Download Human immunodeficiency virus type 1 Vprmediated G2 cell cycle

The EMBO Journal Vol. 19 No. 15 pp. 3956±3967, 2000
- Retracted -
Human immunode®ciency virus type 1 Vpr-mediated
G2 cell cycle arrest: Vpr interferes with cell cycle
signaling cascades by interacting with the B subunit
of serine/threonine protein phosphatase 2A
Mohammed Hrimech, Xiao-Jian Yao,
Philip E.Branton1 and EÂric A.Cohen2
Laboratoire de ReÂtrovirologie Humaine, DeÂpartement de microbiologie
et immunologie, Faculte de meÂdecine, Universite de MontreÂal,
Montreal, Quebec, Canada H3C 3J7 and 1Department of Biochemistry,
McGill University, Montreal, Quebec, Canada H3G 1Y6
2
Corresponding author
e-mail: [email protected]
The Vpr protein of primate lentiviruses arrests cell
cycling at the G2/M phase through an inactivation of
cyclin B±p34cdc2 and its upstream regulator cdc25. We
provide here biochemical and functional evidence
demonstrating that human immunode®ciency virus
type 1 (HIV-1) Vpr mediates G2 arrest by forming a
complex with protein phosphatase 2A (PP2A), an
upstream regulator of cdc25. Vpr associates with
PP2A through a speci®c interaction with the B55
regulatory subunit. This interaction is necessary but
not suf®cient for G2 arrest. Interestingly, we found
that Vpr association with B55-containing PP2A
targets the enzymatic complex to the nucleus and,
importantly, enhances the recruitment and dephosphorylation of the cdc25 substrate. Our data suggest
that Vpr mediates G2 arrest by enhancing the nuclear
import of PP2A and by positively modulating its
catalytic activity towards active phosphorylated
nuclear cdc25.
Keywords: cell cycle/human immunode®ciency virus/
nuclear import/protein phosphatase 2A/Vpr
Introduction
Human immunode®ciency virus type 1 (HIV-1) Vpr is a
small protein (96 amino acids, Mr 14 kDa) that is
incorporated into virions through a speci®c interaction
with the p6 domain of the p55gag precursor protein and was
thus proposed to have a function early in virus replication
(Cohen et al., 1990; Paxton et al., 1993). Two distinct
biological functions are associated with Vpr. First, Vpr has
been found to enhance the nuclear migration of the preintegration complex (PIC) in newly infected non-dividing
cells (Heinzinger et al., 1994). Both the presence of Vpr in
the virion and its function in nuclear import account for the
requirement for Vpr for ef®cient replication of HIV-1 in
non-dividing cell types such as macrophages (Heinzinger
et al., 1994; Subbramanian et al., 1998a). The second
biological activity of Vpr prevents the passage of cells
through mitosis at the G2 stage of the cell cycle (Jowett
et al., 1995; Rogel et al., 1995). Cells expressing Vpr,
from the complete HIV-1 genome or from an expression
plasmid encoding Vpr, do not proliferate but accumulate at
the G2 phase of the cell cycle (He et al., 1995; Re et al.,
3956
1995). Vpr prevents mitosis in human and primate cells
from a variety of tissues, as well as in yeast (Macreadie
et al., 1995; Planelles et al., 1996), suggesting that Vpr
targets a general cellular pathway that controls progression
from G2 to mitosis. The functional role of the G2 cell cycle
arrest mediated by Vpr in HIV-1 pathogenesis is as yet
unclear. Experimental evidence in in vitro systems indicates that the establishment of a cell cycle arrest enables
HIV-1 to optimize virus production and cripple the
immune response, thus facilitating the persistence of the
virus within the infected individual (Stewart et al., 1997;
Goh et al., 1998).
In mammalian cells, the transition from G2 into mitosis
is controlled by activation of the complex between the
cyclin-dependent kinase p34cdc2 and its regulatory partner,
cyclin B. This is achieved by a series of coordinated
phosphorylation and dephosphorylation events (Jackman
and Pines, 1997). Phosphorylation of p34cdc2 on Thr161 by
cyclin-associated kinase (CAK) is one of the ®rst events in
this process. Activation of p34cdc2 is prevented, however,
by additional phosphorylation on Thr14 and Tyr15 by the
protein kinases Wee1 and Myt1. Dephosphorylation of
Thr14/Tyr15 by the protein phosphatase cdc25 eventually
activates the p34cdc2±cyclin B complex, allowing progression to mitosis. Both Wee1/Myt1 and cdc25 activities are
regulated by upstream kinase/phosphatase networks, and
protein phosphatase 2A (PP2A)-like phosphatase activities
have been implicated in keeping Wee1 active and cdc25
inactive during S and G2 phases (Clarke et al., 1993; Tang
et al., 1993).
Cells expressing Vpr have very low or undetectable
levels of cyclin B±p34cdc2 kinase activity (He et al., 1995;
Re et al., 1995). It is unlikely that Vpr blocks the activity
of the cyclin B±p34cdc2 kinase complex directly since
cdc25 phosphatase is found in its inactive, hypophosphorylated state in Vpr-expressing cells (Re et al., 1995),
and forced activation of cyclin B±p34cdc2 by treatment of
cells with the phosphatase (PP1A and PP2A) inhibitor
okadaic acid overcomes Vpr-mediated G2 cell arrest. It is,
therefore, more likely that Vpr acts upstream of cyclin B±
p34cdc2 and cdc25 to affect their coordinate regulation.
Interestingly, recent genetic studies with the ®ssion yeast
Schizosaccharomyces pombe suggest the involvement of
Wee1, PP2A and Rad24 in induction of cell cycle arrest by
HIV-1 Vpr (Masuda et al., 2000). However, the identity of
the cellular pathway(s) or factor(s) targeted by Vpr to
mediate G2 cell cycle arrest is still not clear and remains a
matter of intense research (Withers-Ward et al., 1997;
Mahalingam et al., 1998).
The reversible phosphorylation of proteins, catalyzed by
protein kinases and protein phosphatases (PP), is the key
mechanism for the regulation of diverse cellular functions.
PP2A is one of the four major classes of protein serine/
threonine phosphatase (Hunter, 1995). PP2A is involved in
ã European Molecular Biology Organization
HIV-1 Vpr interacts with the B subunit of PP2A
a broad range of cellular processes, including signal
transduction, transcriptional regulation and control of
DNA replication and cell cycle progression (Lee, 1995;
SchoÈnthal, 1995). This diversity of PP2A functions is
conferred by a diversity of targeting/regulatory subunits
and several levels of post-translational modi®cations. The
diverse heterotrimeric forms of PP2A in vivo are generated
by the association of a ubiquitous core heterodimer,
consisting of a 36 kDa catalytic C subunit and a 68 kDa
structural/regulatory A subunit, with a variable regulatory
B subunit, which binds to the core enzyme yielding the
holoenzyme (Mumby and Walter, 1993). The A and C
subunits are each encoded by two highly related (85 and
97% identity, respectively) and widely expressed genes
that are named a and b. Over 15 different variable B
subunits are expressed in a tissue- and developmentalspeci®c manner. These proteins are generated as isoforms
and splice variants from three unrelated gene families
designated B, B¢ (also called B56) and B¢¢ (Mumby and
Walter, 1993). The B family has three members, Ba, Bb
and Bg, each with a molecular mass of ~55 kDa (Pallas
et al., 1992; Zolnierowicz et al., 1994). The B¢ family
consists of numerous recently identi®ed isoforms and
splice variants whose molecular masses range from 54 to
~70 kDa (McCright and Virshup, 1995). The B¢¢ family
has two members, which have molecular masses of 72 and
130 kDa and are splice variants of the same gene (Hendrix
et al., 1993). The different B subunits interact via the same
or overlapping sites within the A subunit of the AC dimer
so that the binding of different B subunits to AC is
mutually exclusive (Ruediger et al., 1992). B subunits
have important functions in regulating the substrate
speci®city (Kamibayashi et al., 1994) and the subcellular
localization of PP2A (McCright et al., 1996; Zhao et al.,
1997).
To gain insight into the mechanism of Vpr-mediated G2
cell cycle arrest, we examined whether HIV-1 Vpr
interacts with PP2A. Here, we show that HIV-1 Vpr
associates with PP2A through an interaction with the B55
regulatory subunit. This association enhances the nuclear
import of B55-containing PP2A and positively modulates
the enzyme activity. Importantly, Vpr association with
PP2A increases the recruitment and dephosphorylation of
the cdc25 substrate.
Results
Vpr associates with the PP2A holoenzyme
complex through a speci®c interaction with the
Ba subunit
Ruediger et al. (1997) have recently reported that the
balance between PP2A holoenzyme and core enzyme is
important for HIV-1 gene expression and virus production.
This study found that increasing the ratio of PP2A core
enzyme to holoenzyme by overexpression of an
N-terminal mutant of the A subunit (deletion of repeat
5), AD5, which binds the C but not the B subunit, inhibited
transcription from the HIV-1 long terminal repeat (LTR)
and decreased virus production. To investigate the role of
PP2A in the pathway leading to Vpr-mediated G2 cell
cycle arrest, we initially examined whether changes in the
ratio of PP2A core enzyme and holoenzyme interfered
with Vpr-mediated G2 arrest. The results of these experi-
ments indicated that an increase in PP2A core enzyme
levels in cells by overexpression of PP2A Aa subunit
mutant AD5 alleviated the effect of Vpr on cell cycle
progression and consequently provided ®rst evidence that
PP2A holoenzyme was involved in the pathway targeted
by Vpr to induce cell cycle arrest (data not shown).
We next investigated whether Vpr interacted with the
PP2A complex. To test this possibility, we initially
developed a co-precipitation assay in 293T cells that
overexpress tagged versions of Vpr (T7-Vpr) and PP2A
B55a subunit (Flag-B55a) as well as native PP2A Aa or
mutant AD5 subunits. The tagged version of B55a allowed
us to distinguish the exogenous B55a protein from
endogenous B subunits belonging to the three families.
In addition, using a different set of monoclonal antibodies
directed against the native A subunit or mutant AD5
subunit (Figure 1A), we were able to precipitate PP2A
core enzyme or holoenzyme differentially as previously
described (Kremmer et al., 1997).
293T cells were co-transfected with expression plasmids encoding T7-Vpr, Flag-B55a and Aa or AD5 as
depicted in Figure 1A. At 48 h post-transfection, cells were
counted and divided into three equal aliquots, lysed and
incubated with antibodies directed against T7-Vpr (antiT7), Aa (6F9) or AD5 (5H4) subunits, or Flag-B55a
(anti-Flag), respectively (Figure 1A). For aliquots that
were intended to be immunoprecipitated with antibodies
against Aa (6F9) or AD5 (5H4), cells were treated prior to
lysis with the cell membrane-permeable cross-linking
agent
dimethyl
3,3¢-dithiobispropionimidate´2 HCl
(DTBP) as indicated in Materials and methods. The
resulting immunocomplexes were then analyzed further
for the presence of the different components of the
putative Vpr±PP2A complex, including Aa, B55a, C
subunits and Vpr, by western blotting using the appropriate antibodies (Figure 1A).
Antibodies directed against T7-Vpr, Aa or Flag-B55a
all co-precipitated a complex comprising Vpr (14 kDa),
exogenous Flag-B55a (55 kDa), Aa (68 kDa) and
endogenous C subunit (36 kDa) (Figure 1B, lanes 2, 5
and 8). Precipitation of the Vpr±PP2A complex was
observed in conditions where the DTBP cross-linker was
present (Figure 1B, lanes 4±6) or absent (lanes 1±3 and 7±
9). Interestingly, although the 6F9 Aa monoclonal antibody precipitated the endogenous C subunit in mocktransfected 293T cells (Figure 1B, lane 4), the anti-FlagB55a antibody did not precipitate detectable amounts of
endogenous A or C subunits in the presence of AD5 (lane
9). The inability of anti-Flag-B55a antibodies to precipitate endogenous core enzyme (A and C subunits) when
AD5 is expressed probably results from the limiting
amount of endogenous native core enzyme available for
interaction with Flag-B55a. The expression of the AD5
mutant was previously shown to replace the wild-type A
subunit in pre-existing core and holoenzyme and compete
with newly synthesized wild-type A subunit for newly
synthesized C subunit, thereby causing an increase in the
level of AD5±C core enzyme and a parallel decrease in the
level of holoenzyme (Ruediger et al., 1997). Indeed, as
shown later in Figures 1D and 7A, anti-T7 or anti-Flag
antibodies were able to precipitate endogenous PP2A Aa
or C subunits in 293T cells expressing only T7-Vpr or
Flag-B55a (lanes 2 and 3).
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M.Hrimech et al.
Fig. 1. Vpr associates with the PP2A holoenzyme complex through a
speci®c interaction with the B55a subunit. (A) Schematics of PP2A
Aa, AD5, Flag-B55a and T7-Vpr expression vectors. The CMV
promoter is represented by a dark circle. Antibodies used against each
protein in immunoprecipitation (IP) and western blotting (WB) are
indicated. (B) 293T cells (106) were mock transfected (lanes 1, 4 and
7) or co-transfected with different combinations of expression plasmids
encoding T7-Vpr (2.5 mg), Flag-B55a (2.5 mg) and wild-type PP2A Aa
(10 mg) or AD5 mutant (10 mg), as indicated at the bottom of each
panel. At 48 h post-transfection, cells were treated (lanes 4±6) or not
(lanes 1±3 and 7±9) with the membrane-permeable DTBP cross-linker,
lysed and immunoprecipitated with anti-T7 (lanes 1±3), 6F9 or 5H4
(lanes 4±6) or anti-Flag (lanes 7±9) antibodies, as described in
Materials and methods. Immunocomplexes were then separated by
SDS±PAGE and analyzed further by immunoblotting using anti-T7,
anti-Flag, 6G3 or anti-C subunit antibodies to detect T7-Vpr, FlagB55a, PP2A Aa and AD5, or PP2A C subunits, respectively, as
indicated on the right side of each blot. (C) 293T (106) cells were
co-transfected with pcDNA3-Flag-B55a (2.5 mg) and SVCMVT7-VPRmac239 (2.5 mg) or SVCMV-T7-VPXmac239 (2.5 mg),
as indicated at the bottom of the ®gure. Cells were lysed, immunoprecipitated with anti-Flag (upper panel) or anti-T7 (lower panel)
antibodies and the resulting immunocomplexes analyzed by western
blotting using anti-T7 antibodies. (D) 293T cells (106) were mock
transfected (lane 1) or transfected with 2.5 mg of SVCMV-T7-VPR.
Cells were lysed, immunoprecipitated with anti-T7 antibodies and
analyzed further by western blotting using anti-PP2A Aa (6G3) or
anti-C subunit antibodies. The endogenous PP2A Aa and C subunits
are indicated on the right side of the blots.
The data of Figure 1B also indicate that the association
of Vpr with PP2A occurs through an interaction with the
B55a subunit. Immunoprecipitation of protein lysates
from 293T cells overexpressing the mutant AD5, which
binds C but not B subunits, with anti-T7-Vpr or anti-FlagB55a antibodies, respectively, clearly reveals the presence
of Vpr±B55a complexes (Figure 1B, lanes 3 and 9). The A
and C subunits of PP2A do not appear to interact with Vpr,
since immunoprecipitation with the 5H4 monoclonal
antibody, which recognizes both AD5 and endogenous
3958
native A subunit, precipitated the endogenous C subunit
but did not co-precipitate Vpr (Figure 1B, lane 6).
To verify the speci®city of the Vpr±PP2A interaction,
we took advantage of the fact that the cell cycle arrest and
PIC nuclear import functions of HIV-1 Vpr are segregated
in two distinct viral proteins, Vpr and Vpx, respectively, in
simian immunode®ciency virus isolated from macaques
(SIVmac) (Fletcher et al., 1996; Planelles et al., 1996).
Using the transient expression system described above, we
tested whether SIVmac T7-Vpx or T7-Vpr was able to
interact with PP2A B55a subunit. The results in Figure 1C
clearly indicate that while SIVmac T7-Vpr and T7-Vpx
were expressed at similar levels in co-transfected cells
(lanes 2 and 3, lower panel), only SIVmac T7-Vpr coprecipitated with the PP2A B55a subunit (compare lanes 2
and 3, upper panel). All together, these results provide
strong evidence that Vpr associates speci®cally with the
PP2A holoenzyme through a direct or indirect interaction
with the B55a subunit. The interaction of SIVmac Vpr but
not Vpx with B55a strongly suggests that the formation of
a Vpr±PP2A complex plays a critical role in Vpr's ability
to mediate a G2 cell cycle arrest.
To examine whether Vpr can interact with endogenous
PP2A holoenzyme, 293T cells were transfected with
SVCMV-T7-VPR only, and the presence of a Vpr±PP2A
complex was examined by immunoprecipitation using
anti-T7 antibodies and analysis by western blotting as
described above. As shown in Figure 1D, both the
structural/regulatory Aa (68 kDa) and the catalytic C
(36 kDa) subunits were co-immunoprecipitated by the
anti-T7-Vpr antibodies, thus demonstrating that Vpr forms
a complex with endogenous PP2A (lane 2).
Vpr interacts speci®cally with B subunits
belonging to the B family
To test whether the association of Vpr with PP2A occurs
through a speci®c interaction with a particular family of
B subunits, we evaluated the binding of Vpr to different
subunits belonging to B (55a, 55b and 55g), B¢ (56e and
56g) and B¢¢ (PR72). Cells were co-transfected with
plasmids expressing T7-Vpr and Flag-tagged B subunits
belonging to the three gene families. At 48 h posttransfection, cell lysates were immunoprecipitated using
anti-Flag antibodies and the resulting immunocomplexes
were probed by immunoblotting for the presence of T7tagged Vpr. The results shown in Figure 2 reveal that Vpr
interacts speci®cally with the PP2A B subunit belonging to
the B family. Interestingly, the interaction of Vpr with
B55a was found to be more ef®cient than the interaction
with B55b or g (Figure 2, compare lane 1 with lanes 2 and
3) even though the three B subunits were expressed at
similar levels (lanes 1±3). In contrast, no interaction was
detected between Vpr and B subunits belonging to the
B¢ (B56g and B56e) or B¢¢(PR72) families (Figure 2,
lanes 4±6).
Mutations in the C-terminal domain of Vpr abolish
the interaction with PP2A B55a subunit
Secondary structure predictions of Vpr as well as structural studies of Vpr polypeptides by NMR have indicated
the presence of two amphipathic a-helical domains
located between residues 17 and 46 and between residues
53 and 74 (Mahalingam et al., 1995; Yao et al., 1995;
HIV-1 Vpr interacts with the B subunit of PP2A
Fig. 2. Vpr associates speci®cally with B subunits belonging to the
B family. 293T cells (106) were mock transfected (lane 7) or cotransfected with 2.5 mg of SVCMV-T7-VPR and 2.5 mg of pcDNA3Flag-B55a (lane 1), pcDNA3-Flag-B55b (lane 2), pcDNA3-Flag-B55g
(lane 3), pcDNA3-Flag-B56e (lane 4), pcDNA3-Flag-B56g (lane 5)
or pcDNA3-Flag-B¢¢ (lane 6). Cells were lysed, immunoprecipitated
with anti-Flag (upper panel) or anti-T7 (lower panel) antibodies and
analyzed by western blotting for the presence of T7-Vpr or Flag-B
subunits, using anti-T7 or anti-Flag antibodies, respectively, as
indicated.
Subbramanian et al., 1998b; Schuler et al., 1999; Wecker
and Roques, 1999). The protein also contains, between
residues 60 and 81, a hydrophobic leucine/isoleucine-rich
(LR) domain and a partially overlapping C-terminal
arginine-rich region (Di Marzio et al., 1995; Wang et al.,
1996) (Figure 3A). A consensus emerging from structure±
activity studies indicates that three main activities of Vpr
(i.e. virion incorporation, nuclear import and cell cycle
arrest) are encoded by distinct domains of the protein (Di
Marzio et al., 1995; Yao et al., 1995; Mahalingam et al.,
1997; Subbramanian et al., 1998b). Indeed, several studies
have shown that point mutations disrupting the N-terminal
a-helical region preferentially reduced the effect of Vpr on
cell cycle progression and nuclear localization, while
mutations in the second amphipathic helix of Vpr
speci®cally affected PIC nuclear import. Mutations in
the C-terminal basic domain affected both nuclear localization and cell cycle functions, while sparing incorporation of mutant proteins into HIV-1 virions (Di Marzio
et al., 1995; Mahalingam et al., 1997).
To examine the functional relevance of the Vpr±PP2A
interaction, we tested the ability of the B55a subunit to
bind a panel of Vpr mutants, including mutants that are not
able to arrest cells in G2 (Figure 3A). Each mutant Vpr
expression vector was co-transfected in 293T cells with
pcDNA3-Flag-B55a. Cell lysates were ®rst immunoprecipitated with anti-Flag antibodies and the presence
of each Vpr mutant in immunocomplexes was analyzed by
western blotting using anti-T7 antibodies. The results in
Figure 3B (lower panel) reveal that each T7-Vpr mutant
was expressed at comparable levels in co-transfected
cells. Interestingly, two Vpr mutants, VprSR79/80 and
VprR80A, which are defective for cell cycle G2 arrest (Di
Marzio et al., 1995; Forget et al., 1998), were unable to
interact with the B55a subunit (Figure 3B, lanes 7 and 8).
In contrast, mutants such as VprR62P or VprQ65E, which
induce G2 arrest as wild-type Vpr (Forget et al., 1998),
interacted with B55a (Figure 3B, lanes 5 and 6).
Surprisingly, VprE25K and VprA30F mutants that were
previously shown to induce G2 cell cycle arrest, albeit with
reduced ef®ciency (30% of wild-type Vpr; Figure 3A),
interacted with the B55a subunit with ef®ciency comparable to wild-type Vpr (Figure 3B, compare lanes 3 and 4
with lane 2). The same analysis was also carried out by
precipitating the lysates with anti-T7 antibodies and
probing the immunocomplexes with anti-Flag or anti-Aa
subunit (6G3). All mutants that interacted with B55a were
shown to be part of the PP2A holoenzyme complex (data
not shown). These results indicate that the Vpr C-terminal
domain plays a critical role in the interaction of the protein
with PP2A B55a subunit. Furthermore, the data indicate
that the interaction of Vpr with PP2A B55a subunit is
necessary but not suf®cient to induce G2 cell cycle arrest.
Vpr targets PP2A B55a subunit to the nucleus
Vpr has been localized primarily in the nuclear compartment using cellular fractionation approaches as well as
conventional optical immuno¯uorescence and laser confocal microscopy (Subbramanian et al., 1998b; Vodicka
et al., 1998). To assess whether the binding of Vpr to PP2A
Ba subunits modi®es the cellular localization of PP2A, we
co-expressed native Vpr with Flag-tagged B55a in Cos-7
cells and characterized the localization of wild-type Vpr
and B55a by double staining immuno¯uorescence techniques and laser confocal microscopy. As shown in Figure
4, cells expressing native Vpr alone display a pronounced
nuclear staining (Figure 4B). In contrast, cells expressing
Flag-tagged B55a exhibit primarily a cytoplasmic staining
with a clear exclusion from the nucleus (Figure 4C). When
Vpr and B55a were co-expressed in Cos-7 cells, the B55a
cellular localization shifted from the cytoplasm to the
nucleus (Figure 4E) while Vpr localization remained
nuclear (Figure 4D). Highly sensitive confocal laser
sectioning and subsequent computer-assisted image
merging clearly revealed that Vpr co-localized with the
B55a protein (Figure 4F). Not surprisingly, when B55a
and the Vpr R80A mutant that is defective for G2 arrest
and PP2A binding were co-expressed in the same cell, they
failed to co-localize to any appreciable level (Figure 4G±
I). Together, these results strongly suggest that upon
binding to Vpr, the B55a-containing PP2A holoenzyme
complex is targeted from a cytoplasmic to a nuclear
compartment.
Vpr stimulates PP2A activity
To determine whether the association of HIV-1 Vpr with
PP2A in¯uenced the enzyme activity, lysates from 293T
cells transiently expressing Vpr and/or Flag-B55a were
immunoprecipitated with anti-Flag antibodies and the
PP2A-containing immunocomplexes were assayed for
PP2A activity in the presence of increasing concentrations
of a PP2A-speci®c phosphopeptide substrate as described
in Materials and methods. The results depicted in Figure
5A (left panel) clearly show that expression of Vpr
increased the catalytic activity of PP2A by ~5-fold. To
assess whether Vpr stimulation of PP2A activity was
dependent on the interaction of the protein with PP2A, we
performed similar experiments with Vpr mutant R80A,
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M.Hrimech et al.
Fig. 3. The C-terminal domain of HIV-1 Vpr is critical for the interaction of the protein with PP2A B55a subunit. (A) The primary amino acid
sequence of Vpr is shown. The substitution mutations introduced in each Vpr mutant are indicated below. In addition, the relative G2 arrest ability of
each Vpr mutant is also indicated. Wild-type Vpr was set arbitrarily at 100%. (B) 293T cells (106) were mock transfected (lane 1) or co-transfected
with 2.5 mg of Flag-B55a expression vector (pcDNA3-Flag-B55a) and 2.5 mg of plasmid encoding T7-tagged wild-type Vpr (lane 2) or different
Vpr mutants including VprE25K (lane 3), VprA30F (lane 4), VprR62P (lane 5), VprQ65E (lane 6), VprSR79/80ID (lane 7) or VprR80A (lane 8),
as indicated. Cells were lysed, immunoprecipitated with anti-Flag antibodies and analyzed by western blotting with anti-T7 antibodies to reveal
co-precipitated T7-Vpr (upper panel). To verify the expression of each Vpr mutant in transfected cells, each cell lysate was subsequently
immunoprecipitated with anti-T7 antibodies and analyzed by western blotting using anti-T7 antibody (lower panel).
which fails to interact with PP2A and is defective for G2
cell cycle arrest. As shown in Figure 5A, measurement of
PP2A activity in lysates from cells expressing B55a and
R80A reveals that the enzyme catalytic activity remained
unchanged as compared with the control (cells expressing
B55a and Vpr± negative control). In contrast, Vpr mutant
A30F, which retains the ability to bind PP2A B55a but
displays a reduced capacity to mediate G2 arrest, stimulates PP2A activity by ~2-fold (Figure 5A). As shown in
Figure 5B, comparable amounts of B55a, Vpr or Vpr
mutant proteins were detected in each immunocomplex
used to assay phosphatase activity.
To investigate whether recombinant Vpr could increase
PP2A activity in vitro, immunoprecipitated Flag-B55a±
PP2A complexes were incubated with a highly puri®ed
preparation of recombinant Vpr produced from baculovirus-infected insect cells and PP2A activity was determined as described above. The results in Figure 5A (right
panel) reveal that recombinant Vpr is also able to enhance
PP2A catalytic activity towards a synthetic phosphopeptide substrate. This stimulatory effect of Vpr on PP2A did
3960
not result from a contaminating phosphatase activity
carried over by Vpr since the recombinant Vpr preparation
did not display any phosphatase activity. Moreover, when
Vpr was depleted using rabbit polyclonal anti-Vpr antibodies prior to its addition to immunoprecipitated PP2A,
no increase in phosphatase activity was detected (data not
shown). Overall, these results strongly suggest that the
interaction between Vpr and PP2A B55a stimulates the
catalytic activity of the PP2A holoenzyme towards its
substrate.
cdc25c is present in the PP2A±Vpr complex
We next tested whether cdc25, a PP2A cellular substrate
whose phosphorylation pro®le is affected by Vpr (Re et al.,
1995), was part of the Vpr±PP2A complex. 293T cells
were transfected with SVCMV-T7-VPR or pcDNA3-FlagB55a or co-transfected with Vpr and B55a expression
plasmids. At 48 h post-transfection, cells were incubated
with the cross-linking reagent DTBP as described in
Materials and methods. Treated cells were lysed and
immunoprecipitated with anti-cdc25c, anti-T7 or anti-Flag
HIV-1 Vpr interacts with the B subunit of PP2A
Fig. 4. Vpr targets B55a subunit-containing PP2A complex to the nucleus. Cos-7 cells were mock transfected (A) or transfected with 5 mg of
SVCMV-VPR (B) or B55a expressor, pcDNA3-Flag-B55a (C), or co-transfected with 5 mg of Vpr and B55a expressor plasmids (D±F) or Vpr
mutant (R80A) and B55a expressor plasmids (G±I). Following ®xation, cells were incubated ®rst with rabbit anti-Vpr antibodies or goat anti-Flag
antibodies, as indicated, and then labeled with lisamine/rhodamine-conjugated goat anti-rabbit (for Vpr) (B, D and G) or FITC-conjugated anti-goat/
sheep (for the B55a subunit) (C, E and H) antibodies. Labeled cells were then analyzed by confocal laser microscopy. A laser section through a
representative cell is shown for each transfection. The images on the far right column labeled as F and I depict superimposed images of the
Vpr-speci®c (red) and Flag-B55a (green) signals in the same cells.
antibodies, respectively, and the resulting immunocomplexes probed for the presence of cdc25c, PP2A Aa
and C subunits, and Vpr by immunoblotting. The results in
Figure 6A reveal that endogenous cdc25c can be found
associated with the PP2A holoenzyme complex (lane 3).
Indeed, when B55a is expressed alone, the anti-Flag
antibodies precipitate a fraction of the total endogenous
cdc25c (Figure 6A, compare lanes 3 and 1). Surprisingly,
the level of cdc25c associated with the PP2A holoenzyme
increased by ~6-fold when Vpr was co-expressed with
B55a (Figure 6A, compare lanes 4 and 3). The presence of
Vpr changed only the level of cdc25c recruited to the
PP2A holoenzyme complex. The levels of endogenous Aa
or C subunits in the complex did not vary to any signi®cant
extent in the presence or absence of Vpr (Figure 6A,
compare lanes 3 and 4). Furthermore, when endogenous
PP2A was precipitated by anti-T7-Vpr antibodies from
T7-Vpr-expressing cells, a large proportion of endogenous
cdc25c was found complexed with PP2A (Figure 6A,
compare lanes 2 and 1).
In contrast, when similar experiments were performed
with the Vpr mutant R80A, no cdc25c was detected in the
complex precipitated by the anti-T7 antibodies (Figure 6B,
lane 4). However, when the same cells were reacted with
anti-Flag B55a, the resulting PP2A immunocomplex was
shown to contain cdc25c, but at levels that were comparable to those found in cells only expressing Flag-B55a
(Figure 6B, compare lanes 6 and 2).
We next determined the phosphorylation status of the
cdc25c found associated with the Vpr±PP2A complex.
The results in Figure 6C show that in the absence of Vpr,
both hyperphosphorylated and hypophosphorylated forms
3961
M.Hrimech et al.
Fig. 5. Vpr increases the catalytic activity as well as the af®nity of
PP2A holoenzyme towards a speci®c phosphopeptide substrate.
(A) Left panel: 293T cells (106) were co-transfected with 5 mg of
pcDNA3-Flag-B55a and SVCMV-T7-VPR, T7-VPR±, T7-VPRA30F or
T7-VPRR80A, as indicated. Cells were lysed and immunoprecipitated
with anti-Flag antibodies. An increasing concentration of synthetic
phosphopeptide substrate was added to equivalent amounts of each
Flag-B55a-containing PP2A immunocomplex and incubated at 30°C
for 30 min to assay PP2A activity. Right panel: 293T cells (106) were
transfected with 5 mg of pcDNA3-Flag-B55a. Cells were lysed and
immunoprecipitated as described above. Immunocomplexes were
incubated with recombinant Vpr for 2 h at 4°C and phosphatase
activity was monitored. (B) Expression of Flag-B55a and Vpr. Each
transfected cell sample was divided into two aliquots. One was used to
monitor phosphatase activity and the second was treated with DTBP
and immunoprecipitated with 6F9 antibodies. After protein separation
by SDS±PAGE and western blotting, the presence of Flag-B-55a and
Vpr was detected using anti-Flag (upper panel) and anti-T7 (lower
panel) antibodies, respectively.
of cdc25c can be detected in cells (lane 1). Only
hypophosphorylated forms of cdc25c were found complexed to PP2A in B55a-expressing cells (Figure 6C,
lane 2). Interestingly, the pool of hypophosphorylated
cdc25 found associated with PP2A increased substantially
in the presence of Vpr (Figure 6C, lane 3). In addition, a
small fraction of hyperphosphorylated cdc25c, which
probably represents newly recruited cdc25c, was detected
in the Vpr±PP2A complex (Figure 6C, lane 3). As
expected, treatment of cells with aphidicolin, an agent
that causes a block in G1/S phase, led to an inactivation of
cdc25c, which accumulated in a hypophosphorylated form
(Figure 6C, lane 4).
3962
Fig. 6. Association of cdc25c with the Vpr±PP2A complex. (A) 293T
cells (106) were mock transfected (lane 1), transfected with 2.5 mg of
SVCMV-T7-VPR (lane 2) or pcDNA3-Flag-B55a (lane 3), or cotransfected with 2.5 mg of T7-Vpr and Flag-B55a expression plasmids
(lane 4). Cells were treated with the plasma membrane-permeable
DTBP cross-linker, lysed and immunoprecipitated with anti-cdc25c,
anti-T7 or anti-Flag antibodies, respectively, and analyzed by SDS±
PAGE and western blotting using the appropriate antibodies to detect
PP2A Aa subunit (6G3), endogenous cdc25c (anti-cdc25c), PP2A C
subunit (anti-C subunit) and T7-Vpr (anti-T7), as indicated on the right
side of the blots. (B) 293T cells (106) were mock transfected (lane1),
transfected with 2 mg of pcDNA3-Flag-B55a (lane 2), or co-transfected
with 2.5 mg of pcDNA3-Flag-B55a and 2.5 mg of SVCMV-T7-Vpr
(lane 3) or SVCMV-T7-VprR80A (lane 4). Cells were treated with
DTBP, lysed and immunoprecipitated with anti-cdc25c, anti-Flag or
anti-T7 antibodies as indicated. Resulting immunoprecipitates were
then separated by SDS±PAGE and analyzed by western blotting using
anti-cdc25c. Cell lysates similar to those from lanes 3 and 4 were
immunoprecipitated with anti-Flag antibodies and the resulting
immunocomplexes were analyzed for the presence of cdc25c by
immunoblotting using anti-cdc25c antibodies (lanes 5 and 6). (C) 293T
cells (106) were mock transfected (lane 1), transfected with 2.5 mg of
pcDNA3-Flag-B55a (lane 2) or co-transfected with 2.5 mg of pcDNA3Flag-B55a and 2.5 mg of SVCMV-T7-Vpr (lane 3) or treated with
aphidicolin (lane 4). Cells were treated with DTBP, lysed and
immunoprecipitated with anti-cdc25c or anti-Flag antibodies as
indicated. Resulting immunoprecipitates were then separated by 8%
SDS±PAGE and analyzed by western blotting using anti-cdc25c.
Overall, these results indicate that a fraction of cdc25c
present within cells can be found associated with the PP2A
holoenzyme. Moreover, association of Vpr with PP2A
through its interaction with the B55a subunit increases
the recruitment and dephosphorylation of the cdc25c
substrate.
Discussion
Our analyses of physical interactions and cell cycle
modulation lead to the conclusion that PP2A plays a
central role in the G2 cell cycle arrest mediated by the Vpr
HIV-1 Vpr interacts with the B subunit of PP2A
Fig. 7. Model for Vpr-mediated G2 cell cycle arrest. (A) Normally, the active form of cdc25 accumulates in the nucleus and activates cdc2±cyclin B
to trigger mitosis. PP2A holoenzyme containing B55 subunits interacts with cdc25 and regulates its activity. (B) In HIV-1-infected cells, Vpr
associates with PP2A via an interaction with B regulatory subunits. The Vpr±PP2A interaction enhances PP2A catalytic activity and targets the
hyperactive holoenzyme to the nucleus where active cdc25 substrate is located. Inactivation of cdc25, by ef®cient dephosphorylation, keeps
cdc2±cyclin B in its hyperphosphorylated form, thereby stopping cells from entering mitosis.
protein encoded by primate lentiviruses. Speci®cally, our
®ndings provide evidence that HIV-1 Vpr and PP2A
interact in cells and that this interaction has biological
consequences with regard to Vpr-mediated G2 cell cycle
arrest. First, immunoprecipitation experiments with antibodies directed to either T7-Vpr, PP2A Aa or epitopetagged B55a subunits reveal that Vpr exists in a
complex that includes all three subunits of the phosphatase
within Vpr-expressing cells (Figure 1B±D), and that this
complex contains phosphatase activity that is speci®c
towards a PP2A-speci®c phosphopeptide substrate
(Figure 5) and is sensitive to low levels of okadaic acid,
a characteristic of PP2A (data not shown). Furthermore,
similar co-precipitation experiments in cells overexpressing a deletion mutant of the Aa subunit, AD5,
which binds C but not B subunits, reveal that the
association of Vpr with PP2A occurs through a speci®c
interaction with the B regulatory subunit (Figure 1B). Only
B subunits belonging to the B family were found to
interact with Vpr (Figure 2). Interestingly, although the
precise roles of regulatory B subunits are still not clearly
understood, a functional role has been assigned to the
B55a subunit in the regulation of mitosis, as suggested by
the ®nding that deletion of this gene is associated with
cytokinesis defects in both yeast and Drosophila (Healy
et al., 1991; Mayer-Jaekel et al., 1993). It therefore
appears that Vpr, via its ability to bind B55 subunits, forms
a complex with a subspecies of PP2A holoenzyme
involved in the regulation of mitosis.
Secondly, considerable functional evidence correlating
this interaction with Vpr-mediated G2 arrest was obtained.
(i) Overexpression of the PP2A Aa subunit mutant, AD5,
in Vpr-expressing cells, reduces the levels of PP2A
complexed with Vpr and signi®cantly diminishes Vprmediated cell cycle arrest (Figure 1 and data not shown).
Since overexpression of this mutant was shown to reduce
the levels of holoenzyme relative to core enzyme within
transfected cells by at least 2-fold, we believe that this
mutant alleviates Vpr-mediated G2 cell cycle arrest by
reducing the levels of B55 subunit-containing PP2A
holoenzyme available for interaction with Vpr. (ii) Our
data demonstrate that PP2A holoenzyme containing the
B55a subunit interacts with HIV-1 and SIVmac Vpr,
which are both capable of inducing a G2 cell cycle arrest.
In contrast, the holoenzyme was unable to form a complex
with SIVmac Vpx, which mediates the other Vpr function,
i.e. nuclear import of the PIC (Fletcher et al., 1996)
(Figure 1C). (iii) Mutation analysis of Vpr indicates that a
correlation exists between Vpr's ability to bind PP2A and
its capacity to mediate G2 cell cycle arrest. Vpr mutants
containing substitution mutations at amino acid R80 or
S79 and R80 at the C-terminus of the protein, and that are
known to be defective for G2 arrest (Di Marzio et al., 1995;
Mahalingam et al., 1997; Yao et al., 1998), lost their
3963
M.Hrimech et al.
ability to interact with PP2A B55a subunit (Figure 3). In
addition, mutations in the N-terminal ®rst a-helix (E25K
and A30F) affected cell cycle arrest (30% of wild-type
levels) more than they in¯uenced the physical interaction,
thus indicating that the interaction of Vpr with PP2A,
although necessary, was not suf®cient to mediate G2 cell
cycle arrest. These results further suggest the possibility
that other functional domains of Vpr or additional Vprassociated factors may cooperate in facilitating Vprmediated G2 cell cycle arrest. Interestingly, using indirect
immuno¯uorescence and confocal microscopy, we
demonstrate that upon Vpr co-expression, B55a subunits
that have primarily a cytoplasmic localization are targeted
to the nucleus where they co-localize with Vpr (Figure 4).
As expected, co-expression of PP2A B55a subunit with
Vpr mutant R80A, which is unable to form a complex with
PP2A, did not change the cytoplasmic localization of the
B55a subunit (Figure 4). These observations indicate that
Vpr, by binding to the B55a subunit, provides PP2A with
a nuclear targeting component and strongly suggest that
Vpr nuclear localization function is required for G2 cell
cycle arrest.
The third signi®cant ®nding described in this report is
that Vpr stimulates PP2A catalytic activity towards a
PP2A-speci®c phosphopeptide substrate (Figure 5).
Measurement of phosphatase activity in epitope-taggedB55a-containing immunocomplexes isolated from epitope-tagged-B55a- or Vpr/epitope-tagged-B55a-expressing cells reveals that the presence of Vpr stimulates
phosphatase activity by ~5-fold. Furthermore, similar
effects on phosphatase catalytic activity were obtained
when recombinant Vpr was added directly to epitopetagged-B55a immunocomplexes, thus demonstrating that
Vpr can positively modulate PP2A in vitro. Stimulation of
phosphatase activity by Vpr correlated with the protein's
ability to mediate a cell cycle arrest since G2 arrestdefective mutant VprR80A was unable to stimulate
phosphatase activity in this assay. Furthermore, the A30F
mutant, which displayed an impaired G2 cell cycle arrest
(30% of wild type) and a reduced nuclear localization (Yao
et al., 1995) but interacted with PP2A as well as wild-type
Vpr, slightly stimulated phosphatase activity. Thus, these
results suggest that conformational changes in this region
(N-terminal a-helix) of the protein may affect phosphatase
activity once PP2A is bound to Vpr. The availability of an
in vitro assay system should now permit the isolation of
Vpr mutants that bind and stimulate PP2A in vitro but do
not mediate G2 cell cycle arrest in transfected cells. Such
Vpr mutants may correspond to proteins that lost the ability
to target B55±PP2A complex to the nucleus and consequently may help identify nuclear localization domain(s)
relevant to Vpr-mediated G2 cell cycle arrest.
The fourth major ®nding of this study is that the
presence of Vpr in the PP2A complex increases the
recruitment and dephosphorylation of the cdc25c phosphatase substrate in transfected cells (Figure 6). Enhanced
recruitment of cdc25c to the Vpr±PP2A complex is
speci®c since it is not observed with another known
substrate of PP2A, the MAP kinase Raf1, which is known
to interact with PP2A (Dent et al., 1995) (data not shown).
Moreover, it requires the association of Vpr with the PP2A
complex since we did not detect such enhancement of
cdc25c recruitment in the presence of the VprR80A
3964
mutant. These results provide the ®rst evidence that
cdc25c phosphatase is a substrate of PP2A holoenzyme
containing the B55a subunit. Moreover, these ®ndings
suggest that Vpr, by binding to B55a, increases the
speci®city and activity of PP2A towards the cdc25
substrate.
Virus and PP2A
Viruses have evolved a number of mechanisms that allow
them to interact with the normal host cell cycle. Hence, the
ability to negatively modulate the activity of PP2A via the
association of viral protein subunits with the AC core
heterodimer is exploited by the DNA tumor viruses SV40
and polyoma virus, as a mechanism to interfere with
intracellular signaling cascades and promote cell proliferation (Mumby, 1995). In contrast, the Vpr protein encoded
by primate lentiviruses negatively modulates cell growth,
interacts with a complex that includes all of the PP2A
subunits and interferes with signal transduction cascades
regulating G2/M cell cycle progression by conferring the
PP2A enzymatic complex speci®city for the cdc25 regulator. On the basis of its interaction with PP2A, Vpr
resembles a viral protein encoded by the open reading
frame 4 of the adenovirus E4 gene (E4orf4). Like Vpr, the
adenovirus E4orf4 protein forms a complex with the
heterotrimeric form of PP2A in adenovirus-infected cells
(Kleinberger and Shenk, 1993). The interaction between
E4orf4 and PP2A has recently been shown to be important
for the induction of apoptosis by E4orf4 and for downregulation of virally induced signal transduction
(Kleinberger and Shenk, 1993; Shtrichman et al., 1999).
In contrast to SV40 and polyoma small and middle T
antigens, which inhibit PP2A activity, both Vpr and
E4orf4 act as positive regulators of the enzyme activity.
Model of HIV-1 Vpr-mediated G2 arrest
There is increasing evidence that the cell cycle is
controlled in part by localizing speci®c regulators to the
right place at the right time. For instance, Lopez-Girona
et al. (1999) have shown that, in the ®ssion yeast S.pombe,
the cdc25 phosphatase is exported from the nucleus in
response to DNA damage. Indeed, activation of the DNA
damage checkpoint causes the net nuclear export of cdc25
by a process that requires phosphorylation by chk1 kinase
and association with Rad24, a protein that belongs to the
14-3-3 protein family, which acts as an attachable nuclear
export sequence. Export separates cdc25 from its substrate, the cyclin B±cdc2 kinase, thereby stopping cells
from entering mitosis. By analogy, Vpr may mediate a G2
cell cycle arrest by acting at least in part as a PP2A/B55attachable nuclear import signal. Normally, cdc25 continually shuttles between the cytoplasm and the nucleus
where its active phosphorylated form activates cdc2±
cyclin B1 complexes by dephosphorylating cdc2 (Figure
7A). Recent evidence indeed suggests that cdc25 accumulates in the cytoplasm during interphase and progressively
localizes in the nucleus as cells move towards the G2/M
phase of the cell cycle (Dalal et al., 1999) (data not
shown). The cellular location where PP2A interacts with
cdc25 to regulate the levels of active phosphorylated
cdc25 is still not de®ned precisely. However, preliminary
evidence from our laboratory indicates that in Vpr
G2-arrested cells, cdc25 accumulates in the nucleus and
HIV-1 Vpr interacts with the B subunit of PP2A
totally co-localizes with Vpr (data not shown), thus
suggesting that the Vpr±PP2A complex interacts with
cdc25 in the nucleus. Detailed subcellular localization
studies are currently under way to address this important
question.
Overall, our data support a model in which Vpr
mediates G2 cell cycle arrest by ef®ciently targeting
PP2A/B55 to the nucleus and positively modulating the
holoenzyme catalytic activity towards the active phosphorylated nuclear cdc25 substrate (Figure 7B). Ef®cient
recruitment and dephosphorylation of active nuclear cdc25
would in turn lead to a situation where Wee1 and Myt1
kinase activities are greater than cdc25 phosphatase
activity, thus resulting in cdc2±cyclin B inactivation and
G2 cell cycle arrest. The identi®cation of PP2A as the
cellular protein complex targeted by Vpr to mediate G2
cell cycle arrest should provide important information on
how these proteins may regulate and perturb cell growth,
and should present opportunities for the development of
both HIV therapeutics and anticancer strategies.
Materials and methods
Antibodies, recombinant proteins and chemicals
Mouse monoclonal anti-T7-Tag antibodies were obtained from Novagen.
The rabbit anti-Vpr serum was raised against bacterially expressed
recombinant Vpr as described previously (Subbramanian et al., 1998b).
Goat anti-Flag antibodies, rabbit anti-cdc25c or anti-Raf1 sera and goat
anti-human PP2A C subunit were obtained from Santa Cruz. Rat
monoclonal antibodies 6F9, 5H4 and 6G3 were raised against puri®ed
native PP2A Aa subunit (6F9, 5H4) or an 11 amino acid peptide (6G3)
corresponding to the N-terminus of the Aa subunit as described
previously (Kremmer et al., 1997). His-tagged Vpr was expressed in
Sf9 insect cells following infection with a recombinant baculovirus and
puri®cation on an NTA matrix as described previously (Popov et al.,
1998). The cross-linking agent DTBP was from Pierce, the protease
inhibitor cocktail from Boehringer Mannheim, and the propidium iodide
and aphidicolin from Sigma.
Plasmids and cloning strategies
The PP2A Aa subunit expression plasmids pcDNA3-PP2A Aa and
pcDNA3-PP2A AD5 used in this study were described previously
(Ruediger et al., 1997). Plasmid expressor encoding Flag-tagged PP2A
human B (B55a, B55b, B55g) (Mayer et al., 1991), B¢ (B56e, B56g)
(McCright and Virshup, 1995) or B¢¢ (PR72) (Hendrix et al., 1993)
subunits were constructed by fusing a Flag sequence at the 5¢ end of
KpnI±EcoRI-digested cDNA fragments encompassing each of the B
subunit genes. The resulting DNA fragments encoding Flag-tagged B
subunits were inserted in pcDNA-3 to generate pcDNA3-FlagB vectors.
The HIV-1 Vpr expressor plasmid, SVCMV-VPR, and the negative
control plasmid, SVCMV-VPR±, encoding a Vpr gene harboring a point
mutation in the Vpr translation initiation codon, were described
previously (Yao et al., 1995). Plasmids encoding mutant Vpr
(SVCMV-VPRE25K, SVCMV-VPRA30F, VPRR62P, VPRQ65E,
VPRSR79/80ID and VPRR80A) were described elsewhere (Yao et al.,
1995; Subbramanian et al., 1998b). To construct SVCMV-T7-VPR, a
PCR-generated Vpr cDNA derived from the proviral plasmid HxBRU
(Yao et al., 1995) was fused to the C-terminus of the T7-Tag sequence
and inserted into SVCMV-Vpr to replace Vpr. SVCMV-GFP-VPR or
SVCMV-GFP-VPR± were constructed by inserting a cDNA fragment
containing the cytomegalovirus (CMV) immediate early promoter, the
green ¯uorescent protein (GFP) gene and the glutathione terminator
sequence (derived from the pQBI 25 plasmid; Quantum Biotechnologies
Inc.) into the unique BamHI site of SVCMV-VPR or SVCMV-VPR±,
respectively. SVCMV-T7-VPXmac239 and SVCMV-T7-VPRmac239
encoding the Vpx and the Vpr genes, respectively, from SIVmac239
(p239SpSp5¢) (Kestler et al., 1990) were constructed using similar
strategies to those described for the construction of SVCMV-T7-VPR.
Cell lines and transfections
Cos-7 African green monkey kidney cells and human embryonic kidney
293T cells were maintained in Dulbecco's modi®ed Eagle's medium
(DMEM) supplemented with 10% fetal calf serum and 1% penicillin and
streptomycin. All cells were maintained at 37°C and 5% CO2. For all
experiments, we used the standard calcium phosphate co-precipitation
technique to transfect cells, except where indicated.
Cell cycle analysis
Vpr-mediated cell cycle arrest was evaluated by propidium iodide
staining and ¯ow cytometry analysis as described previously (Yao et al.,
1998).
Immunoprecipitations and immunoblotting analyses
293T cells (106) were ®rst transfected or co-transfected with different
plasmids, as indicated in each experiment. At 48 h post-transfection, cells
were washed twice with cold phosphate-buffered saline (PBS) and
subsequently lysed at 4°C for 1 h with NP-40 lysis buffer (50 mM Tris±
HCl pH 7.4, 400 mM NaCl, 0.2% NP-40 and a protease inhibitor
cocktail). Cell lysates were then microcentrifuged at 14 000 r.p.m. for
30 min to remove cell debris. Supernatants were immunoprecipitated with
either mouse anti-T7, goat anti-Flag, rat anti-PP2A Aa (6F9), rat antiPP2A AD5 (5H4) or rabbit anti-cdc25c antibodies as described previously
(Yao et al., 1998). When 6F9 or 5H4 antibodies were used for
immunoprecipitation, cells were treated with the cell membranepermeable cross-linking reagent DTBP prior to lysis. DTBP was
resuspended in PBS buffer pH 7.5 and added to each sample to a ®nal
concentration of 0.25 mM in a 5 ml volume. After a DTBP treatment of 1 h
at 4°C, cells were treated with 1 M Tris buffer pH 7.5 to stop the reaction,
washed three times with PBS to remove excess DTBP, lysed with NP-40
lysis buffer and incubated with the appropriate antibodies overnight at
4°C. Immunocomplexes were recovered by addition of protein A±
Sepharose beads (Pharmacia) for 2 h at 4°C and microcentrifugation.
Beads were washed extensively using lysis buffer and boiled in Laemmli
buffer. After protein separation on SDS±polyacrylamide gels, proteins
were transferred to a nitrocellulose membrane (0.45 mM pore size; BioRad) by electroblotting for 3 h at 30 V in a Bio-Rad Trans Blot cell. The
membrane was then incubated for 1 h in blocking Tris-buffered saline
(TBS) solution containing 1% Tween-20 and 3% non-fat dry milk
(Carnation; NestleÂ) and incubated for an additional 3 h at room
temperature with the appropriate antibody diluted in TBS as follows:
mouse anti-T7-tagged Vpr (1:5000), rat anti-PP2A Aa wild type and AD5
subunit (6G3) (1:10 000), goat anti-PP2A C subunit (1:1000), goat antiFlag (1:1000), anti-cdc25c (1:1000) or anti-Raf1 (1:1000). Bound
antibodies were then probed with horseradish peroxidase-conjugated
anti-mouse (1:7500), anti-rat (1:5000), anti-rabbit (1:7500) or anti-goat
(1:8000) antibodies, respectively, washed extensively and revealed using
a sensitive enhanced chemiluminescence detection system (ECL detection kit; Amersham).
Phosphatase activity assay
293T cells (106) were transfected with pcDNA3-Flag-B55a (5 mg) or cotransfected with pcDNA3-Flag-B55a (5 mg) and SVCMV-VPR or Vpr
mutant expressors (5 mg) using Lipofectamine (Life Technologies)
according to the manufacturer's instructions. A total of 2 3 106 cells
were collected 48 h post-transfection and lysed with NP-40 buffer.
Lysates were microcentrifuged at 14 000 r.p.m. for 30 min and
immediately used for PP2A holoenzyme puri®cation by immunoprecipitation using goat anti-Flag covalently coupled to protein G±Sepharose
beads (Pharmacia). Similar amounts of puri®ed PP2A immunocomplex,
as adjusted by measurement of total protein concentration using the BioRad Protein assay (Bio-Rad), were used for in vitro phosphatase activity
assay. The catalytic activity of PP2A holoenzyme was measured using the
UBI phosphatase assay kit according to the manufacturer's instructions.
This assay used a phosphopeptide (KRpTIRR) as substrate for PP2A
enzyme (Harder et al., 1994). Free phosphate generated by PP2A catalytic
activity was revealed by addition of Malachite green solution, which turns
green in the presence of free phosphate, and quanti®ed using a
spectrophotometer at a wavelength of 650 nm.
Immuno¯uorescence and imaging of cells
Cos-7 cells were co-transfected with 5 mg of SVCMV-VPR or SVCMVVPRR80A and pcDNA3-Flag-B55a (5 mg). Cells were washed with cold
PBS, trypsinized and replated onto glass coverslips 24 h post-transfection.
After a 24 h incubation at 37°C, cells were washed twice with PBS, ®xed
in PBS±4% paraformaldehyde for 5 min, permeabilized in PBS±0.2%
Triton X-100 for 5 min and processed for immunolabeling. For single or
3965
M.Hrimech et al.
double labeling protocols, cells were incubated with the ®rst corresponding antibody diluted in PBS containing 0.03% sodium azide and 2% nonfat dry milk (Carnation; NestleÂ) for 12 h. First antibodies were used at the
following dilutions: rabbit anti-Vpr (1:500) and goat anti-Flag (1:200).
Following several washes, cells were incubated for 3 h with lisamine/
rhodamine-labeled goat anti-rabbit antibodies (1:100) for Vpr detection
and ¯uorescein isothiocyanate (FITC)-labeled anti-goat/sheep antibodies
(1:100) for B55a subunit detection. Confocal laser microscopy was
performed on a Zeiss LSM 410 (Carl Zeiss, Germany) equipped with a
Plan-Apochromat 633 oil immersion objective and an Ar/Kr laser. The
FITC images were obtained by scanning the cells with the 488 nm laser
and ®ltering the emission with a 515±540 nm band-pass. For the lisamine/
rhodamine images, the 568 nm laser was used in combination with a 575±
640 nm band-pass ®lter. For each cell studied and each image, the
additive signal through the whole cell thickness was ®rst digitized. Then
the confocal serial sections were scanned.
Acknowledgements
We thank N.Rougeau for excellent technical assistance. We also thank
G.Walter and R.Rudieger for generously supplying the 6F9, 5H4 and 6G3
monoclonal antibodies as well as the PP2A Aa and mutant AD5
expression vectors, D.M.Virshup and B.A.Hemmings for the B subunit
expression vectors, M.Bukrinsky for the puri®ed baculovirus preparation
of recombinant Vpr, and R.Subbramanian for Vpr mutants. We also thank
R.C.Desrosiers for providing SIVmac proviral clone p293SpS5¢ that was
obtained through the AIDS Research and Reference Reagent Program,
Division of AIDS, NIAID, NIH. We thank G.Lemay and R.Sekaly for
helpful discussion and comments on the manuscript, D.Boivin and
R.Marcellus for advice and important reagents, S.Senechal for FACS
analysis, and H.Dilhuydy for laser confocal microscopy. M.H. is the
recipient of a studentship from the Fonds pour la Formation de
Chercheurs et l'Aide aÁ la Recherche (FCAR). EÂ.A.C. is a Medical
Research Council (MRC) of Canada Scientist. This work was supported
by grants from the MRC and FCAR to EÂ.A.C.
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Received March 20, 2000; revised and accepted June 14, 2000
3967
The EMBO Journal Vol. 21 No. 14 p. 3918, 2002
Retraction
Human immunode®ciency virus type 1
Vpr-mediated G2 cell cycle arrest: Vpr interferes
with cell cycle signaling cascades by interacting
with the B subunit of serine/threonine protein
phosphatase 2A
Mohammed Hrimech, Xiao-Jian Yao,
Philip E.Branton and EÂric A.Cohen
The EMBO Journal, 19, 3956±3967, 2000
In the course of carrying out experiments that were a direct
extension of the above paper, we (the authors) discovered
differences from those presented in the original article
such that the primary conclusions of the paper are in
question. Because of this, we are retracting the entire paper
on the interaction of HIV-1 Vpr and the B55 subunit of
protein phosphatase 2A (PP2A) and its implications on
Vpr-mediated G2 cell cycle arrest. We are deeply regretful
for any scienti®c misconceptions that have resulted from
this study and apologize for any delay that readers may
have incurred in their research.
3918
ã European Molecular Biology Organization