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

268 Abstracts 21146-21150

12th World AIDS Conference

failed to significantly enhance RT activity. In addition, both viable and heat-killed
MAC mediated similar enhancement of HIV LTR-CAT activity, and comparably
induced NF-kB activation. Furthermore, viable and heat-killed MAC comparably
induced HIV p24 antigen upon cocultivation with U1 cells, which are not phagocytic for mycobacteria. HIV activation by MAC in U1 cells could not be detected
by HIV RT assay, which is not as sensitive as HIV p24 antigen. Therefore, the
amount of released p24 reflects the effect of cell membrane exposure to MAC,
and not phagocytosis.
Conclusion: Taken together, these data, in conjunction with our previous findings, indicate that MAC infection plays a significant role in enhancement of HIV
replication, and the induction of either HIV LTR-CAT or NF-kB is not the major
factor in MAC-mediated HIV upregulation. Cellular invasion by mycobacteria is
more important than membrane associated events in relation to HIV upregulation.
21146 HIV-1 strain dependent preferences in Thl- and Th2-type
conditions
Youichi Suzuki1, Naoki Yamamoto', Y. Koyanagi', Y. Tanaka2, T. Murakami1,
T. Kimura', J.A. Hoxie3, W.A. O'Brien4. 'Department of Microbiology Tokyo
Medical and Dental University Yushima Bunkyo-Ku Tokyo 113; 2Department of
Biosciences Kitasato University Sagamihara, Japan; 3Department of Medicine
University of Pennsylvania Medical Center, Philadelphia, PA; 4Department of
Medicine University of Texas Medical Branch, Galveston, TX, USA
Objectives: To investigate the molecular mechanisms of HIV-1 strain-dependent
preferences in Thl- and Th2-type cells.
Methods: We investigated the proliferation of various chimeric HIV-1 strains
derived from macrophage-tropic (M-tropic) and T-cell-line-tropic (T-tropic) strains
in IL-12 (Thl-type) or IL-4 (Th-2 type) stimulated CD4+ T cell culture. Fusion
ability of HIV-1 envelope expressing cells against Thl- or Th2-type cells were
compared. Co-receptor, CCR5 and CXCR4 expression on Thl- and Th2-type
cells were also analyzed by flow cytometry.
Results: M-tropic strains, JR-CSF and JR-FL preferentially replicated in
Thl-type cells. In contrast, T-tropic strains, NL4-3 preferentially infected Th2-type
cells. Additional studies using chimeric viruses identified the V3 region in gp120
as the principal determinant for this effect. Cell-to-cell fusion assays showed that
T-tropic Env expressing cells effectively fused with Th2-type cells. CCR5 and
CXCR4 were highly expressed on Thl- and Th2-type cells, respectively.
Conclusion: In our culture system, M-tropic and T-tropic HIV-1 strains favored
Thl- and Th2-type condition, respectively. This preference was at least partially
determined by V3 loop and co-receptor interaction. Our findings suggest a shift
of Thl- to Th2-type immune responses as a factor involved in the change from
M- to T-tropism by HIV-1 strains in vivo.
13*/ 21147 Thl and Th2 lymphocytes are permissive for CCR5-,
but not for CXCR4-dependent HIV
Guido Poli1, A. Brambilla2, M. Cota2, F. Sinigaglia3, P. Panina Bordigon3,
E. Vicenzi2. 'P2/P3 Laboratories, DIBIT Via Olgettina 58, 20132 Milano;
2DIBIT-HSR, Milano; 3Roche Milano Ricerche, Milano, Italy
Objectives: To investigate the replicative capacity of HIV strains using different
chemokine receptors [R] for entry into Thl and Th2 polarized CD4+ lymphocytes.
Methods: Primary Thl and Th2 cell clones and cord-blood derived polarized
lines were infected with both laboratory-adapted [BaL, LAI, MN, NL4-3, NL(AD8)]
and primary isolates of HIV that had been characterized for chemokine receptor
usage onto U87 CD4+ cell lines. HIV production was monitored by RT assay,
whereas quantitation of proviral DNA was performed by real time PCR (TaqMan).
Expression of chemokines and chemokine R was monitored by RT-PCR and
FACS analysis.
Results: Both primary and laboratory-adapted CCR5-monotropic HIV strains
replicated with comparable efficiency in polarized Thl and Th2 lymphocytic cell
clones and cord blood-derived lines. In contrast, CXCR4-dependent viruses failed
to growth in either Thl or Th2 cells. This pattern was not explained by expression
of the endogenous CXCR4 ligand SDF-1; furthermore, both Thl and Th2 cells
showed comparable levels of CXCR4 on their cell surface, and migrated with
comparable efficiency in response to SDF-1. CXCR4-dependent HIVs readily
infected both Thl and Th2 cells, as demonstrated by the kinetics of proviral DNA
accumulation. However, unlike CCR5-monotropic viruses, they failed to spread efficiently thereafter. Substitution of the gp120 env region of the CXCR4-dependent
NL4-3 virus with its homologous region of the CCR5-using ADA HIV fully restored
the viral replicative capacity in Thl and Th2 cells. In agreement with the recently described selective expression of CCR3 in Th2 cells, a primary dualtropic
(CCR3/CXCR4) HIV isolate replicated in Th2 rather than Thl cells.
Conclusion: Polarization of CD4+ T cells into Thl or Th2 lymphocytes may
play an important role shortly after transmission in the selection process that almost invariably favors the replication of CCR5-dependent, but not CXCR4-using
HIVs.

21148 HIV-1 Nef associates with the tumour suppressor protein
p53 and inhibits apoptosis
Alison Greenway', K. Allen', P. Lambert2, R. Johnstone3, D. McPhee'. 'Acbu,
MacFarlance Burnet Centre for Medical Research, PO Box 254 Fairfield, Victoria,
3078; 3Austin Research Institute, Heidelberg, Victoria, Australia; 2National
Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA
The Nef protein of human immunodeficiency virus type 1 (HIV-1) is essential for
depletion of CD4+ T-lymphocytes, high virus replication and disease progression.
To understand how Nef functions we have used a GST-Nef fusion protein to probe
cellular extracts to identify proteins which bind to Nef. Using this approach we
reported that Nef complexes with multiple host-cell proteins including the tumour
suppressor protein, p53. We now show that Nef associates directly with purified
recombinant p53 in co-precipitation and solid-phase binding assays. Identification of the Nef-p53 interaction during HIV-1 infection of CD4+ T-cells verified
the relevance of our binding studies. Using purified recombinant fragments of
Nef corresponding to amino acid residues 1 to 57, 1 to 79 and 20 to 206 we
have identified that the binding domain for p53 resides in the N-terminal region
of Net. As p53 is known to play a critical role in the regulation of cell cycling
and apoptosis our data suggests that Nef may alter these processes during
HIV-1 infection. To directly address the possibility that Nef acts as an inhibitor of
apoptosis, PBMC and T-cell lines electroporated with purified Net protein were
exposed to UV irradiation, a stimulant of p53-dependent apoptosis and compared
to control cell populations. Quantitation of apoptosis in the Nef-treated and control
cell populations was assessed by annexin V staining, measurement of DNA content by flow cytometry and identification of DNA fragmentation laddering. Using
this approach we show that Nef inhibits apoptosis. The ability of Nef to protect
cells against UV-induced apoptosis also correlated with its ability to abrogate
p53 DNA binding activity, p53-mediated transcriptional activation. Comparison of
apoptotic cell death during in vitro HIV-1 infection of CD4+ T-cells lines confirmed
an anti-apoptotic role for Nef.
Conclusion: These data suggest that Nef binds and modulates the activities
of p53 and is protective against induction of p53-mediated apoptosis. This may
represent a mechanism by which Net acts as a positive factor for virus replication
by enhancing cell viability and allowing increased virus production.
21149 HIV-induced immunodeficiency ex vivo: Role of viral
tropism in pathogenesis in human lymphoid tissue
histoculture
Leonid Margolis, S. Glushakova, J.-C. Grivel, J. Zimmerberg. National Institutes
of Health (NIH), Bethesda, MD, USA
Objectives: To compare pathogenesis of HIV-1 of different tropisms within the
cytoarchitecture of human lymphoid tissue ex vivo. To understand the causative
relationship between the HIV-1 tropism switch and development of immunodeficiency.
Methods: Surgically removed human tonsils or lymph nodes were cultured ex
vivo, infected with laboratory strains and primary isolates of HIV-1 and immunized
with tetanus or diphtheria toxoids. In tissues separately infected with various HIV
isolates we compared virus replication, the ratios of cells of different subsets, and
production of specific antibodies.
Results: Infection with SI/T (CXCR4)-tropic isolates led to almost complete
depletion of CD4+ T lymphocytes in tissues of all tested donors. In contrast, depletion of the CD4+ T lymphocytes in histocultures infected with NSI/M (CCR5)-tropic
isolates was mild. Infection with SI, but not with NSI HIV-1 rendered immunocompetent histocultures immunodeficent. These isolates differently affected the
level of endogenous CC chemokines which may contribute to the pattern of HIV-1
infection.
Conclusions: Using histocultures of human lymphoid tissue which allow the
testing of critical hypotheses regarding HIV pathogenesis in the context of tissue
cytoarchitecture, we demonstrated that viral tropism is the dominant factor in HIV
pathogenesis. There is a causative relationship between tissue infection by T
(CXCR4)-tropic/SI isolates (typical for late HIV infection in vivo) and development
of immunodeficiency.
21150 Cleavage of vimentin and the nuclear mitotic apparatus
(NuMA) protein by HIV-1 protease may play a role in
cytopathogenesis and the development of cancer
Robert L. Shoeman, C. Huttermann, P. Traub. Max-Planck-lnstitutee for Cell
Biology 68526 Ladenburg, Germany
Objectives: To determine if the specific cleavage of host cell proteins by the
HIV-1-encoded protease (HIV-1 PR) leads to alterations in cellular architecture
like those seen during HIV-1 infection.
Design: In vitro assays with purified proteins and microinjection of HIV-1 PR
into tissue culture cells.
Methods: HIV-1 PR was microinjected into tissue culture cells and the alterations in cellular organization were visualized via confocal laser scanning

microscopy and electron microscopy. Proteolysis was monitored by denaturing
gel electrophoresis and immunoblot analysis.
Results: Microinjection of HIV-1 PR into human skin fibroblasts results in a
collapse of the vimentin intermediate filament (IF) network, a disappearance of
stress fibers and alterations in nuclear morphology and chromatin. The nuclear
mitotic apparatus (NuMA) protein is cleaved in these cells, but this does not
appear to be the major cause of the changes seen in nuclear morphology. Exper