involve formation of
G-tetrads (25) so that even though the molecules are desalted
they remain in a complex with the sodium ions. G-tetrads are known to
be stabilized by coordination of guanine O atoms with
alkali cations(25) . Thus GTOs purified using anion exchange
chromatography have an opportunity to form inter- or intramolecular
tetrads even in the absence of added salts.
To investigate the
possibility that the anti-HIV-1 GTOs were folded into inter- or
intrastrand structures which could account for the antiviral activity
(I100-15) or relative inactivity (I100-18) of these
molecules, I100-15, I100-18 (Table 1), and a control
oligonucleotide, Z106-50 (ggttgggggttggg), were analyzed using
nondenaturing polyacrylamide gel electrophoresis. In this assay a
constant amount of oligonucleotide (10M,
total radiolabeled and unlabeled molecules) was suspended in buffer
containing various concentrations of KCl before native polyacrylamide
gel analysis. Under the gel conditions used, electrophoresed at either
4 °C or room temperature, I100-15 migrated as a unique band
faster than expected for a denatured 17-mer (relative to the oligo(dT)
control molecules) independent of the KCl concentration of the buffer.
This is in contrast to I100-18 (16-mer) and Z106-50 which
appeared to migrate as multiple species (aggregates) under the same gel
conditions in a KCl dependent fashion (Fig. 1). In subsequent
studies trace concentrations of
P-end labeled
oligonucleotide was combined with increasing concentrations of
unlabeled molecules in a buffer containing 60 mM KCl as
described under ``Experimental Procedures.'' The results of
these experiments indicated that I100-15 migrated in the native
gel as a unique species independent of the oligonucleotide
concentration while I100-18 and Z106-50 appeared to
aggregate as the concentration of oligonucleotide increased (data not
shown).
Figure 1:
Oligonucleotides
(10M) in buffer containing increasing
concentrations (0, 7.5, 15, 30, 60, and 120 mM) of KCl were
heated to 100 °C and then cooled to room temperature before being
electrophoresed through a 12% native polyacrylamide gel at room
temperature. Lanes 1-6 are samples of I100-15
treated with increasing concentrations of KCl. Lanes 7-12 are the I100-18 samples and lanes 13-18 are
the Z106-50 samples. The nucleotide markers (nt) in the
lanes to the left and right of the oligonucleotide samples are composed
of various lengths of poly(dT).
Taken together these results suggest that I100-15,
which is 10-fold more active than I100-18 (Table 1), folds
into an intramolecular structure, while other G-rich oligonucleotides
(I100-18 and Z106-50) aggregate into higher order
intermolecular structures. It is interesting to note that the PT GTO
compound described by Wyatt et al.(10) , with the
sequence TG
T
, was reported to fold
into an intermolecular tetrad. Therefore, I100-15 (PD backbone)
is structurally and chemically different from the PT oligonucleotide
T
G
T
.
A
three-dimensional model of I100-15 is presented in Fig. 2which is very similar to the intramolecular tetrad
structure deduced from NMR analysis by the Feigon group(26) .
In this model, G-tetrads are formed by stable Hoogsteen H-bonding. Two
such tetrads are linked to form an octet core, by coordination to a
single monovalent cation equivalent per oligonucleotide. Given the
well-defined ion binding stoichiometry of the model of Fig. 2,
and the prediction that 8 stable H-bonds would be induced upon folding,
we have investigated the folding of I100-15 by means of H NMR. Data have been collected, at 2.7 mM in
oligonucleotide strands in 10 mM LiCl (lithium does not
support tetrad based folding) as a function of K
concentration, which is known to selectively stabilize tetrad
folding (26, 27) and as a function of temperature.
Figure 2:
Two-dimensional representation of
I100-15. The guanines involved in the two tetrads are shown
adjacent to each other in the upper and lower planes. The two tetrads
are stabilized by stacking interactions and the presence of a
monovalent cation (Na or K
)
positioned between the planes formed by the tetrads. The two nucleoside
loops are stretched so that the bases in each loop are positioned in an
unusual fashion, facing out into the surrounding medium. The figure is
positioned so that there are two loops on the top of the structure (upper loops) and one at the bottom (lower
loop).
At 300 K, in the absence of added K, imino proton
signals cannot be resolved for I100-15 in the 10-12 ppm
region (Fig. 3A). Subsequent to addition of KCl,
substantial narrowing of imino signals is obtained, saturating at an
added KCl concentration of 3 mM, which is very close to 1
added K
equivalent per octet. At this end point, it
can be seen that at least two classes of imino resonance can be
detected in the 10-12 ppm range with roughly equal intensity: a
broad envelope from 10 to 11 ppm, upon which several sharp resonances
are superimposed in the 11-11.5 ppm region.
Figure 3: One-dimensional NMR analysis of I100-15. A, KCl titration of a solution containing 2.7 mM strands I100-15. B, limited thermal melting analysis, at 2.7 mM in strands, 6 mM KCl, 20 mM LiCl (pH 6.0) over the range from 300 to 350 K. For NMR measurements, I100-15 was synthesized at 15 µM scale using fast deblocking Expedite chemistry on a Milligen synthesizer. Subsequent to purification by denaturing anion-exchange chromatography in base (10 mM LiOH, 0.2-0.7 M NaCl), oligonucleotide purity was confirmed by denaturing gel electrophoresis (7 M urea, 65 °C). The oligonucleotide was then desalted and transferred into 20 mM LiCl adjusted to pH 6.0, in order to minimize tetrad formation.
By analogy with
chemical shifts of other G-tetrad structures(26) , we
tentatively ascribe the sharp H signals to the 8 Hoogsteen
H-bonds of the core octet (guanosine N1). The broad envelope is
ascribed to the G and T imino protons contributed by the loop and
5`-terminal domains.
Substantial line narrowing of the slowly
exchanging imino proton signals is seen upon raising temperature above
300 K, which is accompanied by broadening of the broad imino envelope
at 11 ppm (Fig. 3B). This narrowing gives rise to
7-8 well-resolved imino protons at 320 K. By reference to the NMR
behavior of the other intramolecular tetrads (26) the formation
of 7-8 narrow well-resolved imino resonances at 320 K strongly
suggests that, in the presence of one bound K ion per
octet equivalent, I100-15 has folded into a discrete tetrad
structure, stabilized by the 8 thermally stable Hoogsteen H-bonds
predicted from the secondary structure model in Fig. 2.
In the range from 330 to 340 K (67 °C-77 °C), the imino proton spectrum undergoes an abrupt transition, which is likely to be representative of cooperative unfolding of the octet fold (Fig. 3B). H-bond stability of this kind, accompanied by apparently high thermal cooperativity is very striking indeed, and is generally indicative of a single, well-defined secondary structure for the I100-15 oligonucleotide.
To further study the effect of the intramolecular tetrad on the antiviral profile of I100-15 we substituted deoxyadenosine (A) for G (I100-38 and I100-39, Table 1) at positions which would cause disruptions in either the upper or lower tetrad involved in the octet formation (Fig. 2). We substituted adenosine for guanosine to keep the purine motif at the substituted positions. The antiviral results obtained using these two oligonucleotides demonstrated that reducing the ability of the oligonucleotide to fold upon itself also reduced antiviral activity by approximately 10-fold (Table 1). Molecules with reduced activity displayed an antiviral profile similar to other G-rich PD containing oligonucleotides which have the capacity to form intermolecular aggregates(12) .
To investigate the importance of the bases in the loop structures to the overall antiviral activity we substituted the G in each loop with an ``A'' nucleoside (I100-40, I100-41, and I100-42, Table 1). The data from this experiment indicates that a simple substitution of this nature had dramatic effects on the antiviral profile of the molecule with the most pronounced changes occurring if the G in loop-2 was disturbed in this fashion (Table 1).
To determine that the observed changes in antiviral activity of molecules with G to A substitutions was not due to changes in the half-life of these molecules under tissue culture conditions, the stability of these molecules relative to I100-15 was determined as described previously(11) . The results from these experiments demonstrated that there was no significant difference in the stability of any of the deoxyadenosine-substituted oligonucleotides relative to I100-15 (data not shown).
In separate experiments, HIV-1 infected SUP T1 cells were treated with various concentration of oligonucleotide for 4 days at which time the drug was removed, the cells washed, and further cultured in complete medium without drug. The cells were monitored daily for the appearance of viral-induced syncytium and every third day for viral p24 antigen in the culture medium. In cells treated with I100-15, measurable virus production was suppressed 5-8 days after removal of the drug (5 µM treatment). When cells were treated with the PT version of this molecule, I100-22, comparable virus production was delayed an additional 2-3 days (Fig. 4A). Virus production was observed 2 days after the removal of drug from the infected cell cultures treated with 4 µM AZT (data not shown). The PD version of I100-15 was synthesized with a cholesterol moiety covalently attached to the 3` terminus yielding I100-23. When I100-23 was used in a similar assay, the duration of viral suppression was greater than 21 days after removal of the drug from the infected cell culture (Fig. 4). The enhanced anti-HIV-1 activity observed using I100-23 is in agreement with other studies using cholesterol-modified oligonucleotides to inhibit HIV-1 in culture(28) . On days 4 and 9 post-infection the number of viable cells remaining in the culture medium treated with 5 µM oligonucleotide was determined to be similar to the number of SUP T1 cells found in uninfected untreated cultures, suggesting that the oligonucleotides, including those bearing a cholesterol moiety, were not exerting a toxic effect on the cells.
Figure 4:
Persistent suppression of HIV-1 in
culture. Four days post-infection of SUP T1 cells with HIV-1 the drug
was removed from the infected cultures, the cells were washed,
replated, and monitored for the emergence of HIV-1. A,
prolonged inhibition of HIV-1 induced syncytium formation obtained when
infected cells were treated with 5 µM I100-15
(), I100-22 (
), or I100-23 (
). B, long-term suppression dose-response profile obtained for
I100-23. Drug was removed from cells treated with 5 µM (
), 3 µM (
), 1.5 µM (
), 0.75 µM (
), 0.4 µM (
), 0.2 µM (
), or 0.1 µM (
) of I100-23 on day 4
post-infection.
Figure 5:
Analysis of viral DNA and RNA. A,
HIV-1 infected drug treated SUP T1 cell DNA (100 ng/reaction) was used
as template in the following PCR reactions. AZT, at 0.3 µM (lane 1), I100-15 at 5.0 µM (lane
2), or I100-15 at 0.3 µM (lane 3) were
added to SUP T1 cells at the same time as HIV-1. Lanes 4 (AZT), 5 (5.0 µM I100-15), and 6 (0.3 µM I100-15) are the results of DNA samples
obtained from cells in which drug was added 1 h post-infection. Lane 7 contains DNA from infected but untreated cells and lanes 8-10 contain 10, 100, or 1000 ng of infected but
untreated cellular DNA. In the same reaction mixture a PCR primer set
which would amplify a 220-bp region of the human -actin gene was
used. The 200-bp fragment is the predicted size for the amplified
portion of the HIV-1 genome. B, reverse transcriptase-PCR
analysis of extracted RNA (1 µg/reaction) obtained from SUP T1
cells treated at the same time as virus infection (0 h, lanes
1-3) or 8 h post-infection (lanes 4-6). AZT
(0.3 µM) samples are in lanes 1 and 4.
I100-15 (5 µM) samples are in lanes 2 and 5 and I100-15 (0.3 µM) samples are in lanes 3 and 6. Lanes 7 and 8 are
control HIV-1 infected cell mRNA and lanes 9 and 10 are the results obtained using uninfected untreated SUP T1 cell
mRNA. The same
-actin primers used for the analysis of the DNA
samples were used as internal controls in this experiment. C,
quantification of duplicate reverse transcriptase-PCR experiments
described in panel B. The mRNA extracted from uninfected and
untreated SUP T1 cells was used as a control (100%) value for
-actin levels, while the HIV-1 levels were normalized to the
results obtained using mRNA extracted from infected but untreated
cells.
Total RNA extracted from HIV-1 infected cells was analyzed by reverse transcriptase-PCR. The results of this experiment clearly indicated that a reduced level of HIV-1 specific spliced mRNA transcripts was observed in samples treated with I100-15 in a dose-dependent fashion (Fig. 5B). It was also clear that while samples treated with AZT had reduced levels of viral cDNA, low levels of viral mRNA was still being produced (Fig. 5B). Quantification of radiolabeled reverse transcriptase-PCR generated DNA fragments from two different experiments was performed using a Betascope blot analyzer (Betagen). The results are shown in Fig. 5C.
All DNA and RNA analyses were repeated at least three times. In addition, the same decrease in HIV-1 specific transcription was observed in viral infected cells treated with I100-15 when a PCR primer pair designed to amplify only unspliced mRNA was used (data not shown).
Previously we postulated several possible mechanisms of action to account for the anti-HIV-1 activity of GTOs, including adsorption blocking and inhibition of viral reverse transcriptase(12) . However, in this study, by analysis of intracellular viral DNA and RNA, we show that I100-15 does not inhibit viral entry into cells or affect early virus replication events (reverse transcription). Instead, a reduction of viral specific mRNA was observed which may account for the antiviral mechanism of action of the molecule.
Although most GTOs tested were able to inhibit HIV-1 to some extent, the shortest and most efficacious molecules (I100-15, I100-25, and I100-26) were either capable of forming or predicted to form intramolecular tetrads. The native polyacrylamide gel and NMR analysis suggests that in the presence of monovalent cations, I100-15 is most likely present in the folded state stabilized by tetrad formation similar in general form to the intramolecular tetrad described for the G-rich thrombin binding aptamer by Schultze et al.(29) . Shorter variants of I100-15 or substituted oligonucleotides which were not capable of forming intramolecular tetrads were less active in culture experiments. In addition, the presence of G in the two nucleoside long loops was also very important for the overall antiviral activity of I100-15. Experiments are underway to further investigate and modify the essential elements in this sequence necessary for antiviral activity.
The addition of a sulfur group into the backbone of
I100-15 improved the ED value by a factor of 2 in
the acute assay and allowed for a longer duration of viral suppression
after the removal of the drug from infected cell cultures. It is not
known at this time whether the PT effect was due to enhanced stability
of the oligonucleotide or increased affinity at the point of viral
interdiction. The enhanced antiviral activity when sulfur was present
in the backbones of nuclease-resistant oligonucleotides indicates that
a component of the antiviral activity could be attributed to the
chemical nature of the oligonucleotide backbone and independent of the
oligonucleotide sequence.
Recently Lisziewicz et al.(30) reported the long-term (>80 days) suppression of HIV-1 in culture in which a PT containing ``antisense'' oligonucleotide was continuously present in the culture medium. In the present study we demonstrate the ability to suppress acute HIV-1 infection for at least 7 days after removal of the GTO from the infected cell culture medium. Upon addition of a cholesterol moiety to the 3` terminus of I100-15 the observed long-term suppression increased to greater than 21 days. Both of these studies offer encouragement for the continued development of oligonucleotide-based therapeutics for the treatment of HIV-1 infected individuals.