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

Mo.A. 1022 - Mo.A. 1027

Monday, July 8, 1996

Mo.A. 1022
TARGETED INTEGRATION OF HIV AND HTLV-I PROVIRAL SEQUENCES INTO THE
HUMAN GENOME
Rynditch Alia V*, Zoubak Sergey V*, Bernardi G*. *Institute of Molecular Biology and
Genetics, Kiev, Ukraine; **Institut Jacques Monod, Paris, France
Objective: The aim of present study was to assess if the regional specificity of HIV anid
HTLV-I integration exists and how it may contribute to explain some aspects of HIV and
HTLV-I pathogenesis.
Methods: The distribution of HIV and HTLV-l proviral sequences in DNA from cells in culture, asymptomatic carriers or patients with HIV/AIDS (for HIV) or ATL,TSP/HAM (for
HTLV-I) has been detected using compositional approach. A new procedure involving shallow gradients were adapted to the analysis of DNA f-om the patients. DNA distribution
curves wee constructed by Gaussian decomposition.
Results: In HIVproducing cells the proviral copies were absent in 50% of human genome
(in the frsactions below 39% GC and higher than 50% GC) Complete HIV proviruses which
might be responsible for virus production were found in GC-poor compartments but also
in compartment with higher GC-content. In HTLV-I infected cells as well as in patients with
ATL and TSP/HAM proviral sequences have not been detected in GC poorest 40% of host
genome.Transcriptionally inactive HTLV-I sequences were localized in GC-poorer compartments (comprised between 39% and 44% GC) than transcriptionally active proviral
sequences. A broader distribution of proviral copies were observed in the samples from
asymptomatic carriers.
Conclusions: The results obtained with HIV and HTLV-I are in agreement with non-random
compartmentalized integration of retroviral sequences. As HIV and HTLV-I belong to different compositional classes of retroviruses they were expected to be localized in GC-poor
and GC-rich compartments of human genome respectively While distribution of HTLV-I
shows correlation between proviral transcription and their location in GC-rich compartmetns the significance of broader distribution of complete HIV copies in human genome
needs to be clarified.
A.V. Rynditch, 150 Zabolotnogo Street,Kiev, 252627 Ukraine Telephone: [phone]
Fax [phone] email: rynditch(aimbig.kiev.ua
Mo.A. 1023
HIV-I 5'-LTRs FROM 42 PATIENTS REPRESENTING ALL STAGES OF INFECTION,
DISPLAY A WIDE RANGE OF POLYMORPHISM IN SEQUENCE, TRANSCRIPTION
POTENTIAL AND BINDING OF FACTORS BUT NO CLINICAL CORRELATION
M. C. Estable)3, B. Bell I, A.Merzouki2)..S. Montaner3, M.V. O'Shaughnessy3, I.J. Sadowski I.
U.B.C.,Vancouver, B.C.., IAF, Quebec, CFE HIV/ A.I.D.S., St. Paul's Hospital,Vancouver
B.C.3. Canada
Objective: Despite extensive in vitro studies identifying a myriad of cellular transcription factors that bind the prototypical HIV I 5'-LTR, the relative contribution of these factors to
HIV- I replication in infected individuals remains obscure. We wished to determine what are
the in vivo HIV-I 5' -LTR polymorphisms in sequence, transcription potential and binding of
factors and if any correlate with disease state.
Methods: The HIV- I 5'-LTR (-305 to +80), from PBMC of I0 stage IV, I2 stage III, 13 stage
II and 7 stage I patients were PCR amplified, cloned in front of a reporter gene and 478
LTRs sequenced. Polymorphisms were evaluated with respect to clinical criteria. LTRreporter constructs were assayed for transcription in Jurkat, Jurkat-Tat, U937, U937+Tat.
EMSAs were performed with Jurkat nuclear extracts and synthetic oligos to selected LTR
polymorphisms. 12 LTRs were selected for DNAse I footprinting.
Results: We found highly conserved TATA box, SP- I, NF-kB, Ras-responsive-binding-factor I
(RBF- I) and RBF- 2 sites.The most frequent naturally occurring length polymorphism
(MFNLP) (38% of patients) comprises RBF-2 binding sites in LTRs where these sites are
otherwise ablated and demarcates LTR/ Nef-coding from LTR/ non-coding sequences. No
definitive clinical or transcription MFNLP phenotype was found. Cell type-specific basal-transcription LTRs and TAR-dependent or TAR-independent Tat-unresponsive LTRs were found.
Differential binding of nuclear factor(s), to a 5'-TCTAA-3'TATA box variant, may be the
mechanism for the third. A single point mutation to the RBF- I / Ets core, found in many
LTRs with MFNf.Ps, ablates binding of RBF- I and c-Ets I.
Conclusion: The TATA box, SPI, NF-kB, RBF-I and RBF-2 sites are the major DNA binding
targets contributing to HIV- I replication. HIV- I LTRs contribute to tropism. The MFNLP represents LFR "plasticrty".Tat transactivation requires binding factor(s) to TATAA that don't
bind to T CTAA. Despite striking polymorphism in vivo, at least for the 42 patients in this
study, the LTR is not a determinant of disease state.
Mario Clernente Estable, Dept. Pathology, UBC, 2 146 Health Sciences Mall,Vancouver, B.C.,
V6T- I Z3, Canadi.Tel: [phone]. estable)-rhivnet.ubc.ca /ferrero/unixg.ubc.ca
Mo.A. 1024
PROTECTIVE EFFECTS OF EXOGENOUS COPPER-ZINC SUPEROXIDE DISMUTASE
ON THE TNFa INDUCED OXIDATIVE STRESS AND HIV REPLICATION
Edeas Marvin*. Peltier E', Claise C, Khalfoun Y5, Lindenbaum A. Departements cof
Biochemistry and Pathology* '-H6pital Antoine Bdclhi'e, 921I41I Clarnart-France.
Objective: To examirse the effects of exogenous coppen-zinc SOD on HIV expression and
TNF-indiaced oxiditive stress.
Methods: SOD effects (anti-HIV and oxidative stress) were studied: I -on the TNFa induced
ineox ilteratio n in chronically HIV- I infected promonocytic U I cell line. 2- on HIV replication in peripfreral blood miononuclear cell coculture and 3- on HIV-infected omonocytederived macrophiages. SOD has baern considered not to penetrate the cellular membrane
because of its high molecular weight, so we used heren an rmmunocytochemical method to
visualize Cu/Zn-SOD.
Results: we demoinstrate clearly that SOD, added exogenously, penetrated the cellular
membrane, increased total SOD activity, inhibited cell lipoperoxidation, prevented cellular
GSH consumption and inhibited HIV replication.The anti-HIV efficiency was maximum,
when SOD was added at the initiation and at all time points of cells culture and could not
inhibit HIV expression art later stage of infection.
Conclusion: On the basis of our" present and previous findings, association of SOD and/or
ether- superoxide scavengers with antiviral drugs may be a new therapeutic approach.This

polytherapy, if started early enough after infection. may prolongate the latency period and
limit the ernergenc':, of drg-resistant viral strains.
M Edeas, Laboratc de B ochimie, Hfpital Antoine Beclere. 157 rue de la Porte de
Trivaux, 92 'Ii smart Cedex, France Telephone: [phone] Fax: [phone]
Mo.A. 1025
REACTIVATION BY TAT AND TNF-a OF INTEGRATED HIV- I GENOMES INACTIVATED IN CELL CULTURE BY CPG METHYLATION
Bergmann S, Marschall M, Frohlich U, Lower R, Lower ohannes. Paul-Ehrlich-Institut,
Langen, Germany
Objective: To detect and to characterize intracellular mechanisms which inactivate already
integrated HIV genomes.
Methods: A cell clone (CBH) derived friom the human T-cell line CEM-CM3 has been
selected which contains a single copy of an HIV construct with a positively as well as negatively selectable marker gene (hydroxyguanine phosphoribosyl transferase, HGPRT) in place
of the gag and part of the pol gene without alteration of cis and trans acting regulatory elements.This cellt clone grows in HAT medium only when viral mRNA (including full-length
RNA coding for HGPRT) is expressed. In contrast, only cell derivatives in which the formation of active HGPRT is suppressed can survive in medium containing 8-azaguanine (8-AG).
Results: After incubation of CBH-cells in 8-AG medium, a number of cell clones survive.
Some of them (designated CBHr) regain the ability to produce HGPRT if the medium is
changed to HAT medium, others (CBHi-) do not.While in CBHr cells viral mRNAs can be
detected though at reduced amounts, in CBH- cells viral rrRNA bands are not visible in
Northern blots. In addition, in CBH- cells the viral sequences are methylated to a high
degree.Treating CBH- cells with the demethylating agent 5'-azacytidine allows a number of
cells (CBHi+) to regain the ability to express HGPRT. In CBHi+ cells, the methylation of the
viral genome is diminished and production of viral mRNA nas resumed.The same effect can
be observed when CBHi- cells are treated with TNF-ct or a tat expression plasmid has
been transfected prior to the change to HATm redium.
Conclusions: Methylation may be a mechanism which leads to an inactivation of already
integrated HIV genomes. At least in the cell system presented here the effect of methylation does not depend on the site of integration since all cell clones are derived from a single clone containing a single copy of an integrated HIV genome.Treatment with dthe
cytokineTNF-(s orwith the viral trans-activatorTat leads to a resumption of the production
of viral mRNA accompanied by a reduction in rnethylation of the viral genome. The molecular mechanism of these effects are under study:
J. Lower, Paul-Ehrlich-lnstitut, Paul-Ehrlich-Str: S1-59, D-63225 LangenTelephone: [phone] Fax: +496 I 03-77 1I252 email: loewer(em.uni-frank-urt.de
Mo.A. 1026
INFLUENCE OF THE HIV-I LEADER SEQUENCE ON mRNA STABILITY
Lenz, Christian*, Schaal H*, Scheid A*. * Institut fOr Medizirische Mikrobiologie und
Virologie, Heinrich-Heine-Universitit Dusseldorf, Germany
Objective: Investigation of the influence of the authentic tot mRNA 1.4.7 leader sequence
on human immunodeficiency virus type I (HIV- I) gene expression.
Methods: An expression vector with the bacterial chloramphenicol acetyltransferase (CAT)
open reading frame under transcriptional control of the HIV- I promoter was constructed.
The CAT leader sequence was precisely replaced with that of the at mRNA 1.4.7. Mutants
with complete and partial leader deletions were constructed, as well as a mutant with the
leader in reverse orientation. Following transfection of HeLa-T4+ cells, expression of the
CAT gene was analyzed by ELISA, Northern blot, and nuclear run-on transcription assay.
Results: Quantification of CAT protein showed that the exon I -derived leader sequence
downstream of the US region (lsdU5) largely influences basal protein expression in an orientation-dependent fashion.The dramatic decrease in CAT protein observed when IsdU5 was
deleted was paralleled by a similar- drop in the steady-state level of CAT mRNA. In contrast,
by reversing the leader orientation, distinct amounts of CAT mRNA were detected.The
IsdUS-influence on protein and mRNA expression was independent ofTattransactivation of
the HIV- I LTR. Point mutations of presumable binding sites or nuclear factors DBF I and SpI
in the IsdUS-region had no significant effect on protein expression. Nuclear run-on transcription assays revealed that IsdUS did not increase the rate of transcription.
Conclusions: These results indicate that IsdU5 may function at the post-transcriptional level
as an element to stabilize mRNA.They argue against a role of IsdU5 in the regulation of the
HIV- I promoter activity.
C. Lenz, Institut f. Med. Mikrobiologie und Virologie, Geb. 22.21, Universitdtsstr. I, 40225
Dusseldorf, GermanyTel. +49-21 I-8 I -12393, FAX +49-2 I -81-12227, email:
clenzctrs60OO.virol.uni-duesseldorfde
Mo.A. 1027
ROLE OF VIF IN HIV-I REPLICATION IN NON-PERMISSIVE CELLS
Xiao-Fang Yu, Markus Dettenhofec Xiao-Bo Tang. Department of Molecular Microbiology
and Immunology, Johns Hopkins School of Hygiene and Public Health, Baltimore, MD3 2 1205,
USA
Objective: Replicatom of HIV- I Vif mutants is cell type depnident.Vif is essential for HIV- I
replication in primary targ~et cells and H9 CD4 T cells (ncn-permssrve cells). It is dispensable in many CD4+ T cell lines such as Jurkat (permissive cells).Vif function is required for
generating infectious virus irs thne non-permissive cells duin;g virus production. Detailed
analysis of viral protein synthesis, processing. and virus assembly in Vif mutant infected nonperemissive cells will be critical for the elucidation of Vif function.
Methods: We have developed a strategy to pr-educe large,quantity of Vif mutant infected
H9 cells.Viral protein synthesis, processing, virus assembly, and maturation were studied.
Results: Vif mutant virus produced from H9 cells was eon-infectious even in Jurkat cells.Vrf
mutant virions released fl-em H9 cells contained a higher ratio of unprocessed Gag precursor p55 to mature p24 compared to the Vif positive parental virions. Also, the amounts of
viral Pol proteins BTp66, p5 I, and lNp34 were reduced in SVif mutant virions compared to
the parental virions.The reduced Gag processing and reduced level of Pal proteins were
also observed within Vif mutant infected H9 cells compared to the parental virus infected
H9 cells. Pulse labeling experiments indicated that the synthesis of Gag-Pol ph160 was significantly inhibited in Vif mutant infected H9 cells ccompared to the parental virus.

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