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

12th World AIDS Conference

Abstracts 11144-11148 11

times in culture. The nef/LTR region was PCR amplified from the proviral DNA
in the infected cells using fluorescently labelled primers, and the change in the
proportion of wild type to mutant nef/LTR's quantitated.
Results: Evolution of the nef/LTR region has been observed in isolates and
PBMC samples from the SBBC member C98. The evolved species lacks more
sequence from the nef/LTR region and is now the predominate proviral species.
The fitness of the chimeric virus containing the earlier, larger nef/LTR form was
calculated to be 0.47 times as fit as that of wild type. The competition assays
also show that viruses containing the newly evolved smaller nef/LTR sequences
are also less fit than wild type, and subtle differences in replication are apparent
between chimeras containing the larger and smaller nef/LTR sequences.
Conclusions: We have clearly demonstrated that the defective nef/LTR regions from the SBBC viruses led to decreased replication competency compared
with wild type nef/LTR sequence. The competition assay described here will be
widely applicable to the quantitation of the effects other subtle mutations have on
virus replication.
381 * /11144 Molecular mechanisms of HIV-1 nuclear import:
matrix antigen and Vpr are the key regulators
Michael Bukrinsky, S. Popov. The Picower Institute, 350 Community Dr.
Manhasset, NY 11030, USA
Replication of HIV-1 in non-dividing cells depends on active import of the viral
pre-integration complex (PIC) into the cell nucleus. Earlier studies suggested that
matrix antigen (MA) and Vpr are the principal regulators of HIV-1 nuclear import;
however, controversial results have been reported later regarding the molecular
mechanisms and HIV-1 proteins involved in this process. Here we used an in
vitro transport assay to examine the role of Vpr and MA in the nuclear import of
the viral PIC. In contrast to earlier reports, we find two functional NLSs in MA,
one in the N-terminal (amino acids 27-32), and another in the C-terminal half
(amino acids 110-115) of the protein. Both NLSs required the help from Vpr to
efficiently transport the PIC; no import of Vpr- was detected in vitro. While nuclear
import of the HIV-1 PIC was not greatly impaired by mutation in any one of MA
NLSs when Vpr was present, inactivation of both NLSs knocked out import of
the HIV-1 PIC even in the presence of functional Vpr. Interestingly, cytoplasmic
lysates from HeLa could rescue import of Vpr virus, but only when one of MA
NLSs was intact. Binding studies using recombinant proteins demonstrated that
Vpr interacts with karyopherin a, the cellular receptor for NLSs. This interaction
resulted in approximately 10-fold increase in the affinity of MA-karyopherin a
interaction. In addition, Vpr was required for docking of the HIV-1 PIC to nuclear
envelope and to isolated nucleoporins, thus explaining the stimulating effect of
Vpr on HIV-1 nuclear import.
Conclusions: Our results suggest that Vpr and MA are the major regulators
of HIV-1 nuclear import. While MA provides NLSs that drive the import process,
Vpr functions as an enhancer of karyopherin a/NLS interaction. Presence of Vpr
is required to convert the HIV-1 PIC into a strong karyophile and thus promote its
efficient import to and across the nuclear pore complex.

11146 Diversification of nef and vpu in six hemophilia B patients
after clonal HIV-1 infection
Astrid Klein', B. Kupfer', H.H. Brackmann2, J.K. Rockstroh3, P. Kasper4,
B. Matz', R. Kaiser1. 'Institut F Medizinische Mikrobiologie, Sigmund Freud Str.
25, Bonn; 2lnstitut F Experimentelle Hamatologie, Bonn; 3 Medizinische Klinik,
Bonn; 4Boehringer Mannheim, Penzberg, Germany
Background: In 1989/90 eight hemophilia B patients were clonally infected with
HIV-1 from one lot of clotting factor concentrate. The evolution of these initially
unique viral strains was observed and followed-up by analyses of several viral
sequences (env-V3 and V1/2, vif, and gag-p17).
Objectives: The divergent courses of disease could not be correlated to HIV-1
sequence diversifications as analyzed so far. To investigate, whether the progress
in clinical course is correlated to diversifications in HIV-1 regulatory genes, we
have analyzed the nef-gene and the vpu-gene in six clonally infected patients.
Methods: Nef- and vpu-specific PCR-products were generated from longitudinal samples of proviral DNA and from viral plasma RNA of 6 clonally infected
patients. Genotypes were analyzed by solid phase sequencing of the amplicons.
HIV-1 RNA was quantified using the NASBA HIV-1 QT system. Viral phenotypes
were determined by peripheral blood mononuclear cell (PBMC)-cocultivation.
CD4+ T-cells were quantified by flow cytometry.
Results: Vpu showed a heterogeneous distribution of mutations. Most mutations occurred in the region encoding the transmembrane domain (TMD), however
the hydrophobic character was not affected. The cytoplasmic domains were more
conserved. The TMD and the C-terminus showed a parallel evolution. In 4 of 6
patients an insertion in the nef-gene was observed. In 2 isolates a disrupted nef
open reading frame was noticed. The genetic evolution within the nef-gene was
not directed. No correlation was found between the different genetic variations
and viral load or CD4+-cell count.
Conclusions: Although the patients were clonally infected, the clinical course,
viral load and CD4+-cell count differed significantly. In all patients vpu seemed to
be biologically active. Vpu and nef showed advanced but different diversification.
Within these genes some regions were subjected to a higher selection pressure
than others.
11147 Positive selection of Vpr in human immunodeficiency
virus type 1 infection and disease progression
Nitin Saksenal, B. Wang2, R. Joswiak2. 'Retroviral Genetics Lab 2nd Floor
R=2145 Westmead Hospital WIHR CVR Westmead NSW 2145; 2Westmead
Hospital Retroviral Genetics Lab Westmead, NSW, Australia
Objectives: 1. To study the role of defects in the Vpr gene in long-term nonprogression of HIV disease 2. To detrmine the role of repair of defects and the
positive selection of the Vpr gene in HIV disease progression
Design: Long-term non-progression of HIV disease was studied for the Vpr
gene evolution and repair directly from the PBMCs and plasma of a mother and
child pair which has survived for 15 years with high CD4 T- cell counts since 1983.
Mother acquired the virus through blood transfusion, whereas the child acquired
HIV-1 via breast feeding.
Methods: Peripheral blood cells and plasma were collected from from both
mother and child from 1987-1997. Proviral sequences were amplified in the Vpr
gene from both plasma and PBMCs, and cloning was performed in PGEM-T vector. Multiple clones were sequenced from each time point. Co-culture studies were
carried out on PBMCs and monocyte-derived macrophages for deriving information on replication kinetics and tropism of HIV-1 strains infecting the mother-child
pair.
Results: Molecular analyses of both plasma and PBMC-derived clones from
1987-1996 clearly revealed that overall 77% of the Vpr gene clones carried
defects (deletions/insertions) at the C-terminus of the vpr protein which affected
the size of the vpr gene, and also the frame of tat protein. Between 1987-1996,
both mother and child maintained high CD4 counts (750-800/uL blood), but in
1997 both started showing decline in their CD4 counts (400-450/uL, blood).
Interestingly, the sequencing of the Vpr gene from the PBMCs obtained in 1997
showed no defects at the C-terminus. We propose that the repair of the defects
at the C-terminus may have crucial role in HIV disease progression in these
individuals. In addition, it is known that the defective Vpr can lead to persistent
infection in T-cells, whereas an intact Vpr can cause cell-cycle arrest which may
lead to disease progression. Defects at the C-terminus are important because
they are known to downregulate the cell cycle arrest.
Conclusions: These are the first studies showing sequentially that the positive
selection of the Vpr may have an important role in HIV disease progression. Our
data may have important implications in the designing of therapeutics for HIV
treatment.
S11148 Diversity of HIV-1 long terminal repeat sequences derived
from Mexican donors at different stages of infection
Carmen Soler Claudin', Victor Raul Gomez Roman2, M.C. Basualdo Sigales1.

' Unidad De Investigacion En Retrovirus H. Facultad de Qufmica, Unam/Indre,
SSA, Carpio 470 Mexico, D.F. 11340; 2Universidad Nacional Autonoma de
Mexico, Mexico D.F, Mexico
Objective: To examine the variability of the HIV-1 long terminal repeat's (LTR)
derived from 17 patients at different stages of HIV-1 infection.
Methods: Nested-PCR primers were designed to amplify fragments of the
HIV-1 LTR from peripheral blood samples donated by 17 patients. LTR amplicons

11145

The HIV-1 Nef protein induces CD4+ T-cell apoptosis
through functional upregulation of the CD95/CD95L
pathway

Yves Collette', G. Zauli2, D. Gibellini3, P. Secchiero4, H. Dutartre5, D. Olive5,
S. Capitani2. 'Bd Lei Roure 27, 13009 Marseille; 2University of Ferara, Ferrara;
5INSERM U119, Marseille, France; 3 University of Bologna, Bologna, Italy;
14 University of Maryland, Baltimore, MA, USA
Background: Many viruses have evolved genes encoding proteins which regulate cell death by apoptosis. The Nef protein from primate lentiviruses alters T
cell development and signaling and provides a signal required for optimal viral
replication and pathogenecity in vivo, possibly through interaction with cellular
kinases.
Methods: To analyse interference of Nef with cell survival, we used both regulated and constitutively expressed HIV-1 nef alleles in stably transfected T cell
lines.
Results: Upon Nef expression, cells displayed a marked retardation of cell
growth and were sensitized to cell death by apoptosis which was counter-regulated by growth factors. Apoptosis induced by Nef was exacerbated by an
anti-CD95 (Fas/Apo-1) IgM monoclonal antibody. Indeed, flow cytometric analysis showed that the surface expression of CD95 was upregulated in a Nef
dependent manner, and that upon serum withdrawal, Nef also induced CD95L
expression. Nef-mediated apoptosis could be suppressed by the addition in culture of an anti-CD95L Fab' mAb, which specifically blocks the binding of CD95L.
Lastly, mutation of a proline motif in the core region of Nef, which disrupts its
ability to interact with cellular kinases and reduces HIV-1 replication in vitro, completely abrogated the Nef-mediated induction of apoptosis as well as its ability to
upregulate surface CD95 and to induce CD95L.
Conclusion: Together, our findings unequivocally demonstrate that HIV-1 Nef
is cytotoxic for lymphold T cells via an upregulation of the CD95/CD95L pathway
and provide molecular insights into the role of Nef in T cell depletion observed
in vivo, particularly bystander non-infected cells such as HIV-specific cytotoxic
CD8+ T cells.