Abstracts Vol. 1 [International Conference on AIDS (11th: 1996: Vancouver, Canada)]

Tu.A.2032 -Tu.A.2038

Tuesday July 9, 1996

Tu.A.2032
COMBINED TREATMENT WITH 3-AMINOBENZAMIDE AND N-ACETYLCYSTEINE
INHIBITS HIV REPLICATION IN U937-INFECTED CELLS
R. Rivabene, B.Varano, S. Gessani. L. Conti. G. Rainaldi, Al-M. Gasr,rioli, L,'; h G.
Donelli, W Malorni. Department of Ultrastructures and 'Virology Istituto Srpei(e di
Sanita,Viale Regina Elena 299, 001 61 Rome, Italy
The existence of a significant relationship between oxidai.'stress associated pptoposis and
the increase of viral progeny has been previously demonstrated in HIVchror cilly infected
U937 cells. Moreover, it has been demonstrated that both pheno ena can be modulated
by using some antioxidants, such as N acetylcysteine (NAC). The present inv:stigat ion was
aimed at studying lhe role of the nuclear enzyme poly-(ADP-r bose) polyrn;ase (PADPRP)
in HIV infected cells treated with iTumor Necrosis Factor alpha (TNFu), a cytokine capable
of inducing both apoptosis and intracellular oxygen f-ee radicatl production PADPRP utilizes
NAD+ as substrate to exert its function as DNA excission repair, may be activated as a
result of apoptosis-induced DNA fragrnentation and is associated to glutathione depletion.
For this purpose, we have used the specific PADPRP inhibitor 3-arnrrinobenzamide (3 ABA).
The results obtained by using 3-ABA, NAC and a comrbirned treatment 3-ABA/NAC given
together seem to confirm the role of intracellular redox balance in the modulation of the
HIV expression. In fact, a significant reduction in the number of viral particles was observed
in cultures which have received the combined treatrment I AC/ABA. Furthermore,, in contrast to NAC, 3 ABA exposure seems to play a minor role as apoptotic hindering factorTno
further investigate the role of antaxidants in the regulatio n of HIV expressor additional
experments have been carried out with human peripher albl,;od nsono ytes. These cells
were treated with NAC prior to the infection with a morcytotropic strain of IIV (HIIV
I BaL).The results obtained suggest that antioxidant therapies could be of relevance in the
control of viral replication in the infected cells probably through the aintenance of the
correct redox state.Th's could be important in certaaaes of AIDS, e.g., n asynmptomatic
patients.
Walter Malorni, Dept. Ultrastructures, Ist Sup. Sanita,Viatle Re;gina Elena 299, 00161 Rome,
Italy - tel. ++396/49902905- FAX ++-396/4957634
Tu.A.2033
NEF RECEPTOR RESPONSIBLE FOR CD4+ T CELL DELETION IN VITRO
Fuii Y, COtake K*,Tishiro M", Adachi Akio* ' Nagoya lJrirersity School of Medicine,
"National Institute of Health Japan, ''*Tokushima University School of Medicine,
Tokushima, Japan
Objective: o clarify the function of r ef gene product regard tinyg pathtogenesis of AIDS and
to describe molec ular cloning of Nef receptor.
Methods: MOLT-4 clone no.8 T cells were used for cytotoxrc assay by flow citorctry and
for isolationof a Nef receptor protein. Anti-Nef receptor rAbs vere inewly oiprepared for
inhibition of cytotoxicity with Nef and selection of Nefreceptor DNA clone ba y using panning method. Anti-NefrmAbs E7 and E9, of which epitopes have been described in the pre
vious tConference, were also used.
Results: A 24 kDap rte h t k e protein, which bindinas isolated f roin MO 13-4
cells. A human gene encoding the 24 kDa cellular protein was cloned and sequ;enced. Either
Neft proteincrossainked with anti-NefAbs o rT cells exprlessing Ne f on the cat isurface are
cytotoxic against MOL-4 clone no.8 and Cos7 cells expressn s the 24-kDa protein. Both
anti-Nef mAbs and anti-24 kDa mAbs specifically blocked this cytotoxicity. Some cifference
was noticed in the ability of various Nef proteins to mediate cell binding and cytotoxirc
activities it tio.
Conclusions: We dernonstrate that tire 24 kDa cellular proten binds to Nef protein and
that the interaction of Nef and this 24 kDa Nef receptor leads to cell death i nvitr.
Whether the killing activity by Nef actually occurs in vivo remains to be investigated.
A.Adachi, Departrmentof Virology,Tokushima University S otal of Medicine, Kuranrmolto-cho,
Tokushima 770, Japan.Telephone:[phone] Fax: 0886 /3-080
Tu.A.2034
THE ULTRASTRUCTURE AND ROLE OF THE FIBROUS PROTEIN IN HIV PARTICLES
Takahashi I*,Takamra M*, Ozel M**, Gelderblom H", Ladhff A Mw * University of
Teikyo, Tokyo, Japan;*'"Koch Institute, Berlin, Germany: *"University of Humnboldt, Berlin,
Germany
Objective:To reveal the ultrastructure and role of the fibrous structures existed in HIV particles.
Methods: HIV I particles were obtained from HIV-produc;ng I-I9-clones anid a H-lyr7phocyte line. Siponin-/no saponin-treated samples were fixed. Negatlive stained samples were
stereo -observed by a transmission electron microscope. Irnmuno negative --stained samples
(immunolabeled with anti-p 7-MoAb and coloidal gold) were also observed by the same
technique.
Results: Stereo observations of negative stained images revearled fibrouis structures filing up
the spaces between envelopes and capsids of HIV particles. Immrunio negat ive -,tained
images clarified that these structures were constructed from p1 7.
Conclusions: Gelderblom et al. have already reported on the lateral body t r ated in the
space of matured HIV particles, and others also have reported on the hi membrane like
structure under the envelop'' of immatured HIV particles. o prove the fine structures of
micromolecules such as p l 7, modified ultrastructur al techniques were necess ir y. In this
study, we applied the saponin-detergent technique that's very effective for the observations
of cytoskeletons.The combined technique of this detergent treatment and inmunc negative staining revealed that p1 7 was neither the high electror dense material ior th;ir
raembrane like structure, but was constructed froi fibros structures fillin up the spaces
between envelopes and capsieds both of immatured and matur ed HIV partices as the
matrix protein to suport the viral shape as if cytoskeletons.
Ichiro likahashi, Cent. Lab. EM Teikyo I/niv. Sch.Mred., Kag r 2 I I, Itabsh:-kr, tokyo 173,
Japan Telephone: [phone] I Fax [phone]
Tu.A.2035
DECREASE OF NFKB HIV- I-PERSISTENTLY INFECTED T CELL LINES RESCUED BY
NHS FROM VIURS-MEDIATED CYTOTOXICITY
Nozaki-Renard '*, Amano E' HLirabuki N', Mizuno F. rid i.. ' tokyo Medical Coallege,
Tokyo, Japan; 'Science University 11hiba, Jdpan

Objective: Normal h.smari s:rum (NHS), the environment of HIV-targeed cells, a'mst be a
focal point for uncder -:tanq 'n sthe rnechanism of HIV rinfection. We alre,dy repor ted that the
survivatl of infected ce ls,r-s iossible in the presence of NI IS by the syner gic action of
comnplement factr B, n;'-,r heat-labile cofactor: As follow up, wve examined ictrive
NFKB in the r,scud ', -cur';ed with FNHS to arnve at a better per-ceptio of HIV per sistently infected cells.
Methods: CEM and SP Il i aere infected by IAV I stra i and cltureds withi supplement
of 10% NHS or FC S in standard med;ium (1I0% inactivated FCL S-RPMI 1640).Ten g oft total
nuclear extracts was used to detect p65 with ECL VB reagents, and to measure lthe intensity of resonance to the bsotnilated dsDNA of NFKB binding site with BIAcore 2000.
Results: Surviving cells clearly appeared in CEM and SUPTI within 12 t'o 14 days post incu
bation, after HIV antigen positivity had reached 100% on day 7. and were maintained with
standard medium by splitting. In the case of CEM active NFKB wa s detected 41 days post
incubation in higher quantity in the cells cultured with NHS than in those cultured with
FCS. But on day 7 it was the reverse. In the extracts fromn sirviving cells active NFKB had
markedly decreased. On the other hand, in SUPT I there was no significant difference in the
activation of NF B on dlays 4 and 7 post incubation, though NFKB was hardly detected in
surviving cells.
Discussion: Since no difference of HIV antigen expression was observed in the cultures, we
can surmise that NHS leads, in virus-infected host cells, to signal(s) different fr-om those
induced by FCS and that the activation of NFKB plays a role in HIV-! per sistent infection.
Junko Noza ki -Renard, Deptof Mir obiology okyo Medical College 6-I -1. Shiniuku, ShinjukuWard,Tokyo I60, Jrapan, FAX [phone]-ci03
Tu.A.2036
IN VITRO CORRELATION BETWEEN HIV-I& 2 INFECTIVITY AND HLA CLASS I
AND II WITHIN BANGLADESHIS LIVING IN EAST LONDON.
A.A.AL Jabri, D. Mcloslkey M. Tiaylor, A. Sefton, L.F. Bottazzo, J.S. Oxford. Department of
Medical Microbiology and Department of imnrunologyThe London Hosp tal Medical
College, Univer sity of London. London, U.K.
In order to determine, u inItro, whether a correlation exists between HLA class I and II aleles and srusceptibility or resistance to HIV- I anrid 2 infection, per ipheral b lood rnononurclear
cells (PBMCs) frorn 130 healthy Bangladeshis living in East London were studied. PBMCs
were infected with 4 different isolates of HIV- I & 2 using 10 fold dilutions of each isolate
from 10- to 107. Cultures were maintained for 2 I days with daily inspection for virat
cytopathic effects (C PE).Li -he levels of viral replication (p24 and RT) were determined oan
days 5, 14 and 21 post infection. lILA class I and II tissue typing was performed using star
dard methods. HLA B44 was found to be significantly correlated wths r esistance to infection
among this group. Alleles whici; are statistically correlated with susceptibility to HIV I & 2
infection include A23, B35, B38, B58, B70 and DRI I. l--l A-B52 was found to be correlated
with susceptibility to I-IIV Iinfection and HLA-DR7 was found to be correlated With resistance to HIV-2 infection only. When the Bonferoni Correction factor was ipplied, HL A B58
and B70 were still significantly correlated with susceptlbilty to FI- V-I & 2 infection. Our
results indicate that, H/A alleles of an individual correlate with susceptibl tcor resistance to
HIV- I & 2 infection upon exposure to the virus.
A.A. AI-Jabri, Academic Vrology, 64 Turner Street, Whitechapel, London E 2ADU.K Tel:01 71 -
3777000 XT:2306, Fax:[phone]
Tu.A.2037
INTERFERENCE OF HIVADENOVIRUS AND EPSTEIN-BARR VIRUS UPON MIXED
INFECTION
Dyachenko, Nataa S., Rybalko 5., IFritsakT, Paovnitsa O., Nesterova N, Dyadyun S. Inst. of
Microbiol. & Virotl. Nat. Acad. Sci. of Ukraine, Kiev. Ukraine
Objective: To study the interaction of HIV, Ad and LBV rupon mixed infection in lymphoblast
cells.
Methods: Titers HIV infectivity were determinred in 5 day lysates of studied cells by p24 levels.The levels of p24 HIV (HIV monoclonal "Abbott"), Ad hexon and EBV capside protein
were defined using ELISA.Activity of reverse transcriptase was determined by wel- known
method.
Results: In different van anties successi ve introductions od viruses the considerable reduction of HIV-infectivity titers, Ad hexon and EBV coat protein synthesis were established
upon condition of triple (HIV+Ad+ EBV) on double (HIV +Ad or HIV+EBV) infection.
While expression of p24 s'was unchanged.The reverse transcriptase activity was decreasing
upon superinfection by Ad and EBV of M14/Bill cells.The last chronicaly HIV infected
were producing HIV. Considerable reduction of interferon production under tr-ple infection
of lymphocytes firom HI\-Iinfected patients and MT4 cells was observed.While MT4 cells
were able to synthesise interferone under influence of St. aureus glycoprotein (neuraminine).
Conclusions: The mutual interference of HIV, Ad and EBV were discovered under mixed
infection of lymphocytes that was important for elucidation of HIV infection mechanism.
N S. Dyachenko, 5 I Krasnourreyskaya stn, ap. 27, Kiev, 252005 Ukaine, Fax. +(044) 266
23 79
Tu.A.2038
CALMODULIN ANTAGONISTS INHIBIT SPONTANEOUS APOPTOSIS OF CD.4+
CELLS FROM HIV+ INFECTED PATIENTS
McDonald Ja tl*, Pan G', Zhou Tu, Mountz JE" Radding 'W*, Sag M. rF-e Unversity of
Alabama at Birmingham, Birm nrhan, Alabara, U.S.A.
Objective:To deterrine the effect of calnodulIn antigon sts on n vitro spontaneous apop
tosis of CD4 cells from HIV infected individuals. Increased apoptoss of T cells fiom HIV
infected individuals may play a role in the pithogenesis o the depletion of CD4 T cels in
AIDS.We have demonstrated that calmodulin is involved mr apoptosus induced by an antiFas antibody-activated apoptosis using an n vtro F cell line model transfected writh HIV
recombinant cDNA [Brochem Biophys Res Cr rrnrursn 1996:218:192 197 and Am j Pothol (sub
reitted)].
Methods: PBMCs were obtained fi-om II HIV seropositune indiduals (6 cassiied as CDCB and 5 as CDC-C) and 8 age/sex matched controls.The PBM'Cs were csltured in medium
alone and with 7.5 P of the calmodutn antagonists, tifluoperazine (TFP) or tarnox ifen

41(
4)
12
271