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

Track A: Basic Science

Mo.A.1013 - Mo.A.1020

7lnstitute of Biology, University of Iceland, 108 Reykjavik, Iceland
Masked nucleoside phosphoramidate derivatives of stavudine (diT) such as So324 were
found to show a marked anti-HIV activity in cell culture. So324 poved more inhibitory to
HIV replication in thymidine kinase-deficient CEM/TK cells thar ini wild type cells, whereas
under similar experimental conditions d4T showed a 50- to IOt:, redlv,:i in antiviral
potency in the CEM/TK cells. So324 represents the 5'-monopl I,.it ra dev;tive of d4T
(d4TMP), its phosphate moiety being esterified by a phenyl groul I iI through a
phosphoramidate linkage to the methyl ester of L-alanine. So3, -, i, acellularly
both d4T-MP and alaninyl d4T-MP d4T-MP is further metabolize- i t.' iprhosphate form
(d4T-TP), which is a potent inhibitor of HIV reverse transcriptase. Al iri al 1 lMP accumulates inside the target cells to much higher levels than d4T-TP Aii.ni d. 1 MP was found to
accumulate in all cell types examined (human lymphocytes, epitlihelikJ cei,,and
monocyte/macrophages, and simian, feline, caprine and murine t/ypto. ce and fibroblast
cells), and should be considered as a depot form for the intraceluiw riease lof d4T and
d4T-MPThe masked nucleoside phosphoramidate derivative of di p-i.in,, new perspectives in the design of novel prodrugs of nucleoside analogues.
J. Balzarini, Rega Institute for Medical Research, K.U.Leuven, Mindeirced ersi triat 10, B-3000
Leuven, Belgium.Tel. [phone]. Fax [phone].
Mo.A. 10 13
POSSIBLE REGULATION OF HIV REPLICATION BY A CELLULAR FACTOR BINDING
TO THE PRIMER BINDING SITE
Lai, Derhsing, Guo H-G, Gallo R. C., Reitz M. University of Marylrnd. iBaltimore, Maryland,
USA
Objective: The regulation of HIV replication has been intensively studied One of the early
steps is the reverse transcription of viral RNA into viral DNA. vwhich is then integrated into
the infected host cell genome.The natural primers for reverse tran(_ription are the cell
derived tRNAs. However, the exact interactions of the primer bindie: site (PBS) with the
viral RNA, the tRNA primer, HIV reverse transcriptase and cellular ctor(s) are still unclear.
The present study is intended to help understand these interactions b chracterizing a
possible cellular protein which binds to the PBS on viral RNA.
Methods: The primer binding site (PBS) of HIV-I is an I 8 nucleoti-ie sequeice immediately
3' to the left hand LTR which complements the 3 end of tRNA " s.-I I e pilosphorothiooligonucleotide 24mer (lys3) and different control oligomer we e added so PM-I cells
acutely infected with HIV- I (Bal) or Suptl cells acutely infected with I-iIV-2 Viral expression
was monitored by p24 or p27 antigen capture assays. Gel retardation assays were performed with this oligonucleotide, which was also used as a prolier to screen a humnan lymphocyte cDNA expression library
Results: The PBS oligonucleotide induced HIV- I Bal and HIV-2 expressiof i at irast 8 fold in
vitro. A non-specific induction of cytokines mediated by a reaction to non methylated CpG
pairs was ruled out by use of methylated oligomers.We hypoth'.>izc that this oligonucleotide binds to a cellular factor(s) involved in regulation of the HIV pnrnier binding step,
causes depletion of the cellular factor(s), and thus induces HIV iepicKtion. The gel retardation assays have shown specific binding of cellular protein(s) with ths, oligonucleotide.
Specific clones have been selected from human library
Conclusions: This work may provide evidence for cellular control oi early stages of HIV
replication.
Derhsing Lai, UMBI, 6 I8 West Lombard Street, 2nd Floor, Baltimore Mary,,nd 2 I1201,Tel:
410-706-8614, Fax: [phone] I 84
Mo.A.1014
EVALUATION OF ANTI-HIV-I REVERS-TRANSCRIPTASE (RT) ANTIBODY TITER IN
HIV-INFECTED SUBJECTS
Taka hi Megur o, Satoshi Yamazaki2), Shigenobu Takayama2), Kunio hia2)), Masashi
Takil ), Hironobu Ito'), Shinya Sadamoto4), Hisao Koyanagi4), Hiroshi Kakisihin a4), Kaneo
Yamada1)3). Division of Research Laboratory St. Marianna Univ School Med Yokohama
Seibu Hosp. I), Dep. of Pediatrics St. Marianna Univ. School Med.), ristditute of Medical
Science St. Marianna Univ. School Med.3), Eiken Chemical Co.4)
We tried to study on the antibody titer against reverse transcriptse i(RT) -If HIV and its
clinical significance in 10 cases of HIV-infected hemophiliac patients, 4 case of HIV-infected
homo/heterosexual subjects and 5 I uninfected volunteers.
Methods: Using recombinant RT antigen of HIV- I, RT antibody it. plsma or sear was
assayed with automated chemiluminescent enzyme immunoassry ( IEA which was developed by Riken Co. as AL-- 1000.Titer of anti-HIV- I RT antibody a,,s expresedl by the following formula: individual level of CS/mean level of CS in uninfected vlunters.
Results: The strength of chemiluminescence (CS) was assayed by CL [A in ti e s amples of
HIV uninfected volunteers and HIV positive subjects, being the me,n v,'lues S52~486 (255
CS and 1.02X 105~ 1.26X 105 (I 56 I -5.08 X 105) CS respectively 1 he anti HIV- I RT antibody titer in AC(N=68), ARC(n= 12) and AIDS(n= I I) were 70.;+7-i. 18.1_6.0 and
I I.5+5.7 respectively The titers of anti-HIV- I RT antibody in both ARC,.d AIDS were significantly lower than AC(p<O.001 ). On the other hand, anti-HIV I iRT \ i iii. y titer in the
long term non-progressors was higher than lO0.The correlationii were sinitint in the following relationships between the titers of anti-HIV- I RT antibody ni rius sf (CD4+ lym
phocyte (r-s0.319, n-79). HIV-RNA copies in plasma (r=0.258. n= 1, ani ni MN antibody titers (r=0.773, n=65,p<0.0 I).
Conclusion: Levels of Anti-HIV- I RT antibody titer corresponded vt t er surrogate
markers and clinical progression. Reduction of anti-HIV I RT anti.od. iter was observed
earlier than the decrease of CD4 lymphocyte counts during tte r gre sian of HIV
infected subjects.
These results suggested that the assay of anti-HIV- I RT antibody 1e, t.uld e iignificant
for the evaluation of clinical progression in HIV infected subjects
Takashi MeguroYasashicho I1I97- I, Asahi-ku,Yokohama 2 14, Japai r,pine: P45 366- IIi
ext.3351 Fax: 045-366-i 190

Mo.A. I 0 18
DIFFERENT VIRAL VARIANTS IN CHROMOSOMIC AND EXTRA-CHROMOSOMIC
VIRAL DNA IN AN IN VITRO SUPERINFECTION WITH HIV- I.
Marquina S., Gomez Carrillo M., Galvin V., Libonatti O., Rabinovich R. D. National Reference
Centre for AIDS, Department of Microbiology University of Buenos Aires School of
Medicine, Buenos Aires, Argentina.
Objective: to study the viral DNA in the chromosomic and extrachromosomic fraction and
the viral progenie in a superinfection, with another variant, of a cell line persistently infected
with an HIV- I strain.
Methods: The superinfection system consisted in the H9HTLVIIIB cell line persistently
infected, and the superinfectant strain was the HIV- I MN.The chromnosomic and extrachrornosomic DNA was separated by means of a precipitation technique described by Hirt. In
these fractions and in DNA extracted from infected cells with the viral progenie of the
superinfected cell lines, C2V3 region of the gp120 env gene was amplified by PCR.To distinguish between the genomes of the two strains, PCRs products were cut with Mnl I and
BsaJ I restriction enzymes.
Results: Forty-eight hours post superinfection, both viruses, the persistent and the superinfectant strains, were found in the chromosomic DNA fraction. Interestingly, the superinfectans sitraln was mainly observed in the extrachromosomic viral DNA. Howeve: the viral
product obtained from the superinfection was HIV- I HTLVIIIB.
Conclusions: We report a direct evidence concerning the origin of the extrachromosomic
viral DNA which seems to be produced by reinfection. At least in short time, the virus productiorn comes from the first infectant viral strain.Then, the role of the viral extrachromosomic DNA in the generation of variability should be further studied.
Silvia Marquina. National Reference Centre for AIDS. Paraguay 2 I 55 piso I I, II 2 I Buenos
Aires, Argentina. Phone 54 I 96 I 8124 Fax 54 I 962 5404
Mo.A. 1019
ASSAYING INTEGRATION ACTIVITY OF HIV- I INTEGRASE WITH DNA-COATED
PLATES
Sy u Wan- r, ChangY-C, ChingT-T National Yang-Ming University, Taipei,Taiwan, R.O.C.
Objective: To simplify the measurement of integration reaction, an easy method mimicing
enzyme linked immunosorbent assay (ELISA) procedures was developed.
Methods: Integration of reverse transcribed viral DNA of HIV into host chromosomes is
mediated by the viral enzyme, integrase.This enzymatic activity can be monitored in vitro by
integration of a small labeled DNA (donor) into a second unlabeled DNA (target). DNA
fragments were adsorbed directly on 96-well plates and used as the target DNA.The
donor was a synthetic 2 I-bp DNA duplex of HIV- I U5 LTR; biotin was incorporated into
the 5 end of one strand whose two nucleotides at the 3' end were specifically removed
during the integration.
Results: After integrase reaction, the biotin-labeled donor DNA was joined with the target
DNA and became immobilized on plates.These integration products were then measured
by binding of avidin-alkaline phosphatase on plates.
Conclusions: Previously, microplate-based integration assays have been described. However,
our new method is relatively simple and straightforward. In particular, no complicated procedures for the immobilization of target DNAs onto plates are involved.Thus, this method
should be easily adapted to qualitative screening of integrase inhibitors and quantitative
measurement of the potency of different inhibitors.
WJ. Syu. I 55 Sec. 2, Li-Long Street, Shih-Pai,Taipei, 112 R.O.C.Tel: [phone] Fax [phone] enmail: [email protected]
Mo.A. 1020
ISOLATION AND CHARACTERIZATION OF A NOVEL CLASS OF HUMAN
IMMUNODEFICIENCY VIRUS INTEGRASE INHIBITORS FROM NATURAL
PRODUCT SCREENING
Hazuda, Daria*, Blau C*, Felock P*, Hastings J*, Lineberger D*,Wolfe A*, Goetz M**,
Williams M**, Zink D**, Singh S.**. *Departments of Antiviral Research and ** Natural
Product Chemistry, Merck Research Laboratories, Rahway, NJ
Objective: Integration of a copy of the viral genome into the genome of the host cell is an
essential and defining step in the replication of all retroviruses. Integration is catalyzed by a
virally encoded enzyme, integrase.The absolute requirement for integrase activity in the propagation of HIV- I in cell culture defines the enzyme as a potential biochemical target for HIV I
antiviral chemotherapeutic intervention.Therefore, we have attempted to identify novel
inhibitors of integrase for potential use as chemotherapeutic agents against HIV infection.
Methods: Natural product extracts were randomly screened via a high throughput biochemical assay which measures both the 3' end processing and strand transfer activities of
the enzyme
Results: Equisetin, previously isolated from Fusarium equiseti, was recovered from the fungus
FusariurI hererosporunm and shown to inhibit HIV- I integrase in vitro. Novel compounds
related to equisetin were also isolated and identified from the fungus Phomno sp. Equisetin
and related corpounds inhibited the 3' end-processing, strand transfer and disintegration
activities of the wild type enzyme with comparable potency (5-10 uM). Equisetin also inhibited disintegration catalyzed by the truncated core domain protein (amino acids 50-212).
Binding to the catalytic domain was also demonstrated in competition studies using a radiolabelled ATP afinity analog, fluorosulfonyl benzyl adenosine (FSBA); equisetin was observed
to compete for FSBA binding to both the full length and truncated enzymes.
Conclusions: We have identified a novel class of integrase inhibitors which interact with the
erzyme at the substrate binding site(s).These studies demonstrate that it is possible to isolite sma I molecular weight compounds which act as integrase inhibitors by randomly
screening nitural product extracts using this approach.
Dana Hazuda, Merck Research Laboratories, Bldg. I6- I 0 I, Sumneytown Pike, West Point, PA
19486 USA Telephone: 2 I5-652-79 I 8 Fax: 2 I5-652-0994 email: [email protected]

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