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

10 Abstracts 11138-11143

12th World AIDS Conference

11138 Immunocytochemical localizations of HIV-1 Gag proteins
and formation of O-shaped virion particles associate with
p17
Jaang-Jiun Wang1, C.T. Chiou2, P.H. Chiang2, B. Hortor3, L. Ratner3. 118
Shu-Yuan Rd, Taipei, Taiwan; 2lnst Biol & Anat, Natl Def Med Ctr, Taipei ROC;
3Div Mol Oncol, Wash Univ Med Sch, St. Louis, USA
Objectives: To identify the components and localizations of viral proteins during
HIV-1 assembly.
Design: Transfection of Gag protein p55G1 alone can form virion particles.
Different constructs of Gag/Pol proteins were designed and expressed in human
T cell lines. Intracellular trafficking of the components of virion particles by a
modified immunocytochemical technique was identified.
Methods: Vaccinia virus with pTM3 vector infection/transfection system were
performed with recombined Gag/Pol proteins in Jurkat cells, 293T cells and
BSC40 cells for various time periods. Immunocytochemical data were analyzed
with confocal and immunoelectron microscopy.
Results: High titer of antigenicity of p24 and p17 in the well-fixed and regularly
embedded samples were preserved for the immuno-gold labeling. Budding of
O-shaped virion particles on the cell membrane was found in p55G1 transfections and labeled by p24-Ab conjugated with gold particles. Assembly of p24
occurred in the cytosol on free ribosomal granules, in rER and mitochondrial
membranes but not in the Golgi apparatus. Accumulation of virion particles at
the perinuclear pores indicated that something from the nuclear may joining the
particles. C-shaped free virion particles were found accumulated on the vacuolar
membrane and in the cytosol but not on the cell membrane when transfected with
p39A1, p41A1 and p55A1 omitting whole or part of matrix protein, p17.
Conclusions: Under the vaccinia infection/transfection system, p55G1 particles are assembled among free ribosomes or on membranous structures. The
matrix protein is essential in directing Gag protein to the cell membrane and also
necessary for forming an O-shaped virion particle.
380*/11139 Quantitative molecular analysis of mRNA
expression of HIV-infected T cell using high density
oligonucleotide arrays
Jacques Corbeil1, T.R. Gingeras2, G. Mamtora2, D. Huang2, D.D. Richman1.
University of California San Diego, 9500 Gilman Dr. La Jolla CA 92093-0679;
2Affymetrix, San Jose CA, USA
We have explored how high density arrays (DNA chips) could be used to better
understand how gene expression in HIV-infected cells is globally altered during
the course of infection. Simultaneous mRNA expression levels for the HIV-1
genome and 6640 human genes present in the reporter lymphoblastoid T cell
line CEM-GFP were determined. CEM-GFP were infected with HIV-1LAI at a
multiplicity of infection of 0.5. RNA was prepared from control and HIV-infected
at 2 h, 24 h and 48 h after infection. The percentage of infected and apoptotic
cells were also evaluated to correlate with gene expression. Specific genes were
identified that were expressed only in HIV infected cells (2 h: 261 of 2299; 24 h:
278 of 1538 and 48 h: 319 of 1576) or in the control (2 h: 362 of 2299; 24 h: 293
of 1538 and 48 h: 322 of 1576). A number of genes were specifically modulated
(>3-10 or >10 fold) upon infection.
Hour after infection Downregulation Upregulation
<10X 3-10X 3-10X >10X
2 - 24 41 -
24 1 50 50 1
48 3 81 55 4
Genes known to be involved in pathways associated with apoptosis induction,
G-protein signaling, stress response and cell cycle control were among those
modulated upon HIV infection. The strengths of these analyses are their reproducibility, the ability to measure changes globally and the possibility of identifing
new interactions.
11140 Identification and characterisation of nef deleted viruses
from three long-term non-progressors
David Rhodes1, L. Ashton2, A. Solomon1, A. Carr3, D. Cooper3, J. Kaldor2,
N. Deacon1. 1Macfalane Burnet Centre for Medical Research Yarra Bend Road,
Fairfield Vic 3078; 2National Centre in HIV Epidemiology & Clinical Research
Sydney NSW; 3Centre for Immunology, Sydney NSW, Australia
Background: The lack of disease progression in a small number of people infected with HIV-1 has been attributed to defects in the nef/LTR region of several
HIV strains. We assembled a group of 69 long-term nonprogressors (LTNPs) to
ascertain the frequency at which nef/LTR deletions occur in LTNPs and to screen
for the presence of attenuated strains previously identified in an Australian group
of transfusion recipients and their common donor.
Methods: DNA was isolated from uncultured PBMCs and the nef/LTR region

of the provirus was amplified using triple nested PCR. The products were cloned
and sequenced using a LiCor 4000 L automated sequencer.
Results: In three of nine individuals with viral loads less than 200 RNA
copies/ml, deleted nef/LTR regions only could be amplified. In the remaining 48
individuals with viral loads greater than 200, either mixed populations of wild type
and deleted nef/LTR forms or wild type nef/LTR regions alone were detected. The
three deleted viruses lacked different sequences but all maintained regions known

to be essential to viral replication. The three patients have been infected for at
least 10 years, have CD4 counts higher than 800//I1 and are culture negative.
Conclusions: The proportion of nef deleted viruses in LTNPs with viral loads
<200 copies/m is high in our group of LTNPs. The three patients identified are
most likely LTNPs due to the genomic defects detected in the nef/LTR region. The
three nef deleted viruses do not possess deletions characteristic of the SBBC
strains. These new vaccine candidate strains are important to the furthering of
live attenuated HIV-1 vaccine research.
S11141 Cell cycle G2 arrest by HIV-1 Vpr is not responsible for
Vpr-induced cell death
Yuqi Zhao, R.T. Elder, M. Yu, M.Z. Chen. North Western Univ. Med. School,
Chicago IL, USA
Background: HIV-1 Vpr plays an important role in pathogenesis as it activates viral replication and is involved in the depletion of CD4+ T lymphocytes. Expression
of the HIV-1 vpr gene induces cell cycle G2 arrest and cell death in both human
and fission yeast cells. The target for Vpr-induced cell cycle G2 arrest is the
cyclin dependent kinase p34cdc2 whose activation requires dephosphorylation, a
process that determines the onset of mitosis in all eukaryotic cells. Vpr induces
G2 arrest by preventing dephosphorylation of p34cdc2.
Hypothesis: Vpr-induced cell death is the result of cell cycle G2 arrest
Methods: Genetic and biochemical methods were used to characterize G2
arrest and cell death in fission yeast cells, in which the highly conserved cell cycle
controls are well characterized.
Results: Consistent with Vpr affecting the phosphorylation state of p34cdc2 to
induce G2 arrest, gene mutations that affect phosphorylation in p34cdc2 either
abolished (in cdc2-L7 mutant strain) or severely reduced (in cdc2-1w and cdc2-3w
mutant strains) the ability of Vpr to induce G2 arrest. Our studies further suggest
that the inhibitory effect of Vpr on p34cdc2 is mediated through the well characterized inhibitory phosphorylation site on tyrosine 15 (Tyr15) of p34cdc2. Substitution
of phenylalanine for tyrosine (Y15F) prevents phosphorylation on Tyrl 5 and completely abolishes the ability of Vpr to induce G2 arrest. Interestingly, this Y15F
mutation was unable to abrogate Vpr-induced cell death suggesting that cell
death is independent of G2 arrest. This conclusion was further supported by our
finding that a point mutation in vpr that results in a replacement of histidine with
arginine at amino acid 78 (H78R), ameliorated cell death induced by Vpr while
maintaining its ability to induce G2 arrest.
Conclusions: Induction of cell cycle G2 arrest and cellular death are independent functions of HIV-1 Vpr.
S11142 T cell apoptosis in mice transgenic for HIV-1 Vpr
Yoichiro Iwakura, Tamaki Maiyao. Institute of Medical, Tokyo Minatoku
Shirokanede, Tokyo, Japan
Objectives: To examine the roles of a HIV-1 accessory protein, Vpr, on viral replication in vivo and AIDS pathogenesis, we generated transgenic mice carrying the
vpr gene.
Methods: We produced transgenic mice expressing HIV-1 (NL4-3-2 strain) vpr
under the control of the mouse CD4 promoter/enhancer. To examine whether
Vpr has any effects on T-cell subsets in vivo, FACS analyses was performed on
cells from the thymus and peripheral lymphoid organs of Vpr-transgenic mice.
Cell cycle analysis and detection of apoptotic cells in transgenic thymocytes were
carried out either by propidium iodide (PI) or by Annexin V staining, respectively.
Results: In the thymus of Vpr transgenic mice, high levels of mRNA expression
from the transgene and thymic atrophy were observed. FACS analyses showed
decrease of CD4/CD8 double positive cells and increase of CD4/CD8 negative
cells in the thymus and decrease of T cells in peripheral lymphoid organs. Although
previous studies suggested that Vpr induces G2 arrest of the cell cycle, this was
not observed in Vpr transgenic thymocytes. In contrast, significant increase of
apoptotic cells was observed in the transgenic thymus.
Conclusion: We found that Vpr induces apoptosis of thymocytes in Vpr transgenic mice causing thymic atrophy and T cell depletion in peripheral lymphatic
organs. These results suggest that Vpr play an important role in the CD4 positive
T cell depletion observed in AIDS patients.
| 11143 Sydney blood bank cohort virus evolution and
quantitation of virus attenuation
David Rhodes, A. Solomon, N. Deacon. Macfarlane Burnet Centre for Medical
Research Yarra Bend Road, Fairfield Vic 3078, Australia
Background: The Sydney Bloodbank Cohort (SBBC) is a unique collection of individuals infected with an attenuated HIV-1 virus from a common donor. Molecular
characterisation of viruses from one recipient, C98, shows the loss of sequence
from the nef/LTR region over time indicating continued evolution of the cohort
viruses. We have developed a sensitive replication fitness assay to examine the
effects early and late deletions in the nef/LTR region have on virus replication.

Methods: Proviral DNA was amplified from peripheral blood mononuclear cells
(PBMC's) from cohort members and from virus cultured from PBMC's collected
at different time points. Chimeric viruses were constructed to contain the cohort
nef/LTR sequences in an NL4-3 background. The fitness of the chimeric C98
viruses was calculated relative to wild-type (NL4-3) following growth in a competition assay. Cells, MT-2 or PBMC's, were infected with a mixture of chimeric
C98 and wild-type viruses at low multiplicities of infection, and passaged several