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

12th World AIDS Conference

Abstracts 11113-11116 5

original virus was cocultured with PBMCs from donors (including relatives) who
were normal or homozygous for CCR5A32 mutation.
Results: Recently we have identified a person homozygous for CCR5A32
who was infected with HIV. The homozygous CCR5 mutation in this person was
identical to the published 32 nucleotide deletion found in approximately 1% of
Caucasians. His HIV-ve brother was also homozygous for the A32 mutation,
therefore his cells were utilized as donor cells for culture of defined strains of
virus. The original HIV isolate grew rapidly in cultures from both CCR5 positive
PBMCs and his brother's cells. The virus demonstrated dual tropism growing in
both T cell lines (MT-2) and primary macrophages, exhibiting a similar pattern
of HIV replication to other clinical isolates but with a relatively low productive
infection. Sequencing of the V3 region directly from patient's cellular DNA revealed relatively minor changes compared with B subtype consensus sequence.
Sequencing is currently being repeated with isolates from the cocultures of both
CCR5A32/A32 and CCR5 wild type cells. The envelope region has also been
cloned and expressed into E. coli in attempt to eventually be expressed in CHO
cells. Co-receptor usage by this virus was examined using HOS cells kindly
donated by Dr D. Littman. It predominantly used BONZO (STRL33) but also used
others.
Conclusions: This study demonstrates that homozygosity for CCR5A32 is not
absolutely protective against HIV infection and that infection may occur using
other alternative coreceptors such as STRL33. The viral isolates were confirmed
to be a dual tropic with relatively slow growth in primary cells. No major changes
were found in the sequence of the V3 region of the HIV strain, directly from the
patient's cells.
11113 Coreceptor use and MT2 tropism of HIV-1 chimeric clones
having envelope V3 loop of HIV-1 subtype E
Hironori Sato, K.K. Kato, N.K. Kodaka, Y.T. Takebe. AIDS Research Center,
National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo
162, Japan
Objective: HIV-1 subtype E has phylogenetically distinct class of envelope sequence from HIV-1 subtype B, showing about 60% identity in amino acids of
envelope V3 loop. We examined whether the subtype E V3 loop has a capacity
to specify coreceptor use and MT2 tropism of virus in context of subtype B
background.
Methods: V3 loop coding region of an infectious molecular clone of HIV-1
subtype B (LAI) was replaced precisely by that of HIV-1 subtype E from nonsyncytium-inducing (NSI) variants (NH2 and TH09) or from syncytium-inducing (SI)
variants (NH1 and KH005). Coreceptor use of the chimeric viruses was assessed
by infectivity on HOS-CD4 cells expressing various chemokine receptors.
Site-specific mutagenesis was used to determine minimum requirements in the
subtype E V3 loop for specifying viral phenotypes.
Results: HIV-1LAI-NH2V3 and HIV-1LAI-THO9V3 replicated on HOS-CD4-CCR5,
whereas HIV-1LAI HIV-1LAINH1V3 and HIV-1LAI-KHOO5V3 grew preferentially on cells
expressing CXCR4. HIV-1LAI-NH2V3 and HIV-1LAI-TH09V3 were unable to replicate
on MT2 cells, whereas 1 LA, HIV-1 LAI-NH1V3 and HIV-1LAI-KHO05V3 grew and induced
syncytia. Mutants with at least two naturally-occurring basic substitutions found
in a SI variant grew efficiently on HOS-CD4-CXCR4, whereas acquisition of MT2
tropism required three basic substitutions (positions 8, 11 and 18) in the NSI V3.
Conclusion: The data indicate that HIV-1 subtype E V3 loop can specify
coreceptor use and MT 2 tropism in context of subtype B envelope protein and
that combination of basic substitutions similar but not identical to subtype B can
generate CXCR4 preference for the chimeric virus.
1114 The binding of HIV to permissive cells is coordinated by
interactions of gp120 with both CD4 and V3 loop binding
proteins
Christian Callebaut', J. Blanco', B. Krust', N. Seddiki1, S. Muller2, J.P. Briand2,
A.G. Hovanessian'. 'Institut Pasteur Unite VI.C. 28, Rue du Dr. Roux 75724,
Paris Cedex 15; 2IBMC CNRS, Strasbourg, France
Objectives: To demonstrate the distinct role of the V3 loop of gp120 in the binding
of HIV particles to permissive CD4 positive cells.
Background: Neutralizing mAbs against the V3 loop, block the binding of HIV
virions to permissive cells without affecting the potential capacity of gp120 to
interact with CD4 (J. Virol. 71, 8289, 1997). Interestingly, the anti-HIV pseudopeptide 5[Ki PR]-TASP which mimics the V3 loop (Virology 218, 181, 1996), binds
cell-surface protein(s) other than CD4 and chemokine receptors, and inhibits the
binding of HIV virions to CD4+ target cells (J. Biol. Chem. 272, 7159, 1997).
Results: The pseudopeptide TASP inhibits viral entry in different types of
cells infected by T lymphocyte- and macrophage-tropic HIV-1 and HIV-2 isolates.
TASP has no effect on SIVmac and HIV-1 pseudotypes expressing either Mo-MLV
or VSV-G envelope glycoproteins. By using this specific inhibitor of HIV entry,
nucleolin, PHAP II, and PHAP I were identified as three V3 loop binding proteins (V3-BPs) implicated in the binding of HIV particles to CD4+ cells. Purified
preparations of the V3-BPs, devoid of the CD4 and the fusin/CXCR4 cofactor,

were able to inhibit infection of the CD4+ CEM cells by the lymphotropic HIV-1
Lai isolate. Recombinant gp120 was shown to bind the purified preparation of
V3-BPs with a high affinity, and this binding was inhibited either by TASP or
mAbs specific to the V3 loop. Consistent with the functional association of these
proteins in the same complex, antibodies raised against any one of them inhibited
HIV virion binding and infection of CD4+ cells (CEM, PBMC) by T lymphocyte- or

macrophage-tropic, and as well as primary syncytium-or non-syncytium-inducing
HIV-1 isolates.
Conclusion: Our results suggest that these three V3-BPs serve as a potential
V3 loop receptor which plays an essential role in the binding of HIV particles
to permissive cells. Consequently, the binding of HIV virions is coordinated by
two complementary interactions, one through the CD4 and the other one through
such a V3 loop receptor; both interactions being necessary before the membrane
fusion event mediated by the chemokine receptors.
111 Coreceptor utilization by divergent human
immunodeficiency viruses involves a highly conserved
envelope residue
Wei-Kung Wang, T. Dudek, Y.J. Zhao, H.G. Brumblay, M. Essex, T.H. Lee. l/D,
Harvard School of Health, FXB, Rm 304B, 651 Huntington Ave, Boston MA. USA
Background: Several seven-transmembrane chemokine receptors were recently
found to double as a coreceptor for a genetically diverse family of human and
non-human primate lentiviruses. Paradoxically, the main region believed to be
involved in coreceptor utilization was mapped to the hypervariable region 3, or
V3, of gp120. The question of whether functional convergence in coreceptor
utilization is mediated by some V3 residues that are highly conserved among
HIV-1, HIV-2, and SIV was addressed.
Methods: Site-directed mutagenesis was carried out to introduce substitutions
to highly conserved V3 residues in infectious molecular clones of HIV-1. The
effect of substitution on envelope expression, processing and incorporation into
virions was examined by Western blot. The effect on coreceptor utilization was
monitored by the ability of the mutant viruses to infect cell lines stably transfected
with various chemokine coreceptors.
Results: A highly conserved arginine residue at position 298, Arg298, in the
V3 of gp120 was found to have an important role in coreceptor utilization. The
inability of Arg298 mutants to use chemokine coreceptors was not attributed to
global alteration of gp120 conformation. Neither the expression, processing, and
incorporation of mutant envelope proteins into virions, nor CD4 binding were
significantly affected by the mutations. Substitutions introduced to adjacent V3
residues that are not as highly conserved as Arg298 had no discernible effect on
coreceptor utilization.
Conclusions: Functional convergence in chemokine coreceptor utilization by
genotypically divergent HIV-1 involves at least one highly conserved residue in
the V3. Such highly conserved residues may represent a new class of targets
for future anti-viral designs aimed at blocking interaction between HIV-1 and
chemokine coreceptors.
11116 Tropism and co-receptor usage of tissue-derived HIV-1
primary isolates obtained from patients at the terminal
stage of disease
Hassan Naif, Shan Li, M. Alali, N. Wasr, A. Cunningham. Centre for Virus
Research Westmead Hospital, Westmead 2145, Sydney, NSW, Australia
Objectives: To investigate the tropism and replication characteristics of HIV-1
isolates derived from different tissues (brain, CSF, lung, and spleen) in human
monocytes and in vitro cultured monocyte-derived macrophages (MDM). Differential tropism and level of replication of these isolates was analysed in association
with the co-receptor usage and the state of maturation/differentiation of monocytes
into MDM.
Methods: 10 tissue-derived HIV-1 isolates from brain, spleen, lung and cerebrospinal fluids and HIV-1 BaL were subcultured on PBMCs then used to infect
fresh monocytes, monocyte-derived macrophages (MDM) and primary T lymphocytes. Replication kinetics of HIV-1 isolates was measured by p24 antigen
ELISA and hot-PCR. V3 regions from viral DNA outputs were amplified using
nested-PCR. Co-receptor usage was monitored by HOS cells transfected with
cDNA for chemokine receptors.
Results: Three levels of replication were observed: high, low or non-productive
in both monocytes and MDM while only two levels (high and low) were observed
in T lymphocytes. Most isolates (7/10) replicated with productive infection in both
monocytes and MDM as tested by the extracellular p24 antigen. Full length HIV
DNA was correlated with the level of extracellular p24 antigen in most isolates,
as detected by highly sensitive hot-PCR. The level of restriction of low replicating isolates was prior to reverse transcription. V3 sequences revealed that
these isolates had similar genotypes to those of non-syncytium inducing (NSI)
macrophage-tropic strains and phylogenetic analysis demonstrated a clear segregation of isolates grown on different cell types especially MDM. Most isolates
used the chemokine receptor 5 (CCR5) to infect cells as determined by infection
assay using HOS cells.
Conclusions: Tissue-derived HIV isolates replicated either at low or high level
of replication irrespective of the state of cell maturation. High replicating isolates
showed no restriction at any stage of replication and low replicating isolates restricted at the stage of pre-reverse transcription. Most isolates used CCR5 as the
main co-receptor to enter cells although they have been obtained at the terminal

stage of disease.