©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Suppression of Human Immunodeficiency Virus Type 1 Activity in Vitro by Oligonucleotides Which Form Intramolecular Tetrads (*)

(Received for publication, August 22, 1994; and in revised form, November 9, 1994)

Robert F. Rando (1)(§) Joshua Ojwang (1) Ahmed Elbaggari (2) Gregory R. Reyes (1)(¶) Robert Tinder (3) Michael S. McGrath (2) Michael E. Hogan (3)

From the  (1)From Triplex Pharmaceutical Corp., The Woodlands, Texas 77380, the (2)Department of Laboratory Medicine, University of California at San Francisco, San Francisco General Hospital, San Francisco, California 94110, and (3)The Center for Biotechnology, Baylor College of Medicine, The Woodlands, Texas 77381

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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) (^1)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 alpha(2, 4) , beta, 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 times 10M. 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(2)G(4)T(2)) 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.


EXPERIMENTAL PROCEDURES

Oligonucleotide Synthesis and Modification

Oligodeoxynucleotides were synthesized on an Applied Biosystems Inc. (ABI) DNA synthesizer model 380B or 394, using standard phosphoramidite methods or using fast deblocking ``Expedite'' chemistry on a Milligen synthesizer as described previously(11, 12) . The hydroxyl terminus of oligonucleotides was blocked to reduce degradation by cellular exonucleases by the covalent attachment of a propanolamine group to the 3`-hydroxyl group (13, 14, 15, 16) or a triglycyl-linked cholesterol moiety(17) . PT-containing oligonucleotides were prepared using the Beaucage sulfurizing agent described by Iyer et al.(18) .

Structural Analysis of I100-15

Oligonucleotides were 5`-end labeled using [-P]ATP and T4 polynucleotide kinase(19) . Trace concentrations of radiolabeled oligonucleotides (10M) were combined with increasing concentrations of unlabeled oligonucleotide (up to 10M in a total volume of 20 µl) in 25 mM Tris-HCl (pH 7.4), 2.5 mM EDTA, and 60 mM KCl, heated to 100 °C for 5 min, cooled to room temperature, and then analyzed using a 12% native polyacrylamide gel (200 V for 7 h at either room temperature or 4 °C). In another experiment oligonucleotides (10M total labeled and unlabeled molecules) were suspended in the same buffer containing variations in the KCl concentration (0-120 mM) before gel analysis as described.

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. ^1H NMR at 500 mHz was measured in H(2)0, employing a Redfield pulse sequence to saturate the water resonance, as described previously(20) .

Cytotoxicity and Stability Assays

Cytotoxicity was assayed using the CellTiter 96 Aqueous Non-radioactivity Cell Proliferation Assay (Promega). Viable H9, Vero, SUP T1, or NIH-3T3 cells were dispensed in quadruplicate into a 96-well microculture plate at a concentration which assured for log phase cell growth. The degree of cell proliferation was determined according to the manufacturer's protocol. The stability of oligonucleotides under tissue culture conditions was determined as described previously(11) .

Inhibition of Acute HIV-1 Infections

Various concentrations of oligonucleotides or AZT were tested for dose-dependent inhibition of syncytium formation or viral p24 antigen production in an acute infection assay(21) . Briefly, HIV was used to infect the SUP T1 cell line using 0.1 tissue culture median infectious doses for 1 h at 37 °C prior to washing and resuspension in media containing increasing concentrations of the test oligonucleotides. The SUP T1 cells (2 times 10^4 cells/well) were inoculated in triplicate in 200 µl of RPMI 1640 containing 10% fetal calf serum. Four days post-infection the total number of syncytia per well were calculated and expressed as percent inhibition compared to the untreated control infected cells. Supernatants from wells scored for syncytium formation were analyzed for the presence of HIV p24 antigen using the Coulter p24 antigen capture kit. Zidovudine (3`-azido-3`-deoxythymidine, AZT) was obtained from Pharmatec.

Analysis of HIV-1 RNA and DNA

Total RNA and DNA were extracted from SUP T1 cells 36 h after infection with a 0.1 multiplicity of infection of HIV-1. The infected cells were treated with I100-15 or AZT at various time points before, during or after infection. Nucleic acids were extracted using the Snap-o-sol kit (Biotecx), deproteinized using a 50:50 mixture of phenol:chloroform, and precipitated in ethanol.

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 beta-actin gene was used. The beta-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. beta-Actin primers were used as internal controls in this experiment.


RESULTS

Structural Analysis of I100-15

Recently we reported on the anti-HIV-1 activity of a series of G-rich oligonucleotides (GTOs) with unmodified PD backbones(12) . One of the sequence motifs (I100-07, Table 1) was 10-fold more active at inhibiting HIV-1-induced syncytium formation and viral p24 production than the other motifs tested. Analysis of six I100-07 size variants revealed that I100-15 (17-mer, 3`-end deletion) was as effective at reducing syncytium formation as I100-07 (45-mer), while I100-18 (16-mer 5`-end deletion) was less active than I100-07(12) . This observation is of interest since the first 14 nucleotides from either end of I100-07 are direct inverted repeats (Table 1).



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^6 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 T(2)G(4)T(2), was reported to fold into an intermolecular tetrad. Therefore, I100-15 (PD backbone) is structurally and chemically different from the PT oligonucleotide T(2)G(4)T(2).

NMR Analysis of I100-15

Most eukaryotes possess a repeating G-rich sequence of the form (T/A)(n)G(m), where n = 1-4 and m = 1-8. Of particular interest to the study of the I100-15 class of GTO is the structure of the telomere sequence repeat T(4)G(4), first detected in Oxytricha. Structures related to the Oxytricha repeat have been studied in oligonucleotides by NMR (26) and crystallographic methods(27) . Both the NMR and crystallographic studies suggest that folding is mediated by square planar Hoogsteen H-bonding among G residues, with overall antiparallel orientation of the four-strand equivalents comprising the tetrad fold. As expected, the crystallography has confirmed that the structure is stabilized by tight binding of a small monovalent cation (especially K) at the interface between adjacent G-tetrads, by means of coordination to the O^6 oxygen of guanosine.

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 ^1H 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 ^1H 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.

Cytotoxicity Assays

GTOs with PD backbones such as I100-15 inhibited cell growth in culture by 50% (TC) in the range of 40-100 µM (depending upon the cell line used in the assay) while GTOs with PT backbones (such as I100-22) had TCs in the 15-30 µM range (data not shown). The TC for AZT in SUP T1 cells was determined to be 10 µM. The results of these assays, taken together with the results of viral inhibition experiments, allowed for the calculation of the therapeutic indices for assays performed in SUP T1 cells found in Table 1.

Inhibition of Acute HIV-1 Infections

Various concentrations of oligonucleotides or AZT were tested for dose-dependent inhibition of syncytium formation or viral p24 antigen production in an acute infection assay in which drug was added to cells after viral infection (12, 21) . In previous studies (12) the anti-HIV-1 activity of the PD GTO I100-07 (45-mer) was superior to other GTO sequence motifs tested and that the antiviral activity of I100-07 was maintained when the length of the molecule was reduced to 17 (I100-15) by deleting segments from the 3`-end (I100-15, -16, -17) but not when deletions from the 5`-end were used (I100-18, -19, -20) (Table 1). It is also apparent from the native gel and NMR analysis presented above that I100-15 (ED 0.1 µM) is capable of folding into an intramolecular tetrad while I100-18 (ED 4.5 µM) has a tendency to exist as an intermolecular aggregate (Table 1). To further confirm that the core octet involved with tetrad formation (Fig. 2) was important to the enhanced antiviral activity of I100-15 as compared with I100-18, we synthesized size variants of I100-15 (I100-25 to I100-30, Table 1). The data suggest that as long as the nucleotides involved in tetrad formation were intact (Fig. 2) the antiviral activity remained in the 0.1 µM range while perturbations in the core tetrads decreased the antiviral efficacy of the molecule by a factor of at least 10 (Table 1). For example, I100-25, which replaces the 3`-T of I100-15 with a G and is predicted to be able to form an intramolecular tetrad, and I100-26, which removes the 3`-terminal T of I100-15 and can form an intramolecular tetrad (data not shown), both have ED values in the 0.1-0.2 µM range while all other size variants tested were too short to form intramolecular tetrads and had ED values in the 5 µM or higher range (Table 1, Fig. 2).

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 (box), I100-22 (), or I100-23 (circle). B, long-term suppression dose-response profile obtained for I100-23. Drug was removed from cells treated with 5 µM (box), 3 µM (), 1.5 µM (circle), 0.75 µM (up triangle), 0.4 µM (bullet), 0.2 µM (), or 0.1 µM () of I100-23 on day 4 post-infection.



Analysis of HIV-1 RNA and DNA

The nucleic acids from SUP T1 cells were harvested 36 h post-infection with a 0.1 multiplicity of infection of HIV-1. Total DNA was analyzed for the presence of viral cDNA by amplification of viral specific sequences. The results of the DNA analysis are shown in Fig. 5A. As expected for cells treated with AZT, there was a marked decrease in viral DNA synthesis when drug was added up to 4 h post-infection. However, no observable decrease in viral DNA synthesis was observed at any time point tested when I100-15 was added to infected cells (Fig. 5A). This observation indicated that I100-15 did not inhibit virus entry into the cells, even in samples treated with I100-15 before (not shown) or at the same time as virus infection (Fig. 5A), and therefore suggests that I100-15 has a different mechanism of action from the G-rich PT oligonucleotide reported by Wyatt et al.(10) . Furthermore, it suggests that I100-15 did not interfere with HIV-1 reverse transcriptase and therefore has a different mechanism of action compared to AZT.


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 beta-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 beta-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 beta-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).


DISCUSSION

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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Triplex Pharmaceutical Corp., 9391 Grogan's Mill Rd., The Woodlands, TX 77380. Tel.: 713-363-8761; Fax: 713-363-1168.

Current address: Ingenex, Inc. 1505 O' Brien Drive, Menlo Park, CA 94025.

(^1)
The abbreviations used are: PT, phosphorothioate; PD, phosphodiester; HIV, human immunodeficiency virus; GTO, G/T oligonucleotide; AZT, 3`-azido-3`-deoxythymidine; PCR, polymerase chain reaction; bp, base pair(s); poly(SdC), oligodeoxycytidine with phosphorothioate linkages.


ACKNOWLEDGEMENTS

We thank Dr. P. Cossum for critical reading of this manuscript, Sean Smith for computer graphics, and R. Unger for editorial assistance. M. E. H. acknowledges the support of the National Institutes of Health and Bruker Instruments for access to their AMX 500 spectrometer.


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