Protective activity of tetracycline analogs against the cytopathic effect of the human...

~ I~ST~TUT PA.STEug/ELs~VIE~ Pads 1990

Ra=r. V.;yoL t99(% 14t~ 5-i6

PROTECTIVE ACTI~ITY OF TETRACYCLINE ANALOGS

AGA~!ST "fHE CY~I"OPATHIC EFFECT

OF THE HUMAN 13OtUNODEFICIENCY VIRUSES IN CEM CELLS

M. Lemaitrf (~), D. Gu4tard 6~), Y. H4r~n (.z)~ L. Montagnier (2) and A. Zeriai (~) (*)

(~) Rh6ne-.9ou~enc Santg, Centre de Recherche de ~qtr3"~ Ddparteme,'u de- Biologic, 13, Quai Yu;es (iuesde~

94403 /itry-sur-Seine Ceder (Francc}, .a, ad (z) Unitd d'Oncoiogie Virate,

h,stitut Pzsieur (UR CNRS 1157). 75724 Paris Cedex 15

SUMMARY

Tetracycline analogs were evaluated for anti-HIV activky in CEM :zils; mino¢:yclm.e and dox-?c.vcline were the most active of these in inhibiting the ~Srus-/nduced c.vtopathic effect bem'een 7 and I4 days post-infecfon° The aaive concentrations (O.3-J .5 ~tg/mt) were devoid of toxicity in unix~ected cultures. VL-~x~ production, h owe'mr, was not inhibited, indicating a dissociation between protection against celI death and suppression of virus growth. These protected cells coukl be maintained in culture for 6-7 weeks, even in the ab~nce of the compounds. After that period, ,¢Lru s production ceased and ceils could then be cultivated for severat months without loss of viabifity or reappearance of virus producEon. As HIV stocks produced in the presence of tetracycline aaatogs were unable to induce ceii death, we suggest that the ~'topathogenicity c~f HIV may be due in some cases to the presence of tetracycline-sensitive contaminating microorganisms.

F,.F¢-WORD3: AIDS, C~opathology: T 1}m'tphocyte, Tetracycline, HIV; HIV-I, HIV-2, CEM cell Hne.

Submited January 3, 199Q_, accepted January 9, 1990.

(*) To whom reprint requ~ts sho-ald be addressed.

6, M. L~E;vlAITRE E T AL.,

To date, the o~tly drug approved for therapy of AIDS is AZT (3'-azido-2',3'- dideoxb<hymidine). This compound acts as a reverse transcdptase (RT) in- hibitor after its 5 '-phosphorytatien inside the cell (Furman etaL, 1986) and has been reported to improve the ctinicai and irnmunotogicai status of patients with AIDS and AIDS-related complex (Fischi et aL., 1987).

Unfortunately, severe tor:icity, particularly bone marrow suppression: has oeen observed following AZ'r administration (Richman etaL, 1987). Toxic side effects were aIso found in patients treated with other compounds, indicating the need to search for more potent and selective compou;As and possibly other than nucleoside analogs.

In the course of our search for anti-HIV compounds, we observed that tetracycline was abIe to inhibit the cytopathic effect (CPE) of HIV-1 i1~ CEM ceils. Tiais activky, as measured 7-t5 days post-infection, was confirmed with other tetracyc!Jne analogs and for another human retrovirus (HIV-2). ttowever, protection against the CPE was not accompanied by inhibition of virus production, indicating an uncoupling ~,c~ween cytoprotccdve and antiviral activity. These compounds may" provide a too! tbr the study of cytopathogenicity associated with HIV replication in T lymphocytes.

M&TERIALS AND METHODS

Cells.

r CEM clone 13, a .ubc~one enriched iv. CD4 receptors, was obtained from the CEM T-Ib~phoblastoid turnout co!! i~ne (Monmgnier etaL, 1985) originally isoiar.ed from a child with acute lymphocVic !eukaemia (Folley etaL, I965). Cells were grown at 37¢'C in a CO 2 incubator (5 %) in RPMt-I640 medium, supplemented, with 10 % heat-inactivated foetal ca!f serum, peNciNn (IC£t tU/ml), s~reptomyc~rt (100 ~g/ml) and potybrene (S iva , 2 ~tg/rcd).

Viruses.

The LAV-Bru strain of HIV-! (Barrd-Sinoussi etaL, 1983) and the Red strain of HIV-2 (Clavel et aG 1986) were used in our experiments. Virus stocks were oh-

A I D S = a c q u i r e d i m m u n e d e f i c i e n c y ~ 'n f l rome .

A Z T = 3' -az ide -2' ,3 ' -dideox-ythym~dine. C C ~ = cul ture- inhibi t ing dose 50:7e. C C I D , , = ceU~culture-';nfeetlve dose 50 ga. C P E = cy topa th ic effect . ~ m = counts per minute . D M F = d ime thy l fo rmamide .

H i V - i , H I ~ - 2 = h u m a n -Lmmunodef~cie~cy v4~as D~2~. i

a n d 2. IL2 = 'nr.erteukin 2. P B L = per iphera l h~oed i-':rapk,c~cT~e. P B S = phospha~e-huf~'ered sM';r~e. P H A = pl '}aohaemaggludn~n. RT = reverse t raascr i~tase .

T E ~ , ~ C Y C L I N E S VS. t H V CYTOPA THO TV 7

t~;ned from filtered (0.45-~m pore size) suparnazan~s of CEM 13 ¢e~s. The t~r::s of the different HtV-I prepara.'..~ons ran~ed between ! and 8 .~ lO ~" 50 % ~ze~!-cuh~,'z¢- Snfeetive doses (CC~D~t~)!'mL ' lhe titres of the HIV-2 prei~a~a~o~s were ~t~:,r~dar~. ed using reverse tra~;scr~p~.as~: ~ ac~ivi:y of stock su~:~na ta~ (5,Y9¢90,3 G;.m/zM).

Compounds.

Tetracycline and chtonetracyctine were obta'~ned from R~6n~-?o~!en¢ San,~.

t~on was made in PBS to obtain a stock solution ~ was k~:~ z.z ~ C. ~ c i u ~ ~ were thawed and d~Iuted ~n PBS just before ~se,

"-J 2', '"i "

.II ~ ~'~,~ ~'~'c~'~ ::

.4

C F

Cell ~dab~ty was determined at day 7 ~ost*infe~on by- MTT absarban~ z,.: 5 ~ n~..

8 M : L E ~ I A I T R E E T A L .

Anli-Hl~--1 assay on CEM cells.

Compounds were tested for cytc~oxicky and for t.ileir abilffy ~c~ inMi2i~ ~:aP. cytopath!c effect induced by HIV.d in:::cctiop,+

in the micropiate (96-well) tests, 25 #.l of each compound dilution or PBS ;fione were distributed in. triplicate in each welt; 125 FI of CEM ceils (5 × I0: c¢l!s/ml) ;~re then added and suspensions were incubated for t h at 37°C (5 % CO2). Ceils were infected with 100 ~l of virus suspension (10G200 CCID.+e) and cultured for at least 7 days. Mock-infected cultures were carried out in paral~eI to determine the cyto- toxicity o f the compounds. To assess cei~ v'iabiliv, 100 M of celt sust~er~sion were collected and mixed witk t0 ¢ of a solution of (4,5-dimethylthiazoI-2-yi)-2,5-dil~henyi tetrazolium bromide (MTT) at 7 gg/mt in PBS (Schwartz er aL, 1988). AAer 3-h in- cubation at 37°C, most of the supeme_tant was removed and the formazan p.recip;.tate dissolved in !(~? ~I of 0.04 M HCt in 2-propanol. The absorbance at 540 nm was measured. Longer (> 7 days) incubations were carried out by adding ig~- a!/welI of fresh medium (containk'~g or not the compounds) to the remairAng ce!l sas~ension. This operation was repeated evely 4 days. RT activity in supernatants was determined in order to follow HIV repIicatiom For the RT determination, the micromethod

t2~ A D , ¢ _ . -

=. i 4 ( ~ , %-

e' + . . . . ] pg[mi

T o ~ . . . . . . e ' . + . . _ = . _ _ , i ~ . ~ _ . # g f m ~

, e l # 2 "k"i I 0 .~'~ .!~. £ t G-

Fro. 2. - - Activity q f doxycycline (-4, t7, C) and minoeycEne CD, E, F) oa HIV-h~fect.ed ~<~,+l and mock-infected ( 0 ) CEAf ceils+ . . . . . .

Cell viability was determined at dab's 9 (A:.+ D) 14 (B:, E) and 18 (C:, F) days ~osl-i~:cc~:ic/.~ by MTT adsorbance at 540 nm.

T E T R A C Y C L L N E S VS. H I V C Y T O P A T H O G E N I C I T Y 9

Mread7 9ubhshed was used (Schwartz ez aL, 1988). B~effy, 50 ~i of each supern> rant w~re di!uted whh 10 ~I o f buffer A (0.5 M KCI, 50 mM ~!fffio~hreito.',: Q5 % Trhon-Xttg)) and 40 ~,t reagent buffer (t .25 rnM EGTA, O. 125 M 'Iris~HC~ pH 7..~ ~2~5 mM MgCt2, 3 p£i of {methyl-SH)dTTP at 30 Ci/mmoI~ 0 . ( ! 5 0 . D i-:o?y('.:A).- oligo(dT:e)).

The plate~ were inc'abated for I h at 37°C. The reaction ;va~ a~.opI:~a~ " ~ b',. . . . . a~,~'~;';.,:,~ .... of 20 :al o f ice-cold I20 mM Na~P20: in 60 % ~richloroacedc acid. Ra.d~c.acti'dr!:y -~,::as cofiected on g.Ia.ss fiber filters using a celt hap,'ester apparatus (Skatrcn). RT &ct+e~ W was expresse~ m cpm.

AntI-HIV-2 assay on CEM cells,

CEM !3 ceils were infected with HtV-2 ( t0 a cpm RT/10 s cei!s) ar mock-;.infec:e:d fc)r 1 h at BT°C. Cel!s >-ere cemrKuged, washed twi~.e with fresh medium a::d s:e.:ied

_~_.-cm- culture flasks at a density of i o6 × 10~ ce.I}s/4 m~/iia~k. 2',:[i~o=3=cYr~e i "'~ or 3 ag/m]) or PBS was added. Cel} viability was de:.erm-~ne.d by z<,,l.,a~ b?xe e,:ziu~cn twice a~ week ; at d~e same time, ceils were readjested to a denshy of 1.8 "< !0Q/4 m:.-' a~:d further incubated with or without the compound. RT was deten~ined a.~ the t!mes bldic.ated in figure 3.

RESULTS

Cyto~rotecl ive activity of tetracycline analogs in HIV-l- infe , : ted CEM ceils.

In a f i rs t set o f e x p e r i m e n t s , C E M c lone I3 cells ~ micropia te~ were in- cub_~2ed wi th tetra%vcline, me tacyc l ine , doxycycI~ne, c51onetracyci~ne, bTnecyc l ine o r minoc:ecl ine a t di f feren~ c o n c e n t r a t i o n s and then i n f e c t e d or no~ .,-,.:h H t V - t . " '"~- " Re~,~,c are s h o w n L~ = . . . . . , , ~ . I t'A, B, C , D . E., F) : o r each

T,'-.~,'..~ I. - - Effect of letracycline analogs o , the CPE and virus pr.oduction in HIW-l-infected CEM cells.

V~rus detern2:J t:2aticn Protect ion agains~ C P E : % living ceils compared. to ~nh2fec~ed cuh~res a~ %1: CPE

day RT a.t dflutlon: Compotmd 7 13 !~ cpm×10." I 0 - : t0 -z 10 -s

~*{~rloc)'c~.{~:a 1.2~g/Iri~ 55 I (~ 90 3.55 70 50 0 O~ 9 " 55 1 ~ i.C~3 5.20 7.5 50 10

Dc, xyc,~ ;.~iae i .2 ~g/mt 50 i.0~ IC~3 3.36 72 50 0 " ~, ~. ~ .36 50 I0 O 0.39 ~0 "~ 25

None (; 50 I0 3 0.4 50- 10 0

via~i~ky a~ day~ 7, ~a ar.-:d I6. Su~ma-t,an~ a~ day ~6 w.ere ~..es-;ed 5at RT arid ~factkJD'; by l~termin{n~ a~ i0-L 10 -a and 18 -~ ~!='J~ns the de aT.~ of CPE ].n =-e=~pient CEM ce?2s.

10 ;t,f, LgJv fAITRE E T AL .

compound, respect[vely. In infected untreated cuk.ures; the CPE at day 7 post- infection was 50-70 %~ T~tracyclhie :rod ch~crtet, raeyc!ine reduced CPE at concentrations which sligh~.iy affected viabi[ky in u~infected cel~s, but. the odx¢; four compounds were a,.;t[ve at cor~centr.~. -;-.us totally devoid of cyto~oxicity.

In order to evaluate the ton.g4erm effic;;~%' of these compounds, in ar;.otlv.~r experiment, cells treated with doxycycline or minocycline were passaged at days 7, 10 and t4 and examined for ceK viability at days 9, i4 and 18. The results are shown in figure 2 (doxycycline: fig. 2A, B, C; or mfnocycline: fig. 2D, E, F). Fuit protection was obtained at day t4 with t~1 ~zg/ml doxycyctine (fig. 2B) and 0.37 and 1.1 ~g/rnI minocycline (fig. 2E). At day I8, the protection persisted, although for this particular experiment the action of doxycycline sIight!y decreased.

"A~en ceils were also pretreated for i0 days with minocycline (.0.2, I or 3 g.g/ml) before infection, no difference in the exk.~:t and time course of protection was noticed compared to celts treated only after infection (resuks not shown).

In summary, in the case of doxycyctine and minocydir~e, thc extent of" pro-. teetion at day 7 couId vary from one experiment to another, but protection was complete (t00 % i~s;~:ibition of CPE, Le. t00 %~ viable ceils compared to uninfected ae.d treated ce£ls) at day 14 post-infection. The concentration which inhibited col1 growth by 50 % (CCsa) ~as .8 ± 1.2 F.g/mt in the case of doxycycline (n = 5) and 2.8 ± 0.8 ~g/mI ( n = 6) in the case of m.inocyclinc. Results were cont~-med by counffng (trypan blue exclusion) exponemialIy grow- ing CEM ceUs. The average concentrations which inhibited the CPE by 50 eZ-, (ECHO were 0.314-0.12 ag/mI (n = ;~) and 0.2 + 0.1 !xg/ml (n = 4), respectively. Thus protection against CPE was seleoth.--e and the ratio CCsc./ECs0 was

HIV produced:

TABL~ II. - - Infectivity- (RT) and CPE of l i D stock-s prepared in the presence and in the absence of mittocyetine.

Eilect of HIV ~fecti.oa on. CEM ce~i~s- pretreated with mh;ocyc[ine withou~ I~re~reatment

day 7 day 18 day 7 day 18

without CPE 25 % 88 % 50 ;~ 92 % minocycline RT I00:CK~o N D 40,(~1~ N D

~-iti~ 2.4 ~g /ml CPE 0 -% 0 % 0 % 0 Z:, of minocycline RT 120:000 89,C~ I 6 0 ~ 96,cXG ~

t~rV was produced. L'~ CEM ¢¢1!~ after ~ d-zys (~Stho~t c-ompo~md) or afk~r 2i days ([~ th~ cr,za'.z~ of mino,~-c~e). Rec~pieat CEM ce.~ were pretrea_re5 or ~o~ for 21 da:,;s w~.h 2.4 gg./=a[ ~.i~tc=:..:~f~:¢l no compound was addezi af!er inf~den~

ND-= not done.

T E T R A CYCLI_,VE.S ,;~. h T V C Y T O P A THOGE,N,¥CIT-Y 11

approximately t0. In these experimental conditions:. AZT, ~ake~ a5 rcfere~oe compom~d, displayed an ECs0 of 64 ng/m! aact a ra~io CC.-UEC~.:~ ef }.5@.

It should be noted that the observed pro~:ctivc acd'~,ky ?~gai~}~ ,'-_iw CPi£ does not include inhibition of syncy[ia formation by the vku~. Ho;s<::ver, i:'~ CEM ceils and in our experimental conditions, the ex:en: of <,';~cy:ia ap- p.earance was negligib!e.

CelIs protected against CPE by tetracycline analogs do prod, ce virus in their supernatants.

In the experiment described in table t, CEM cN~s Sn 2~.--vd~ . . . . with minocycline at t.2 and .0.39 F~g/rnI or with do×ycF.cilne a~ i .2. ;s,~/m} wer<- 100 % protected against CPE from day t3. At day 16, ~.t~e: :?K~te.c:%::, ',an5 maintained, while infected u~'reated cultures were 97 7::~ ~ysett~ A.: "...i~!~ dr=e. 3.55 × IO s and 5.2× 105 cpm/mt of RT activity were found iii cukures r.rea.~:.~d whh minocycIine at, respectively, 1.2 and 0.39 tag/m!. Doxycyc!!::e-treated cultures also produced high levels of RT activkv.. ~ ,_.~.~.~."-v 3.!6 >< ~c'5.~ a:x! 1.36 × 105 cpm/mI at, respectively, 1.2 and 0.39 ~.g/m}. U.ntr.eatel i.'.:~fe-:;¢,:; control cultures produced only 4× 10 ~ cpm/mi...%t] @ese suIe:::-na;acts ',re.re: found Lo be infectious when titrated in either CEM (table I) o:7 M o b 4 cd?.s (no~ shown.)" 50 % cell lysis was obtained a.t a I 0 - : d.iiu:io.n: ind~ca.:ic:fi/ha; production of RT activity was indeed accompanied by infecfivky. Suw,r?,a<r->:s of prote~ed CEM ce~ recovered on day 25 post-infection were ~es~e~ 4 ~.n hum.an peripheral b!ood l}wnphoc?~es (PBL) for ~he presence of in£ectbus viru.s~. Target PBL were stimulated for 3 days with PI-k-k and in.cuba.ted i_n :he ;..resenc..e ,~-'~.~ 20 U/mA IL2 ~i th a ~'L~as stock (com.aimng 8 × 104 c~m RT/St ~1) or :.;:kh supematants of minocyciine(2. !. x i(14 cprn./50 ?J)- or doxycydke.2~9 × !04 cpm/50 ?.!)-treat.ed (I .2 pg/ml) CEM cukures. PBL cukures were foi!ow~z..c2 for 2I days. A peak of RT activity with similar cpm values was obsen:~d :.:or all samples tested at day 7 post-infection, ind~ca~g a .JmHar degre.e c~f ~ h> fectivitv. Thus. tetracycline analoes can prod, oct CKM ~"~'~ ~.:'~,,~, the cytopathic effect of HIV, but are unable ,*.o .~u r '~.~ "

Cytoproteciive aetiviiy of minocy_elioe ii~ HtV.2-infe~ied CEM c.el[so

HtV-2 is anoti~er humm ~. lemiv[rus wh.ich is a,.]se a.s._~oda.¢ed wkh A/,DS a::~ has similar biological properties, including tropism and a CPE for CD4 + t}~mphocytes. HIV-2 differs from HIV-I by a~ much as 60 % in, its nucleoffde sequence (ClaveI el aL, I986; Guyader e t a L , t987).

As shown in figure 3A, the HIV-2-infected CEM ceiA ~:.:ea:.ci ,~'2~ minocydine at i Ftg/mt d[splaTed a transient !oss of cell vi~bi]i~:,' at day i(} post-infection which was overcome tater on. In contrast,, v:~ab!.[hy was dramatically lost in un.treated HIV-2-infec~ed ceils. Withdra~vaI of ~he compound at day t 5 post-infeaion did not F.rovoke resumption of ~'ropatho~enid-

12 M . L E M A I T R E E T A L .

ty, a phenomenon also observed in the case of HIV-l-infected cells (see below). A similar pattern was observed at 3 t~g/ml (not shown), but at this concentration, the magnitude was less pronounced because of the toxicity of the compound.

Howew ,', minocycline-treated ce!is displayed prolonged important production of v~;~s, as indicated by the high RT activity in supernatant of infected treated cells (fig. 3B). At day 7 pest-infection, 3 × 10 5 cpm/m! were measured for untreated infected cultures compared to 5.5 x 10 5 for the treated one. At day 12 post-infection, RT ac:ivity of control cultures decreased to 1.Sz 10 5 cpm/ml, while it increased dramatically to 2.7× 10 6 cpm/ml in minocyclineotreated cultures.

The cytoprotective activity of tetracycline analogs is long lasting°

In the experiment reported in table I, HIV-l-infected cells treated with 1.2 ~tg/ml of either minocycline or doxycycline were allowed to grow, at day 18 post-infection, in 25-cm 2 culture flasks. Each sample was divided into two aliquots: one was continously treated with the compound and the other was grown in its absence. Cells were cultured, assayed for RT production and

4 8 12 1G 20 24 28 3

O i i | i J ;

4 8 12 16 20 24 28

days poet-Infection

FIG. 3. -- Cell viability (A) and R T activity (B) in control (0) and minocyeline-treated CEM cells" (O) as a function of time after infections with HIV-2.

Compound was present from day 0 to 15 at 1 ~tg/ml.

T E T R A C Y C L I N E S VS. H I V C Y T O P A T H O G E N I C I T Y !3

counted by trypan blue exclusion for more than 2 months. CPE did not emerge in cultures, even when the compounds were whhdrawn.

All four cell cultures displayed similar cell viability, ir~dicating that this concentration was well-tolerated in long lasting cell cultures. As shown in figure 4, virus productien continued and no significant difference in RT activity could be detected among the 4 sets of cultures (around 6× 105 cpm/ral). Between days 50 and 60 post-infection, RT activity gradually decreased, later reaching background levels. Reappearance of RT was not obse~ed thereafter. Cukuring was then discontinued and cells were frozen and stored in liquid nitrogen.

These long-term experiments, in which cells maintained viability even in the absence of the compounds and despite virus production, indicate that tetracycline analogs are capable of promoting the emergence of chronically infected cells.

Studies on the possible mode of action of tetracycfine analogs.

As cells protected against the CPE can be maintained alive even when tetracyclines are withdrawn (fig. 4), the mechanism of these compounds must certainly involve the permanent inactivation of a key component of the lytic effect of this virus. One possibility is that tetracyclines affect the viral and/or

RT { ~m.|0-31

4o[

32

' " ~ l " A . . . . . . . ~o . . . . i ; ' ' ~ 80 days post-IflfectloP.

FIG. 4. -- RT activity (expressed as cpm/50 ~0 in the supernatants o f C'E~I cells obtained at different days post-infection.

A total of 1.1 ~tg/ml minocycline (ff]) or doxycycline ((3) was present from day 0 to 16 or from day 0 to the end ( I , 0, respectively).

14 M. LEAIAITRE ET AL.

the cellular genomes or their expressiom Another hvj~othesis is that tetraeyciines might eliminate: a microorganism which potentiates HIV for ceil lysis. If this hypothesis is cc;rrect, one would expect that the virus produced in ",.~septic conditions", e.g. in the presence of high concentrations of minocyc!ine, would be unable to lyse CEM cells. As shown in table II, virus produced in the presence of 2.4 ~g/ml minocycline, when added to CEM ceils, wa~; found to be infectious, as determined by RT production, but was unabte to produce a CPE. CEM cells pretreated or not with ininocyc!ine did not differ in their CPE, indicating that a possible microorganism sensitive to minocycline was brought into our system by the HtV stock.

DISCUSSION

The results reported here indicate that tetracycline analogs, at subtoNc concentrations, are able to protect cetts from HIV-induced cytopathogenicity m~.cl to promote the establishment of chronicaliy infected cells. The most surprising observation was that, desp.~te their cytoprotective activity, these compounds were unable to block cat1 infection or giam ceil formation and to atop virus multiplication.

The only published report on tetracycline analogs and HtV concerns their inhibition of viral[ RT activity (Wondrak et at., 1988)o Doxycycline and chtortetracycline were the most active of these analogs, but their active concentration was high (IDa0 >t 17 !xg/ml) and their setecfi'Aty incomplete, since DNA polymerase ~ was also affected. The lack of antiviral activky at non- toxic concentrations which we describe in the present study is therefore con- sistent with the fact that high concentrations are required to inhibit the en- ~'me (Wondrak etaL, I988; and our own unpunished data).

Effective concentrations for inhibition of the CPE (0.3-1.5 ~g/mi) were devoid ef cytotoxicRy in uninfected cells. The tack of toxicity at these concentrations is also supported by the fact that they are easily reached in plasma of humans after oral administration of either n-Krlocycline or doxycycIine (.Jonas et aL, I984).

Cells which escape initial Iysis thro~:gn ;he action of these compounds can then be cultured far weeks in a chronic infectious state. Non-cytopathic persistent infections have already been described in macrophages (Gart ner etaL,. t986) and in some human T-cell tines (Hoxie etaL, 1985). Furthermore, once the chronic state is established, the compound can be removed without reappearance of the CPE. This long-Iastir~g effect can be explained through a direct interaction of tetracyclines with the virus and/or the cel!uIar genomes. However, this hypothesis is unlikely, as these compounds do not appear to interact directly with DNA (Jonas et aL, 1984)~ A more tike!y possibility is that a tetracycline-sensitive microorganism play~; the role of cofactor in HtV- induced cell Iysis. In contrast to the virus produced under standard conditions, viiias stocks prepared after long-terme treatment with high concentra,

TETRACYCLINES VS. H I V CYTOPA THOGENICITY !5

tion of minocycline were indeed unable to cause a CPE in redpieuc CEM ce~s. This suggests that a contaminating microorganism potentiates HtV for ~ts rapkI induction of ceil tysis (more than 50 % CPE at 7 days postqnfection).

Mycoplasmas are the likely candidates, as (1) we failed to de~ect visible contaminants, (2) they are known to be frequent and subtle contaminants of laboratory ceIi cultures, and (3) they are sensitive to tetracycline anaiogs. That HIV is also required to induce cell lysis is indicated by the fac~ that wdl- est~'Nished anti-retrovirus compounds are also abte to suppress CPE (De Clercq, 1988; and our own unpunished results). Therefore, the mos~ ~5ke!y explanation for the results presented here is that both HtV and a microorganism act synergistically to induce celt Ix, sis. Further experiments are presently being undertaken to isolate and identify the microorgaNsm and to investigate its role in HIV-induced pathogenicity.

RESUM£

ACTIV;Tg PROTECTRICE D'M,~ALOGUES DE LA TETRACYCL?_NE CONTRE L'Ev.-FET CYTOPATHOGENE DES V~RUS DE L'iMMUNODEY~CIENCE !~UMAI.'-,E

DANS LES CELLULES CEM

Les t~tracyctines, en particutier la minocycline e~ ia doxycydA.ne, inhibenz 1'effet cytopathoga~ne (ECP) de VIH-! et 2 en culture de cellu!es CEM. Cet~e act!vk~ a, exe> c~e/t des concentrations non cy~otoxiques, n'e-:.t toutefois pas accompaga~-e d'un offer ir~hibiteur de ia mukiplication virale. Los cel!ules i:.rot~gaes init~alerr:ent centre i'ECP peuvent 8tre cultiv~es durant plusieurs semaines, ma~me en Fabsence des preduits, tout en continuaet ~ excreter du virus dons ie surnageant. Apr~s 6-7 semaines, !a production virate pout s'mrrSter spontan~ment. Des stocks de vh-us pr@ar = & en prel- sence de t~traeycHnes se sont r~v ~i~s infectieux ma~s incapables de produ~re un ECP. Cette observation sugg~re que I'ECP des VIH est dfi, dons ce~ains cos, E ia presence concomitante de mfcroorganismes se~ibles aux t6trac:;clines.

MOTS.CLES; SIDA, V!H, T~tracycIine, Effet cs~opathog.,Sne, Lb..~phec?~e T; VIH-1, VIH-2, Lignde CEM.

ACKNOWLEDGEMENnYS

We thank Genevi&ve Co!son and Cbamal Ferrieux for exce~en~ technical a~s~s~ap.ce, Nad~ne Thomne!in for editoriai heip and Charles Dauguet for hdpfu[ d~scusqon°

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