Bridging the Gap: Conference Record [Abstract book, International Conference on AIDS (12th: 1998: Geneva, Switzerland)]

8 Abstracts 11127-11132

12th World AIDS Conference

11127 Inactivation and reactivation of integrated HIV-1 genomes
in cell culture by CpG methylation
Johannes Loewer, Mareike Marschall, R. Loewer, S. Bergmann.
Paul-Ehrlich-lnstitut Paul-Ehrlich-STR.51-59; 63225 Langen, Germany
Objectives: To detect and characterize intracellular mechanisms which are able
to inactivate integrated HIV genomes.
Methods: A human T-cell line CEM-CM3 (CBH) stably transfected with a single copy of an HIV construct was used. Instead of gag/pol genes the construct
carries a marker gene (HPRT) for selection in HAT medium. HPRT expression
coupled to viral mRNA expression enables survival of the cell clone (CBH+).
Cell derivatives in which expression is suppressed (CBH-) are able to grow in
8-azaguanine-medium.
Results: Some 8-azaguanine resistent cell clones regain the ability to produce
HPRT in HAT medium (CBHr+), but fourteen percent of the clones (CBHi-) are
irreversibly inactivated and can not adapt to HAT medium again. In contrast to
CBHr+ cells, viral mRNAs were not detected in CBHi-cells by Northern Blot analysis. Southern blots with methylationsensitive restriction enzyms correlate with the
degree of methylation and the type of inactivation. The irreversible inactivation
can be obviated by treatment with the demethylating agent 5'-azacytidine, with
TNF-a and after transfection with a tat expression plasmid. After change to HAT
medium, CBHi+ cells survive, produce viral mRNA again and methylation of the
viral genome is diminished.
Conclusion: Further studies to define in detail the methylation pattern of the reversibly, the irreversibly inactivated and the reactivated integrated HIV genomes
by bisulphite based cytosine methylation analysis will show its contribution to
regulation of the HIV construct by methylation.
S11128 Potential protective effect of antibodies to P66 during the
course of HIV-1 infection
Philippe Moja', V. Cheynet2, T. Bourlet1, F. Mallet1, F. Lucht1, B. Pozzetto1,
C. Genin1. 1Gimap University of St.-Etienene 15 Rue A. Pare 42023 St.-Etienne
Cedex2; 2CNRS-Biomerieux Ecole Normale Superieure, Lyon, France
Background: This study was performed to estimate the changes in levels of
antibodies to reverse transcriptase during the course of HIV-1 infection.
Methods: Sera from 89 HIV+ patients classified into 3 groups according to
CD4+ cells counts, were analysed. Group 1: 30 patients with a CD4+ cells count
higher than 500/mm3, group 2: 28 patients with a CD4+ cells count between 200
and 500/mm, 3 group 3: 31 patients with a CD4+ cells count lower than 200/mm3.
30 seronegative subjects matched for age, were used as controls. Specific IgA
and IgG to 3 HIV antigens (gag p25, pol p66 and gp120 V3 loop) were measured
by ELISA using recombinant proteins and a synthetic peptide. IgA dosage was
performed after absorption of IgG.
Results: Our study confirms a relationship between the decrease of CD4+
cells counts and the IgG to p25 levels (comparison group 1/group 2 p = 0.02,
group 1/group 3 p = 0.01). In the same way but more significantly, IgG to p66
levels decreased with the CD4+ cells numbers (comparison group 1/group 2 p
= 0.02, group 1/group 3 p = 0.001). A decrease in IgA to p66 levels was also
demonstrated but as they were not present in some patients, the value are not
conclusive. As shown previously, IgG and IgA to V3 levels remained stable during
the course of increase immune deficiency.
We selected 20 patients followed during 5 years: a group of 10 patients termed
"evolutive" with a decrease in CD4+ cells counts over this period and a group of 10
"non evolutive" patients with a stable CD4+ cells counts. The dosage of specific
IgG and IgA to the three previous HIV antigens in 3 sera sampled collected during
this period confirmed the higher prognostic value of IgG to p66 than of IgG to
p25. Moreover IgG to p66 level was inversely related to the viral load in evolutive
patients.
Conclusion: The presence of anti p66 antibodies seems to have a good prognostic value in HIV+ patients. These results may suggest that anti-p66 have a
protective effect against HIV.
11129 Heat shock modulation of HIV-1 infection
Edward V. Karamov1, A.N. Yudin1, M.B. Evgen'ev2, A.V. Abelian1,
G.V. Kornelayeva1, A.V. Kozlova1, Kh.A. Ulmasov3. 1lvanovski Institute of
Virology, Gamaley 16, 123098 Moscow; 2nstitute of Molekular Biology, Moscow;
3Moscow State University, Moscow, Russia
Objectives: To study participation of heat shock proteins (HSPs) in HIV-cell
intimate relationships.
Methods: H9 lymphoblastoid cell line, infected with HIV-1 HTLV-IIIRF NIH 1983.
Western blot with monoclonal antibodies against different families of human HSPs.
Heat shock induction (heat treatment at 41.5 C for 60 min). Quantitative DNA-PCR
on HIV provirus. ELISA for anti-HSP antibodies.
Results: H9 cells infected with HIV-1RF, produce additional HSPs 75, 40 and
32 kD. First one resembles to GRP. Heat shock induction increases number of
HIV provirus copies. Amplification has two phases: 1. Immediate; it takes 3-5 h

after heat treatment (3 times difference) and 2. Late; after 24-36 h (up to 6 times
difference). Second is probably due to preferable proliferation of HIV-infected
cells. Some HIV-infected persons (up to 25%) have serological reactivity towards
HSP 60.
Conclusions: HIV-1 infection changes profile of HSPs synthesized in the cell
which leads to higher resistance to alteration and induces anti-HSP antibody
production.

377*/11130 HIV-1 Vif is an endogenous inhibitor of HIV-1
protease and peptide derivatives of Vif inhibit
Gag-Pol processing and HIV-1 replication
David Volsky1, P. Sova1, M. Simm1, C. Krachmarov1, T. Muir2, R.J. Potash1.
1Molecular Virol, Lab. Columbia St. Luke's-Roosevelt Hosp. Ctr, New York
10019; 2Rockefeller University, New York, NY USA
Objectives: To study the role of Vif in Gag-Pol processing, to define Vif function
as a PR inhibitor, and to identify PR-inhibitory Vif peptides which block HIV-1
infection.
Design: Effect of Vif and Vif fragments on HIV-1 PR activity and Gag-Pol
processing was studied in a bacterial two-plasmid expression system, during
co-expression of Gag-PR and Vif in eucaryotic cells, and in cell-free proteolytic
assays of synthetic peptide substrate and Gag-containing virus-like particles
(Gag-VLP). Vif peptides were tested for PR inhibition in vitro and for inhibition of
HIV-1 infection in peripheral blood lymphocytes (PBL).
Results: Vif and N-terminal, but not C-terminal, fragment of Vif inhibited PRmediated processing of Gag-PR precursor polyprotein in bacteria as determined
by visualization of processed Gag-PR products. Vif and N'Vif, but not C'Vif, inhibited Gag-PR processing in eucaryotic cells, resulting in production of virus-like
particles lacking mature p24. In an in vitro model, the processing of a synthetic
peptide substrate by purified recombinant protease was inhibited by purified recombinant Vif at equimolar concentrations. Furthermore, Vif but not BSA inhibited
proteolytic digestion of Gag-VLP by rPR in vitro. Peptide fragments from the
N'terminal half of Vif were synthesized by improved Merrifield solid phase synthesis. Vif-based peptides but not control peptides blocked Gag-VLP proteolysis
by HIV-1 PR in vitro and inhibited blocked the production of HIV-1 by infected
PBL by four orders of magnitude but had no effects on the viability or mitogenic
response of PBL.
Conclusion: These results describe a novel mechanism of action of Vif as
an endogenous regulator of HIV-1 PR. Vif-based peptides are extremely attractive candidates for treatment of HIV-1, utilizing the endogenous and essential
interaction of Vif with protease.
379*/11131 The impact of interactions between nucleocapsid
protein, viral RNA and protease of HIV-1 on
infectious virion assembly
Susan Erickson-Viitanen, D. Ozturk, H.K. Choi, R. Tritch, R. Meade,
A. Draganescu, P. Cawood. Dupont Merck Pharm. Co., Wilmington DE
19880-0336, USA
Objective: The nucleocapsid protein (NC) of HIV plays several roles critical to
the assembly of mature, infectious virions. We have previously demonstrated that
production of mature NC by the viral protease is RNA dependent. We seek to
identify specific interactions between nucleocapsid precursors, the viral protease
and the viral RNA critical for cleavage and virion assembly for rational drug
design.
Methods: The ability of model RNAs and DNAs to bind to Gag p15 and
mediate cleavage to yield mature NC was assessed with a panel of mutant
oligonucleotides using binding, cleavage, footprinting and affinity labeling assays. Electron Microscopy and light scattering were used to monitor formation of
complexes between mature NC and RNA or DNA fragments.
Results: RNA or DNA oligonucleotides of 21-4 residues in length which form
stem loop structures appear sufficient to mediate enhanced cleavage of NC
precursors by the viral protease. Nucleotide substitutions or deletions in regions
that affect stem-loop stability disrupt binding and cleavage properties of DNA
or RNA oligonucleotides. Footprinting assays suggest some interaction between
the loop regions and the 5' stem forming portion. Site directed mutagenesis of
p15 NC has indicated several discrete regions of p15 are involved in binding
and cleavage interactions: basic amino acids near the first zinc finger are critical, but the residues within the zinc fingers are not. Methionine at residue 46
and arginine at position 52 are also critical for cleavage efficiency. When purified
p15NC protein, model oligonucleotide and protease are combined, thick filaments
composed of a compact helical arrangement of small beads were observed by
EM. Such filaments, which bear resemblance to nucleocapsid complexes isolated
from other retroviruses, do not form when any of the components are omitted,
nor if an inhibitor of the viral protease is present. An increase in light scattering
can also be used to compare rates of complex formation with similar results as
observed by electron microscopy.
Conclusions: Using a model system comprised of purified p15 NC precursor
protein, the viral protease and RNA or DNA oligomers we have identified structural features at both the protein and oligonucleotide level that are involved in
the interactions leading to efficient processing to yield mature NC, and formation
of complexes similar to those observed in intact retrovirions. This understanding
should aid in the design of HIV assembly inhibitors.
S11132 Transmission of immature HIV-1 during protease inhibitor
treatment or by mutants in the viral protease

Mary Jane Potash, C. Krachmarov, G. Bentsman, D.J. Volsky. St
Lukes-Roosevelt/Columbia University, New York, USA
Objective: To evaluate the requirement for mature HIV-1 for cell-cell virus transmission.
Methods: HIV-1 chronically infected cells were cultured in the presence or
absence of the viral protease (PR) inhibitor, Ro 31-8959 (saquinavir), and trans