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

6 Abstracts 11117-11121

12th World AIDS Conference

11117 A small molecule CXCR4 inhibitor that blocks T-cell
line-tropic HIV-1 entry
Tsutomo Murakami1, Naoki Yamamoto1, T. Nakajima2, M. Waki3,
H. Tamamura4, N. Fujii4. 1Depart. of Microbiology, Tokyo Medical and Dental
University, 1-5-45 Yushima Bunkyo-Ku, Tokyo 113; 2Shionogi Institute for
Medical Science, Settsu, Osaka 566; 3Seikagaku Corporation, Chyuo-Ku, Tokyo
103; 4Kyoto University, Sakyo-Ku, Kyoto 606-01;, Japan
Objectives: To investigate whether anti-HIV peptide T22 specifically inhibits T
cell line-tropic (T-tropic) HIV-1 infection mediated by its coreceptor CXCR4.
Methods: We examined the effect of T22 on HIV-1 infection mediated by
CD4- and coreceptor-expressing U87MG and HOS cells. Cell fusion assay was
performed by coculture of HIV-1 Env-expressing HeLaS3 cells and CD4- and
coreceptor-expressing NIH3T3 cells using a-complementation of b-galacotosidase. Interaction of T22 with a CXCR4 molecule was examined by effect of
T22 on Ca2+ mobilization induced by PBSF/SDF-1 in CXCR4-expressing CHO
cells, 12G5, an anti-CXCR4 monoclonal antibody binding to CXCR4-expressing
MOLT-4 cells, and PBSF/SDF-1-induced chemotaxis of PHA-activated PBMC.
Results: T22 specifically inhibited CXCR4-mediated HIV-1 infection and cell
fusion. It also blocked Ca2+ mobilization induced by PBSF/SDF-1, 12G5 binding
to CXCR4-expressing cells, and chemotaxis induced by PBSF/SDF-1 in a dose
dependent manner.
Conclusion: We can described T22 as a small molecule CXCR4 antagonist
and a potent inhibitor of T-tropic HIV-1 infection. This peptide represents a useful
tool for investigating the entry mechanism of T-tropic HIV-1.
11118 The common molecular determinants of HIV-1 tropism for
human colonic cells and astrocytes
J. Roberto Trujillo1, B. Meek2, J.D. Brain2. 1665 Huntington Avenue 1-1304,
Boston, MA; 2Harvard School of Public Health, Boston, MA, USA
Background: Infection of CD4+ T lymphoyctes by HIV-1 requires the presence
of CD4 surface receptor. The specific domains on both gp120 and CD4 that are
involved in this envelope-receptor interaction have been well defined. However,
HIV-1 infects some cells that do not express CD4, including glial and colonic epithelial cells. Studies in both cells have identified galactosyl ceramide as a potential
receptor of HIV-1. However, it appears that the interaction between galactosyl ceramide and gp120 involves multiple sites of the envelope glycoprotein. Recently,
we have identified that V3 loop serves as a primary viral determinant for infectivity
of CD4-neuronal cells (Trujillo et al, Virology, 217: 613-617, 1996). In addition,
preliminary data suggests that some HIV-1 isolates derived from epithelial colonic
cells and brains of AIDS patients may have similar V3 loop sequences. The
objective of this study is to determine if the same amino acid changes of HIV-1
gp120 V3 loop affect the tropism of CD4-negative human colonic epithelial cells
and glial cells.
Methods: Tumor cell lines glial-U373-MG and epithelial colonic-HT29 were
obtained from the American Type Culture collection. SupT1 was obtained from
the AIDS Research NIH. In order to study the molecular determinant of gp120 of
HIV-1, we first analyzed an HIV-1/Illb-derived molecular clone, HxB2RU3. Using
site directed mutagenesis, we constructed a mutant proviral clone, HxB2RU3CI,
which contained the consensus V3 sequence on the background of HxB2RU3.
Infection dose was based on quantification of virus by a 50% tissue culture
infectious dose.
Results: Our data demonstrates that the amino acid sequence of HxB2RU3
(positive charge residues) is critical for infectivity of these CD4-negative colonic
epithelial and glial cells. When the HxB2RU3CI (negative charges residues) was
tested, it did not infect the colon or glial cells.
Conclusions: This data suggest that the V3 loop also serves as a primary viral
determinant for infectivity of both colonic and glial CD4-negative cells. Preliminary
data suggest that virus derived from colon and brain of AIDS patients present
similar V3 sequences. Our findings may have implications in HIV-1 pathogenesis
of ADC and HIV-1 wasting syndrome.
| 11119 MIP-1a, MIP-1/3 and RANTES /3-chemokines increase the
replication of T-tropic HIV strains through CXCR4
stimulation
Antonina Dolei1, Adriana Biolchini1, C. Serra1, S. Curreli1, E. Gomes1,
M. Ziccmeddu1, F. Dianzi2. 1Dept. Biomedical Sciences, Univ. Sassari Viale San
Pietro 43B, 107100 Sassari; 2lnst. Virology, La Sapienza University, Rome, Italy
Objective and Design: It has been reported that fp-chemokines interfere in T
lymphoid cells with the replication of monocytotropic (M-tropic) HIV-1 strains, but
not with that of T-tropic strains, through their occupancy of the CCR5 receptor,
used by M-tropic strains as co-receptor, and consequent perturbation of the process of HIV fusion with the plasmamembrane. Aim was to analyse the effects
of MIP-lca, MIP-1l and RANTES /t-chemokines on the replication in T cells of
lymphocyte-tropic (T-tropic) HIV-1 strains.
Methods: T lymphoid cells (freshly PHA-activated peripheral blodd lymphocytes

(PBL) and cultured PHA-activated T cells from healthy volunteers as well as the
C8166 T cell line) were treated overnight with graded amounts of /-chemokines
before infection with T-tropic HIV-1 isolates, or HTLV-IIIB. HIV replication was
followed as production of infectious particles, p24 antigen and detection of viral
DNA and RNA sequences. CXCR4 expression was followed by detection and
quantification of specific transcripts.

Results: Pretreatment of T cells with MIP-la, MIP-1/l and RANTES can affect
also T-tropic strains, as it increases dose-dependently the replication of HIV-1p1
and HIV-1 RPdT strains, as well as virus absorption and provirus DNA accumulation.
These findings are associated to increased accumulation of CXCR4 transcripts,
and mediated by the protein tyrosine kinase signalling. Moreover, 3-chemokines
stimulate PBL proliferation.
Conclusions: At variance with published papers, our results show that
/-chemokines increase the adsorption and replication of at least some T-tropic
HIV-1 strains, and this is related to stimulated expression of the CXCR4 co-receptor.
Work supported by ISS-Progetto AIDS 1997 grant n. 40A.0.41
11120 Actin dependent dual receptor HIV-1 infection vs. single
receptor chemotaxis induction by HIV-1 envelope
David H. Schwartz, Sujatha lyengar, J.E.K. Hildreth, J.B. Mitchell. MMI, Johns
Hopkins School Public Health, 615 n. Wolfe St., Baltimore, MD, USA
Background: CD4 independent cultured HIV isolates have been described.
Given the mutability of HIV-1 envelope, why do almost all isolates retain a requirement for dual receptor binding? Do similar requirements apply to gp120
induced chemotaxis?
Objectives: To understand envelope-receptor interactions of HIV infection and
HIV induced chemotaxis.
Methods and Results: Peripheral blood mononuclear cells (PBMCs) were
activated with PHA and exposed to 100 TCID50 HIV-1MN. Transient 1 hr pretreatment with 1 uM Cytochalasin D (CyD), which disrupts cytoskeletal actin
filament attachment to transmembrane proteins, blocked infection at the entry
step. This was demonstrated by decreased p24 internalization @ 1 hr, reverse
transcribed DNA @ 8 hrs, and virus production @ day 7. Cell division and viral
budding were unaffected by CyD at this dose. Confocal fluorescent microscopy
of PHA stimulated PBMCs revealed polarized co-capping of CD4 and CXCR4
(60') and pseudopod formation (90') induced by MN rgpl20. These membrane
changes were blocked by pretreatment with CyD. Staining with B4 mAb (UBI,
Inc.) specific for a conformational epitope dependent on CD4-chemokine receptor
(CKR) heterodimerization demonstrated equal or greater expression after Cy D
treatment. In contrast, chemotaxis induced by HIV-1MN or MN rgpl20 was CD4
independent, requiring only gp120-CXCR4 binding. Native, heat denatured, and
reduced carboxymethylated gp120 were all chemotactic for both CD4+ or CD8+
lymphocytes. Chemotaxis was blocked by mAb to CXCR4 but not by mAb to CD4
which prevented gp120-CD4 binding.
Conclusions: Beyond heterodimerization, the requirement of actin dependent
CD4 - CKR cocapping for HIV entry may ensure that virions do not enter cells
in early apoptosis (which lose the ability to form cytoskeletal actin filaments).
Movement of cells along a gradient of gp120 also requires intact actin, but only
gp120 engagement of fusin. The selective value to HIV of attracting CD4 negative
cells is unclear, but CD8+ T cell migration to germinal centers is thought to be
important in pathogenesis.
11121 | Mutations in the leucine zipper-like heptad repeat
sequence of HIV-1 gp41 dominantly interferes with
wild-type virus infectivity
Steve L. Chen, S.F. Lee, H.J. Hao, L.K. Lauang. 128 Yen-Chiu-Yuan Road
Section 2, Institute of Biomedical Science Taipei, Taiwan
Objectives: We have previously shown that a proline substitution for the conserved leucine or isoleucine residues located in the leucine zipper-like heptad
repeat seqeuence of HIV-1 gp41, whose feature is also conserved among a
number of other viruses, renders viruses noninfectious and the envelope (Env)
protein unable to mediate membrane fusion. The objective of this study was to
utilize the HIV-1 gp41 heptad repeat sequence as a model system to determine
the feasibility of developing an antiviral strategy targeting this highly conserved
sequence.
Methods: The interference effect conferred by mutants on infectious virus
production was examined by cotransfection of cells with wt and mutant proviruses
followed by measurement of the infectivity of the resulting supernatant viruses
on SupT1 cells or on a MAGI indicator cell line. The interference conferred by
the mutant Env upon the ability of wt protein to mediate virus entry into cells
was determined by an env trans-complementation assay using a defective HIV-1
reporter vector.
Results: Viruses obtained from coexpression of wt provirus with mutant 566
or 580 provirus inhibited more potently the production of infectious virus than did
viruses generated from cotransfection of wt provirus with other mutant proviruses.
Nevertheless, all viruses produced from mixed transfections showed decreased
infectivity compared with that of the wt virus when MAGI assay was performed.
The ability of wt Env to induce cytopathic effects was inhibited by coexpression
with mutant Env. Coexpression of mutants inhibited the ability of the wt protein to
mediate virus-to-cell transmission.
Conclusions: Our study demonstrates that mutations in the leucine zipper-like

heptad repeat sequence dominantly inhibit infectious virus production. These
results suggest that the heptad repeat sequence of gp41 can be targeted by
trans-dominant negative mutant-based anti-HIV strategies.