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

12th World AIDS Conference

Abstracts 21141-21145 267

Results: HIV-1 envelope interaction with CD4 triggered activation of lck, mek
(probably due to upstream activation of ras by lck and raf by ras), and erk which
in turn stimulates AP-1 (probably due to the activation by erk of rsk and c-fos).
AP-1 activation is likely required for induction of Go/G1 transition to the SG2M
phase of the cell cycle observed following HIV-1 envelope interaction with CD4.
We also demonstrated that activation of the erk pathway by HIV-1 triggers NF-KB
nuclear translocation. These activation signals require the integrity of the CD4
cytoplasmic domain for their induction, and pretreatment of the viral ligand with
soluble CD4 inhibited this process. In contrast, HIV-1 induced apoptosis was observed in cells expressing either wild type or a truncated form of CD4 suggesting
that the apoptotic signal does not involve the CD4 cytoplasmic domain.
Conclusion: HIV-1 envelope interaction with CD4 activates the erk pathway
which in turn induces cell cycle progression (and virus expression in latently
infected cells), whereas interaction with CXCR4 could be required for stimulation
of apoptotic processes. Our results may contribute to understand better some
aspects of the physiopathology of HIV-1 infection.
21141 HIV-1 Tat protein promotes the synthesis of
platelet-activating factor by human monocytes
Luigi Biancone, A. de Martino, A. Del Sorbo, P.G. Conaldi, A. Toniolo,
G. Camussi. Dpt. Clinical and Biological Sciences, Viale Born 57-21100, Varese,
Italy
Objectives: HIV-1 Tat protein has been shown to induce chemotaxis and invasive
behavior of monocytes and to possess potent angiogenic properties. The aim of
the present study was to evaluate whether Tat was able to induce the synthesis by monocytes of platelet-activating factor (PAF), a phospholipid mediator of
inflammation involved in leukocyte-endothelial interaction and extravasation as
well as in neoangiogenesis induced by certain cytokines.
Methods: Human peripheral blood monocytes were purified with the help
of anti-CD14 mAb-coated magnetic beads or by differential centrifugation and
adhesion to plastic. Monocytes were stimulated with various doses of either recombinant HIV proteins (gp41, gp120, Tat) or immunocomplexes produced with
the corresponding mAbs. At different times post-stimulation, lipids were extracted
and PAF was purified TLC and HPLC. PAF levels were measured either by
bioassay or incorporation of 3H-acetate.
Results: The results obtained demostrate that Tat - but not gp41 or gp120
- induces a rapid synthesis of PAF from monocytes which peaks at 15 min
to decrease thereafter. This effect of Tat is detectable at doses as low as 0.1
ng/ml. Monocytes stimulate with immunocomplexes containing gp41 or gp120
also stimulated the synthesis of PAF. Immunocomplexes contained Tat enhanced
and anticipated the kinetics of synthesis. PAF synthetized from monocytes remains associated to the cell-surface. The analysis of molecular species of PAF
synthetized by monocytes performed by TLC analysis of 1-radyl-2-acetyl glycerols obtained from phospholipase-C-treated 3H-PAF synthetized by monocytes
indicate a prevalence of the alkyl species of PAF.
Conclusions: These results indicate that Tat may stimulate the synthesis of
PAF by monocytes and suggest the direct involvement of this mediator in the
biological properties of Tat. The synthesis of PAF may be also triggered by
immunocomplexes containing HIV glycoproteins.
21142 Characterisation of human immunodeficiency virus type
1 Nef protein quasispecies from long term
non-progressors (LTNP)
Dale McPhee1, G. Holloway1, A.L. Greenway', C. Chatfield1,
C. Raynes-Greenow2, G. Smith2, J. Learmont2. 1MacFarlene Burnet Centre for
Medical Research, PO Box 254 Fairfield, Victoria, 3078; 2New South Wales Red
Cross Blood Transfusion Service, Sydney, NSW Australia
Objectives: To develop an in vitro expression system for HIV-1 Nef derived from
LTNPs to rapidly assess open reading frame (ORF) abnormalities and obtain
protein products for further characterisation.
Design: Retrospective, controlled study.
Methods: nef genes were amplified directly from LTNP and control patient
peripheral blood mononuclear cells (PBMC) or in vitro infected PBMCs using
double nested PCR and cloned. PCR directly from lysed transformed bacterial
colonies using a 5' primer containing the T7 promoter sequence was then performed. These products were then added as templates to a coupled in vitro
transcription/translation system utilising T7 RNA polymerase and incubated with
35S-methionine at 30 degrees celsius for 90 minutes. Products were then electrophoresed in polyacrylamide gels and visualised by autoradiography.
Results: Translation products from most clones showed two bands corresponding to translation from the first and second initiation codons in the nef coding
region, as determined by immunoblot analysis using an N-terminal and C-terminal
antibody to Nef. For LTNPs intrapatient variability in protein size was seen consistently and several products showed gross defects. Such variability and defects
were not seen in the control nef clones derived from matched progressors.
Conclusion: Our in vitro expression system is an efficient method for screening

nef ORFs for possible defects. Greater size variability and level of gross defects
in LTNPs compared to controls may suggest a link between Nef abnormalities
and non-progression in the patients tested. Synthesis of Nef proteins using this
system allows further characterisation directly in functional assays.

211431 Saliva neutralizes HIV-1 infection by displacing envelope
gp120 from the virion
Daniel Malamud1, T. Nagashunmugan2, H.M. Friedman2, C.A. Davis2,
W.R. Abrams2. 1Dept. Biochemistry Univ. Penn Dental Med. 4001 Spruce St
Phila PA 19104-6003; 2Div. of Infectious Disease U Penn Med Philadelphia, USA
Background: Incubation of HIV-1 with human saliva decreases infectivity. This
inhibition is specific for HIV-1, with no effect on adenovirus, HIV-2 or SIV and
appears to work at the level of the virus rather than the host cell. We have now
identified an active protein fraction and provide evidence that the mechanism of
action involves stripping of gp120 from the virus.
Methods: HIV-1 (laboratory strains and primary isolates) was grown in PBMCs
and purified by centrifugation and chromatography on Sephacryl 1000. Submandibular saliva from seronegative donors, or fractions obtained after anion
exchange chromatography, were incubated with HIV-1, and then tested for infectivity with HeLa CD4 cells or PBMCs as compared to virus incubated with media
only. To test for effects of salivary proteins on gp120-CD4 binding, gp120 binding
to immobilized CD4 (NEN-drugquest) was utilized. To detect gp120 stripping,
virus treated with media or salivary proteins was analyzed after sucrose gradient
centrifugation (10-60% sucrose) or centrifugation at 145,000 x g on a 5% sucrose
cushion. Supernatant and pellet were analyzed by ELISA and Western blotting
using antibodies to p24 and gp120.
Results: Submandibular saliva did not block the binding of gp120 to immobilized CD4. Incubation of saliva with laboratory strains or primary isolates of
HIV-1 resulted in a shift of approximately 50% of the gp120 from the viral pellet to
the supernatant. After anion exchange chromatography of submandibular saliva
we identified a fraction which inhibited HIV-1 infectivity. This fraction contained
two high molecular weight sialyated glycoproteins, and several lower molecular
weight proteins. This active fraction also stripped gp120 from the virus.
Conclusion: The specific inhibition of HIV-1 infectivity by human submandibular saliva is associated with removal of gp120 from the virus. The active fraction
contains several proteins, including two high molecular weight glycoproteins.
121144 Chemotactic cytokines and their receptor expression in
adult Simian astrocytes cultures
Juliana Croitoru, G. Guillemin, F.D. Boussin, S. Lebel-Binay, R. Le Grand,
G. Gras, D. Dormont. Service de Neurovirologie, DSV/DRM/IPSC BP6, CEA,
92265 Fontenay-Aux-Roses Cedex, France
Background: Chemokines expressed in the CNS are emerging as primary mediators in acute et chronic non-immunological inflammatory reactions. It could be
anticipated that astrocytes might participate in chemokine synthesis and in cell
recruitment during pathological processes of CNS inflammation or virus dissemination in the brain. In this study, we have investigated chemokine production in
adult simian astrocytes, that occurs after stimulation by TNFU and IFNy.
Material and Methods: Pure primary astrocyte cultures from simian brains were
obtained from adult rhesus macaques (Macaca mulatta). Detection of chemokines
and their receptors mRNAs was performed using specific RT-PCR amplifications
after in vitro stimulation with TNFu and IFNy. Proteins were detected by ELISA
and immunofluorescence techniques.
Results: Among the chemokines tested on 3 different cultures of adult simian
astrocytes, we found an enhanced expression of RANTES, IP10, MCP-1 and
HuMIG mRNA 72 hours after stimulation with TNFa. In addition, pretreatment
with IFNy significantly increased production of these chemokines. Corresponding
protein synthesis was also evidenced in astrocyte culture supernatants, at abundant levels. Furthermore, we have detected the expression of mRNA encoding
for receptors CXCR4 (LESTR/fusin), GPR1, BOB and Bonzo, cofactors for fusion
and entry of HIV-1 and SIV. The membranous expression of fusin was confirmed
by immunofluorescence studies using a monoclonal antibody against CXCR4.
Conclusions: Adult simian astroglial cells can be an important source of
chemokines in the CNS and express chemokine receptors involved in both inflammation regulation and HIV-1/SIV infection mechanisms. Our observations
suggest the possibility that astrocyte-derived cytokines might play key roles in the
accumulation of leukocytes at the site of the lesions into the CNS parenchyma.
211 45 The effect of mycobacteria on enhancement of HIV
replication is mainly at the level of infection as heat-killed
organisms mediate significantly less HIV-1 upregulation
Mahmood Ghassemi, M. Richard Novak. 808 S. Wood Chicago IL, USA
Objective: To compare the effect of viable and heat-killed Mycobacterium avium
complex (MAC) on HIV upregulation
Design: The macrophage-like cell line, U937 and its latently HIV infected clone,
U1 were used for these in vitro studies
Methods: U937 and U1 cells were infected with HIV in the presence or absence of the same concentration of viable or heat-killed MAC, and the rate of
HIV infection was monitored by HIV reverse transcriptase (RT) and p24 antigen
capture assays. In addition, a full-length wild-type HIV-1 LTR-CAT (long terminal

repeat linked to chloramphenicol acetyl transferase) was transiently transfected
into U937 cells, and then the cells were exposed to live or heat-killed MAC; after
48 h, the cell extracts were analyzed for CAT activity. Similarly, the induction
of NF-kB, a cellular transcription factor implicated in HIV pathogenesis, was
measured by electrophoretic mobility shift assays (EMSA).
Results: Exposure of U937 cells to viable MAC consistently increased HIV RT
activity more than three fold in repeated experiments. Heat-killed MAC, however,