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

Track A: Basic Science

Mo.A.1028 - Mo.A.1035

Conclusions: Vif is required for efficient -I frameshifting to generate Gag-Pol in non-permissive cells.The reduced level of Gag-Pol explains the reduced cleavage of the Gag precursor,
reduced amounts of viral Pol products, and the lack of infectivity ofVif mutant virions generated from H9 cells.
Xiao-Fang Yu, Dept. Molecular Microbiology and Immunology, Jonhs Hopkins School of Hygiene,
6 15 N. Wolfe Street, Baltimore, MD 2 I 205, USA Tel: [phone] Fax: [phone]
Mo.A. 1028
INDUCTION OF NITRIC OXIDE SYNTHASE (iNOS) GENE IN H9 BY ANTI-HIV-I
COMPOUND 3-DEAZA-NEPLANOCIN A
Chiang Peter K., Burke Donald, S *Kutty, R. Krishnan, Mayers Douglas L, Pardhasaradhi K.
Division of Retrovirology Walter Reed Army Institute of Research, Washington, DC 20307 -5100, USA;*NEI, NIH, Bethesda, MD 20892
Issue: 3-Deaza-naplanocin A (DZNep) has been shown to exhibit potent anti-viral activity
against clinical strains of HIV- I in phytohemagglutinin (PHA)-stimulated peripheral blood
mononuclear cells (PBMC) and other cell lines [Mayers DL et al. Proc. Natl. Acad. Sci. USA,
92, 2 I 5 (95)]. However, the mode of action of this class of antiviral compounds remains
largely undefined.
Project: Since nitric oxide (NO) has been shown to attenuate viral infectivity, we examined
whether DZNep could induce the expression of nitric oxide synthase (iNOS) in human Tcell lymphoma cell line (H9). Moreover DZNep has been shown to be capable of activating
gene expression in cells [Chiang PK et al.j Biol. Chem. 267, 4988-499 I (92)].The objective
was to identify the possible role of nitric oxide in the anti-HIV- I activity of DZNep.
Results: H9 cells were treated with 60 nM DZNep for 24 hours before harvesting and isolating the total mRNA. Reverse transcriptase polymerase chain reaction (RT-PCR) was
employed to study the induction of iNOS gene by the drug. Primers were designed from
the reported cDNA sequence for the iNOS (inducible nitric oxide synithase) gene.The
PCR reaction showed a band corresponding to the expected size (-500 bp) in the
DZNep-tireated cDNAs.The sequence of the amplification product was found identical to
the reported for iNOS cDNA sequence. Northern blot analysis using the amplified product
showed a band at --4.4 kb in the DZNep treated H9 cells after 24 hours.This indicated
that DZNep is upregulating iNOS in the H9 cells.Thus, it would be interesting to investigate
further the significance of the induction of iNOS and the role of NO in the anti-HIVI /anstiviral activity of DZNep.
Dr Peter K. Chiang, Walter Reed Army Institute of Research,Washington, DC 20307-5100,
USA 202 782-6361; fax [phone]
Mo.A. 1030
MODIFICATION OF THE PLASMA MEMBRANE OF CELLS INDUCED BY HIV-I
GrigorievV.B., Kadochnicov U.P, Vorcunova N K., Klimenko S.M. The D.I. Ivanovsky Institute
of Virology, RAMS, Moscow, Russia.
Objective: The aim of present investigation was to study distribution of integral proteins in
hidrophobic zone of the plasma membrane of HIV-I infected cells.
Methods: Important structural markerso ne for membrane functions are the intramembrane
particles (IPMs) seen on membrane fructure faces.The presence of IPMs correlates with
the presence of integral ierbrane proteins.MT-4 and Jucut-tat at 24-48 h.postinfection
were used in experiments.To reveal topography of the hydrophobic zonee of the plasma
membrane freeze- fructure and fructure-label methods were applied.The serum fom
patients was used for fructure-labeling.
Results: We showed that transmembrane hoist proteins formed clusters on the P fiucture
face of the plasma mIembrane in infected cells whereas their distribution was random in
unifeted cells. Places of HIV budding were devoid of IMPs. Fructuie-label experiments
showed that hidrophobic domens of glicoproteins gp4 I are located on outer leaflet of the
plasma maembrane.
Conclusions: Redistribution of IMPs is probably related to insertion of virus-coded proteins
in hidriphobic part of the host cell membrane bilayer.The localization of gp4I hidrophobic
domens explains a weak anchorage of the surface glicoproteins in mature HIV virions and
their following spontaneous shedding.
V.B.Grigoriev, 16 Gamaieya street, Moscow, 123098,RussiaTelephone: [phone];
Fax: [phone]
Mo.A. 1032
P 15 RNA BINDING: A POTENTIAL TARGET IN AIDS TREATMENT
Ozturk Lt, Frickson-Viitarien, S.The DuPont Merck Pharmaceutical Company, Wilmington,
DE, USA
Introduction/objectives: One of the steps in HIV- I virus replication is the cleavage of gag
gene pr-oduct Pr55 by HIV- I protease.The Pr55 processive events by the protease are
ordered and time dependent, and yield proteins necessary for infectious viral assembly. One
of the Pr55 cleavage products is p I5 protein, which is further processed by HIV- I protease
to yield p7. p6 and p I products. In the viral assembly process, timely cleavage of p 15
requires the piesnce of viral RNA [Sheng and Erickson-Viitanen,(J.Virol., 68, 6207, 1994]. A
specific 24-urier RNA oligo derived fr-om the viral RNA sequence and its 21-.mer antisense
DNA oligo define the minumuua oligonucleotide size requiied fcc coiirplete cleavage of p I 5,
sod it ippeurs thait the stern-loop structure of these oligos dicta tes their binding specificity
to plIS.We wish to leari which amino acids within the p1I5 precursor ice involved in specific iinteiractions with the viril RNA.
Results: We are runrently in the process of identifying the interaction site(s) of the oligo
with p Il5 by affinity labeling sod crystallography.We have developed Letter exparession and
purification schemes to obtain >95% pure p I15 in 8- 10 milligram levels for crystallization
iad aftinuty libeling studies. Foi- iffinity labeling, a single phosphorethunate moiety at various
positions ina the 2 I-mei DNA oligonucleotide was converted ite thio((3-broirno-2 -oxo)pr-opyl( as the covalent piobe. In addition, 21 -mer DNA oligo having 2'deoxy-5 -iodociracil it various positions is being employed as a photoaffirity probe. Initial results suggest that the derivatized oligos react covalently with p1l 5.
Conclusions: The binding of a specific oligonuclentide structure that is required for the specific cleavage of gag p IS5 by the viral protease may define a novel target for* the inhibition of
HIV replication.

Derya Ozturk DuPont Merck Pharmaceutical Co. PO Box 80400 Wilmington, DE 19880 -0400 USA
Mo.A. 1033
GENOMIC DEFECTS IN ATTENUATED HIV- I FROM THE SYDNEY BLOODBANK
LONG-TERM NON-PROGRESSORS.
Solomon, Aantha*/, Smith K1, Ludford-Menting M I, Hooker D I,Tsykin A1, McPhee D I,
Chatfield C, Ellett A1, Greenway A1, Crowe S, Learmont j2, Deacon N 1. Macfarlane
Burnet Centre for Medical Research I, PO Box 254, Fairfield Vic 3078, Australia. NSW Red
Cross Blood Transfusion Service2, Sydney NSW 2000, Australia.
Objective: Between April 198I and July 1984 seven people were infected with HIV-I by
transfusion of blood from a single HIV- I infected donor. All cohort members (the Sydney
Blood bank long-term non-progressor cohort) have remained free from AIDS or any HIV- I
related illness for up to 14 years. Since cohort members could share a common attenuated
HIV- I strain, characterising the HIV- I strains from the cohort could provide important
insights into HIV- I pathogenesis.
Methods:To characterise the HIV- I strain associated with the cohort we employed PCR
amplification, molecular cloning and DNA sequencing techniques using either patient
peripheral blood mononuclear cell (PBMC) DNA or HIV- I isolate-infected PBMC DNA as
template.The HIV- I DNA copy number in patient's PBMCs (in 3 cases < 10O copies per 105
CD4 cells) provided a technical challenge that was met by developing nested booster PCR.
Results: Comparison of the partial DNA sequence obtained from the donor and recipient
indicated a common strain of HIV- I distinct from the infectious molecular clone HIV- I
NL43 and forom clinical isolates associated with progressive infection.Three common features were observed in the nef-LTR amplimers: (a) deletion of various lengths from the nefalone region, (b) deletion from at least one part of the nef-LTR overlap region and (c)
duplications and rearrangement of NFand Sp I sequence upstream of the basal promotor
(Sp I) region of the LTR.
Conclusion: As these features are common to all the cohort sequence studied and there
appears to be no other siginificant genomic abnormalities this data provides the first
unequivocal evidence for the transmission of an attenuated strain of HIV- I that does not
cause disease.
Ajantha Solomon, Macfarlane Burnet Centre for Medical Research, PO Box 254, Fairfield Vic
3078, Australia.Tel: [phone]; Fax: [phone] Email:
[email protected]
Mo.A. 1034
PCR CLONING AND SEQUENCING OF THREE HYBRIDOMA CELL LINES
PRODUCING MONOCLONAL ANTIBODIES AGAINST THE SAME REGION OF
NEF PROTEIN
Chang, Alex H.,* ** Leslie, Kevin,** Hoxie, James,*** Cassol, Sharon.*** *B.C. Center for
Excellence in HIV/AIDS, Canada; **University of British Columbia, Canada; ***University of
Pennsylvania, USA
Objective:To determine the sequences of the antibody variable regions of three hybridoma
cell lines, all of which produce monoclonal antibodies against the C' terminus of Nef protein, and to construct anti-Nef single-chain antibodies (SFV) in order to study the intracellular role of Nef in HIV- I infection.
Methods: The polymerase chain reaction (PCR) has been adopted to directly clone the VL
and VH-specific moieties from hybridoma cell linesThese sequences were then inserted
into a plasmid vector and sequenced using ABI's automated sequencerThe sequences were
analyzed using Blast similarity search against the comabined Non-redundant Nucleotide
Database at National Center for Biotechnology Information (NCBI) Blast network server.
The nucleic acid sequences of the variable regions (including both VL and VH) of hybridoma
cell lines were also compared to each other by Fasta analysis. SFv were then constructed by
connectingVL with VH, using a 15 amino-acid peptide linker (GGGGS)3.The SFv were
expressed in pET- 1 9b (Novagen) E. coli expression system, and the immunological activities
were tested against the Nef protein.
Results: The sequences fi-om the variable regions, VL and VH of the three hybridoma cell
lines, are unrelated to the sequences found in NCBIs database.There was 97.8% similarity
(among 774 overlapping nucleotides) between AGI I and AE6, which were raised in the
same fusion. But only 76. 1% similarity (among 66I overlapping nucleotides) was found
between AG I I and EH I. All of the three SFv expressed by E. coli could recognize recombinant Nef protein by ELISA assay.
Conclusion: The recombinant SFv retained the immunological activities of their corresponding parent monoclonal antibodies.The similarity of the 3-dimensional conformation of the
antibody variable regions is believed to be contributing to the EH I monoclonal antibody
recognizing an overlapping epitope with AG I I and AE6, rather than their linear amino-acid
sequence similarities. By intracellularly expressing anti-Nef SFv in CD4 and monocytic cell
lines, the SFv can be used to study the intracellular role of Nef in HIV- I infection and to set
up a gene therapy model for the potential treatment of AIDS.
A.H. Chang, 6 13- I 08 I Burrard St.,Vancouver B.C.V6Z I Y6 Canada Tel: [phone] Fax:
604-631-5646 email:[email protected]
Mo.A. 1035
MOLECULAR BIOLOGY OF THE HUMAN ENDOGENOUS RETROVIRUS FAMILY
HERV-K:TRANSACTIVATION BYVIRAL PROTEINS
Lower RoswithaThelen K, Hasenmaier B, Knbjl M, Kurth R, Ldwer J. PauI-Ehrlich-lnstitut,
Langen, Germany
Objective: no determine whether viral trans-activators (including HIV Tat) can modulate the
expression of a human endogenous retuovirus family which gives rise to virus particles in
teratocarcunoma cell lines.
Methods: In the human genome, the endogenous retrovirus family HERV-K is represented
by approximately 50 copies of full length proviruses. In teratocarcinoma cell lines, they are
expressed in a comnplex mRNA pattern comprising full length mRNA, subgenomic eev
RNA, and two small mRNAs one of which is doubly spliced.The protein coded for by this
mRNA resembles H Rev asm s it contains a nucleolar localization signal and a
leucine rich donaain. However a Rev-like function has not yet been demonstrated. HERV-K
mRNA expression can also be detected in a series of other cell lines and tissues, but the

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