www.elsevier.com/locate/yviro

Virology 334 (20

Potent inhibition of HIV-1 entry by (s4dU)35

Andras Horvatha,b, Szilvia T}ookesa,b, Tracy Hartmanc,1, Karen Watsonc,1, Jim A. Turpinc,2,

Robert W. Buckheit Jr.c,1, Zsolt Sebestyenb,d,e, Janos Szfll}oosib,d,e,Ilona Benk}oof, Thomas J. Bardosg, Joseph A. Dunnh, Laszlo Fesqsa,b,

Ferenc D. Tothi, Janos Aradia,b,TaDepartment of Biochemistry and Molecular Biology, University of Debrecen, H-4012 Debrecen, Nagyerdei krt. 98, HungarybResearch Center for Molecular Medicine, Medical and Health Science Center, Faculty of Medicine, University of Debrecen,

H-4012 Debrecen, Nagyerdei krt. 98, HungarycSouthern Research Institute, Infectious Disease Research, Frederick, MD 21701, USA

dDepartment of Biophysics, University of Debrecen, H-4012 Debrecen, Nagyerdei krt. 98, HungaryeCell Biophysics Research Group of the Hungarian Academy of Sciences, University of Debrecen, H-4012 Debrecen, Nagyerdei krt. 98, Hungary

fDepartment of Pharmacology and Pharmacotherapy, University of Debrecen, H-4012 Debrecen, Nagyerdei krt. 98, HungarygDepartment of Chemistry, State University of New York, Buffalo, NY 14260, USA

hOmniPharm Inc. 255 Great Arrow Avenue Buffalo, NY 14207, USAiDepartment of Medical Microbiology, University of Debrecen, H-4012 Debrecen, Nagyerdei krt. 98, Hungary

Received 22 November 2004; returned to author for revision 4 January 2005; accepted 26 January 2005

Abstract

We have previously reported the potent in vitro HIV-1 anti-reverse transcriptase activity of a 35-mer of 4-thio-deoxyuridylate [(s4dU)35].

In efforts to define its activity in a more physiological system, studies were carried out to determine the stage of viral infection that this

compound mediates its anti-viral effect. Results of the studies reported herein show that (s4dU)35 is nontoxic and is capable of inhibiting both

single and multi-drug resistant HIV strains (IC50: 0.8–25.4 Ag/ml) in vitro. Besides its previously reported anti-RT activity, (s4dU)35 mediated

its antiviral action by preventing virus attachment (IC50: 0.002–0.003 Ag/ml), and was stable in vitro and slowly degraded by DNAses.

Competition studies and fluorescence resonance energy transfer (FRET) experiments indicated that (s4dU)35 preferentially binds to CD4

receptors, but not to CD48. Confocal laser scanning microscopy (CLSM) studies showed that (s4dU)35 did not penetrate into the cells and

colocalized with cell surface thioredoxin. Our studies identify (s4dU)35 as a potential novel HIVentry inhibitor that may have utility as either

a systemic antiretroviral or as a preventing agent for HIV transmission.

D 2005 Elsevier Inc. All rights reserved.

Keywords: HIV-entry; Inhibition; Oligonucleotide; Redox process; CD4

0042-6822/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.virol.2005.01.033

* Corresponding author. Department of Biochemistry and Molecular

Biology, University of Debrecen, H-4012 Debrecen, POB. 6. Hungary.

Fax: +36 52 314 989.

E-mail address: aradi@indi.biochem.dote.hu (J. Aradi).1 Current address: Genelogic, 18761 N. Frederick Avenue, Gaithersburg,

MD 20879, USA.2 Current address: Henry M. Jackson Foundation, 6700 B Rockledge

Drive, Bethesda, MD 20892, USA.

Introduction

The development and discovery of inhibitors of human

immunodeficiency virus type 1 (HIV-1), the etiological

agent for HIV/AIDS, has been dominated by inhibitors of

the HIV enzymes reverse transcriptase (RT) and protease

(De Clercq, 1993; Richman, 2001). However, as issues of

patient compliance, cost and access to therapies and the

emergence of single- and multi-drug resistant strains have

come to the forefront of HIV/AIDS therapy, the need for

05) 214–223

A. Horvath et al. / Virology 334 (2005) 214–223 215

more potent and novel antivirals as well as new antiviral

targets has arisen. Part of the need for new antiviral targets

has been met by the identification of many of the processes

involved in HIV entry into cells (Cooley and Lewin, 2003;

Dimitrov, 1997; Doms and Moore, 2000; Eckert and Kim,

2001; Kilby and Eron, 2003; LaBranche et al., 2001).

The HIV-1 envelope protein is a trimer of gp120–gp41

heterodimers on the virion surface. The multi-step HIVentry

process is initiated by the highly specific interaction

between gp120 HIV envelope glycoprotein and the CD4

molecule, its primary receptor. This interaction induces

conformational alterations in gp120, enabling it to sub-

sequently interact with the co-receptor (CCR5 or CXCR4)

(Eckert and Kim, 2001; LaBranche et al., 2001). Following

binding of gp120 to CD4 and a chemokine receptor, a

further conformational change occurs in the gp120–gp41

oligomer that leads to insertion of the hydrophobic N-

terminal peptide sequence of gp41 into the membrane of the

host cell bringing about the fusion process. Recently,

Enfuvirtide (T20), an inhibitor of gp41 and virus entry,

has been approved for use in combination with other anti-

HIV medications, providing access to a new class of drugs

as antiviral targets for the treatment of HIV/AIDS (Kilby

and Eron, 2003).

In addition to the above conformational changes, a

chemical process, that is, redox changes of cell surface

proteins and the viral envelope glycoprotein (gp120) are

required for successful entry of HIV-1 into cells. It was

recently shown that the 2nd domain (D2) disulfide bond of

CD4 is redox-active and the redox process is controlled by

cell surface thioredoxin, secreted by the T cell (Hogg, 2003;

Matthias and Hogg, 2003, Matthias et al., 2002). Protein-

disulfide isomerase (PDI) is another cell surface protein that

is known to catalyze redox changes, and thus, may also be

involved in the control of cell surface redox chemistry

required for HIV-1 entry. It was reported that purified PDI

can cleave disulfide bonds in recombinant gp120 and two of

the nine disulfides of gp120 are reduced during interaction

with the lymphocyte surface on CD4+ cells (Barbouche et

al., 2003; Gallina et al., 2002) and anti-PDI agents

effectively block CXCR4 Env-mediated fusion (Markovic

et al., 2004). Localization of CD4 receptors in cholesterol

and glycosphingolipid-rich lipid rafts, present in the external

leaflet of the plasma membrane, also appear indispensable

for successful HIV-1 entry (del Real et al., 2002; Popik et

al., 2002), although these data remain a subject of debate

(Percherancier et al., 2003). Further studies are needed to

elucidate the detailed functional connection between the

well-established conformational changes and the disulfide

exchange processes involved in the entry of HIV-1 into

target cells. However, these two findings, the cell surface

reducing power, that is, thioredoxin and perhaps PDI, as

well as the localization of the CD4 receptors in lipid rafts,

together may represent new information which can be

exploited as potential targets in the inhibition of HIV-1

entry.

Although oligonucleotides with demonstrated activity

against various viral structural and enzyme targets have

been reported, no oligonucleotide drug has yet been

approved for routine therapeutic use against HIV infection.

Both antisense and triple-helix forming oligonucleotides

exert their inhibitory activities by sequence-specific nucleic

acid interactions (Agrawal et al., 1989; Inagawa et al., 2002;

Kinchington et al., 1992; Tsukahara et al., 1997). In vitro

oligodeoxycytidine containing a phosphorothioate backbone

binds to CD4, blocking the interaction between gp120 and

CD4. In addition, it has also been shown to inhibit

polymerase activity of purified RT enzyme (Majumdar et

al., 1989; Matsukura et al., 1987; Stein et al., 1991).

Guanine-rich oligonucleotides with phosphorothioate inter-

nucleotide linkages also exert anti-HIV activity (Buckheit et

al., 1994; Jing and Hogan, 1998; Kuwasaki et al., 2003;

Ojwang et al., 1995). We have previously shown that 5-

mercaptopyrimidine-containing oligo- and polynucleotides

are potent inhibitors of RT enzyme activity and HIV-1

replication in vitro (Bardos et al., 1992; T}ookes and Aradi,

1995). In the studies described herein, we have conducted

further detailed studies aimed of the characterization of the

precise stage of the viral life cycle which is the target of the

anti-HIV activity of a 35-mer homo-oligonucleotide com-

posed of 4-thio-deoxyuridylates, (s4dU)35.

While this 35-mer oligonucleotide was previously shown

to be a potent inhibitor of cell-free HIV-1 RT and telomerase

in vitro (Szatmari et al., 2000; T}ookes and Aradi, 1996),

results of the studies reported herein utilizing a more

physiological assay for the measurement of anti-viral

activity provide data that supports the view that this novel

oligo-compound (s4dU)35 primarily mediates its anti-viral

effect by inhibiting virus entry.

Results

Antiviral activity of (s4dU)35

(s4dU)35 was assessed for inhibition of HIV-1 replication

in a standard cell-based assay (Buckheit et al., 1993; Rice

et al., 1997; Weislow et al., 1989) using both wild-type and

viruses expressing mutations known to confer resistance to

nucleoside (NRTI) and non-nucleoside (NNRTI) reverse

transcriptase inhibitors (Buckheit et al., 1995a, 1995b).

Inhibitory concentration 50% (IC50s) ranged from 0.8 to

25.4 Ag/ml (Table 1). Minor 0.9- to 3.3-fold variations in

(s4dU)35 IC50s were noted for non-nucleoside reverse

transcriptase inhibitor (NNRTI) and nucleoside reverse

transcriptase inhibitor (NRTI) resistant viruses compared

to corresponding wild type strains (NL4-3 and IIIB); but

these changes are not statistically significant in the bioassay.

There was no detectable evidence of cytotoxicity (morpho-

logically or by formazan dye reduction) when doses as high

as 100 Ag/ml of (s4dU)35 were utilized in these assays. The

results presented in Table 1 are representative of multiple

Table 1

Antiviral activity of (s4dU)35

Virus

isolate

Resistant to IC50a

(s4dU)35(Ag/ml)

TC50b TIc

TC50/

IC50

Fold

resistance

IC50mutant/

IC50wild type

Wild type

RF 0.8 N50 N62

IIIB 4.4 N100 N23

NL4-3 7.8 N100 N13

NL4-3 mutant

K103N NNRTI 25.4 N100 N4 Sensitive; 3.3

Y181C NNRTI 6.7 N100 N15 Sensitive; 0.9

4X AZTd NRTI 24.5 N100 N4 Sensitive; 3.1

IIIB mutant

DPS-Re NNRTI 10.5 N100 N10 Sensitive; 2.4

a 50% inhibition of virus replication.b 50% reduction in cell viability.c In vitro therapeutic index.d Contains mutations D67N, K70R, T215Y and K219Q to express

complete resistance to AZT.e Generated by in vitro selection against diphenylsulfone (Buckheit et al.,

1995b).

A. Horvath et al. / Virology 334 (2005) 214–223216

antiviral assays with values shown which are mean of

triplicates. We have demonstrated that the standard error

between multiple antiviral assays averaged less than 15% of

respective IC50 values. AZT and nevirapine were included

as positive controls in each assay and showed the expected

fold- resistance (16- to 333-fold) for the mutant viral strains

(data not shown). These data suggest that (s4dU)35 is

inhibitory for both wild-type and RT resistant viruses as

defined by the results with this assay.

Mechanism of action

Initial enzyme-based assays using recombinant HIV-1

RT identified (s4dU)35 as a potent competitive inhibitor

(T}ookes and Aradi, 1996) of reverse transcriptase enzyme in

vitro. Since oligonucleotides may exert their anti-HIV

Fig. 1. Studies on the mechanism of action of (s4dU)35. (a) Effect of the time of a

activity in the culture fluid of infected MT4 cells. (b) Effect of the chain length of t

by measuring the RT activity in the culture fluid of infected MT4 cells. Values a

activity through various modes of action, it was reasoned

that a more detailed study is required to specifically identify

the stage(s) of the viral life cycle targeted by (s4dU)35. We

first examined the time of addition of (s4dU)35 relative to the

time of HIV infection (Fig. 1a). The results indicate that the

maximal inhibitory activity of (s4dU)35 was observed when

the oligonucleotide was added prior to HIV-1 infection.

When the (s4dU)35 was added 2 h post-infection its

inhibitory activity was in fact reduced by 50%, suggesting

that the inhibition was mediated during the early stage of

viral entry. The RT inhibitory activity of (s4dU)35 has been

previously observed to be a chain length-dependent

phenomenon (T}ookes and Aradi, 1996). Similarly, antiviral

activity correlated with the length of the oligonucleotide

(Fig. 1b).

We next assessed the direct effects of (s4dU)35 on virus

entry. Inhibition of virus binding to cells was estimated by

ELISA quantitation of cell-associated p24, while inhibition

of virus entry was assessed after 48 h by h-galactosidasereporter expression (Buckheit et al., 1994). Inhibition of

both virus binding and entry by (s4dU)35 were equivalent

(IC50: 0.003 and 0.002 Ag/ml, respectively). We next

determined the effect of (s4dU)35 on cell fusion using co-

culture of HeLa CD4 LTR h-gal (providing CD4 and LTR-

h-galactosidase reporter) and HL2/3 cells (providing HIV

Tat and Env). The two cell lines were co-cultured in

the continuous presence of (s4dU)35 for 48 h and h-galactosidase reporter activity determined. Gp120-CD4

mediated fusion was inhibited with an IC50 of 8.75 Ag/ml. Chicago Sky Blue was used as a positive control

(Clanton et al., 1992). The results demonstrate that the

antiviral activity of (s4dU)35 is associated with early stages

of virus-cell binding and entry (Table 2).

Interaction of (s4dU)35 with CD4 receptor

Since the primary receptor for HIV-1 gp120 protein is the

CD4 molecule, we performed binding competition and flow

cytometric FRET studies to determine whether the binding

ddition of (s4dU)35 on the anti-HIV activity, assessed by measuring the RT

hiolated oligonucleotides on the anti-HIVactivity of the molecules, assessed

re means F SE of three independent experiments.

Table 2

Assessment of (s4dU)35 for inhibition of virus-cell attachment, entry and

fusion

IC50a TC50

b TIc IC50a TC50

b TIc

(s4dU)35(Ag/ml)

Chicago Sky Blue (Ag/ml)

Virus cell

association

0.003 N100 N33,000 1.20 N10 N8.3

Virus entry 0.002 N100 N50,000 0.04 N10 N250

Cell–cell

fusion

8.750 N100 N12 0.67 N10 N15

a 50% inhibition of virus replication.b 50% reduction in cell viability.c In vitro therapeutic index.

A. Horvath et al. / Virology 334 (2005) 214–223 217

site of (s4dU)35 was physically associated with the CD4

molecule. Results obtained showed that the unlabeled

(s4dU)35, when added simultaneously with a fluorescently

tagged anti-CD4 mAb (MEM115), served as a partial

competitor for anti-CD4 mAb. In contrast, the binding of

anti-CD48 mAb (MEM102) to cells was not significantly

competed by the addition of unlabeled (s4dU)35, denoting a

degree of specificity for the inhibition of the binding of

(s4dU)35 to the CD4 molecule (Fig. 2a).

The specificity of the association of (s4dU)35 with CD4

was then measured more directly by FRET analysis. This

technique determines if the donor Cy3-labeled anti-CD4

mAb and the acceptor Cy5-labeled (s4dU)35 are within close

proximity. The molecular distance between the two dyes can

be calculated by the energy transfer efficiency, which is

extremely sensitive to changes in the range between 1 and

10 nm. Energy transfer (ET) efficiencies (above 5%) reflect

physical association of the donor and acceptor labeled

molecules. The calculated ET values for the association of

(s4dU)35 and the CD4 molecule were between 10 and 25%

indicating that a portion of the CD4 and (s4dU)35 molecules

were in close proximity (Fig. 2b). In order to prove the

specificity of the interaction between CD4 and (s4dU)35 and

to rule out the role of so-called chance transfer, we

performed FRET analyses between Cy3 labeled CD48

mAb and Cy5 labeled (s4dU)35. Since the number of bound

Fig. 2. Study the interaction of CD4 and CD48 molecules with (s4dU)35. (a) Compe

monoclonal antibodies. Values are means F SE of five experiments. (b) Flow cyt

anti-CD4 mAb, acceptor: Cy5-(s4dU)35 (black bars); donor: Cy3-anti-CD48 mA

experiments.

anti-CD48 antibody was comparable to that of anti-CD4,

FRET values were not affected by any differences in donor

to acceptor ratios. Low ET (1–5%) efficiency values were

found between CD48 mAb and (s4dU)35 (Fig. 2b), indicat-

ing negligible physical association. These observations

together with the competition data strongly suggest that

(s4dU)35 is associated with CD4 molecules but not CD48.

Localization of (s4dU)35 and thioredoxin

Since CD4 receptors are subject to oxidoreduction

during HIV-1 entry, mediated by cell surface thioredoxin

(Matthias et al., 2002), and thioredoxin is a chemically

feasible target for (s4dU)35, we studied the respective

cellular localization of (s4dU)35 and thioredoxin by CLSM.

Results of these studies showed that (s4dU)35 was found

only on the cell surface in well defined clusters and patches,

and the different labels (Cy3, Cy5) did not modify the

binding sites of the oligonucleotide (Fig. 3a). Of importance

was the finding that thioredoxin (labeled with Cy3-

monoclonal antibody) co-localized with Cy5-(s4dU)35 in a

very reproducible fashion (representative data illustrated in

Figs. 3b, c).

Stability and toxicity of (s4dU)35

The results above suggest that (s4dU)35 could be a

potential inhibitor of viral entry and serve as an anti-HIV-1

agent. Two important requirements for the successful use of

(s4dU)35 as an antiviral therapeutic are the stability of the

oligonucleotide and potential limited if any levels of cellular

toxicity.

Many oligonucleotide-based drugs require an intact

structure for full biological activity; therefore, we

assessed the stability of (s4dU)35 and its parent oligonu-

cleotide, (dC)35 to nuclease digestion. Preliminary experi-

ments conducted by incubation of (s4dU)35 in either

human serum, FCS containing tissue culture media and/or

cell extracts showed that this compound to be highly

stable (data not shown). In order to more rigorously

tition between (s4dU)35 and anti-CD4 (black bars) or anti-CD48 (open bars)

ometric fluorescence resonance energy transfer measurements; donor: Cy3-

b, acceptor: Cy5-(s4dU)35 (open bars). Values are means F SE of three

Fig. 3. Confocal laser scanning microscopy studies on the localization of

(s4dU)35 on the surface of Kit225 K6 cells. (a) Effects of different

fluorescent labels on the localization of (s4dU)35; (1) Cy5-(s4dU)35, (2)

Cy3-(s4dU)35, (3) overlay. (b) Colocalization of Cy5-(s4dU)35 with cell

surface thioredoxin labeled with monoclonal antibody conjugated to Cy3

(cross-section); (1) (s4dU)35, (2) thioredoxin, (3) overlay. (c) Colocalization

of Cy5-(s4dU)35 with cell surface thioredoxin labeled with monoclonal

antibody conjugated to Cy3 (upper slice); (1) (s4dU)35, (2) thioredoxin, (3)

overlay.

A. Horvath et al. / Virology 334 (2005) 214–223218

assess the stability of (s4dU)35, we mixed it with DNase I

(25 Ag/ml) and snake venom diesterase (50 Ag/ml) and

measured the terminal phosphate groups formed on

digestion (Fig. 4a). The thiolated oligonucleotide

[(s4dU)35] proved to be approximately 40-fold more

stable than its unthiolated parent, (dC)35. These results

demonstrate that the base modification alone, that is,

introduction of 4-thiono group, converts the parent

natural oligonucleotide, (dC)35, to an oligonucleotide

which is highly stable against nucleases. Although our

Fig. 4. Stability and toxicity of (s4dU)35. (a) Comparison of stability of (s4dU)35 [

colony formation of human granulocyte-macrophage progenitor cells. Colonies/1

highest concentration of (s4dU)35 was 180 Ag/ml (15.8 AM). Values are means F

in vitro antiviral assay (Table 1) results identified

(s4dU)35 as a relatively non-cytotoxic oligonucleotide,

we further investigated its toxicity by performing an

assay in which colony formation of human granulocyte-

macrophage progenitor cells were studied as described in

the methods section. Results of these studies showed that

(s4dU)35 had negligible effect on colony formation at

doses as high as 180 Ag/ml (Fig. 4b). These data strongly

suggest that (s4dU)35 is a highly stable and non-cytotoxic

drug and represents an interesting candidate for further

studies aimed at defining its potential efficacy in

inhibiting viral infection in an in vivo model of HIV-1

infection such as the SIV nonhuman primate model.

These studies are in progress.

Discussion

We have demonstrated herein that the thiolated homo-

oligonucleotide, (s4dU)35, acts extracellularly to prevent

virus entry, although previous experiments had shown it to

be a potent inhibitor of RT (T}ookes and Aradi, 1996). Time-

of-addition experiments indicated an early target for

(s4dU)35 in virus replication, which corresponded to virus

attachment (Fig. 1a). This was further confirmed using

specific in vitro assays to assess inhibition of virus attach-

ment, entry and cell-fusion (Table 2). Furthermore, (s4dU)35when added to cell cultures was demonstrated to be

associated with the cell membrane and was not found to

be localized intracellularly by CLSM (Fig. 3). Due to the

extracellular localization of (s4dU)35, it is unlikely that in

physiological systems it mediates its antiviral activity by

inhibition of reverse transcription or any intracellular

processes in the life cycle of HIV, in spite of the fact that

it is a potent inhibitor of purified RT enzyme (T}ookes and

Aradi, 1996). However, internalization of low amount of

adsorbed (s4dU)35, under the detectable level by CLSM,

cannot be excluded completely inhibiting the reverse tran-

scription. The highly potent inhibition of virus entry (Table

–E–] and (dC)35 [–x–] in a mixture of nucleases. (b) Effect of (s4dU)35 on

05 nucleated bone marrow cells are shown as fraction of the control. The

SE of three experiments.

A. Horvath et al. / Virology 334 (2005) 214–223 219

2) indicates that the inhibition of reverse transcription

cannot be the main mechanism of the anti-HIV action of

(s4dU)35.

(s4dU)35 inhibited the in vitro replication of various

laboratory strains of HIV, including resistant mutants to

NRTI and NNRTI (Table 1), with IC50s in the 0.8 to 25.4

Ag/ml range (0.07-2.23 AM), which is better or comparable

to the activity of other reported oligonucleotide based anti-

HIV agents (Agrawal et al., 1989; Bardos et al., 1992;

Buckheit et al., 1994; Inagawa et al., 2002; Jing and Hogan,

1998; Kinchington et al., 1992; Kuwasaki et al., 2003;

Matsukura et al., 1987; Ojwang et al., 1995; Tsukahara et

al., 1997; Yamaguchi et al., 1997). (s4dU)35 was also shown

to be noncytotoxic at concentrations 50 to 1000 times its

IC50 and exhibited negligible effect on colony formation of

granulocyte-macrophage progenitor cells (Fig. 4b). These

experimental data identify (s4dU)35 as a nontoxic anti-HIV

agent, acting as an entry inhibitor.

The potent inhibition of virus entry (IC50 = 0.002 Ag/ml)

and the inhibition of the virus replication at much higher

concentrations, as both of them are documented herein

(Tables 1 and 2), is an apparent contradiction. However,

similar observations have also been reported in the literature

(Chang and Bewley, 2002). It is potentially possible that the

reasons for this differential outcome could be due to the fact

that whereas the assay for entry determines the effect of one

round of infection, the antiviral assay measures multiple

rounds of infections.

Chemically, at least three features of (s4dU)35 could be

important for its antiviral activity. First: the 4-thiono group

increases the hydrophobic character of the base (T}ookes andAradi, 1996). Second: the 4-thiono group has a propensity

toward tautomeric conversion (Simuth et al., 1970) to form

reactive -SH groups at position 4. Third: the 4-thiolation

converts the natural oligonucleotide to a highly nuclease

resistant molecule (Fig. 4a). The dependence of antiviral

activity on the length of the thiolated oligonucleotides

indicates that the presence of the 4-thiono or 4-mercapto

group alone were not sufficient to mediate inhibition of

virus entry. Chain-length dependency further suggests an

aptamer-like effect or a size dependent affinity for the

antiviral target(s) requiring hydrophobic and/or ionic inter-

action. A similar chain length-dependent effect was

observed for other oligonucleotide-based entry inhibitors

(Bardos et al., 1992; Matsukura et al., 1987).

A significant difference between (s4dU)35 and many

other previously reported oligonucleotides with anti-HIV

activity is that (s4dU)35 does not penetrate into the cells.

This feature may be partly responsible for its low toxicity.

On the other hand, the nontoxic nature of (s4dU)35 could be

due to the fact that its biological effect is mediated at the

cell surface with specificity for CD4 and perhaps other yet

to be identified proteins involved in HIV-1 entry. It should

be noted that 4-thiouracil is a naturally occurring base

(Lipsett, 1965), and that disulfide linkage can be formed

between two 4-thiouridines (Shefter and Kalman, 1968),

and poly-4-thiouridylic acid could support the synthesis

(Fiser et al., 1974) of polyphenylalanine as an artificial

mRNA.

Since the 4-thiono group has a propensity toward

tautomeric conversion (Simuth et al., 1970) to form reactive

-SH groups at position 4, intervention by (s4dU)35 during

the cell surface disulfide exchange processes is chemically

feasible and if it precedes the cell-virus interaction could be

the bases for its antiviral activity.

Redox change within the second domain of CD4

molecule is required for successful HIV-1 entry, mediated

by cell surface thioredoxin (Matthias et al., 2002). Therefore

we first studied the interaction between of (s4dU)35 and CD4

and thioredoxin. The results of CD4/(s4dU)35 and CD48/

(s4dU)35 competition studies together with the data of FRET

analysis strongly suggest that (s4dU)35 is associated with

CD4 molecules but not with CD48. On addition, the CLSM

study (Fig. 3) suggests that (s4dU)35 may interact with cell

surface thioredoxin, too. These proteins, with essential roles

in the entry process, carry free -SH groups. Our results and

the physico-chemical nature of -SH groups are consistent

with the formation of disulfide linkage between -SH

carrying proteins and thiolated oligonucleotide. While this

is conceptually possible, it is important to specifically

demonstrate that this indeed occurs. Thus, further studies are

needed to determine the nature of these interactions and to

show whether any covalent interactions could occur

between (s4dU)35, CD4, thioredoxin and other proteins,

subjects of redox reactions and involved in HIV entry. It has

to be noted that another redox process between PDI and

gp120 is also required for HIV-1 entry (Barbouche et al.,

2003; Gallina et al., 2002) which perhaps may serve as

additional targets for -SH interacting agents.

In summary, (s4dU)35 is a thiolated oligonucleotide with

low in vitro cytotoxicity and potent inhibition of HIV entry.

The results presented herein further demonstrate that the

entry process of HIV can be specifically interdicted with

low cellular toxicity, identifying (s4dU)35 as a new member

of entry inhibitors, with a potential as either a systemic

inhibitor of HIV replication or for use in prevention of HIV

transmission.

Materials and methods

Synthesis and characterization of (s4dU)35

We synthesized (s4dU)35 according to our earlier

developed method (T}ookes and Aradi, 1996), by conversion

of (dC)35 to (s4dU)35. This method is based on published

procedures of the synthesis of 4-thio-uridylate containing

oligonucleotides (Miura et al., 1973) and of the conversion

of polycytidylic acid to poly-4-thiouridylic acid (Hochberg

and Keren-Zur, 1974).

For preparation of (s4dU)35 the starting material, chro-

matography-purified (dC)35, was purchased from IDT

A. Horvath et al. / Virology 334 (2005) 214–223220

(Coralville, IA). The thiolated oligonucleotide was isolated

by two precipitations with 2 volumes of alcohol from a

solution containing 0.3 M Na-acetate (pH 5.4). The

precipitates were washed twice with 80% alcohol, dissolved

in water and purified in 6 mg portion by ion exchange

chromatography (Horvath and Aradi, 2005). Most of the

batches proved to be homogenous by ion exchange

chromatography; therefore, the purification by chromato-

graphy could have been omitted.

The ratio of thiolated and unmodified bases in the final

product was determined after complete degradation of the

thiolated oligonucleotide to nucleosides (4-thio-deoxyuri-

dine and deoxycytidine) by phosphodiesterase and phos-

phatase followed by HPLC analysis, as we have described

earlier (T}ookes and Aradi, 1996).

The homogeneity of (s4dU)35 was further proved by

denaturing gel electrophoresis; 0.5, 1.0, 2.0, 4.0 Agoligonucleotide was applied to denaturing polyacrylamide

gel, and visualized by silver staining (PlusDNARAmersham-Pharmacia).

The (s4dU)35 used in this study was homogenous in

length and 98.64% of the bases were thiolated and 1.36%

were unmodified. The theoretical molecular mass of

(s4dU)35-Na salt is 11,403 kDa.

The fluorescently labeled (s4dU)35 was synthesized

using an automated oligonucleotide synthesizer (Pharma-

cia Gene-Assembler Plus) by standard phosphoramidite

chemistry in our laboratory. The 4-thio-deoxyuridine

phosphoramidite and Cy3 or Cy5 phosphoramidite were

purchased from Glen Research (Sterling, VA) and

Amersham-Pharmacia, respectively. Crude products were

purified by ion-exchange chromatography (Amersham-

Pharmacia Akta Purifier, on a RESOURCE Q column)

using Na-perchlorate as an eluent (Horvath and Aradi,

2005).

Cells and viruses

The CEM-SS, HL2/3 and HeLa CD4 LTR h-gal cell lineswere obtained from the NIH AIDS Research and Reference

Reagent Program (Bethesda, MD). CEM-SS cells were

maintained in RPMI 1640 medium supplemented with 10%

Fetal Calf Serum (FCS), 2 mM l-glutamine, and antibiotics.

HL2/3 and HeLa CD4 LTR h-gal were maintained in

DMEM 10% FCS, 2 mM l-glutamine, and antibiotics.

Kit225 K6 human T lymphoma cells were cultured in

RPMI 1640 medium supplemented with 10% FCS and

antibiotics in the presence of human recombinant IL-2.

Human immunodeficiency virus type 1 (HIV-1) strains

RF, IIIB and the molecular clones of NL4-3 were

obtained from the NIH AIDS Research and Reference

Reagent Program (Buckheit et al., 1995a). 4X AZT

contains mutations at D67N, K70R, T215Y and K219Q to

express complete resistance to AZT. DSP-R, a viral strain

resistant to NNRTIs, was generated as described earlier

(Buckheit et al., 1995b).

Antiviral assay based on RT activity measurement

Antiviral activity in the indicated experiments was

determined by measuring the RT activity in the culture

fluid of infected MT-4 cells after 96 h of infection (Hoffman

et al., 1985). The protocol is summarized briefly below.

To determine the anti-HIV activity of thiolated oligo-

nucleotides with various chain-length, 2.5 � 105 MT-4

cells were treated with the oligonucleotide inhibitors (in

300 Al) for 1 h before infection then infection was made at

a MOI (multiplicity of infection) of 0.01 with a virus stock

of HIV-1IIIB (100 Al) and after 1 h 600 Al of complete

tissue culture medium was added to get the final volume of

1 ml. Eventually, the treated and infected cells were grown

at 37 8C in RPMI 1640 medium, supplemented with 10%

heat-inactivated FCS, 2 mM l-glutamine and antibiotics.

The RT activity was determined 96 h of postinfection as

described (Hoffman et al., 1985). The indicated oligonu-

cleotide concentration was present in the 300 Al cell

culture during the pre-incubation of the cells with (s4dU)n.

The inhibitors and unabsorbed viruses were not removed

after the infection. Uninfected and untreated samples

(negative control), infected and untreated samples (positive

control) were included in each experiment.

In the time-of-addition assay, the above described

protocol was used, except that the time of infection varied

relative to the addition of (s4dU)35.

Antiviral assay by determination of cytoprotection

The HIV-inhibitory activity of the compounds on CEM-

SS cells was evaluated as described previously (Buckheit et

al.,l 1993; Rice et al., 1997; Weislow et al., 1989) in a

microtiter anti-HIV assay which measures the ability of the

compound to inhibit HIV-1 induced cell killing. Zidovudine

(AZT, Sigma Chemical Co., St. Louis, MO) or nevirapine

were used as positive controls.

HIV attachment, binding and inhibition of cell–cell fusion

assays

Attachment and fusion assays were performed as we

described earlier (Buckheit et al., 1994) with minor

modifications. Briefly, the virus attachment and entry assays

were performed following the interaction of HIV-1IIIB with

HeLa CD4 LTR h-gal cells for 1 h at 37 8C, washing to

remove unbound virus and immediately quantitating cell-

associated p24 (virus binding) by ELISA (Coulter) or

culturing the cells for an additional 48 h and assessing

h-galactosidase reporter expression (virus entry). The h-galactosidase enzyme expression was detected by Tropix

Gal-screen (Tropix, Bedford, MA).

Inhibition of fusion was quantitated by coculture of HL2/

3 (expressing HIV Tat and Env proteins) and HeLa CD4

LTR h-gal cells for 48 h in the presence of drug and

determining h-galactosidase reporter expression measured

A. Horvath et al. / Virology 334 (2005) 214–223 221

by Tropix Gal-screen. Chicago Sky Blue (CSB), a poly-

sulfonic acid dye inhibitor of HIV attachment and fusion

was used as a positive control for all mechanism assays

(Clanton et al., 1992).

Methods for study the interaction between CD4 receptor

and (s4dU)35

The MEM115 monoclonal antibody (mAb) against CD4

molecule and MEM102 mAb against CD48 molecule was a

kind gift from Vaclav Horejsi (Institute of Molecular

Genetics, Prague, Czech Republic).

For labeling of the antibodies with fluorescent probes,

purified Abs were conjugated with succinimidyl ester of

Alexa 546 (Molecular Probes, Eugene, OR) or with

sulfoindo-cyanin succinimidyl bifunctional ester derivative

of Cy3 and Cy5 (Amersham, Braunschweig, Germany) as

we described earlier (Matko et al., 2002).

For labeling of cell-surface antigens, about 106 cells in 50

Al of PBS were incubated with fluorescently tagged label-

ing molecules for 30 min on ice in the dark. The applied

antibody concentration (50–100 Ag/ml) saturated all avail-

able binding sites. The excess mAb was removed by washing

the cells twice in PBS. Antibodies were centrifuged at

100,000 � g for 30 min before use to remove aggregated

molecules. Flow cytometric measurements were carried out

using a Becton Dickinson FACScalibur flow cytometer ac-

cording to our earlier published method (Matko et al., 2002).

For competition assays, cells were incubated with the

mixture of fluorescently conjugated mAb and unlabeled

(s4dU)35. Competition was calculated from the fractional

decrease of the fluorescence of the antibody in the presence of

the (s4dU)35.

For FRET measurements, cells were labeled simulta-

neously with the mixture of donor Cy3-labeled mAb and

with acceptor Cy5-labeled (s4dU)35. Four fluorescent

intensities were detected. Three were excited at 488 nm

and detected at 530 F 30 nm, 585 F 42 nm and above

670 nm, respectively, and the fourth was excited at 635 nm

and detected at 661 F 16 nm. Correction factors for the

spectral overlap between the different fluorescence channels

were obtained from data measured on unlabeled and single-

labeled cells. Forward and side angle light scatterings were

used for gating out debris and dead cells. We calculated the

energy transfer efficiency on a cell-by-cell basis from the

four fluorescence intensities on 10,000 cells using list mode

data (Sebestyen et al., 2002).

The calculated energy transfer efficiency was expressed

as the ratio of the number of excited donor molecules

tunneling their excitation energy to the acceptor and the

number of all excited molecules.

Confocal laser scanning microscopy analysis

The CLSM studies were performed as we have described

earlier (Sebestyen et al., 2002). Briefly, monoclonal anti-

body against cell surface thioredoxin (Pharmingen) was

labeled with sulfoindo-cyanin succinimidyl ester derivative

of Cy3 (Amersham). For labeling of cell-surface, about 106

cells in 50 Al of PBS were incubated with Cy3 and Cy5

tagged molecules for 30 min on ice, in the dark, the cells

were washed twice before analysis. The microscope was a

Zeiss, LSM 510 model.

DNase stability of (s4dU)35

Briefly, DNase stability was determined in a final volume

of 500 Al. The final reaction mixture contained 100 mMTris–

HCl (pH 8.0), 5 mMMgCl2, 1,250 Ag/ml (s4dU)35, 50 Ag/ml

phosphodiesterase I (0.16 U/mg, Sigma), 25 Ag/ml deoxy-

ribonuclease I (3000 Kunitz U/mg, Sigma) and 0.025%

NaN3. The reaction mixture was incubated at 37 8C, 40 Alaliquots were removed at the times indicated, and then the

terminal phosphate hydrolyzed by alkaline phosphatase (E.

coli A19, Amersham) and determined (Tokes and Aradi,

1996).

Toxicity of (s4dU)35 on human bone marrow

granulocyte-macrophage progenitor cells

The effect of (s4dU)35 on the colony formation of human

granulocyte-macrophage progenitor cells was determined

using published methods (Benk}oo et al., 1999). Management

of patients conformed to the Helsinki Declaration. The

protocol was approved by the Regional and Institutional

Ethics Committee of University of Debrecen, Hungary

(DEOEC RKEB/IKEB-2055/2003).

Human bone marrow samples, derived from adults,

were aspirated for diagnostic purposes and included in

these experiments only if subsequent morphological

examination failed to find any major abnormality (from

three patients). The mononuclear cell fraction was

separated by Ficoll-Iodamide gradient centrifugation

(1.077 g/ml). Methyl cellulose cultures were made by

using inocula of 2 � 105 bone marrow cells/ml in Petri

dishes and were grown in McCoy’s 5A modified medium

(GIBCO, Grand Island, NY) supplemented with amino

acids, Na pyruvate, NaHCO3 and antibiotics as described

(Benk}oo et al., 1999), using 1.2% Methyl cellulose

(Methocel, Fluka Chemie AG, Buchs). Mononuclear bone

marrow cells were grown in the presence of (s4dU)35(highest concentration: 180 Ag/ml, 15.8 AM) incubated

for 14 days at 37 8C at 100% relative humidity in an

atmosphere containing 5% CO2. Colonies, defined as

groups of at least 50 cells, were counted under a

dissecting microscope at the end of the incubation period.

Acknowledgments

This work was supported by a Hungarian Science Foun-

dation (OTKA) grant T 038163 to J.A., grant T 043061 to J.S.

A. Horvath et al. / Virology 334 (2005) 214–223222

References

Agrawal, S., Ikeuchi, T., Sun, D., Sarin, P.S., Konopka, A., Maizel, J.,

Zamecnik, P.C., 1989. Inhibition of human immunodeficiency virus in

early infected and chronically infected cells by antisense oligodeoxy-

nucleotides and their phosphorothioate analogues. Proc. Natl. Acad.

Sci. U.S.A. 86, 7790–7794.

Barbouche, R., Miquelis, R., Jones, I.M., Fenouillet, E., 2003. Protein-

disulfide isomerase-mediated reduction of two disulfide bonds of HIV

envelope glycoprotein 120 occurs post-CXCR4 binding and is required

for fusion. J. Biol. Chem. 278, 3131–3136.

Bardos, T.J., Schinazi, R.F., Ling, K.H., Heider, A.R., 1992. Structure-

activity relationships and mode of action of 5-mercapto-substituted

oligo- and polynucleotides as antitemplates inhibiting replication of

human immunodeficiency virus type 1. Antimicrob. Agents Chemother.

36, 108–114.

Benk}oo, I., Hernadi, F., Megyeri, A., Kiss, A., Somogyi, G., Tegyey, Z.,

Kraicsovits, F., Kovacs, P., 1999. Comparison of the toxicity of

fluconazole and other azole antifungal drugs to murine and human

granulocyte-macrophage progenitor cells in vitro. J. Antimicrob.

Chemother. 43, 675–681.

Buckheit, Jr., R.W., Hollingshead, M.G., Germany-Decker, J., White, E.L.,

McMahon, J.B., Allen, L.B., Ross, L.J., Decker, W.D., Westbrook, L.,

Shannon, W.M., Weislow, O., Bader, J.B., Boyd, M.R., 1993.

Thiazolobenzimidazole: biological and biochemical anti-retroviral

activity of a new nonnucleoside reverse transcriptase inhibitor. Antiviral

Res. 21, 247–265.

Buckheit, Jr., R.W., Roberson, J.L., Lackman-Smith, C., Wyatt, J.R.,

Vickers, T.A., Ecker, D.J., 1994. Potent and specific inhibition of

HIV envelope-mediated cell fusion and virus binding by G quartet-

forming oligonucleotide (ISIS 5320). AIDS Res. Hum. Retroviruses

10, 1497–1506.

Buckheit, Jr., R.W., Fliakas-Boltz, V., Decker, W.D., Roberson, J.L., Stup,

T.L., Pyle, C.A., White, E.L., McMahon, J.B., Currens, M.J., Boyd,

M.R., Bader, J.P., 1995a. Comparative anti-HIV evaluation of diverse

HIV-1-specific reverse transcriptase inhibitor-resistant virus isolates

demonstrates the existence of distinct phenotypic subgroups. Antiviral

Res. 26, 117–132.

Buckheit Jr., R.W., Kinjerski, T.L., Fliakas-Boltz, V., Russell, J.D., Stup,

T.L., Pallansch, L.A., Brouwer, W.G., Dao, D.C., Harrison, W.A.,

Schultz, R.J., Bader, J.P., Stringner, Y.S., 1995b. Structure-activity and

cross-resistance evaluation of a series of human immunodeficiency

virus type-1-specific compounds related to oxathiin carboxanilide.

Antimicrob. Agents Chemother. 39, 2718–2727.

Chang, L.C., Bewley, C.A., 2002. Potent inhibition of HIV-1 fusion by

cyanovirin-N requires only a single high affinity carbohydrate binding

site: characterization of low affinity carbohydrate binding site knockout

mutants. J. Mol. Biol. 318, 1–8.

Clanton, D.J., Moran, R.A., McMahon, J.B., Weislow, O.S., Buckheit Jr.,

R.W., Hollingshead, M.G., Ciminale, V., Pavlakis, B.K., Bader, J.P.,

1992. Sulfonic acid dyes: inhibition of the human immunodeficiency

virus and mechanism of action. J. Acquired Immune Defic. Syndr. 5,

771–781.

Cooley, L.A., Lewin, S.R., 2003. HIV-1 cell entry and advances in viral

entry inhibitor therapy. J. Clin. Virol. 26, 121–132.

De Clercq, E., 1993. HIV-1-specific RT inhibitors: highly selective

inhibitors of human immunodeficiency virus type 1 that are

specifically targeted at the viral reverse transcriptase. Med. Res. Rev.

13, 229–258.

Del Real, G., Jimenez-Baranda, S., Lacalle, R.A., Mira, E., Lucas, P.,

Gomez-Mouton, C., Carrera, A.C., Martınez-A., C., Manes, S., 2002.

Blocking of HIV-1 infection by targeting CD4 to nonraft membrane

domains. J. Exp. Med. 196, 293–301.

Dimitrov, D.S., 1997. How do viruses enter cells? The HIV coreceptors

teach us a lesson of complexity. Cell 91, 721–730.

Doms, R.W., Moore, J.P., 2000. HIV-1 membrane fusion: targets of

opportunity. J. Cell Biol. 151, F9–F13.

Eckert, D.M., Kim, P.S., 2001. Mechanisms of viral membrane fusion and

its inhibition. Annu. Rev. Biochem. 70, 777–810.

Fiser, I., Scheit, K.H., Stfffler, G., Kuechler, E., 1974. Identification of

protein S1 at the messenger RNA binding site of the Escherichia coli

ribosome. Biochem. Biophys. Res. Commun. 60, 1112–1118.

Gallina, A., Hanley, T.M., Mandel, R., Trahey, M., Broder, C.C.,

Viglianti, G.A., Ryser, H.J.-P., 2002. Inhibitors of protein-disulfide

isomerase prevent cleavage of disulfide bonds in receptor-bound

glycoprotein 120 and prevent HIV-1 entry. J. Biol. Chem. 277,

50579–50588.

Hochberg, A.A., Keren-Zur, M., 1974. The synthesis of poly-4-thiouridylic

acid and its effect on protein synthesis in a cell free system derived from

rat liver. Nucleic Acids Res. 1, 1619–1630.

Hoffman, A.D., Banapour, B., Levy, J.A., 1985. Characterization of the

AIDS-associated retrovirus reverse transcriptase and optimal conditions

for its detection in virions. Virology 147, 326–335.

Hogg, P.J., 2003. Disulfide bonds as switches for protein function. Trends

Biochem. Sci. 28, 210–214.

Horvath, A., Aradi, J., 2005. Advantages of sodium-perchlorate solution as

mobile phase for purification of synthetic oligonucleotides by anion-

exchange chromatography. Anal. Biochem. 338, 341–343.

Inagawa, T., Nakashima, H., Karwowski, B., Guga, P., Stec, W.J., Takeuchi,

H., Takaku, H., 2002. Inhibition of human immunodeficiency virus type

1 replication by P-stereodefined oligo(nucleoside phosphorothioate)s in a

long-term infection model. FEBS Lett. 528, 48–52.

Jing, N., Hogan, M.E., 1998. Structure-activity of tetrad-forming oligonu-

cleotides as a potent anti-HIV therapeutic drug. J. Biol. Chem. 273,

34992–34999.

Kilby, J.M., Eron, J.J., 2003. Novel therapies based on mechanism of HIV-1

cell entry. N. Engl. J. Med. 348, 2228–2238.

Kinchington, D., Galpin, S., Jaroszewski, J.W., Ghosh, K., Subasinghe, C.,

Cohen, J.S., 1992. A comparison of gag, pol and rev antisense

oligodeoxynucleotides as inhibitors of HIV-1. Antiviral Res. 17, 53–62.

Kuwasaki, T., Hatta, M., Takeuchi, H., Hiroshi, T., 2003. Inhibition of

human immunodeficiency virus 1 replication in vitro by a self-stabilized

oligonucleotide with 2V-O-methyl-guanosine-uridine quadruplex motifs.

J. Antimicrob. Chemother. 51, 813–819.

LaBranche, C.C., Galasso, G., Moore, J.P., Bolognesi, D.P., Hirsch, M.S.,

Hammer, S.M., 2001. HIV fusion and its inhibition. Antiviral Res. 50,

95–115.

Lipsett, M.N., 1965. The isolation of 4-thiouridylic acid from the soluble

ribonucleic acid of Escherichia coli. J. Biol. Chem. 240, 3975–3978.

Majumdar, C., Stein, C.A., Cohen, J.S., Broder, S., Wilson, S.H., 1989.

Stepwise mechanism of HIV reverse transcriptase: primer function of

phosphorothioate oligodeoxynucleotide. Biochemistry 28, 1340–1346.

Markovic, I., Stantchev, T.Z., Fields, K.H., Tiffany, L.J., Tomic, M., Weiss,

C.D., Broder, C.C., Strebel, K., Clouse, K.A., 2004. Thiol/disulfide

exchange is a pre-requisite for CXCR4-tropic HIV-1 envelope-mediated

T-cell fusion during viral entry. Blood 103, 1586–1594.

Matko, J., Bodnar, A., Vereb, G., Bene, L., Vamosi, G., Szentesi, G.,

Szfllfsi, J., Gaspar Jr., R., Horejsi, V., Waldmann, T.A., Damjanovich,

S., 2002. GPI-microdomains (membrane rafts) and signaling of the

multi-chain interleukin-2 receptor in human lymphoma/leukemia T cell

lines. Eur. J. Biochem. 269, 1199–1208.

Matsukura, M., Shinozuka, K., Zon, G., Mitsuya, H., Reitz, M., Cohen,

J.S., Broder, S., 1987. Phosphorothioate analogs of oligodeoxynucleo-

tides: inhibitors of replication and cytopathic effects of human

immunodeficiency virus. Proc. Natl. Acad. Sci. U.S.A. 84, 7706–7710.

Matthias, L.J., Hogg, P.J., 2003. Redox control on the cell surface:

implication for HIV-1 entry. Antioxid. Redox Signal. 5, 133–138.

Matthias, L.J., Yam, P.T.W., Jiang, X.-M., Vandegraaff, N., Li, P.,

Poumbourios, P., Donoghue, N., Hogg, P.J., 2002. Disulfide exchange

in domain 2 of CD4 is required for entry of HIV-1. Nat. Immunol. 3,

727–732.

Miura, K., Shiga, M., Ueda, T., 1973. Nucleosides and nucleotides: VI.

Preparation of diribonucleoside monophosphates containing 4-thiour-

idine. J. Biochem. 73, 1279–1284.

A. Horvath et al. / Virology 334 (2005) 214–223 223

Ojwang, J.O., Buckheit, R.W., Pommier, Y., Mazumder, A., De Vreese, K.,

Este, J.A., Reymen, D., Pallansch, L.A., Lackman-Smith, C., Wallace,

T.A., De Clercq, E., McGrath, M.S., Rando, R.F., 1995. T30177, an

oligonucleotide stabilized by an intramolecular guanosine octet, is a

potent inhibitor of laboratory strains and clinical isolates of human

immunodeficiency virus type 1. Antimicrob. Agents Chemother. 39,

2426–2435.

Percherancier, Y., Lagane, B., Planchenault, T., Staropoli, I., Altmeyer, R.,

Virelizier, J.-L., Arenzana-Seisdedos, F., Hoessli, D.C., Bachelerie, F.,

2003. HIV-1 entry into T-cells is not dependent on CD4 and CCR5

localization to sphingolipid-enriched, detergent-resistant, raft membrane

domains. J. Biol. Chem. 278, 3153–3161.

Popik, W., Alce, T.M., Au, W.-C., 2002. Human immunodeficiency virus

type 1 uses lipid raft-colocalized CD4 and chemokine receptors for

productive entry into CD4+ T cells. J. Virol. 76, 4709–4722.

Rice, W.G., Turpin, J.A., Huang, M., Clanton, D., Buckheit Jr., R.W.,

Civell, D.G., Wallqvist, A., McDonnell, N.B., DeGuzman, R.N.,

Summers, M.F., Zalkow, L., Bader, J.P., Haugwitz, R.D., Sausville,

E.A., 1997. Azodicarbonamide inhibits HIV-1 replication by targeting

the nucleocapsid protein. Nat. Med. 3, 341–345.

Richman, D.D., 2001. HIV chemotherapy. Nature 410, 995–1001.

Sebestyen, Z., Nagy, P., Horvath, G., Vamosi, G., Debets, R., Gratama, J.W.,

Alexander, D.R., Szfll}oosi, J., 2002. Long wavelength fluorophores and

cell-by-cell correction for autofluorescence significantly improves the

accuracy of flow cytometric energy transfer measurements on a dual-laser

benchtop flow cytometer. Cytometry 48, 124–135.

Shefter, E., Kalman, T.I., 1968. The molecular structure of the disulfide of

the t-RNA constituent, 4-thiouridine. Biochem. Biophys. Res. Com-

mun. 32, 878–884.

Simuth, J., Scheit, K.H., Gottschalk, E.M., 1970. The enzymatic synthesis

of poly 4-thiouridylic acid by polynucleotide phosphorylase from

Escherichia coli. Biochim. Biophys. Acta 204, 371–380.

Stein, C.A., Neckers, L.M., Nair, B.C., Mumbauer, S., Hoke, G., Pal,

R., 1991. Phosphorpthioate oligodeoxycytidine interferes with

binding HIV-1 gp120 to CD4. J. Acquired Immune Defic. Syndr. 4,

686–693.

Szatmari, I., T}ookes, S., Dunn, C.B., Bardos, T.J., Aradi, J., 2000.

Modified telomeric repeat amplification protocol: a quantitative

radioactive assay for telomerase without using electrophoresis. Anal.

Biochem. 282, 80–88.

T}ookes, S., Aradi, J., 1995. Inhibition of moloney murine leukemia virus

reverse transcriptase by partially 5-thiolated polyuridylic acid. Biochim.

Biophys. Acta 1261, 115–120.

T}ookes, S., Aradi, J., 1996. (s4dU)35: a novel highly potent oligonucleotide

inhibitor of the human immunodeficiency virus type 1 reverse

transcriptase. FEBS Lett. 396, 43–46.

Tsukahara, S., Suzuki, J., Hiratou, T., Takai, K., Koyanagi, Y., Yamamoto,

N., Takaku, H., 1997. Inhibition of HIV-1 replication by triple-helix-

forming phosphorothioate oligonucleotides targeted to the polypurine

tract. Biochem. Biophys. Res. Commun. 233, 742–747.

Weislow, O.S., Kiser, R., Fine, D.L., Bader, J., Shoemaker, R.H., Boyd,

M.R., 1989. New soluble-formazan assay for HIV-1 cytopathic effects:

application to high-flux screening of synthetic and natural products for

AIDS-antiviral activity. J. Natl. Cancer. Inst. 81, 577–586.

Yamaguchi, K., Papp, B., Zhang, D., Ali, A.N., Agrawal, S., Byrn, R.A.,

1997. The multiple inhibitory mechanisms of GEM 91R, a gag

antisense phosphorothioate oligonucleotide, from human immunodefi-

ciency virus type 1. AIDS Res. Hum. Retroviruses 13, 545–554.