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Blood, Vol. 93 No. 3 (February 1), 1999:
pp. 1000-1010
By
From the Human Anatomy Section, Department of Morphology and
Embryology, University of Ferrara, Ferrara; Institute of Microbiology,
University of Bologna, Bologna, Italy; Institute of Human Virology,
University of Maryland at Baltimore, Baltimore, MD; and Unité 119 Istitut National de la Santé et de la Recherche Medicale
(INSERM), Université dela Mediterrainée, Marseille, France.
Many viruses have evolved genes encoding proteins that regulate cell
death by apoptosis. The human immunodeficiency virus type 1 (HIV-1) Nef
protein alters T-cell development and signaling and is required for
optimal viral replication and pathogenicity in vivo. To analyze the
interference of Nef with cell survival, we used both regulated and
constitutively expressed nef alleles in stably transfected
T-cell lines. Nef-expressing cells were sensitized to cell death by
apoptosis, which was specifically exacerbated by an anti-CD95 IgM
monoclonal antibody (MoAb). Flow cytometric analysis showed that the
surface expression of both CD95 and CD95 ligand (CD95L) was upregulated
by endogenous Nef expression. Nef-mediated apoptosis was almost
completely suppressed by the addition in culture of an anti-CD95 Fab'
IgG MoAb, which specifically blocks CD95/CD95L interactions. Lastly,
mutation of a proline motif in the core region of the nef gene,
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 CD95L. These findings may provide molecular
insight into the role of endogenous Nef in the T-cell depletion
observed in vivo, particularly HIV-specific cytotoxic
CD8+ T cells.
APOPTOTIC CELL DEATH has been proposed as
one of the key mechanisms involved in T-cell depletion during the
course of human immunodeficiency virus type 1 (HIV-1)
disease.1-3 It has also been shown that CD95 (Fas/Apo-1)
antigen stimulation induces marked apoptosis of T lymphocytes in
HIV-1-infected carriers,4,5 and the lack of chronic immune
activation in HIV-infected chimpanzees correlates with the resistance
of T cells to CD95-induced apoptosis.6
In vivo studies have clearly demonstrated that an intact nef
gene is critical to attain high virus loads and the development of an
acquired immune deficiency syndrome (AIDS)-like illness in rhesus
monkeys infected with simian immunodeficiency virus (SIV).7-10 A similar requirement for the maintenance of a
high virus load has been demonstrated for HIV-1 using the severe
combined immunodeficiency (SCID-Hu mouse) model,11 and
deletions of nef sequences were consistently identified in some
long-term nonprogressors with HIV-1 infection.12 Of note,
the Nef proteins of SIV and HIV are functionally
interchangeable.13 Enhanced virus replication and
infectivity are associated with nef expression upon propagation of HIV-1 in peripheral blood mononuclear cell (PBMC) cultures in
vitro.14 An increased inoculum of nef-deleted virus
can overcome the decreased infectivity of the viral progeny but still
does not induce illness in infected animals, suggesting a role for nef in HIV-1 pathogenicity.15 Indeed, mice
expressing a lymphoid-targeted nef transgene develop severe
T-cell depletion with altered T-cell function.16
Besides the enhancement of virus replication, in vitro studies also
indicate that the regulatory HIV-1 Nef protein plays a key role in AIDS
pathogenesis. One function of Nef is to mediate downregulation of cell
surface CD4, the major HIV-1 receptor.17 Nef also induces
dramatic effects on T-cell activation,18-21 leading to a
specific Th1 cytokine impairment, possibly via binding to cellular
kinases, including serine/threonine and tyrosine
kinases.21-27 Moreover, different investigators have
reported difficulties in establishing cell lines constitutively
expressing Nef protein. In fact, it has been shown that Nef may be
either cytotoxic or cytostatic when expressed in transfected cell
lines.28-30 In this context, we were able to establish
stably transfected clonal T-cell lines that can be propagated in
culture and allow for the controlled expression of an HIV-1-derived
nef allele.21 Taken together, these observations
imply that Nef has a role in perturbing T-cell activation pathways,
which are presumed to influence viral replication in the host and
possibly cause a dysfunction of cells in the immune system.
Virus-specific cellular immune responses were also
studied. They were barely detectable, if at all, in
macaques infected with pathogenic SIV, whereas they were strongly
induced in animals infected with the nef-deleted
virus.31,32 In this last group of animals, increased Fas
expression and apoptotic cell death were noted on T-lymphocyte
populations.32 Furthermore, in vitro infection of macaque
PBMCs was associated with increased Fas ligand (FasL) expression in a
nef-dependent manner. It was thus proposed that expression of
FasL may protect infected cells from cytotoxic T lymphocyte (CTL)
attack, killing viral-specific CTLs in the process.
Whether nef itself can induce FasL expression and apoptosis
thus remains to be established. Here, we investigated the effect of
endogenous Nef on the degree of apoptosis and CD95/CD95 ligand (CD95L)
expression and function in a set of stably transfected T-cell
lines.21
Cell lines.
JH6.2 and JBru.2 are CD4+ lymphoblastoid T stably
transfected clonal cell lines that have been previously
described.21 JBru.3 and JBru.mut.8 clonal cell
lines were obtained similarly in another round of stable transfections.
JBru.mut.8 was obtained by stable transfection of the Nef Bru
P72A-P75A mutant (Dutartre et al, manuscript
submitted for publication). Nef Bru
P72A-P75A was generated by substitution of Pro
residues 72 and 75 with Ala residues and yielded a construct allowing
for the expression of a stable Nef protein (data not shown). Cells were
cultured in RPMI 1640 (GIBCO, Grand Island, NY) plus 10% fetal calf
serum ([FCS] GIBCO) at an optimal cell density of 0.3 × 106 to 1 × 106/mL. In most experiments,
exponentially growing JH6.2, JBru.2, JBru.3, and JBru.mut.8
cell clones were cultured in the presence of low serum levels
(RPMI + 0.1% FCS) for up to 24 hours, and cell aliquots were
harvested at various time points to measure the percentage of apoptosis
and the expression of CD95, CD95L, and Nef proteins by flow cytometry.
Western blot analysis.
Samples derived from 2 × 106 cells, containing
approximately 100 µg protein, were migrated in 10% acrylamide gels
and blotted onto nitrocellulose filters. Blotted filters were blocked
for 30 minutes in a 3% suspension of dried skimmed milk in
phosphate-buffered saline (PBS) and incubated overnight at 4°C with a
1:200 dilution of anti-Nef MoAb (Transgene, Strasbourg, France) or
1:1,500 dilution of anti-tubulin MoAb (Sigma, St Louis, MO). The
filters were washed and further incubated for 1 hour at room
temperature with a 1:1,500 dilution of peroxidase-conjugated anti-mouse
IgG (Sigma) in 1% bovine serum albumin. Specific reactions were
revealed with the ECL Western blotting detection reagent (Amersham
Corp, Arlington Heights, IL).
Flow cytometric analysis of surface CD95 and CD95L and intracellular
Nef.
The detection of CD95 and CD95L surface expression was
performed on aliquots of 3 × 105 cells using unconjugated
anti-CD95 IgM MoAb (dilution 1:100; Immunotech) or anti-CD95L rabbit
polyclonal antibody (dilution 1:40; Santa Cruz Biotechnology, Santa
Cruz, CA), respectively, at 4°C for 30 minutes. After two washings
with PBS, the cells were stained with a polyclonal goat anti-mouse IgG
covalently linked to fluorescein isothiocyanate (GAM-FITC, dilution
1:100; Becton Dickinson, San Jose, CA) or a polyclonal goat anti-rabbit IgG covalently linked to FITC (GAR-FITC, dilution 1:100;
Becton Dickinson), respectively, at 4°C for 30 minutes. Nonspecific
fluorescence was assessed using irrelevant isotype-matched controls
(IgM for anti-CD95 MoAb or normal rabbit IgG for anti-CD95L) followed
by GAM-FITC or GAR-FITC.
Determination of apoptosis.
Apoptosis was evaluated as previously described34 by
combining two independent methods: (1) propidium iodide (PI) staining followed by flow cytometry and (2) the TdT-mediated d-UTP-biotin nick
end labeling (TUNEL) technique. For the first procedure, cells were
harvested and fixed in 70% ethanol for at least 1 hour at 4°C. They
were then treated with 0.5 µg RNase/mL (Type I-A; Sigma) and
resuspended in PBS containing 50 µg/mL PI. Analysis was performed by
FACScan with the FL2 detector in logarithmic mode, using Lysis II
software (Becton Dickinson). The threshold of PI fluorescence was
triggered on the Fl2 signal, where a clear-cut
Statistical analysis.
The data are expressed as the mean ± SD for three or more experiments
performed in duplicate. Statistical analysis was performed using the
two-tailed Student's t-test.
HIV-1 Nef induces apoptosis of Jurkat cells that is counterregulated by
growth factors.
In this study, we aimed to characterize the mechanisms
underlying in vitro cytopathic effects mediated by endogenous
Nef.28-30 For this purpose, we used the HIV-1
nef-transfected JBru.2 and JBru.3 CD4+
lymphoblastoid T-cell clones and compared them with the control JH6.2
transfected with the backbone empty vector.21 Nef
expression is constitutively high in JBru.3 cells, whereas it is almost
undetectable in JBru.2 cells under basal conditions (Fig
1). We have previously shown21
that nef expression sharply increases in JBru.2 cells upon
treatment with FK, phorbol esters, or Ca2+ ionophores, due
to the presence of cAMP and NF-kB responsive elements in the human
immediate early cytomegalovirus promoter regions driving nef
expression in these cells.33 As in preliminary experiments,
FK alone did not modify the percentage of apoptosis in JH6.2 cells, at
variance with phorbol esters and Ca2+ ionophores, and this
agonist was chosen to induce Nef expression in JBru.2. The very weak or
absent Nef expression in JBru.2 readily increased (P < .01)
after stimulation of the cells for 5 hours with 10
HIV-1 nef-expressing cells are sensitized to CD95-induced apoptosis
and express high levels of CD95.
Fas is an important intermediate in T-cell apoptosis, and peripheral
blood mononuclear cells from HIV-infected patients demonstrate enhanced
CD95 expression that is correlated with an enhanced susceptibility to
the induction of apoptosis with anti-CD95 antibodies.37 For these reasons, we investigated CD95-mediated apoptosis in
nef-transfected cells. JH6.2, JBru.2, and JBru.3 were cultured
under low growth factor concentrations and in the presence of anti-CD95
IgM MoAb, which induces apoptosis in cells expressing functional CD95
(Fig 3A). The percentage of apoptotic cells
showed a progressive increase in all cell lines following the addition
of anti-CD95 IgM in culture. However, the kinetics of apoptosis was
much faster and reached significantly (P < .05) higher
levels in JBru.3 and FK-treated JBru.2 cell clones versus untreated
JBru.2 and JH6.2.
HIV-1 Nef induces CD95-dependent apoptosis through induction of CD95L
membrane expression.
CD95 triggers apoptosis upon oligomerization by CD95L.38 We
thus determined CD95L expression at the surface of JH6.2, JBru.2, and
JBru.3 cells (Fig 4). In cells cultured in the presence
of low serum (RPMI + 0.1% FCS) for 24 hours, membrane-bound CD95L was clearly detectable. However, the expression was significantly (P < .01) higher in JBru.3 and FK-treated JBru.2 versus
untreated JBru.2 or control JH6.2 cells (Fig 4). These findings
suggested that an upregulated expression of both CD95 and CD95L may be
involved in the nef-dependent apoptosis observed in
nef-expressing Jurkat cells.
Mutation in the core sequence of Nef abrogates its ability to induce
apoptosis and CD95/CD95L upregulation.
It has been previously shown that both HIV-1 and SIV Nef interact with
src-like tyrosine kinase(s), and with a member of the p21-activated kinase (PAK) family of
kinases.24 Moreover, it has been suggested that the
association of SIV Nef with these kinases is important for the
development of AIDS in rhesus macaques and may provide a novel target
for clinical intervention.24,40,41 Therefore, in this group
of experiments, the induction of apoptosis upon serum withdrawal was
evaluated in a Jurkat cell clone stably transfected with a
nef-expressing plasmid in which the nef core region has
been mutated by substitution of Pro residues 72 and 75 by Ala residues
(JBru.mut.8). This mutant is unable to activate src-like tyrosine kinase(s) or PAK (Dutartre et al, manuscript submitted for publication). Nef expression in
JBru.mut.8 was very low under basal conditions, whereas it
rapidly and significantly (P < .01) increased upon
treatment with FK, reaching levels similar to those observed in
FK-treated JBru.2 (Fig 6A and B). The
percentage of apoptosis in both untreated and FK-treated
JBru.mut.8 cultured in RPMI + 0.1% FCS was very low (Fig
7A). When anti-CD95 IgM was added to the
culture, a progressive increase of apoptosis was noted (Fig 7B),
reaching values similar to those previously observed in JH6.2 control
cells (Fig 3A). Moreover, the surface expression of both CD95 (Fig 7C)
and CD95L (Fig 7D) in JBru.mut.8 was significantly (P < .05) lower versus FK-treated JBru.2 or JBru.3 cells
(Figs 3 and 4), while the CD4 surface expression was similar to that of
JBru.2 and JBru.3 (data not shown). Thus, the Nef core domain responsible for the interaction with src-like tyrosine
kinase(s) and PAK is critical for the ability of Nef to induce
apoptosis and upregulate CD95/CD95L.
The loss of functional immune cells is a hallmark of AIDS. Although the
magnitude of the viral burden increases with disease progression42 and much emphasis has been placed on the
direct cytopathic effect of a productive HIV-1 infection of
CD4+ T lymphocytes,43,44 a considerable loss of
uninfected or abortively infected bystander T lymphocytes occurs in
HIV-infected individuals.
Submitted May 1, 1998; accepted September 30, 1998.
Supported by the AIDS Project of the Italian Ministry of Health, the
Association Nationale de Recherche sur le syndrome d'Immunodeficience Acquise, and the INSERM, and in part by a fellowship from the European
Community (ERB-CHRX CT94-0537 to Y.C.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Giorgio Zauli, MD, PhD, Human Anatomy
Section, Department of Morphology and Embryology, University of
Ferrara, Via Fossato di Mortara 66, 44100 Ferrara, Italy; e-mail:
zlg{at}dsn.unife.it.
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Top
Abstract
Introduction
5 mol/L forskolin (FK), a potent protein
kinase A activator, which increases Nef expression due to the presence
of CREB consensus sequences in the cytomegalovirus
promoter
converting enzyme (ICE).