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Inhibition of HIV-1 reverse transcriptase and HIV-1 integrase and

antiviral activity of Korean seaweed extracts

Mi-Jeong Ahn1, Kee-Dong Yoon1, Chul Young Kim1, So-Young Min1, Yong-ung Kim1,

Hyun Jin Kim2, Jeong Ha Kim2, Cha-Gyun Shin3, Chong-Kyo Lee4, Tae Gyun Kim5,Seung Hee Kim5, Hoon Huh1 and Jinwoong Kim1,*1College of Pharmacy, Seoul National University, Seoul, 151742, Korea; 2Department of Biological Sciences,

Sungkyunkwan University, Suwon, 440746, Korea; 3Department of Biotechnology, Chung-Ang University,

Anseong, Kyungki-Do, 456756, Korea; 4Korean Research Institute of Chemical Technology (KRICT), Taejon,

305600, Korea; 5National Institute of Toxicological Research, Korea Food and Drug Administration, Seoul,

122-020, Korea; *Author for correspondence (e-mail: jwkim@snu.ac.kr; fax +82-(2)-8878509)

Received 12 November 2001; accepted in revised form 19 December 2001

Key words:Antiviral activity, HIV-1 integrase, HIV-1 reverse transcriptase, Macroalgae, Seaweed extracts

Abstract

Forty-seven species of marine macroalgae from the coast of Korea have been screened for the presence of in-

hibitory compounds against human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and HIV-1

integrase (IN). One of 4 Chlorophyta, 8 of 17 Phaeophyta and 6 of 26 Rhodophyta showed inhibitory activity

against HIV-1 reverse transcriptase. Five species (Ecklonia cava,Ishige okamurae,Sargassum confusum,Sar-

gassum hemiphyllum,Sargassum ringgoldianum) belonging to Phaeophyta showed to inhibit the 3-processing

activity of HIV-1 integrase. In cell-based assays, the methanol extracts ofBossiellasp. andChondria crassicau-

lisinhibited cytopathogenecity of HIV-1 at a concentration below that cytotoxic for MT4 cells.

Abbreviations: CCID50

50% cell culture inhibitory dose, CHAPS 3-[(3-Cholamidopropyl)dimethylammo-

nio]-1-propane-sulfonate, DMSO dimethylsulfoxide, IN integrase, MOI multiplicity of infection, MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide, PMSF phenylmethylsulfonyl fluoride, RT re-

verse transcriptase

Introduction

Human immunodeficiency virus type 1 (HIV-1) is the

cause of acquired immunodeficiency syndrome

(AIDS) which has been a major human viral disease.

The development of anti-HIV agents has been con-

sidered to be one of the most important approaches

toward effective therapy for AIDS. The three viral

enzymes encoded by thepolgene of HIV-1 have been

regarded as the appropriate targets for the antiretrovi-

ral agents, since these enzymes play key roles in the

virus replication cycle. HIV-1 reverse transcriptase

(RT) converts the single-stranded (+) viral RNA ge-

nome into double-stranded proviral DNA prior to its

integration into the host genomic DNA (Rey et al.

1984). HIV-1 integrase (IN) is an enzyme that incor-

porates the double-stranded DNA product resulting

from the reverse transcription of viral RNA into a host

genome (LaFemina et al. 1992; Andrake and Skalka

1996). These enzymes along with retroviral protease

are not indigenous to the host and therefore represent

attractive targets for new anti-HIV agents.

The aim of this study was to screen a wide range

of seaweeds harvested in Korea for their ability to in-

hibit HIV-1 reverse transcriptase and HIV-1 integrase

and for their antiviral activity.

325Journal of Applied Phycology 14: 325329, 2002.

2003Kluwer Academic Publishers. Printed in the Netherlands.

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Materials and methods

Sampling and extraction

Seaweed samples were collected from the coasts of

Korea including Sungsanpo, Wando and Namhaedo

from January 1999 to June 2000. After cleaning thesurface of thalli with sea water to remove visible epi-

phytes and dirt, samples were dried at 60C for 12 h

in an oven, and then ground in a coffee grinder. A

1020 g sample was used for the extraction process,

which involved sonication three times with ca. 200

mL 100% MeOH,filtration and vacuum evaporation.

The exact mass was determined after complete lyo-

philization. Stock solutions were prepared in DMSO

and kept at 4C until appropriate dilutions of solu-

tion were used in each assay.

Assay procedures

The recombinant HIV-1 RT protein and the RT non-

radioactive assay kit were purchased from Roche Di-

agnostics GmbH (Mannheim, Germany). Nevirapine,

a reference compound, (Viramune, Boehringer-In-

gelheim Pharma KG, Germany) was provided from

Boehringer-Ingelheim, Korea. A 20 L of the reac-

tion mixture containing a template-primer hybrid and

10 M dUTP/dTTP was added to the wells of a

streptavidine coated microtiter plate which contained

20 L of 3 concentrated test samples and 4 ng of

the HIV-1 RT in 20 L lysis buffer. The reaction was

carried out at 37 C for 1 h and followed by the ad-dition of 200 L anti-digoxigenin-peroxide solution

and 200 L ABTS substrate solution. The absorbance

of each well was recorded at 405 nm with the refer-

ence wavelength, at 490 nm.

The preparation of recombinant HIV-1 integrase

and two radiolabeled 20-mer oligonucleotides and the

standard HIV-1 integrase assay (3- processing assay)

were performed as described in Kim et al. (1998). A

standard reaction assay of endonucleolytic activity

was carried out in the presence of potential inhibitor

containing 0.1 pmol of duplex oligonucleotide sub-

strate (K16 (U5-LTR, +strand), 5-[32P]TGTG-

GAAAATCTCTAGCAGT-3, and K17 (U5-LTR,

-strand), 5-ACTGCTAGAGATTTTCCACA-3) and

15 pmol of HIV-1 integrase in 15 mM Tris-HCl (pH

7.4), 100 mM NaCl, 1 mM MnCl2, 2 mM 2-mercap-

toethanol, 2.5 mM CHAPS, 0.1 mM EDTA, 0.1 mM

PMSF, 1% glycerol, and 10 mM imidazole in a total

volume of 10 L. Reaction mixtures were incubated

at 33 C for 90 min and stopped by the addition of 4

L of 95% formamide, 20 mM EDTA, 0.05% bro-

mophenol blue and 0.05% xylene cyanol FF. The re-

actions were heated to 90 C for 3 min and electro-

phoresed on a 20% denaturing polyacrylamide gel.

Reaction products were visualized by autoradiogra-

phy of the wet gel. IC50 values were calculated byscanning bands on Kodak-5film (Image Master VDS,

Pharmacia Biotech.).

A standard virus-induced cytopathic effect (CPE)

inhibition assay (Pauwels et al. 1988) was used for

anti-HIV-1 assays. MT4 cells (provided by Dr. N

Yamamoto at Tokyo Medical and Dental University,

Japan) at the log phase were pelleted and infected

with virus at an multiplicity of infection (MOI) of 100

CCID50

per well. The cells were immediately resus-

pended with RPMI 1640/10% fetal bovine serum at a

concentration of 106 cells/ml. One hundred micro-li-

ter of the resuspended cells were dropped into the

wells of a 96 well plate which initially contained 100l of 2 concentrated test samples. After 5 days of

incubation at 37 C, the cells were observed micro-

scopically and quantified using the MTT assay. Cyto-

toxicity was evaluated based on the viability of mock-

infected cells, monitored by MTT method. 2,3-

dideoxycytosine (ddC) (Sigma) and 2,3-

dideoxyinosine (ddI) (Sigma) were used as reference

compounds.

Results

Inhibition of HIV-1 RT by seaweed extracts

In an attempt to screen for antiviral activity, we eval-

uated the effect of the methanol extracts of Korean

seaweeds on the HIV-1 RT enzyme. Table 1 summa-

rizes the results of HIV-1 RT inhibition of 47 extracts

studied. At the concentration of 200 g mL1 of the

extracts, one (Monostroma grevillei) of 4 Chloro-

phyta, 8 (Ecklonia cava,Ishige okamurae,Myelophy-

cus simplex, Padina crassa, Sargassum confusum,

Sargassum hemiphyllum, Sargassum muticum, Sar-

gassum thunbergii) of 17 Phaeophyta and 6 (Acroso-

riumsp.,Dumontia simplex, Gloiopeltis furcata, Hyp-

nea japonica, Neorhodomela munica, Schizymenia

dubyi) of 26 Rhodophyta showed inhibitory activity

against HIV-1 RT. Among these, E. cava, I. okamu-

rae, S. confusum and S. hemiphyllum which all be-

long to Phaeophyta were found to inhibit HIV-1 RT

more potently than other 11 species. In the experi-

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ment using the nonradioactive RT kit, the IC50

value

of nevirapine was 0.097 g mL1.

Inhibition of HIV-1 IN by seaweed extracts

HIV-1 IN initially removes two nucleotide bases from

the 3-end of the linear viral DNA (3-processing).Then it cuts the host DNA and transesterifies its 5-

end to the viral DNAs newly shortened 3-end (inte-

gration). In this study, radiolabeled oligonucleotides

were used to measure the inhibitory effects on this

3-processing activity of HIV-1 IN by the methanol

extracts of Korean seaweeds.

Among the macroalgae tested, the methanol ex-

tracts of only five Phaeophyta species (E. cava, I.

okamurae,S. confusum, S. hemiphyllum,S. ringgold-

ianum) showed the inhibition of the 3-processing ac-

tivity of HIV-1 IN at the concentration of 100

g mL

1

(Table 1).E. cavashowed the strongest in-hibition of the IN activity. The IC50

value of baica-

lein, a reference compound, was 1.2 g mL1 in this

test.

Antiviral effects

In a cell-based assay, we measured the ability of the

test compounds to inhibit the HIV-1-induced cyto-

pathogenicity in HIV-1 strain IIIBinfected MT4 cells.

Bossiella sp. and Chondria crassicaulis showed a

weak prevention of the virus-induced cytopathogenic

effect at concentrations below that which is cytotoxic

for MT4 cells (Table 2).

Discussion

Recently, inhibitory activities of algal extracts have

been investigated against major enzymatic mecha-

nisms of HIV. Scheffler and Krylov (2000) reviewed

anti-HIV activity of algal extracts and the compounds

isolated from the algae. Anti-RT compounds includ-

ing Peyssonol A and KM043 have been reported by

several researchers (Talpir et al. 1994; Ohta et al.

1998). However, to our knowledge, this study is the

first report in screening inhibitory activity of marine

algae against HIV-1 integrase. For Korean seaweeds,

there have been a few previous attempts to screen

potential antiviral activities from the extracts. The in-

hibitory activity of Korean seaweeds on Tag DNA

polymerase, which is related to antiviral activity, has

been evaluated (Jin et al. 1997). Kim et al. (1997) also

reported the anti-Herpes simplex virus type 1

(HSV-1) and anti-Sindbis virus (SINV) activities of

27 Korean seaweeds.

In this study, E. cavaappeared to be a potent spe-

cies, which inhibited both the HIV-1 reverse tran-

scriptase (RT) and integrase (IN). This macroalga has

also been reported to show inhibitory activity against

TaqDNA polymerase (Jin et al. 1997). I. okamurae

andS. confusum, which displayed anti-HIV-1 RT andIN activities, could be another interesting species.

Kim et al. (1997) reported that I. okamuraeexhibited

antiviral activity against SINV. The inhibitory effect

ofS. confusumonTaqDNA polymerase has also been

identified (Jin et al. 1997). In anti-HIV-1 RT activity

test with organic fractions of these three species, the

ethylacetate fractions showed the most potent inhibi-

tory activity (Table 3). This result suggests that re-

sponsible compound(s) involved in these activities

may not be polysaccharides.

Extracts ofBossiellasp. and Chondria crassicau-

lis, which lowered virus cytopathogenecity in de no-

vo-infected MT4 cells, did not inhibit HIV-1 RT or

IN. Although Codium fragile, Ulva pertusa, Scytoci-

phon lomentaria, Undaria pinnatifidaand Carpopel-

tis affnisdid not show the anti-HIV-1 RT or IN ac-

tivity in this study, the methanol extracts of these

Table 2. Antiviral activity against HIV-1 by the methanolic ex-

tracts ofBossiellasp. andChondria crassicaulis.CC50

means con-

centration required to reduce the viability of MT4 cells by 50% as

determined by the MTT method, whereas EC50

denotes concentra-

tion required to prevent the cytopathic effect of HIV-1 by 50%. SI

stands for selective index (EC50/CC

50).

Species CC50

(g mL1) EC50

(g mL 1) SI

Bossiella sp. 120.4 49.2 2.5

Chondria crassicaulis 135.7 19.9 6.8

ddI > 100 7.15 > 14.0

ddC 3.54 0.06 61.6

Table 3. Inhibition of HIV-1 reverse transcriptase (RT) by the or-

ganic fractions (100 g mL1) of three Korean seaweed extracts.

+/, + and ++ indicate 5570%, 7085% and 85% inhibition of

HIV-1 RT, respectively.

Species CH2Cl

2 EtOAc BuOH

Ecklonia cavaKjellman ++ ++ +

Ishige okamuraeYendo ++ ++ ++Sargassum confusumC. Agardh +/ ++ +/

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seaweeds together withS. thunbergiiwere reported to

inhibit HSV-1 or SINV (Kim et al. 1997).

In conclusion, E. cavais one of the most promis-

ing species due to its inhibitory strength and the avail-

ability of sampling, and further investigations focus-

ing on identifying chemical compounds of valuable

fractions from several potent species, including E.cava, are in progress. The present study provides an

additional information of inhibitory activities of sea-

weeds against HIV-1, which can be useful for the

discovery of natural products for this particular virus.

Acknowledgements

We wish to thank S. M. Boo and Y. S. Oh for the

identification of seaweeds used in this study. This re-

search was supported by a grant from the Ministry of

Maritime Affairs & Fisheries of Korea (19980024).

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