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Laboratories of
*
Virology and
Ultrastructures, Istituto Superiore di Sanità, Rome, Italy; and
Shionogi Institute for Medical Science, Osaka, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Apoptosis is a regulated mechanism of cell suicide that is essential for normal development and homeostasis in multicellular organisms and provides a defense against virus invasion and oncogenesis (27). Recent evidence suggests that most eukaryotic cells respond to viral disruption of cellular homeostasis by undergoing apoptosis (28). In this regard, a variety of viral products, endowed with antiapoptotic activity, have been identified in many animal virus genomes (29).
The important issue of whether HIV-1 infection results in direct killing of infected cells or induces death of bystander uninfected cells remains a matter of debate. In this regard, many studies have identified apoptosis as a major mechanism involved in both direct and indirect HIV-mediated T cell killing, ultimately leading to CD4+ T cell depletion in AIDS (30, 31, 32, 33, 34, 35, 36).
In this study, we have investigated the role of vpr, physiologically expressed during the course of an acute HIV-1 infection, in the response of infected cells to apoptotic stimuli as well as on the HIV-induced apoptosis. We report that, at early time postinfection, HIV-1 productively infected cells were protected from apoptosis induced by exogenous stimuli with respect to uninfected cells. The suppression of vpr expression by antisense oligodeoxynucleotides targeted at two different regions of the vpr mRNA completely abrogated this protective effect. Notably, cell cultures infected with a vpr-mutated virus did not exhibit any reduction in the percentage of apoptotic cells with respect to uninfected cultures. In contrast, at later time postinfection, infected cells underwent spontaneous apoptosis that was, at least in part, due to vpr expression. These results indicate that HIV-1 vpr plays opposite roles, negative vs positive, in the regulation of apoptosis. The dual function of vpr represents a novel mechanism in the complex strategy evolved by HIV to influence the turnover of T lymphocytes, leading to either viral persistence or virus release and spreading.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TNF-
, PHA, and cycloheximide
(CHX)3 were purchased
from Sigma (St. Louis, MO). IL-2 was obtained from Chiron (Emeryville,
CA). mAb to the HIV-1 capsid protein p24 was obtained from Coulter
Pharmaceutical (Palo Alto, CA) (clone KC57-RD1). Phosphorothioate
oligodeoxynucleotides targeted at the vpr mRNA have been synthesized
according to previously described sequences (37).
Antisense oligodeoxynucleotides (AO) 2 and 3 (27 bases long) were
complementary at the genomic region encoding only vpr
(37). The control sense oligodeoxynucleotide (SO) 4 was
derived from the complementary strand of AO3.
Cell cultures
The Jurkat human T cell line (clone E6-1; obtained from the National Institutes of Health AIDS Research and Reference Reagent Program, Bethesda, MD) and CEM T cells were maintained in RPMI 1640 with 10% heat-inactivated FCS and penicillin/streptomycin sulfate in 5% CO2 atmosphere. Human PBMC were isolated from the peripheral blood of healthy donors and stimulated with PHA (5 µg/ml) in endotoxin-free RPMI 1640 containing 20% FCS. After 24 h, IL-2 (25 U/ml) was added, and cells were further cultured for 4 days.
Production of viruses and infection
Supernatants from acutely infected C8166 cells were used as the sources for HIV-1NL432. Wild-type and vpr-mutated (Af2) HIV-1NL432 (38) were obtained by transfection of 293 cells with 10 µg of proviral DNA by using the calcium phosphate coprecipitation method. Forty-eight hours posttransfection, cell supernatants were collected and subjected to titer determination as previously described (39). Jurkat and CEM cells were infected with HIV-1NL432, either produced in acutely infected C8166 or in 293 transfected cells, or Af2 virus as previously described (39). Briefly, cells collected during the logarithmic growth were incubated with the virus, at the multiplicity of infection (moi) of either 0.1 or 1, for 1 h at 37°C. Cells were then extensively washed to remove the unabsorbed virus and seeded in fresh medium at the concentration of 5 x 105/ml. The expression of vpr protein during the course of infection was monitored by immunoprecipitation as described previously (25). Activated PBMC were infected at day 5 of culture with both wild-type and vpr-mutated (Af2) HIV-1NL432 at the moi of 1. After 2 h of virus adsorption at 37°C, cells were washed and seeded at the concentration of 1 x 106 cells/ml in RPMI 1640 containing 20% FCS. Culture medium containing IL-2 was replaced every 3 days.
Induction and evaluation of apoptosis
At the indicated time points after infection, cells were
stimulated to undergo apoptosis by treatment with CHX/TNF- as
described elsewhere (25). For time-course experiments,
infected as well as uninfected cells were counted every 24 h and
reseeded at the same density. Treatment of cells with phosphorothioate
oligodeoxynucleotides targeted at vpr was conducted as previously
described (25). The oligodeoxynucleotides were added every
24 h and 1 h before CHX/TNF- treatment. For studies on the
HIV-induced apoptosis, infected cells were treated for 48 h with
the specific oligodeoxynucleotide before flow cytometry analysis.
Control uninfected cultures were subjected to the same treatments.
Quantitative evaluation of apoptosis was performed by using the
following flow cytometry methods: 1) decreased DNA content (subdiploid
peak characteristic of DNA fragmentation) by using propidium iodide
(Molecular Probes, Eugene, OR) as previously described
(40), and 2) TdT incorporation of labeled nucleotides into
DNA strand breaks (TUNEL; Boehringer Mannheim, Milan, Italy). For
double-staining experiments, cells were fixed with 4% formaldehyde in
PBS for 1 h. After washing in the same buffer, samples were
permeated with 70% ice-cold ethanol for 1 h at 4°C. After
washing, cells were incubated with p24-PE mAb for 1 h at 4°C,
washed, and then incubated with TUNEL reaction mixture according to
manufacturer instructions. Samples were then analyzed by a FACScan flow
cytometer (Becton Dickinson, San Jose, CA) equipped with a 488-nm argon
laser. Data were recovered by a Hewlett Packard computer using the
LYSIS II software.
Statistical analysis
All values are given as mean value ± SD from three or more separate experiments performed in duplicate. Student’s t test for correlated samples was used. Values of p < 0.001 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have evaluated the capacity of infected cells to undergo
apoptosis induced by exogenous stimuli. Jurkat cells were infected with
HIV-1NL432 at different moi (0.1 and 1) and
stimulated to undergo apoptosis by a combined treatment with
CHX/TNF- at different times postinfection. A comparable number of
apoptotic cells was detected 24 h postinfection in control and
infected Jurkat cultures after stimulation with CHX/TNF-,
independently of the moi (Fig. 1
A). In contrast, a marked
decrease in the number of apoptotic cells was observed in
CHX/TNF--stimulated Jurkat cells, infected at the moi of 0.1 and 1,
48 h postinfection with respect to control uninfected cultures.
Interestingly, the resistance to apoptosis found in infected cultures
was a transient phenomenon and completely disappeared 72 h
postinfection (Fig. 1A). Similar results were obtained in
CEM cells infected with HIV-1NL432 at the moi of
0.1, although the protective effect was better observed 24 h
postinfection. At this time point, 48% of apoptotic cells were
detected in uninfected control cells after CHX/TNF- treatment,
whereas only 20% of cells underwent apoptosis in infected cultures.
Likewise, at higher moi (1), infected cultures were still
protected from induction of apoptosis (15% of apoptotic cells) with
respect to uninfected control cells (35% of apoptotic cells).
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We previously reported that the constitutive expression of low
levels of the HIV-1 vpr protein renders Jurkat cells less susceptible
to undergo apoptosis induced by different stimuli (25). We
thus conducted experiments aimed at investigating the role of this
protein in the reduced susceptibility to apoptosis of infected cells
48 h postinfection. As shown in Fig. 1
B, the addition
of antisense oligodeoxynucleotides (AO3) targeted at vpr soon after
HIV-1 infection, rendered infected cells 48 h postinfection as
susceptible to apoptosis as control cultures. Similar results were
obtained by treatment with a different antisense oligodeoxynucleotide
(AO2, data not shown). In contrast, the same treatment did not modify
the apoptotic response of infected cells at 24 and 72 h
postinfection (Fig. 1B). Likewise, no changes were observed
in the presence of control sense oligodeoxynucleotides (SO4) either in
control or infected cells at any experimental time. Notably, treatment
of infected cultures with both sense and antisense
oligodeoxynucleotides did not affect viral replication at any time
point, as similar levels of viral p24 expression were detected in
HIV-infected oligodeoxynucleotides treated cells with respect to
untreated cultures (see Figs. 3 and 6). To further establish the
crucial role of vpr in the control of apoptotic response of
HIV-infected cells to exogenous stimuli, Jurkat cells were infected
either with wild-type HIV-1NL432 or with the Af2
variant, which carries a deletion in the vpr gene resulting in the
absence of a functional protein (T. Ogawa and A. Sato, unpublished
results). The replication of wild-type and Af2
HIV-1NL432 viruses was comparable as a similar
percentage of p24-positive cells was detected 48 h postinfection
(data not shown), confirming that vpr was dispensable for efficient
replication within these cells (11, 12). Although the
cells had similar levels of virus replication, a significantly higher
percentage of cells undergoing apoptosis was found in cultures infected
with the vpr-mutated Af2 virus with respect to cultures infected with
the wild-type counterpart (Fig. 2). As
expected, a marked decrease in the apoptotic response of cells infected
with wild-type virus with respect to control uninfected cultures was
detected (Fig. 2).
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-induced apoptosis selectively occurs in bystander
uninfected cells
To address the question of whether apoptosis occurred in infected
or uninfected cells, we conducted experiments of single-cell analysis
in HIV-infected cultures to simultaneously identify apoptotic and
infected cells. Therefore, Jurkat cells were infected with
HIV-1NL432 at the moi of 1 and stimulated to
undergo apoptosis 48 h postinfection. At this time point, cells
were immunostained for HIV capsid protein p24. Concurrently, cell death
was assessed by TUNEL. As shown in Fig. 3, treatment of control uninfected
cultures with CHX/TNF- (b) resulted in >50% of
apoptotic cells. In contrast, only 25% of apoptotic cells were found
in infected cultures stimulated under the same experimental conditions
(d). Notably, despite the presence of about 40% of
productively infected (p24-positive) cells in these cultures
(d), the majority of TUNEL-positive cells turned out to be
p24-negative (only 2.3% of cells were both TUNEL- and p24-positive)
(Fig. 3). Interestingly, the percentage of p24-positive cells
undergoing apoptosis (i.e., TUNEL-positive) markedly increased (about
30%) when infected cells were maintained in the presence of antisense
oligodeoxynucleotides targeted at vpr (e), whereas no
changes were observed in the percentage of TUNEL-positive/p24-negative
cells (e). In contrast, treatment of infected cultures with
sense oligodeoxynucleotides did not modify the profile of cells
undergoing apoptosis (f). Moreover, the addition of
either sense or antisense oligodeoxynucleotides to control uninfected
cultures did not affect the susceptibility of these cells to undergo
apoptosis (data not shown). Notably, cultures infected with the
vpr-mutated virus displayed the same phenotype as cells infected with
the wild-type virus and cultured in the presence of antisense
oligodeoxynucleotides (data not shown).
To verify whether the anti-apoptotic effect of vpr observed in T
cell lines could be reproduced in a more physiological cell model,
experiments with human PBMC were performed. Activated PBMC from two
different donors were infected with either wild-type or vpr-mutated
HIV-1NL432 and stimulated to undergo apoptosis by
a combined treatment with CHX/TNF- at different times postinfection.
As shown in Fig. 4, in spite of some
variability among donors, a markedly lower percentage of apoptotic
cells was consistently detected in cultures infected with the wild-type
virus (donor 1, 13.8%; donor 2, 30.7%) as compared with control
uninfected cultures (donor 1, 47.3%; donor 2, 56.2%) at day 4
postinfection. In contrast, the percentage of apoptotic cells found in
vpr-mutated infected cultures (donor 1, 50.3%; donor 2, 60.7%)
overlapped that determined in control cultures. Notably, the efficiency
of infection was identical for both viruses. Double-staining analysis
for both apoptotic and p24-positive cells clearly indicated that
apoptosis predominantly occurred in uninfected cells (Fig. 4).
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As shown in Fig. 5, the percentage
of cells spontaneously undergoing apoptosis markedly increased at late
time postinfection. The enhancement of spontaneous apoptosis started to
be detected around day 7–8, when about 80% of cells were productively
infected and further increased at later times (day 14; >90% of
infected cells). These high levels of apoptosis were directly related
to HIV infection, because the uninfected cultures kept under the same
experimental conditions exhibited very low levels of spontaneous
apoptosis (<5%, Fig. 5). The addition of antisense
oligodeoxynucleotides targeted at vpr to infected cultures 48 h
before apoptosis evaluation strongly reduced the percentage of
apoptotic cells 8 days postinfection. Likewise, cells infected with the
vpr-mutated virus exhibited a lower level of spontaneous apoptosis with
respect to cultures infected with the wild-type strain (data not
shown). Notably, although there was only a 2-fold increase in the
number of infected cells at this time postinfection with respect to
earlier stages (80% vs 40% at 48 h), the levels of expression of
vpr protein in cultures infected with the wild-type virus increased by
8- to 10-fold (data not shown). The effect of the antisense
oligodeoxynucleotide was less marked at day 14 of infection, suggesting
that other viral factors, in addition to vpr, were likely involved in
the HIV-induced apoptosis. The treatment of infected cells with control
sense oligodeoxynucleotides did not modify the levels of HIV-induced
apoptosis at days 8 and 14 of infection. Similar results were also
obtained in HIV-1-infected CEM cells, although the appearance of
spontaneous apoptosis was already detected at day 6 and became massive
at day 9 (data not shown). In contrast to Jurkat cells, HIV-induced
cell death in CEM cells was not entirely due to apoptosis, and a minor
component of necrotic cells was also observed (data not shown).
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To determine the phenotype (infected vs uninfected) of cells
undergoing apoptosis at late stages of infection, p24/TUNEL
double-staining experiments were conducted at 8 and 14 days
postinfection. As shown in Fig. 6A, a high percentage (30%)
of apoptotic cells was detected in control HIV-infected cultures
(b) 8 days after virus challenge, as compared with
uninfected cultures (a, 2.0%). Notably, all the
TUNEL-positive cells were found to be productively infected
(p24-positive) at this time of infection (b). Interestingly,
the addition of antisense (c), but not of control sense
(d), oligodeoxynucleotides targeted at vpr to HIV-infected
cultures almost completely inhibited the virus-induced apoptosis (8%
vs 30%). As shown in Fig. 6B, at later times of infection
(i.e., 14 days) when the majority of cells (91%) were productively
infected (p24-positive), a further increase in the number of apoptotic
cells was observed in these cultures (f, 50%). In
contrast, only 3% of control uninfected cells underwent apoptosis
under the same experimental conditions (e). Again, all the
apoptotic cells were found to be p24-positive (f).
Interestingly, when the infected cultures were treated with vpr
antisense (g), but not with control sense (h),
oligodeoxynucleotides, there was also a reduction of virus-induced
apoptosis. This inhibitory effect of antisense oligodeoxynucleotides
was somehow less marked than that obtained in infected cells at day 8
postinfection (Fig. 6, a and c), further
suggesting the involvement of other viral factors in HIV-induced cell
death at late stages of infection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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with
respect to control uninfected cells. This protective effect was found
to be transient and restricted to a defined time point postinfection.
In fact, a lower percentage of apoptotic cells was found in
HIV-infected CEM and Jurkat cells, respectively, at 24 and 48 h
postinfection, and this difference disappeared later on (48 and 72
h, respectively). Protection from apoptosis induction was related to
the expression of vpr as its genomic deletion or functional
inactivation by antisense oligodeoxynucleotides targeted at two
different regions of the vpr mRNA completely restored the sensitivity
of infected cells to apoptosis. Interestingly, single-cell analysis of
HIV-infected cultures, aimed at simultaneously identifying apoptotic
and infected cells, showed that apoptosis induced by CHX/TNF- only
occurred in uninfected cells. Noteworthy, a similar vpr-dependent
transient protection from apoptosis was also observed in HIV-1-infected
PBMC. In keeping with our results showing protection from apoptosis
induction in infected cells, it has been previously reported that
apoptosis is rarely observed in productively infected cells in the
lymph nodes of HIV-infected individuals, whereas it occurs
predominantly in bystander uninfected cells (34).
Likewise, it has also been reported that coculture of HIV-infected with
uninfected cells results in cell death by apoptosis of uninfected cells
(35). Moreover, recent studies aimed at analyzing
apoptosis in infected vs uninfected cells have concluded that death can
also occur in infected subsets (36, 41, 42). Furthermore,
death of infected cells was found to be dependent on the viral strain
used (43). We also report that high levels of spontaneous apoptosis can be detected in HIV-infected cultures at late times postinfection (8–14 days). Notably, the majority of cells undergoing apoptosis were found to be productively infected (p24-positive). In contrast to the protective effect exerted by vpr on the apoptosis induced by exogenous stimuli at early times of infection, at later stages, vpr resulted to be a major effector of the viral-induced apoptosis. However, we have been unable to detect any cell cycle perturbation in HIV-infected cells at different times postinfection (data not shown). In this regard, a number of studies have demonstrated that vpr induces apoptosis following cell cycle arrest (14, 22, 23, 24, 44). The apparent discrepancy between our results and those reported by others could be mostly explained by the experimental approaches used for achieving vpr expression. It is conceivable to assume that our experimental conditions, under which vpr exhibited its effect on the regulation of apoptosis (i.e., acute infection of T lymphocytes with T tropic infectious viruses at low moi), can mimic events occurring during the course of a natural HIV infection.
In conclusion, this study provides the first evidence indicating that
the HIV-1 vpr protein exerts a dual role in the regulation of
apoptosis in the experimental context of HIV infection of T cells.
Our results clearly indicate that, at early time postinfection (24–48
h), vpr expression protects cells from apoptosis induced by exogenous
stimuli. At later times postinfection, the same viral product promotes
spontaneous apoptosis of infected cultures (8 days), subsequently
cooperating with other viral and/or cellular factors (14 days). This
dual function of vpr cannot be simply explained by differences in the
percentage of productively infected cells detected at early stages
(
45% at 48 h) vs later times postinfection (80–90% at days 8
and 14, respectively). In fact, double-staining experiments, in which
we characterized at a single cell level the phenotype of apoptotic
cells (infected vs uninfected), clearly show that the shift in vpr
effect on apoptosis (protection/induction) only occurs in productively
infected p24-positive cells. Although our results do not provide a
direct evidence for a relationship between vpr intracellular
concentration and vpr activity, it is reasonable to hypothesize that
the dual function of vpr in the regulation of apoptosis may be somehow
related to its intracellular concentration achieved during the course
of HIV infection in individual cells. This hypothesis is at least in
part supported by the observation that the levels of expression of vpr
protein were very low at early stages of infection, when cells were
protected from apoptosis, whereas an 8- to 10-fold increase was found
in concomitance with the appearance of spontaneous apoptosis (data not
shown). The increase in vpr expression cannot be simply explained by an
enhanced number of infected cells (40% at 48 h vs 80% at 8 days)
and it is likely due to an increased intracellular expression of vpr at
single cell level.
The dual function of vpr may represent a useful strategy developed by
HIV for manipulating the turnover of T cells to its own advantage, thus
ensuring viral survival, persistence, and spreading. Although further
studies are needed to fully elucidate the mechanism by which vpr can
exert opposite functions, it is conceivable to assume that a variety of
factors (i.e., vpr concentration, physiological state of T cell,
environmental conditions) likely contribute to determine the different
vpr effects at a single cell level. The capacity of vpr to increase the
survival of infected cells by subtracting them to the physiological
cell death would provide a great advantage for viral survival and
spreading. Conversely, under different circumstances, vpr may
contribute, to different extent and likely in cooperation with other
viral/cellular factor(s), to the viral-induced apoptosis, thus
facilitating virus release and spreading. Recent studies have
demonstrated that the turnover of both virions and infected T
lymphocytes is extremely rapid in HIV-infected individuals. In
particular, it has been estimated that the half-life of an infected T
cell is only of 1 day, thus indicating that T cells could not
proceed through more than two complete cell cycles after viral
production has started (45, 46, 47). Moreover, results
obtained by Finzi and coworkers (48), showing the
persistence of T cell harboring infectious virus even after prolonged
antiretroviral therapy, argue that productive infection does not kill
all infected cells. In this regard, our finding that vpr acts as a
negative regulator of apoptosis for a period of time almost overlapping
the half-life of an infected lymphocyte strongly suggests that vpr may
play a key role in HIV pathogenesis by interfering with the turnover of
infected T lymphocytes. We suggest that the vpr-mediated enhancement of
T cell survival may favor harboring of virus during the course of
therapy.
Last, the finding that the same viral product can determine opposite effects in T cells, depending on the cellular context in which it is expressed, resolves the controversy on the negative vs positive role of vpr in apoptosis and moves the field ahead highlighting a new mechanism evolved by HIV to regulate T lymphocyte functions, driving the cell fate from survival (persistence) to cell death (viral spreading).
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Sandra Gessani, Laboratory of Virology, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.
3 Abbreviations used in this paper: CHX, cycloheximide; AO, antisense oligodeoxynucleotides; moi, multiplicity of infection; SO, sense oligodeoxynucleotides.
Received for publication March 13, 2000. Accepted for publication June 21, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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helix 2 of human immunodeficiency virus type 1 Vpr contains a determinant which is responsible for the nuclear translocation of the proviral DNA in growth-arrested cells. J. Virol. 72:4104.
B. Nat. Med. 3:1117.[Medline]
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