JOURNAL OF VIROLOGY, Dec. 2009, p. 12253–12265
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 83, No. 23
The CDK Inhibitor p21Cip1/WAF1Is Induced by Fc?R Activation and
Restricts the Replication of Human Immunodeficiency Virus
Type 1 and Related Primate Lentiviruses
in Human Macrophages?
Anna Bergamaschi,1Annie David,1Erwann Le Rouzic,2,3Se ´bastien Nisole,2,3
Franc ¸oise Barre ´-Sinoussi,1and Gianfranco Pancino1*
Institut Pasteur, Unite ´ de Re ´gulation des Infections Re ´trovirales, Paris, France1; Institut Cochin, Universite ´ Paris Descartes,
CNRS (UMR 8104), De ´partement des Maladies Infectieuses, Paris, France2; and INSERM, U567,
27 Rue du Faubourg St. Jacques, 75014 Paris, France3
Received 7 July 2009/Accepted 10 September 2009
Macrophages are major targets of human immunodeficiency virus type 1 (HIV-1). We have previously shown
that aggregation of activating immunoglobulin G Fc receptors (Fc?R) by immune complexes inhibits reverse
transcript accumulation and integration of HIV-1 and related lentiviruses in monocyte-derived macrophages.
Here, we show that Fc?R-mediated restriction of HIV-1 is not due to enhanced degradation of incoming viral
proteins or cDNA and is associated to the induction of the cyclin-dependent kinase inhibitor p21Cip1/WAF1
(p21). Small interfering RNA-mediated p21 knockdown rescued viral replication in Fc?R-activated macro-
phages and enhanced HIV-1 infection in unstimulated macrophages by increasing reverse transcript and
integrated DNA levels. p21 induction by other stimuli, such as phorbol myristate acetate and the histone
deacetylase inhibitor MS-275, was also associated with preintegrative blocks of HIV-1 replication in macro-
phages. Binding of p21 to reverse transcription/preintegration complex-associated HIV-1 proteins was not
detected in yeast two-hybrid, pulldown, or coimmunoprecipitation assays, suggesting that p21 may affect viral
replication independently of a specific interaction with an HIV-1 component. Consistently, p21 silencing
rescued viral replication from the Fc?R-mediated restriction also in simian immunodeficiency virus SIVmac-
and HIV-2-infected macrophages. Our results point to a role of p21 as an inhibitory factor of lentiviral
infection in macrophages and to its implication in Fc?R-mediated restriction.
Macrophages are targets of human immunodeficiency virus
(HIV) infection and play crucial roles in viral dissemination
and pathogenesis (23, 24, 70). HIV-infected macrophages con-
tribute to HIV spread to CD4 T lymphocytes and to the es-
tablishment of cellular virus reservoirs (2, 25, 48, 60). Identi-
fication of the mechanisms controlling HIV-1 replication in
macrophages may lead to new therapeutic strategies.
Microenvironmental stimuli can both enhance and inhibit
HIV-1 replication in macrophages (29). Several cellular factors
have been suggested to restrict HIV-1 infection in undifferen-
tiated monocytes or to reduce macrophage permissivity to in-
fection, including members of the APOBEC3 cytidine deami-
nase and TRIM families, the small isoform of the transcription
factor C/EBP? (CCAT enhancer-binding protein ?), PPAR
(for peroxisome proliferator-activated receptor), and more
recently, microRNAs (8, 51, 61, 64, 71, 74). Despite these
findings, no restriction factors that can be manipulated to
render macrophages resistant to HIV-1 replication have
We have previously shown that the engagement of activating
immunoglobulin G Fc receptors (Fc?R) by immune complexes
(IC) on monocyte-derived macrophages (hereafter called mac-
rophages) restricts HIV-1 reverse transcription and integra-
tion, whereas viral entry, nuclear import, and gene expression
from integrated proviruses are not inhibited (19, 52). Interest-
ingly, Fc?R-mediated inhibition is not limited to HIV-1 but
also includes target related lentiviruses such as HIV-2, simian
immunodeficiency virus (SIV)mac, and SIVagm, suggesting that
this may be a common mechanism of lentivirus control (19).
Engagement of activating Fc?R on macrophages triggers sig-
naling pathways, including phospholipase C, phosphatidylino-
sitol-3 kinase, and mitogen-activated protein kinase/extracel-
lular signal-regulated kinase (19). This leads to intracellular
calcium augmentation, cytoskeleton remodeling and phagocy-
tosis, as well as activation of transcription factors such as NF-
?B, NFAT, and AP-1 (21, 33). Therefore, Fc?R-mediated sig-
naling could affect postentry steps of HIV-1 replication by
modulating the expression of genes encoding molecules that
interfere with reverse transcription or genome integration
and/or by acting on the incoming virus through modifications
of the cellular environment.
The present study was designed to test these hypotheses. We
examined whether Fc?R aggregation by IC could modulate the
expression of host factors that can interfere with early posten-
try steps of HIV replication. We studied restriction factors of
the APOBEC3 and TRIM families (43, 63, 65), as well as host
proteins recruited in the HIV-1 reverse transcription and pre-
integration complexes (RTC/PIC). We found that the cyclin-
* Corresponding author. Mailing address: Institut Pasteur, Unite ´ de
Re ´gulation des Infections Re ´trovirales, 25 Rue du Docteur Roux,
Paris, France. Phone: 33-1-4568 8738. Fax: 33-1-4568 8957. E-mail:
?Published ahead of print on 16 September 2009.
dependent kinase inhibitor (CKI) p21Cip1/Waf1(hereafter re-
ferred to as p21) is induced by Fc?R aggregation. Interestingly,
p21 has been recently involved in the resistance to HIV infec-
tion in primitive hematopoietic cells (81). We show here that
p21 inhibits the replication of HIV-1 and related primate len-
tiviruses in macrophages.
MATERIALS AND METHODS
Monocyte-derived macrophages. Buffy coats from healthy HIV-seronegative
donors were obtained through the French blood bank (Etablissement Franc ¸ais
du Sang [EFS]) as part of the EFS-Institut Pasteur Convention. Written in-
formed consent was obtained from each donor to use the cells for clinical
research, in accordance with French law. Monocytes were isolated from buffy
coats and differentiated into macrophages as previously described (19). Briefly,
monocytes were separated from peripheral blood mononuclear cells by adher-
ence to plastic and then detached and cultured for 7 to 11 days in hydrophobic
Teflon dishes (Lumox; D. Dutscher) in macrophage medium (RPMI 1640 sup-
plemented with 200 mM L-glutamine, 100 U of penicillin, 100 ?g of streptomycin,
10 mM HEPES, 10 mM sodium pyruvate, 50 ?M ?-mercaptoethanol, 1% min-
imum essential medium, vitamins, 1% nonessential amino acids) supplemented
with 15% of human AB serum. For experiments, macrophages were harvested
and resuspended in macrophage medium containing 10% heat-inactivated fetal
calf serum. Macrophage purity was assessed by flow cytometry, based on side and
forward scattering and immunofluorescence staining. Cells obtained with this
method are 91 to 96% CD14?and express CD64, CD32, and CD16 Fc?R.
For Fc?R activation, macrophages were seeded in culture plates precoated
with immune complexes formed by dinitrophenyl-conjugated bovine serum al-
bumin (BSA-DNP) and anti-DNP. Briefly, the plates were coated with 0.1 mg of
BSA-DNP/ml for 2 h at 37°C, saturated with 1 mg of BSA/ml in phosphate-
buffered saline, and then incubated for 1 h at 37°C with 30 ?g of rabbit anti-DNP
antibodies (Sigma)/ml to form IC. All reagents were lipopolysaccharide (LPS)-
Cell treatment with MS-275 (Alexis) or MC 1568 (kindly provided by A. Mai
and L. Altucci) was performed by adding the reagents at the indicated concen-
trations to culture medium 24 h before infection.
Cell infection. For single-round infections we used HIV-1 particles containing
the luc reporter gene and pseudotyped with the VSV-G envelope protein (HIV-
1VSV-G) that permits HIV receptor-independent entry into cells. HIV-1VSV-G-
pseudotyped viruses were produced by transient cotransfection of HEK293T
cells with proviral pNL4-3Nef?Env?Luc?DNA and the pMD2 VSV-G ex-
pression vector. Supernatants containing pseudotyped viruses were harvested
48 h after transfection, passed through 0.45-nm-pore-size filters, and stored at
?80°C. The viral stocks were titrated on HeLa P4P cells by measuring luciferase
activity (relative light units per second), and HIV-1 p24 antigen was quantified
with a commercial enzyme-linked immunosorbent assay (ELISA) kit (Zeptome-
trix Corp.). Macrophages were seeded in untreated or IC-coated culture plates
(105cells/well in 96-well plates or 0.5 ? 106cells/well in 12-well plates) in
HIV-1VSV-Gsuspension (90 ng of p24 per 106cells) and infected by spinoculation
(1 h of centrifugation at 1,200 ? g at room temperature, followed by 1 h of
incubation at 37°C). In experiments with PCR detection of HIV DNA, viral
preparations were pretreated with DNase I (Roche Diagnostics) for 1 h at room
For productive infection we used the strains HIV-1Bal, SIVmac251, and HIV-
2GHpropagated in phytohemagglutinin-activated human PBMC. Culture super-
natants were collected at the times of peak p24 and p27 production, respectively.
Viral stocks were titrated on human CD4?T cells. p24 and p27 were measured
in the stocks and supernatants by using commercial ELISA kits (Zeptometrix
Corp.). In these experiments macrophages were infected by spinoculation with
0.1 or 0.05 50% tissue culture infective doses of HIV-1Balor SIVmac/106cells,
respectively. HIV-2GHwas used at 230 ng of p27 per 106cells. Culture super-
natants were harvested at various times after infection, and p24 and p27 were
measured by ELISA.
Quantification of HIV-1 cDNA by quantitative PCR (qPCR). Total DNA in
infected macrophages was purified 72 h postinfection (p.i.) by using a DNeasy kit
as recommended by the manufacturer (Qiagen). Cytoplasmic DNA was selec-
tively extracted with a mitochondrial/cytoplasmic viral DNA purification kit
(V-GENE) as recommended by the manufacturer. Briefly, macrophages were
collected in M-A lysis buffer and left for 5 min on ice in the presence of RNase.
Nuclei were pelleted by centrifugation at 1,500 rpm for 5 min at 4°C, the
cytoplasmic fraction was clarified by centrifugation at 5,000 rpm for 5 min at 4°C,
and then the DNA was recovered by phase separation. Quantitative real-time
PCR analysis of late (U5-Gag) forms of viral DNA and two long terminal repeat
(2-LTR) circles were carried on an ABI Prism 7000 sequence detection system as
previously described (19). Standards for U5-Gag amplification products were
generated by serial dilution of DNA extracted from HIV-1 8E5 cells containing
one integrated copy of HIV-1 per cell. 2-LTR copies were quantified from
standard curves generated by serial dilution of DNA extracted from CEM cells
infected with HIV-1NL4-3. Integrated HIV-1 DNA was quantified by real-time
Alu-LTR nested PCR using the primers and probes described elsewhere with
some modifications (19, 78). Briefly, the first round of amplification was per-
formed on a GeneAmp PCR system 9700 (Applied Biosystems). Integrated
HIV-1 sequences were amplified by using an Expand High Fidelity kit (Roche)
using two Alu primers (Alu F and Alu R) and an LTR primer extended with an
artificial tag sequence at the 5? end of the oligonucleotide (NY1R). Real-time
nested PCR was run on the ABI Prism 7000 system using 10 ?l of a 1/10 dilution
of the first-round PCR product as a template (primers NY2F and NY2R; probe
NY2Alu). The integrated HIV-1 DNA copy number was determined with ref-
erence to a standard curve generated by concurrent amplification of HeLa R7
Neo cell DNA (10). A nested PCR conducted in parallel without the Alu primers
in the first round gave a very weak background signal. The number of integrated
HIV-1 DNA copies was obtained by subtracting the copy number measured
without the Alu primers in the first round from the copy number measured in the
full reaction. The amount of viral DNA was normalized to the endogenous
reference gene albumin (for total DNA extracts) or to mitochondrial DNA (for
cytoplasmic DNA). Standard curves were generated by serial dilution of a com-
mercial human genomic DNA (Roche).
We used a previously described qPCR method to measure HIV-1 cDNA
degradation (79). Briefly, macrophages were transduced with strain HIV-1VSV-G
and refed with medium containing zidovudine (AZT) at 10 ?M 30 h after
infection. Total DNA from treated and untreated macrophages was extracted 30,
48, 72, and 96 h after infection. The number of cDNA molecules per cell treated
with AZT was divided by the number of cDNA molecules per untreated cells
(percentage of remaining cDNA).
siRNA transfection. Small interfering RNA (siRNA) duplexes for p21 were
obtained as follows: siRNAs n.9 and n.12 and the SMARTpool for p21 were
purchased from Dharmacon, and negative control siRNA was synthesized by
Qiagen from the sequence proposed by Zhang et al. (81). A p21-specific siRNA
sequence described by Zhang et al. (81) was also used in some experiments (not
shown). The SMARTpool for p53 from Dharmacon was used for p53 silencing.
Macrophages were plated in 12-well plates (0.5 ? 106cells/well) in 500 ?l of 10%
fetal bovine serum-supplemented medium or in 96-well plates (105cells/well) in
100 ?l of the same medium. siRNA transfection was then performed with
InterferIN (PolyPlus Transfection), according to the manufacturer’s instructions.
Briefly, the siRNA was diluted in OptiMEM medium and mixed with InterferIN
transfection reagent at a ratio of 0.15 nmol of siRNA/10 ?l of transfection
reagent. The siRNA reagent mixture was incubated for 10 min at room temper-
ature and then added dropwise into wells at a final concentration of 100 nM
siRNA. Macrophages were then incubated at 37°C for 24 h. The medium was
replaced with fresh 10% fetal bovine serum medium before infection. Cell lysates
were assayed for protein expression by Western blot and for mRNA expression
by reverse transcriptase qPCR (RT-qPCR) to determine the efficiency of gene
knockdown at the moment of infection.
RT-qPCR analysis. Total RNA from macrophages was extracted with the
RNeasy kit (Qiagen) and treated with DNase according to the manufacturer’s
instructions. RNA was quantified by GeneQuant (Amersham), and equal
amounts (1 ?g) were reverse transcribed with SuperScript II RT (Invitrogen).
We used custom RT2Profiler PCR arrays (SABiosciences) to quantify TRIM
transcrits, allowing us to detect TRIM5, TRIM11, TRIM19/PML, TRIM22,
TRIM26, TRIM31, and GAPDH (glyceraldehyde-3-phosphate dehydrogenase)
simultaneously. PCRs were performed with the RT2Realtime SYBR green PCR
mix (SABiosciences) according to the manufacturer’s instructions on a Light-
Cycler 480 (Roche Diagnostics). The amplification program consisted of 10 min
at 95°C, followed by 45 cycles at 95°C for 15 s and 60°C for 1 min. For other
transcripts, PCR amplification of cDNAs was carried out in duplicate in
MicroAmp Optical 96-well reaction plates (30 ?l/well), using 25 ?l of Taq-
Man Universal Master Mix, 0.2 mM TaqMan, and 1.5 ?l of Assays-on-
Demand gene expression assay premade mix (GAPDH, Hs99999905_m1;
p21, Hs00355782_m1; LEDGF/p75, Hs01045714_g1; BAF, Hs00427805_g1;
Ini1, Hs00996890_m1; Gemin2, Hs01031721_m1; p53, Hs00153349_m1; and p27,
Hs00153277_m1). The amplification conditions were as follows: 50°C for 2 min
and 95°C for 10 min, followed by 45 cycles at 95°C for 15 s and 60°C for 90 s, on
an ABI Prism 7700 sequence detector (Applied Biosystems). The data were
analyzed with the cycle threshold (CT) method, and the amount of target mRNA
in each sample was normalized to GAPDH mRNA as an endogenous reference.
12254 BERGAMASCHI ET AL.J. VIROL.
All results were expressed relative to unstimulated macrophages (nonactivated
control macrophages) as 2???CT, where ??CT? ?CT-sample– ?CT-controland
where ?CT? CT-target gene– CT-GAPDH.
Measure of proteasome activity. Macrophages were cultured in 96-well plates
with or without epoxomycin (50 nM) for 48 h. They were then washed once with
phosphate-buffered saline and lysed in 150 ?l/well of lysis buffer (10 mM
HEPES, 10 mM NaCl, 0.1 mM EDTA, 1 mM dithiothreitol, 1% Triton X-100).
The fluorogenic substrate Suc-LLVY-AMC was then added at 50 ?M to start the
proteolysis reaction. The mixture was incubated at 37°C for 2 h, and AMC
release was detected by measuring fluorescence emission at 450 nm (excitation
385 nm) with a Victor-2 fluorometer (Perkin-Elmer).
Western blot. Macrophages cultured in 12-well plates were lysed in 80 ?l of
M-PER lysis buffer (Pierce) containing Complete protease and phosphatase
inhibitor cocktail (Roche). Protein was quantified with the BCA kit (Pierce), and
samples were then diluted to 1 ?g/?l with Laemmli buffer, boiled at 95°C for 5
min, and loaded in NuPAGE gel 4 to 12% (Invitrogen) for electrophoretic
separation. Proteins were then blotted onto Immobilon-P membranes (Milli-
pore). After blocking with 5% skimmed milk, the membranes were incubated
with the primary antibodies as indicated, followed by secondary horseradish
peroxidase-conjugated anti-rabbit or anti-mouse antibodies (Sigma). The pro-
teins were revealed on Hyperfilms (Amersham) by using the ECL chemilumi-
nescent substrate (GE Healthcare) and X-Omat films (Kodak). The anti-p21
mouse monoclonal antibody (1:500) was purchased from Santa Cruz, anti-
GAPDH (1:5,000) was from Abcam, and mouse monoclonal anti-?-actin (1:
2,000) was obtained from Sigma.
Plasmid construction. (i) Two-hybrid expression vectors. The complete cDNA
of human p21 was ligated into the yeast two-hybrid prey vector pGad-GE, while
the cDNA of proliferating cell nuclear antigen (PCNA) was cloned into the bait
vector pLex10. Sequences encoding the HIV-1 proteins matrix p17 (MA), inte-
grase (IN), Vpr, and RT p66 (RT) were fused to the LexA DNA-binding domain
(LexABD) of the pLex10 vector.
(ii) Mammalian expression vectors. p21 was also ligated into the mammalian
glutathione S-transferase (GST)-tagged expression vector pCMV-GST
(GeneCopoeia). Vectors for the expression of hemagglutinin (HA)-tagged PCNA,
IN, MA, and Vpr were constructed by inserting the corresponding cDNA into the
pAS1b vector, as described elsewhere (59).
Pulldown assay. HeLa cells were seeded in 10-cm-diameter plates at a density
of 1.5 ? 106cells/plate the day before transfection. Transfection was performed
with the Lipofectamine reagent (Invitrogen) and 4 ?g of plasmid according to
the manufacturer’s instructions. Cells were then cultured for 48 h before being
lysed for 10 min on ice in a buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM
NaCl, 0.5% Triton X-100, and an anti-protease cocktail (Sigma). Equal quanti-
ties of lysate were incubated with 25 ?l of glutathione-Sepharose beads (vol/vol)
for 1 h at 4°C. The beads were extensively washed in lysis buffer and resuspended
in 4? LDS sample buffer (Invitrogen). Samples were loaded onto NuPAGE
Bis-Tris gels (Invitrogen) and then blotted onto a nylon membrane (Hybond-P;
GE Healthcare). The membrane was saturated for 1 h at room temperature with
5% nonfat dry milk in Tris-buffered saline containing 0.5% Tween 20 and then
with the primary antibody (anti-HA [clone3F10 from Roche] or anti-GST [clone
GST-2 from Sigma]) for 1 h in the blocking solution. The membrane was then
incubated with a horseradish peroxidase-conjugated secondary antibody in Tris-
buffered saline–Tween, and proteins were detected with an ECL kit (GE Health-
Yeast two-hybrid assay. The yeast reporter strain L40 containing the two
LexA-inducible genes HIS3 and LacZ was cotransformed with the indicated
LexABD and Gal4AD hybrid expression vectors. Cotransformed yeasts were
plated on selective medium lacking tryptophan and leucine. Double transfor-
mants were patched on the same medium and replica plated on selective medium
lacking tryptophan, leucine, and histidine for auxotrophy analysis and on What-
man 40 filters for ?-galactosidase (?-Gal) activity assay.
Statistical analyses. Analyses were performed by using the Mann-Whitney test
and the Wilcoxon signed-rank test.
Fc?R-mediated restriction of HIV-1 replication does not
depend on increased degradation of incoming virus products.
We have previously reported that macrophage stimulation by
IC inhibits the accumulation of both viral reverse transcripts
and integrated forms after HIV-1 infection (19). In this previ-
ous study, quantification of viral cDNA was performed on
whole-cell DNA extracts, and we were thus unable to precisely
evaluate the degree of inhibition of reverse transcription be-
fore nuclear import of the viral cDNA. Here, we monitored the
accumulation of newly synthesized viral cDNA in the cyto-
plasm of IC-activated and unstimulated macrophages in single-
round infections with HIV-1VSV-G.Full-length HIV-1 cDNA
was measured by qPCR in purified cytoplasmic fractions, by
comparison with total cell extracts. Viral cDNA accumulated
in unstimulated macrophages, reaching maximal levels at 72 h
in the cytoplasmic fraction and still increasing at 96 h in the
whole-cell extract (Fig. 1A). As expected, HIV-1 cDNA levels
were strongly decreased in total extracts of IC-activated mac-
rophages (70% reduction at 72 and 96 h compared to unstimu-
lated macrophages). A substantial reduction in HIV-1 cDNA
was also observed in the cytoplasmic fraction of IC-activated
macrophages (50% reduction at 72 h in comparison with un-
stimulated macrophages) (Fig. 1A). This confirms that a major
block occurs during the reverse transcription process.
The reduction in viral cDNA in the cytoplasm of IC-acti-
vated macrophages could result from enhanced degradation of
incoming viral products, i.e., newly synthesized cDNA or viral
proteins. We first compared the rate of degradation of HIV
cDNA in unstimulated and IC-activated macrophages by de-
termining HIV-1 cDNA stability after treatment with an RT
inhibitor. Macrophages were infected with HIV-1VSV-G, and
AZT was added to the medium 30 h later in order to block
further accumulation of reverse transcripts. The viral cDNA
level showed a similar pattern of decline in IC-stimulated mac-
rophages and in unstimulated macrophages (Fig. 1B). We then
investigated whether IC stimulation could induce an increased
degradation of incoming viral proteins by the proteasome,
which is the main proteolytic complex operating in the cytosol
(58, 73). The catalytic activity of the proteasome was evaluated
by measuring hydrolysis of the fluorogenic peptide Suc-LLVY-
MCA added to macrophage cell lysates. Degradation of the
fluorogenic substrate was not significantly different between
IC-stimulated and control macrophages (Fig. 1C), indicating
that Fc?R-mediated activation does not modulate proteasome
activity. In the presence of epoxomycin, a selective irreversible
inhibitor of chymotrypsinlike proteasome activity the catalytic
capacity of the proteasome was strongly reduced (80%) in both
activated and control macrophages (Fig. 1C), whereas cell vi-
ability measured at the same time was not affected (data not
shown). Epoxomycin treatment of HIV-1VSV-G-infected mac-
rophages did not restore the loss of HIV-1 reverse transcrip-
tion products and integrated forms (?55% and ?84%, respec-
tively) in IC-stimulated macrophages (Fig. 1D). Accordingly,
HIV gene expression, reflected by luciferase activity in cell
extracts, was not increased by epoxomycin in either IC-stimu-
lated or control macrophages (data not shown). Altogether,
these results strongly suggest that the decrease in viral cDNA
induced by Fc?R aggregation is not caused by increased deg-
radation of reverse transcripts or viral proteins.
Fc?R engagement triggers p21 gene expression. To deter-
mine whether Fc?R engagement can induce factors that have
been implicated in the restriction of early postentry steps of
HIV-1 replication we examined gene expression of members of
the APOBEC3 and TRIM families, including APOBEC3A,
APOBEC3F, APOBEC3G, TRIM5, TRIM11, TRIM19/PML,
TRIM22, TRIM26, and TRIM31 (4, 6, 30, 41, 50, 67). We
VOL. 83, 2009 p21-MEDIATED HIV RESTRICTION IN MACROPHAGES12255
measured their expression levels by RT-qPCR in IC-stimulated
macrophages in comparison with unstimulated macrophages
from three different donors. The target mRNA levels in
IC-activated macrophages were normalized relative to the
GAPDH mRNA level in each sample and were expressed as
relative levels compared to unstimulated macrophages (Fig.
2A and B). APOBEC3A mRNA was very low or undetectable
in unstimulated macrophages (data not shown) and its ex-
pression was not increased by IC stimulation (Fig. 2A).
APOBEC3G and APOBEC3F mRNAs expression were down-
regulated by IC stimulation (Fig. 2A). None of the TRIM
genes was upregulated by IC stimulation, and TRIM11,
TRIM26, and TRIM22 expression was downregulated (Fig.
2B). Therefore, an increased expression of these restriction
factors cannot account for the Fc?R-mediated HIV-1 restric-
Alteration of host cell components of RTC/PIC may affect
the formation or the stability of these complexes and thereby
have a negative impact on viral replication (22, 31). We there-
fore measured the expression of host factors associated with
the RTC/PIC that might interfere with either reverse transcrip-
tion or integration, including lens epithelium-derived growth
factor (LEDGF)/p75 (12, 40), integrase interactor 1 (Ini1) (34,
45), barrier-to-autointegration factor (BAF) (39), Gemin2
(27), and p21(81), after IC stimulation. Although variations in
basal gene expression were observed among the donors, IC
FIG. 1. Degradation of incoming viruses does not account for the
defective HIV-1 reverse transcription. (A) Macrophages plated in un-
treated (unstimulated, US) or IC-coated plates (stimulated, S) were
infected with HIV-1VSV-G. Late reverse transcription products (U5-
Gag) in total DNA extracts or in cytoplasmic fractions of infected
macrophages were analyzed by qPCR at 5, 24, 72, and 96 h p.i. U5-Gag
copies in each sample were normalized to mitochondrial DNA.
(B) Unstimulated (US) or IC-simulated (S) macrophages were in-
fected with HIV-1VSV-G. At 30 h p.i., the medium was replaced, and 10
?M AZT was added. The number of U5-Gag copies in infected cells
was measured by qPCR at the indicated times, and the amount of
remaining viral DNA (%) was calculated as the number of U5-Gag
copies in AZT-treated macrophages divided by the number of U5-Gag
copies in untreated macrophages. The error bars are the standard
deviation of triplicate values of a representative experiment (n ? 3).
(C) Proteasome activity was measured in total cell extracts of unstimu-
lated (US) or IC-simulated (S) macrophages treated or not with 50 nM
epoxomycin, as described in Materials and Methods. (D) Unstimulated
or IC-simulated macrophages, treated or not treated with epoxomycin,
were infected with HIV-1VSV-G. U5-Gag, and integrated forms of
HIV-1 (Alu-LTR) were quantified 72 h p.i. by qPCR. Viral cDNAs
were normalized to the albumin gene content in each sample. The
error bars indicate the standard deviations of triplicate values of a
representative experiment (n ? 3).
FIG. 2. Fc?R aggregation induces p21 mRNA expression. (A and
B) APOBEC3 and TRIM gene expression was measured in macro-
phages after 24 h of IC activation. Total RNA from macrophages was
extracted and reverse transcribed, and APOBEC3 and TRIM expres-
sion was analyzed by RT-qPCR. The data are means ? the standard
deviations of results obtained with macrophages from three different
donors. (C) Macrophages were stimulated with IC, and after 24 h the
total RNA was analyzed by RT-qPCR. The LEDGF/p75, Ini1,
Gemin2, BAF, and p21 expression levels were normalized to the
GAPDH gene and are presented relative to unstimulated macro-
phages (LEDGF/p75, P ? 0.08; p21, P ? 0.04, Wilcoxon signed-rank
test). All results are expressed relative to unstimulated macrophages,
as 2???CT, where ??CT? ?CT-sample– ?CT-controland ?CT?
CT-target gene– CT-GADPH. The data are means ? the standard devia-
tions of results obtained with macrophages from five different donors.
12256 BERGAMASCHI ET AL.J. VIROL.
stimulation induced no significant change in the expression of
Ini1, Gemin2, or BAF relative to control macrophages (Fig.
2C). LEDGF/p75 mRNA expression was slightly reduced in
IC-activated macrophages from four out of five donors, but the
difference did not reach significance (P ? 0.08). In contrast,
p21 expression was significantly upregulated by IC in all of the
donors (P ? 0.04, Fig. 2C).
Fc?R engagement upregulates p21 specifically and irrespec-
tive of p53 modulation. We then examined the impact of IC
stimulation on p21 protein expression compared to LPS stim-
ulation that also inhibits early steps of HIV-1 replication by
reducing reverse transcription (83) but via signaling pathways
different from those activated by Fc?R engagement. We found
that p21 protein was strongly induced by IC stimulation (Fig.
3A). In contrast, LPS reduced p21 expression (Fig. 3A). Mac-
rophage infection with HIV-1VSV-Gdid not further modulate
p21 expression in either IC-stimulated or unstimulated mac-
rophages (Fig. 3A). p21 induction by IC stimulation was con-
centration dependent (Fig. 3B), and the protein level was
clearly increased 6 h after IC stimulation and then accumu-
lated during the time of monitoring (Fig. 3C).
Usually, p21 expression is transcriptionally regulated by p53
(20), although it can be modulated by p53-independent mech-
anisms (7). We therefore examined the effect of IC stimulation
on p53 expression. We also examined whether IC stimulation
could induce other members of the Cip/Kip family of CDK
inhibitors, to which p21 belongs, such as p27Kip1(5). We mea-
sured p53 and p27Kip1transcript levels in IC-stimulated mac-
rophages from four different donors, in parallel with p21. In
contrast to p21, neither p53 nor p27 mRNA levels were in-
creased by IC stimulation (Fig. 3D), and they were even down-
regulated in some donors, suggesting that Fc?R engagement
induces p21 specifically and irrespective of p53 modulation. To
gain further insight on the role of p53 in the Fc?R-mediated
induction of p21, we knocked down p53 expression in macro-
phages by siRNA transfection. We then analyzed the effect of
p53 reduction on p21 expression in unstimulated macrophages
and in macrophages activated by IC. In siRNA-untreated mac-
rophages, IC stimulation resulted in an induction of p21 and a
decrease of p53 expression (a representative example of ex-
periments with macrophages from three donors is shown in
Fig. 3E). At 24 h posttransfection with specific siRNA, p53
mRNA levels were reduced of 51 and 49% in unstimulated and
IC-stimulated macrophages, respectively, compared to macro-
phages transfected with nonspecific siRNA (Fig. 3E left). p53
silencing led to a decrease in p21 expression both at mRNA
and protein levels (Fig. 3E middle, right). However, p21 in-
duction by IC was not modified by p53 silencing: the p21
FIG. 3. Fc?R aggregation induces p21 protein expression specifically and irrespective of p53 expression. (A) Uninfected and HIV-1VSV-G-
infected macrophages were either left untreated (US) or stimulated with IC or LPS (100 ng/ml) for 48 h before total protein extraction. Proteins
were separated by electrophoresis, and p21 was revealed with a monoclonal anti-p21 antibody. ?-Actin was used as a control. (B) Macrophages
were activated with increasing concentrations of IC (3 ?g of anti-DNP/ml was added to wells coated with 0 to 30 ?g of DNP-BSA/ml). Total cell
lysates were extracted 48 h later and analyzed by electrophoresis and Western blotting to reveal the p21 protein. (C) Macrophages were stimulated
with IC and collected at the times indicated. p21 protein was then analyzed by Western blotting as reported above. (D) Macrophages were
stimulated with IC, and total RNA was extracted 24 h later, reverse transcribed, and analyzed by RT-qPCR to determine the mRNA levels of p21,
p27Kip1, and p53. Values were normalized to the GAPDH gene and are reported relative to unstimulated macrophages. Macrophages from four
different donors were analyzed. (E) Macrophages were transfected with p53-specific siRNA or a scrambled siRNA (si-Neg) or were mock treated
(Ctrl) and seeded in the presence (S) or absence (US) of IC. After 24 h, p53 and p21 mRNAs were quantified in each sample by RT-qPCR,
normalized to GAPDH, and expressed relative to US control macrophages (left and center). Proteins were analyzed by Western blotting. p21 and
GAPDH (control) were detected with specific monoclonal antibodies (right). Consistent results were obtained with macrophages from three donors.
VOL. 83, 2009 p21-MEDIATED HIV RESTRICTION IN MACROPHAGES12257
mRNA in IC-stimulated macrophages was increased 2.4- and
2.6-fold, respectively, after p53 and nonspecific siRNA trans-
fection with a corresponding increase in protein levels (Fig. 3E,
middle, right). With the caution that p53 knockdown was
not complete, these results suggest that p21 expression is
partially regulated by p53 both in unstimulated and in IC-
stimulated macrophages, but other pathways may contribute
to its induction by IC.
Fc?R-mediated induction of p21 restricts HIV-1 replication
in macrophages. To determine whether p21 expression exerts
antiviral activity in macrophages, we knocked down p21 ex-
pression and monitored HIV-1 replication in p21 silenced mac-
rophages. Transfection with specific siRNAs reduced both
mRNA and protein levels of p21 in both unstimulated and
IC-activated macrophages (Fig. 4A). p21 mRNA and protein
levels consistently remained slightly higher in siRNA-treated
IC-stimulated macrophages than in siRNA-treated unstimu-
lated macrophages, suggesting an increase in their stability in
activated macrophages (Fig. 4A). Unstimulated p21 knock-
down macrophages were infected with HIV-1VSV-G24 h after
siRNA transfection, and the luciferase activity was then mea-
sured at various times. p21 silencing enhanced HIV-1 replica-
tion (Fig. 4B). Compared to cells treated with nonspecific
siRNA, the median increase in luciferase activity in p21 si-
FIG. 4. p21 silencing enhances HIV-1 replication in macrophages. (A) Macrophages were seeded in the presence (S) or absence (US) of IC
and immediately transfected with p21-specific siRNA duplexes n.9 and n.12 or SMARTpool for p21 (Dharmacon), or a scrambled siRNA (si-Neg)
or were mock treated (Ctrl). Cells were cultured for 48 h and then lysed. p21 mRNA was quantified in each sample by RT-qPCR, normalized to
GAPDH, and expressed relative to US control macrophages (left). Proteins were analyzed by Western blotting. p21 and GAPDH (control) were
detected with specific monoclonal antibodies (right). (B) Unstimulated macrophages were transfected with p21 si-RNA or an irrelevant siRNA
(si-Neg) and infected with HIV-1VSV-G24 h later. The luciferase activity was measured at 24, 48, and 72 h p.i. The data are means ? the standard
deviations of triplicate wells. (C) Macrophages were transfected with a p21-specific siRNA or an irrelevant siRNA (si-Neg) in the presence (S) or
absence (US) of IC. Cells were infected with HIV-1VSV-G24 h after siRNA transfection. At 72 h p.i. the cells were lysed for luciferase assay and
RNA extraction. Consistent results were obtained with macrophages from five different donors. (D) Macrophages were seeded in the presence
(S) or absence of IC (US) and were transfected with p21-specific siRNA duplexes (si-p21) or mock treated (Ctrl). At 24 h after siRNA transfection,
the macrophages were infected with HIV-1Bal, and p24 capsid protein was quantified in the supernatants at the indicated times. Consistent results
were obtained with macrophages from three donors.
12258 BERGAMASCHI ET AL. J. VIROL.
lenced macrophages from five donors was 4.8-fold (range, 1.3
to 14.9, P ? 0.016). Remarkably, p21 silencing also rescued
HIV-1 replication in IC-stimulated macrophages to the levels
seen in siRNA-untreated unstimulated macrophages (Fig. 4C).
Compared to macrophages treated with control siRNA, the
median increase of luciferase activity in IC-activated macro-
phages from five donors was 4.5-fold (range, 3.3 to 19.6, P ?
0.016). Similar results were obtained using three different se-
quences of p21-specific siRNAs for p21 depletion (data not
To determine the impact of p21-mediated restriction on
productive HIV-1 infection, we treated unstimulated and IC-
stimulated macrophages with p21 siRNA or control siRNA
and then infected them with HIV-1Baland monitored super-
natant p24 levels. HIV-1Balinfection was strongly suppressed
in IC-activated macrophages (2-log reduction on day 14), and
p21 silencing rescued HIV-1 replication by 1.5 log in these
macrophages (Fig. 4D). A small and transient increase (0.2
log) was observed in p21 siRNA-treated unstimulated macro-
phages on day 4 p.i., suggesting that p21 silencing in unstimu-
lated macrophages that have low levels of p21 expression and
are permissive to HIV-1 infection may not have significant
effects on multiple cycles of infection. Nonspecific siRNA did
not affect either p21 expression or HIV-1 replication in either
unstimulated or IC-stimulated macrophages (data not shown).
Together, these results strongly suggest that p21 is a limiting
factor for HIV-1 replication in macrophages and that it largely
accounts for Fc?R-mediated HIV-1 inhibition.
p21 restricts HIV-1 reverse transcription and integration.
To go further in the characterization of the mechanisms of
HIV-1 restriction mediated by p21 in macrophages, we inves-
tigated which steps of HIV-1 replication are affected by p21.
We measured reverse transcripts and integrated DNA by
qPCR in unstimulated and IC-activated p21 knockdown mac-
rophages infected with HIV-1VSV-G. p21 silencing increased
the level of late transcription products in both unstimulated
and IC-stimulated macrophages (by two- and fourfold, respec-
tively, in the experiment shown in Fig. 5A). The integrated
forms of HIV-1 increased 2.6-fold in unstimulated macro-
phages and were rescued in activated macrophages, from an
undetectable level to levels higher than those in untreated
unstimulated macrophages (Fig. 5B). These results therefore
indicate that p21 affects the same steps of HIV-1 replication as
those restricted by Fc?R engagement in macrophages.
p21 induction in macrophages by other stimuli is also as-
sociated with reduced permissivity to HIV-1 infection. To as-
sess whether the effect of p21 on HIV-1 replication was specific
to Fc?R-mediated restriction, we induced p21 expression in
macrophages by treatment with phorbol myristate acetate
(PMA) and the histone deacetylase (HDAC) inhibitor MS-
275, both of which have been reported to induce p21 (54, 56,
57). As expected, PMA increased p21 expression in macro-
phages (Fig. 6A, inset), and treatment of macrophages with
PMA before HIV-1VSV-Ginfection strongly inhibited viral rep-
lication, as reflected by luciferase activity decrease (Fig. 6A).
Of note, we have previously shown that PMA treatment after
macrophage infection, when HIV-1 integration is completed,
leads to an enhancement of viral gene expression, owing to
stimulation of HIV-1 transcription (55). These results suggest
that the viral inhibition caused by PMA treatment before in-
fection occurs at a preintegration step.
Treatment of macrophages with increasing concentrations of
MS-275 increased p21 expression by up to fourfold compared
to untreated macrophages in a concentration-dependent man-
ner (Fig. 6B). Remarkably, when MS-275-treated macrophages
were infected with HIV-1VSV-G, viral replication fell as p21
expression rose (Fig. 6B). Since HDAC inhibitors have several
effects on cell biology and may thus affect HIV-1 replication
next to their effect on p21, we also used a class II HDAC
inhibitor, MC 1568 (42, 47) that does not induce p21. Macro-
phage treatment with MC 1568 did not upregulate p21 and did
not affect HIV-1 replication (Fig. 6C). MC 1568 activity in
macrophages was assessed by measuring the acetylated tubulin
(Fig. 6C, inset). Cell viability was not reduced by the chosen
concentration ranges of either MS-275 or MC 1568 (data not
shown). The association between p21 upregulation by different
stimuli and reduced permissivity to HIV-1 further supports a
negative effect of p21 on HIV-1 replication in macrophages.
Since the inhibition of HIV-1 replication by MS-275 may
affect different steps from those targeted upon Fc?R engage-
ment, we measured HIV-1 cDNA in MS-275-treated macro-
phages at 96 h after HIV-1 infection. MS-275 caused a dose-
dependent reduction of viral cDNA, concomitant to p21
increase, corroborating evidence for the implication of p21 in
a preintegration block (Fig. 6D).
FIG. 5. p21 restricts HIV-1 reverse transcription and integration in
macrophages. Macrophages were transfected with a p21-specific
siRNA or an irrelevant siRNA (si-Neg) in the presence (S) or absence
(US) of IC. Cells were infected with HIV-1VSV-G24 h after siRNA
transfection. Late reverse transcription products (U5-Gag) (A) and
integrated forms (Alu-LTR) (B) were quantified by qPCR in DNA
extracted from infected macrophages at 72 h p.i. The data were nor-
malized to the albumin gene. These results are from the experiment
shown in Fig. 4C. The error bars represent standard deviations of
triplicate wells. Similar data were obtained with macrophages from
VOL. 83, 2009 p21-MEDIATED HIV RESTRICTION IN MACROPHAGES12259
Interactions between p21 and viral proteins of the HIV-1
RTC/PIC were not detected by yeast two-hybrid and pulldown
assays. Coimmunoprecipitation assays in human megakaryo-
blastic leukemia and ACH2 cell lines have suggested that p21
is associated with the HIV-1 PIC (75, 81). To determine
whether p21 could inhibit preintegration steps of HIV-1 rep-
lication by direct interaction with viral proteins of the RTC/
PIC we used a yeast two-hybrid assay to test p21 binding to
HIV-1 IN, MA, RT, and viral protein R (Vpr). p21 protein was
fused to Gal4AD and tested for interactions with PIC-associ-
ated viral proteins fused to LexABD in the L40 yeast strain,
which contains the two LexA-inducible reporter genes LacZ
and HIS3. We used LexA-PCNA fusion as a positive control,
since the interaction with p21 has been previously reported
using a similar system (72). None of the tested viral proteins
reacted with p21, as revealed by the absence of growth on
FIG. 6. PMA and the HDAC inhibitor MS-275 induce p21 expression and inhibit HIV-1 replication in macrophages. (A) Macrophages were
treated with PMA (30 and 100 ng/ml) and infected with HIV-1VSV-G. The luciferase activity was measured 72 h p.i. For the inset, macrophages
were treated with PMA (30 ng/ml) for 48 h before lysis and Western blot detection of p21. ?-Actin was used as a control. (B and C) Macrophages
were treated with the HDAC inhibitors MS-275 (B) or MC 1568 (C) at the indicated concentrations for 24 h and then infected with HIV-1VSV-G.
Total RNA was extracted at 48 h after treatment and p21 mRNA expression was measured by RT-qPCR. Values were normalized to the GAPDH
gene and expressed relative to untreated macrophages. The luciferase activity in cell lysates of infected macrophages was measured at 72 h p.i. The
data are means ? the standard deviations of triplicate wells. Similar data were obtained with macrophages from three donors. (D) Macrophages
were treated with MS-275 at the indicated concentrations for 24 h and then infected with HIV-1VSV-G. Total RNA was extracted at 48 h after
treatment, and p21 mRNA expression was measured by RT-qPCR. Values were normalized to the GAPDH gene and are expressed relative to
untreated macrophages. Total HIV-1 DNA was measured 96 h p.i. in DNA extracts from infected macrophages and normalized to the albumin
gene. (C) Macrophages were treated with the indicated concentrations of MC 1568. At 24 h after treatment, macrophages were lysed, and the
amount of acetylated tubulin was measured by Western blotting. ?-Actin protein was used as a control.
12260 BERGAMASCHI ET AL.J. VIROL.
medium without histidine (?His) and expression of ?-Gal
activity (Fig. 7A). The fusion Gal4AD-p21 protein was effi-
ciently expressed, since it yielded positive signals, growth on
medium without histidine, and expression of ?-Gal activity in
the presence of the LexA-PCNA fusion protein. Expression of
the viral proteins in yeast cells was checked by using specific
partners of each viral protein (data not shown). We confirmed
the lack of p21 interaction with HIV-1 proteins by using a
Gal4BD-p21 fusion protein and Gal4AD-fused IN, MA, and
Vpr (not shown).
We further investigated the possible p21 interaction with
HIV-1 proteins in pulldown assays after coexpression of each
viral protein with GST-p21 in HeLa cells. GST-tagged proteins
(GST alone or GST-p21) were precipitated from cell lysates
with glutathione-Sepharose beads, and the precipitates were
analyzed by Western blotting with an anti-HA antibody. MA,
IN (Fig. 7B, lanes 4 and 5), Vpr (Fig. 7C, lane 9), and RTp66
(not shown) were not precipitated with GST-p21, whereas
PCNA was efficiently precipitated (Fig. 7B, lane 6, and Fig. 7C,
lane 10). Analysis of cellular lysates indicated that all of the
proteins (MA, IN, Vpr, RTp66, and PCNA), were correctly
expressed in the transfected cells (Fig. 7B and C, bottom pan-
els, and results not shown). Moreover, no signal was detected
after coimmunoprecipitation of Flag-tagged IN or Vpr and
HA-tagged p21 coexpressed in HeLa cells, whereas p21 was
immunoprecipitated with Flag-PCNA (data not shown).
Therefore, in keeping with the yeast two-hybrid assay results,
we detected no interaction between p21 and IN, Vpr, or MA in
pulldown assays nor between p21 and IN or Vpr in coimmu-
noprecipitation experiments. These results suggest that p21
may interfere with RTC/PIC functions independently of a spe-
cific interaction with HIV-1 proteins.
p21-mediated inhibition affects HIV-1-related primate len-
tiviruses in macrophages. To determine whether p21-medi-
ated restriction was or not virus specific, we examined the
effect of p21 silencing on the replication of HIV-1 related
lentiviruses SIVmacand HIV-2. Macrophages were transfected
with p21-specific siRNAs and then challenged with SIVmac251
or and HIV-2GH24 h later. The levels of viral replication,
evaluated by measuring supernatant p27 levels at day 7 p.i.,
were reduced by 80 and 48% for SIVmacand HIV-2, respec-
tively, in IC-activated macrophages (Fig. 8). p21 silencing
increased SIVmacand HIV-2 replication 3.4- and 2.8-fold,
respectively, in IC-activated macrophages compared to mac-
rophages treated with nonspecific RNA (Fig. 8). Thus,
Fc?R-mediated inhibition of SIVmacand HIV-2 was sub-
stantially reversed by p21 silencing.
FIG. 7. p21 protein interaction with viral components of the HIV-1 PIC is not detected in yeast two-hybrid or in vitro. (A) Two-hybrid assay
between p21 and viral components of the PIC. The yeast reporter strain L40, expressing the indicated pairs of hybrid proteins, was plated in
medium with histidine (?His) or without histidine (?His) or replica plated on Whatman filters and tested for ?-Gal activity (?-Gal). Growth in
the absence of histidine and development of a blue color in the ?-galactosidase assay both indicate interaction between hybrid proteins. (B and
C) In vitro binding analysis of the interaction between p21 and viral proteins of the PIC. HeLa cells were cotransfected with 2 ?g of plasmids for
expression of GST (lanes 1 to 3, 7, and 8), GST-p21 (lanes 4 to 6, 9, and 10), HA-tagged MA (lanes 1 and 4), IN (lanes 2 and 5), Vpr (lanes 7
and 9), or PCNA (lanes 3, 6, 8, and 10). Lysates of transfected cells were incubated with equal amounts of glutathione-Sepharose beads. Bound
proteins and cell lysates (3% of the total cellular extract) were resolved by NuPAGE 10% Bis-Tris gel and immunoblotted with anti-HA or
anti-GST. Protein markers are shown in kilodaltons on the left (PageRuler; Fermentas).
VOL. 83, 2009p21-MEDIATED HIV RESTRICTION IN MACROPHAGES 12261
Together, these results show that p21 inhibits not only
HIV-1 but also other primate lentivirus replication in macro-
phages restricting preintegration steps of viral cycle, a finding
consistent with its implication in Fc?R-mediated restriction
We have previously shown that IC activation of human mac-
rophages through Fc?Rs inhibits the replication of HIV-1 and
other primate lentiviruses, reducing both reverse transcript
and provirus levels (19). We show here that this antiviral ac-
tivity involves the CDK inhibitor p21. The main findings that
support a role of p21 in Fc?R-mediated lentiviral restriction
are as follows: (i) the inhibition of HIV-1 replication induced
by IC activation of macrophages was accompanied by in-
creased p21 mRNA and protein expression and (ii) siRNA
silencing of p21 rescued HIV-1, SIV, and HIV-2 replication in
IC-activated macrophages by increasing reverse transcript and
integration levels in IC-activated macrophages. Our results
also suggest that p21 is a limiting factor to HIV infection in
macrophages. Its depletion enhanced HIV-1 replication not
only in IC-activated macrophages but also in unstimulated
macrophages. In addition, p21 induction in macrophages by
different stimuli, including PMA and the HDAC inhibitor MS-
275, was associated with preintegration restrictions of HIV-1
replication. The degree of viral inhibition exerted by p21 in
macrophages depends on its intracellular concentration and
might thus vary according to macrophage cellular microenvi-
ronments in body tissues, including cytokine patterns, and
other stimuli (76).
p21 belongs to the Cip/Kip family of CKIs (26, 28, 77).
Although it was first described as a cell cycle inhibitor, blocking
cell cycling at the G1/S interface and playing a critical role in
the control of cell growth, p21 has also been shown to be
involved in the regulation of apoptosis and differentiation (16,
62). It has been reported that p21 exerts a protective role
against apoptosis in macrophages and that the antiapoptotic
activity of p21 in monocyte differentiation is determined by its
cytoplasmic localization (3, 76). In fact, the activities of p21
depend on the cell type, its subcellular (nuclear or cytoplasmic)
location, and its expression level and phosphorylation status (3,
13, 46). p21 expression is regulated by both p53-dependent and
p53-independent mechanisms (20, 82). The increase in p21
expression induced by Fc?R cross-linking in macrophages was
not accompanied by an induction of p53, since p53 expression
was either unaffected or downregulated by IC stimulation.
siRNA-mediated p53 silencing decreased p21 expression in
both unstimulated and IC-activated macrophages but did not
block p21 induction by IC. Altogether, these results suggest
that while p21 expression in macrophages is modulated by p53,
other pathways may contribute to its induction by Fc?R cross-
Fc?R cross-linking activates several signaling pathways in
macrophages, including PKC and ERK1/2 (19), both of which
have been implicated in PMA induction of p21 in myeloid cells
(18, 49, 57). Although further studies are needed to precisely
identify the signals involved in p21 induction by IC, they are
likely to occur at the transcriptional level, as p21 mRNA ex-
pression increased after IC stimulation. In addition to tran-
scriptional activation, stabilization of p21 mRNA and/or pro-
tein may contribute to the IC-mediated enhancement of p21
expression (32, 49) since, after p21 siRNA treatment, p21
mRNA and protein levels remained higher in IC-activated
macrophages than in unstimulated macrophages. In keeping
with this observation, p21 silencing did not restore HIV-1 gene
expression or cDNA levels in IC-activated macrophages to the
levels achieved by p21 silencing in unstimulated macrophages
(Fig. 4C and 5A). We cannot, however, rule out the possibility
that the residual inhibition of HIV-1 replication observed in
IC-stimulated macrophages after p21 silencing was due to ad-
ditional factors. The decrease in LEDGF/p75 expression after
IC stimulation in macrophages from some donors might, for
example, contribute to reducing viral integration (68).
Conflicting results have been reported with respect to p21/
HIV-1 interaction. HIV-1 infection of T lymphocytes was
found to be associated with a loss of p21 expression (15), while
two studies based on transcriptome analysis showed either an
increase or no change in p21 expression in HIV-1-infected
macrophages (9, 69). Vazquez et al. reported that p21 enhance
HIV-1 infection in macrophages 12 to 14 days after viral chal-
lenge (69). Our data seem to be at odds with these results.
Whereas Vazquez et al. detected no change in HIV-1 DNA
levels during the first 2 days of infection, our knockdown ex-
periments showed an inhibitory effect of p21 on the reverse
transcription and integration steps of HIV-1 replication. How-
ever, we used one-cycle infection and real-time PCR, while
Vazquez et al. used a replicative strain of HIV-1 and nonquan-
titative PCR, which are not ideally suited to analyzing the first
steps of viral replication. The increase of p21 expression at late
times (14 days) after HIV-1 infection reported by Vazquez et
FIG. 8. p21 silencing rescues SIVmacand HIV-2GHreplication in
IC-stimulated macrophages. (A and B) Macrophages were seeded in
the presence (S) or absence of IC (US) and were transfected with
p21-specific siRNA duplexes (si-p21) or with an irrelevant siRNA
(si-Neg) or mock treated (Ctrl). At 24 h after siRNA transfection,
macrophages were infected with SIVmac251or HIV-2GH, and p27 cap-
sid protein was quantified in the supernatants at 7 days p.i. Consistent
results were obtained with macrophages from three donors.
12262 BERGAMASCHI ET AL.J. VIROL.
al. may be linked to the accumulation of Vpr that stimulates
p21 gene expression in infected cells (1, 14, 17, 69) or may be
a cell response against stress and apoptotic stimuli associated
to infection (3, 76). p21 might also have different impacts on
HIV-1 infection of macrophages depending on the time since
infection: a block of early stages of HIV-1 replication in acute
infection, as we show here, or an activation of HIV-1 gene
expression, synergistically with Vpr, in chronic infection (17).
However, we did not observe an inhibitory effect of p21
depletion on productive HIV-1 infection of unstimulated
macrophages. Methodological differences, including mono-
cyte differentiation into macrophages (fetal bovine serum in
Vazquez’s study versus human serum in our study), might also
account, at least in part, for the discrepancy between the re-
sults of the two studies.
Zhang et al. reported that p21 knockdown enhances human
hematopoietic stem cell (HSC) sensitivity to transduction with
pseudotyped HIV-1 vectors (80). More recently, they showed
that p21 knockdown permits productive HIV-1 replication in
human HSC by enhancing HIV-1 integration (81). Whereas
p21-mediated restriction targeted only viral integration in
HSCs, our results indicate that p21 interferes already with the
cytoplasmic phases of viral replication in macrophages inhib-
iting the accumulation of reverse transcripts. This could be
related to p21 translocation from the nucleus to cytoplasm
during monocyte differentiation (3). Differences in p21 local-
ization and cellular context might subtend the observed differ-
ences in the restriction phenotypes in macrophages and HSCs.
p21 restriction in macrophages was not mediated by either
enhanced expression of APOBEC3 or TRIM restriction fac-
tors or increased degradation of nascent reverse transcripts or
incoming viral proteins. Since reverse transcription, as well as
integration, depend on a functional RTC/PIC, we examined
potential interactions between p21 and viral components of the
RTC/PIC that could inhibit their activity. It has previously
suggested that p21 may inhibit HIV-1 integration in HSC by
complexing with IN (81). However, we did not detect p21
interactions with RTC/PIC-associated HIV-1 proteins, includ-
ing IN and Vpr, either in yeast two-hybrid assays, in pulldown
or in immunoprecipitation experiments. Although an interac-
tion of p21 with Vpr has been previously reported (17), we did
not detect it by any of the experimental approaches that we
used. Using the same yeast two-hybrid system, interactions of
Vpr with three different cellular partners: the nucleoporin
hCG1, DCAF1/VprBP, and the uracyl DNA glycosylase UNG
have been detected in previous studies (37, 38). In addition,
these interactions have also been visualized by pulldown ex-
periments with HA-tagged Vpr (11, 38), confirming that Vpr
protein is well folded in our assay. Although we cannot for-
mally exclude interactions either in the context of the RTC/
PIC, since viral proteins were analyzed for their binding to p21
separately, or in vivo in infected cells, our results suggest that
p21 may interfere with HIV-1 RTC/PIC activities indepen-
dently of a direct interaction with its viral components. p21
might modify the cellular context of the PIC, affecting either its
stability or its interactions with other host factors important for
its function. An antiviral activity independent of a specific
interaction with an HIV-1 protein may underlie the inhibitory
effect of p21 on other lentiviruses. Indeed, p21 affected not
only HIV-1 but also SIVmacand HIV-2 replication in macro-
phages, in line with our previous results on the Fc?R-mediated
restriction of primate lentiviruses (19). On the contrary, p21
silencing did not modify HSC restriction of SIVmacreplication
(81). However, macrophages are susceptible to HIV and SIV
infections, whereas HSCs are resistant to both viruses: differ-
ent host cell-virus interactions might subtend the differences in
the spectrum of lentiviral restriction in the two cell types.
Further work will be required to determine the precise
mechanisms responsible for p21-mediated restriction of HIV-1
replication in macrophages and other cells. Our findings point
to a role of p21 as an inhibiting factor of primate lentivirus
replication in macrophages, suggesting its relevance in viral
control in a cellular compartment that is critical to HIV infec-
tion and pathogenesis. p21 has already been proposed as a
target for anticancer therapy (36, 44, 66) and can be induced by
pharmacological compounds, including HDACi, that are stud-
ied as adjuvants to highly active antiretroviral therapy to erad-
icate HIV-1 cellular reservoirs (35, 53). If p21 acts indeed as an
inhibitor of HIV infection, this could have implications for
antiretroviral therapy research.
We thank Y. Xiong and E. Warbrick for providing the plasmid
containing p21 and PCNA cDNA and A. Mai and L. Altucci for the
gift of MC 1568. We thank P. Versmisse and L. Carthagena for their
technical help and F. Letourneur, N. Lebrun, and A. Vigier from the
sequencing facility of Institut Cochin. We are grateful to Asier Saez-
Cirion for helpful discussions and to A. Saïb for critical reading of the
This study was supported by Agence Nationale de la Recherche sur
le SIDA et les He ´patites Virales and by Sidaction.
A.B., G.P., E.L.R., and S.N. conceived and designed the experi-
ments. A.B., A.D., and E.L.R. performed the experiments. A.B., G.P.,
A.D., F.B.-S., E.L.R., and S.N. analyzed the data, and A.B. and G.P.
wrote the paper.
1. Amini, S., M. Saunders, K. Kelley, K. Khalili, and B. E. Sawaya. 2004.
Interplay between HIV-1 Vpr and Sp1 modulates p21WAF1gene expression
in human astrocytes. J. Biol. Chem. 279:46046–46056.
2. Aquaro, S., P. Bagnarelli, T. Guenci, A. De Luca, M. Clementi, E. Balestra,
R. Calıò, and C. F. Perno. 2002. Long-term survival and virus production in
human primary macrophages infected by human immunodeficiency virus.
J. Med. Virol. 68:479–488.
3. Asada, M., T. Yamada, H. Ichijo, D. Delia, K. Miyazono, K. Fukumuro, and
S. Mizutani. 1999. Apoptosis inhibitory activity of cytoplasmic p21Cip1/WAF1
in monocytic differentiation. EMBO J. 18:1223–1234.
4. Barr, S. D., J. R. Smiley, and F. D. Bushman. 2008. The interferon response
inhibits HIV particle production by induction of TRIM22. PLoS Pathog.
5. Besson, A., S. F. Dowdy, and J. M. Roberts. 2008. CDK inhibitors: cell cycle
regulators and beyond. Dev. Cell 14:159–169.
6. Bishop, K. N., M. Verma, E. Y. Kim, S. M. Wolinsky, and M. H. Malim. 2008.
APOBEC3G inhibits elongation of HIV-1 reverse transcripts. PLoS Pathog.
7. Blundell, R. A. 2006. The biology of p21Waf1/Cip1Am. J. Biochem. Biotech-
8. Bouazzaoui, A., M. Kreutz, V. Eisert, N. Dinauer, A. Heinzelmann, S. Hal-
lenberger, J. Strayle, R. Walker, H. Ru ¨bsamen-Waigmann, R. Andreesen,
and H. von Briesen. 2006. Stimulated trans-acting factor of 50 kDa (Staf50)
inhibits HIV-1 replication in human monocyte-derived macrophages. Virol.
9. Brown, J. N., J. J. Kohler, C. R. Coberley, J. W. Sleasman, and M. M.
Goodenow. 2008. HIV-1 activates macrophages independent of Toll-like
receptors. PLoS ONE 3:e3664.
10. Brussel, A., and P. Sonigo. 2003. Analysis of early human immunodeficiency
virus type 1 DNA synthesis by use of a new sensitive assay for quantifying
integrated provirus. J. Virol. 77:10119–10124.
11. Chen, R., E. Le Rouzic, J. A. Kearney, L. M. Mansky, and S. Benichou. 2004.
Vpr-mediated incorporation of UNG2 into HIV-1 particles is required to
modulate the virus mutation rate and for replication in macrophages. J. Biol.
VOL. 83, 2009 p21-MEDIATED HIV RESTRICTION IN MACROPHAGES12263
12. Cherepanov, P., G. Maertens, P. Proost, B. Devreese, J. Van Beeumen, Y.
Engelborghs, E. De Clercq, and Z. Debyser. 2003. HIV-1 integrase forms
stable tetramers and associates with LEDGF/p75 protein in human cells.
J. Biol. Chem. 278:372–381.
13. Child, E. S., and D. J. Mann. 2006. The intricacies of p21 phosphorylation:
protein-protein interactions, subcellular localization, and stability. Cell Cycle
14. Chowdhury, I. H., X. F. Wang, N. R. Landau, M. L. Robb, V. R. Polonis,
D. L. Birx, and J. H. Kim. 2003. HIV-1 Vpr activates cell cycle inhibitor
p21Waf1/Cip1: a potential mechanism of G2/M cell cycle arrest. Virology
15. Clark, E., F. Santiago, L. Deng, S. Chong, C. de La Fuente, L. Wang, P. Fu,
D. Stein, T. Denny, V. Lanka, F. Mozafari, T. Okamoto, and F. Kashanchi.
2000. Loss of G1/S checkpoint in human immunodeficiency virus type 1-in-
fected cells is associated with a lack of cyclin-dependent kinase inhibitor
p21Waf1. J. Virol. 74:5040–5052.
16. Coqueret, O. 2003. New roles for p21 and p27 cell-cycle inhibitors: a function
for each cell compartment? Trends Cell Biol. 13:65–70.
17. Cui, J., P. K. Tungaturthi, V. Ayyavoo, M. Ghafouri, H. Ariga, K. Khalili, A.
Srinivasan, S. Amini, and B. E. Sawaya. 2006. The role of Vpr in the
regulation of HIV-1 gene expression. Cell Cycle 5:2626–2638.
18. Das, D., G. Pintucci, and A. Stern. 2000. MAPK-dependent expression of
p21WAFand p27Kip1in PMA-induced differentiation of HL60 cells. FEBS
19. David, A., A. Saez-Cirion, P. Versmisse, O. Malbec, B. Iannascoli, F. Her-
schke, M. Lucas, F. Barre-Sinoussi, J. F. Mouscadet, M. Daeron, and G.
Pancino. 2006. The engagement of activating Fc?Rs inhibits primate lenti-
virus replication in human macrophages. J. Immunol. 177:6291–6300.
20. el-Deiry, W. S., T. Tokino, V. E. Velculescu, D. B. Levy, R. Parsons, J. M.
Trent, D. Lin, W. E. Mercer, K. W. Kinzler, and B. Vogelstein. 1993. WAF1,
a potential mediator of p53 tumor suppression. Cell 75:817–825.
21. Eng, E. W., J. Ibrahim, and R. E. Harrison. 2007. MTOC reorientation
occurs during Fc?R-mediated phagocytosis in macrophages. Mol. Biol. Cell
22. Fassati, A., and S. P. Goff. 2001. Characterization of intracellular reverse
transcription complexes of human immunodeficiency virus type 1. J. Virol.
23. Gonza ´lez-Scarano, F., and J. Martin-Garcia. 2005. The neuropathogenesis
of AIDS. Nat. Rev. Immunol. 5:69–81.
24. Gorry, P. R., M. Churchill, S. M. Crowe, A. L. Cunningham, and D.
Gabuzda. 2005. Pathogenesis of macrophage tropic HIV-1. Curr. HIV Res.
25. Groot, F., S. Welsch, and Q. J. Sattentau. 2008. Efficient HIV-1 transmission
from macrophages to T cells across transient virological synapses. Blood
26. Gu, Y., C. W. Turck, and D. O. Morgan. 1993. Inhibition of CDK2 activity in
vivo by an associated 20K regulatory subunit. Nature 366:707–710.
27. Hamamoto, S., H. Nishitsuji, T. Amagasa, M. Kannagi, and T. Masuda.
2006. Identification of a novel human immunodeficiency virus type 1 inte-
grase interactor, Gemin2, that facilitates efficient viral cDNA synthesis in
vivo. J. Virol. 80:5670–5677.
28. Harper, J. W., N. Wei, K. Keyomarsi, and S. J. Elledge. 1993. The p21
Cdk-interacting protein Cip1 is a potent inhibitor of G1cyclin-dependent
kinases. Cell 75:805–816.
29. Herbein, G., A. Coaquette, D. Perez-Bercoff, and G. Pancino. 2002. Macro-
phage activation and HIV infection: can the Trojan horse turn into a for-
tress? Curr. Mol. Med. 2:723–738.
30. Holmes, R. K., K. N. Bishop, and M. H. Malim. 2007. APOBEC3F can
inhibit the accumulation of HIV-1 reverse transcription products in the
absence of hypermutation: comparisons with APOBEC3G. J. Biol. Chem.
31. Iordanskiy, S., M. Altieri, F. Kashanchi, and M. Bukrinsky. 2006. Intracy-
toplasmic maturation of the human immunodeficiency virus type 1 reverse
transcription complexes determines their capacity to integrate into chroma-
tin. Retrovirology 3:4.
32. Jascur, T., I. Salles-Passador, V. Barbier, A. El Khissiin, B. Smith, R.
Fotedar, and A. Fotedar. 2005. Regulation of p21WAF1/CIP1stability by
WISp39, an Hsp90 binding TPR protein. Mol. Cell 17:237–249.
33. Joshi, T., J. P. Butchar, and S. Tridandapani. 2006. Fc? receptor signaling
in phagocytes. Int. J. Hematol. 84:210–216.
34. Kalpana, G. V., S. Marmon, W. Wang, G. R. Crabtree, and S. P. Goff. 1994.
Binding and stimulation of HIV-1 integrase by a human homolog of yeast
transcription factor SNF5. Science 266:2002–2006.
35. Keedy, K. S., N. M. Archin, A. T. Gates, A. Espeseth, D. J. Hazuda, and
D. M. Margolis. 2009. A limited group of class I histone deacetylases acts to
repress human immunodeficiency virus type 1 expression. J. Virol. 83:4749–
36. Kraljevic Pavelic, S., T. Cacev, and M. Kralj. 2008. A dual role of p21waf1/cip1
gene in apoptosis of HEp-2 treated with cisplatin or methotrexate. Cancer
Gene Ther. 15:576–590.
37. Le Rouzic, E., N. Belaidouni, E. Estrabaud, M. Morel, J. C. Rain, C. Transy,
and F. Margottin-Goguet. 2007. HIV1 Vpr arrests the cell cycle by recruiting
DCAF1/VprBP, a receptor of the Cul4-DDB1 ubiquitin ligase. Cell Cycle
38. Le Rouzic, E., A. Mousnier, C. Rustum, F. Stutz, E. Hallberg, C. Dargemont,
and S. Benichou. 2002. Docking of HIV-1 Vpr to the nuclear envelope is
mediated by the interaction with the nucleoporin hCG1. J. Biol. Chem.
39. Lin, C. W., and A. Engelman. 2003. The barrier-to-autointegration factor is
a component of functional human immunodeficiency virus type 1 preinte-
gration complexes. J. Virol. 77:5030–5036.
40. Llano, M., D. T. Saenz, A. Meehan, P. Wongthida, M. Peretz, W. H. Walker,
W. Teo, and E. M. Poeschla. 2006. An essential role for LEDGF/p75 in HIV
integration. Science 314:461–464.
41. Luo, K., W. T., B. Liu, C. Tian, Z. Xiao, J. Kappes, and X. F. Yu. 2007.
Cytidine deaminases APOBEC3G and APOBEC3F interact with human
immunodeficiency virus type 1 integrase and inhibit proviral DNA forma-
tion. J. Virol. 81:7238–7248.
42. Mai, A., S. Massa, R. Pezzi, S. Simeoni, D. Rotili, A. Nebbioso, A. Scog-
namiglio, L. Altucci, P. Loidl, and G. Brosch. 2005. Class II (IIa)-selective
histone deacetylase inhibitors. 1. Synthesis and biological evaluation of novel
(aryloxopropenyl)pyrrolyl hydroxyamides. J. Med. Chem. 48:3344–3353.
43. Mangeat, B., G. Caron, M. Friedli, L. Perrin, and D. Trono. 2003. Broad
antiretroviral defence by human APOBEC3G through lethal editing of nas-
cent reverse transcripts. Nature 424:99–103.
44. Marlow, L. A., L. A. Reynolds, A. S. Cleland, S. J. Cooper, M. L. Gumz, S.
Kurakata, K. Fujiwara, Y. Zhang, T. Sebo, C. Grant, B. McIver, J. T.
Wadsworth, D. C. Radisky, R. C. Smallridge, and J. A. Copland. 2009.
Reactivation of suppressed RhoB is a critical step for the inhibition of
anaplastic thyroid cancer growth. Cancer Res. 69:1536–1544.
45. Maroun, M., O. Delelis, G. Coadou, T. Bader, E. Segeral, G. Mbemba, C.
Petit, P. Sonigo, J. C. Rain, J. F. Mouscadet, R. Benarous, and S. Emiliani.
2006. Inhibition of early steps of HIV-1 replication by SNF5/Ini1. J. Biol.
46. Morisaki, H., A. Ando, Y. Nagata, O. Pereira-Smith, J. R. Smith, K. Ikeda,
and M. Nakanishi. 1999. Complex mechanisms underlying impaired activa-
tion of Cdk4 and Cdk2 in replicative senescence: roles of p16, p21, and cyclin
D1. Exp. Cell Res. 253:503–510.
47. Nebbioso, A., F. Manzo, M. Miceli, M. Conte, L. Manente, A. Baldi, A. De
Luca, D. Rotili, S. Valente, A. Mai, A. Usiello, H. Gronemeyer, and L.
Altucci. 2009. Selective class II HDAC inhibitors impair myogenesis by
modulating the stability and activity of HDAC-MEF2 complexes. EMBO
48. Orenstein, J. M., C. Fox, and S. M. Whal. 1997. Macrophages as a source of
HIV during opportunistic infections. Science 276:1857–1861.
49. Park, J. W., M. A. Jang, S. H. Baek, J. H. Lim, T. Passaniti, and T. K. Kwon.
2001. Arsenic trioxide induces G2/M growth arrest and apoptosis after
caspase-3 activation and bcl-2 phosphorylation in promonocytic U937 cells.
Biochem. Biophys. Res. Commun. 286:726–734.
50. Peng, G., T. Greenwell-Wild, S. Nares, W. Jin, K. J. Lei, Z. G. Rangel, P. J.
Munson, and S. M. Wahl. 2007. Myeloid differentiation and susceptibility to
HIV-1 are linked to APOBEC3 expression. Blood 110:393–400.
51. Peng, G., K. J. Lei, W. Jin, T. Greenwell-Wild, and S. M. Wahl. 2006.
Induction of APOBEC3 family proteins, a defensive maneuver underlying
interferon-induced anti-HIV-1 activity. J. Exp. Med. 203:41–46.
52. Perez-Bercoff, D., A. David, H. Sudry, F. Barre-Sinoussi, and G. Pancino.
2003. Fc?-mediated suppression of human immunodeficiency virus type 1
replication in primary human macrophages. J. Virol. 77:4081–4094.
53. Reuse, S., M. Calao, K. Kabeya, A. Guiguen, J. S. Gatot, V. Quivy, C.
Vanhulle, A. Lamine, D. Vaira, D. Demonte, V. Martinelli, E. Veithen, T.
Cherrier, V. Avettand, S. Poutrel, J. Piette, Y. de Launoit, M. Moutschen, A.
Burny, C. Rouzioux, S. De Wit, G. Herbein, O. Rohr, Y. Collette, O. Lam-
botte, N. Clumeck, and C. Van Lint. 2009. Synergistic activation of HIV-1
expression by deacetylase inhibitors and prostratin: implications for treat-
ment of latent infection. PLoS ONE 4:e6093.
54. Rosato, R. R., J. A. Almenara, and S. Grant. 2003. The histone deacetylase
inhibitor MS-275 promotes differentiation or apoptosis in human leukemia
cells through a process regulated by generation of reactive oxygen species
and induction of p21CIP1/WAF1. Cancer Res. 63:3637–3645.
55. Saez-Cirion, A., M. A. Nicola, G. Pancino, and S. L. Shorte. 2006. Quanti-
tative real-time analysis of HIV-1 gene expression dynamics in single living
primary cells. Biotechnol. J. 1:682–689.
56. Saito, A., Y. Mariko, Y. Nosaka, K. Tsuchiya, T. Ando, T. Suzuki, T. Tsuruo,
and O. Nakanishi. 1999. A synthetic inhibitor of histone deacetylase, MS-
27-275, with marked in vivo antitumor activity against human tumors. Proc.
Natl. Acad. Sci. USA 96:4592–4597.
57. Schwaller, J., T. Pabst, G. Niklaus, D. E. Macfarlane, M. F. Fey, and A.
Tobler. 1997. Up-regulation of p21WAF1expression in myeloid cells is acti-
vated by the protein kinase C pathway. Br. J. Cancer 76:1554–1557.
58. Schwartz, O., V. Marechal, B. Friguet, F. Arenzana-Seisdedos, and J. M.
Heard. 1998. Antiviral activity of the proteasome on incoming human im-
munodeficiency virus type 1. J. Virol. 72:3845–3850.
59. Selig, L., J. C. Pages, V. Tanchou, S. Pre ´ve ´ral, C. Berlioz-Torrent, L. X. Liu,
L. Erdtmann, J. Darlix, R. Benarous, and S. Benichou. 1999. Interaction
12264 BERGAMASCHI ET AL.J. VIROL.
with the p6 domain of the gag precursor mediates incorporation into virions
of Vpr and Vpx proteins from primate lentiviruses. J. Virol. 73:592–600.
60. Sharova, N., M. Sharkey, and M. Stevenson. 2005. Macrophages archive
HIv-1 virions for dissemination in trans. EMBO J. 24:2481–2489.
61. Skolnik, P. R., J. M. Mathys, and A. S. Greenberg. 2002. Stimulation of
peroxisome proliferator-activated receptors alpha and gamma blocks HIV-1
replication and TNF? production in acutely infected primary blood cells,
chronically infected U1 cells, and alveolar macrophages from HIV-infected
subjects. J. Acquir. Immune Defic. Syndr. 33:1–10.
62. Steinman, R. A., and D. E. Johnson. 2000. p21WAF1prevents down-modu-
lation of the apoptotic inhibitor protein c-IAP1 and inhibits leukemic apop-
tosis. Mol. Med. 6:736–749.
63. Stremlau, M., M. J. Perron, M. Kiessling, P. Autissier, and J. Sodroski.
2004. The cytoplasmic body component TRIM5? restricts HIV-1 infection in
Old World monkeys. Nature 427:848–853.
64. Sung, T. L., and A. P. Rice. 2009. miR-198 inhibits HIV-1 gene expression
and replication in monocytes and its mechanism of action appears to involve
repression of cyclin T1. PLoS Pathog. 5:e1000263.
65. Takeuchi, H., and T. Matano. 2008. Host factors involved in resistance to
retroviral infection. Microbiol. Immunol. 52:318–325.
66. Tanaka, T., K. S. Suh, A. M. Lo, and L. M. De Luca. 2007. p21WAF1/CIP1is
a common transcriptional target of retinoid receptors: pleiotropic regulatory
mechanism through retinoic acid receptor (RAR)/retinoid X receptor
(RXR) heterodimer and RXR/RXR homodimer. J. Biol. Chem. 282:29987–
67. Uchil, P. D., B. D. Quinlan, W. T. Chan, J. M. Luna, and W. Mothes. 2008.
TRIM E3 ligases interfere with early and late stages of the retroviral life
cycle. PLoS Pathog. 4:e16.
68. Vandekerckhove, L., B. Van Maele, J. De Rijck, R. Gijsbers, C. Van den
Haute, M. Witvrouw, and Z. Debyser. 2006. Transient and stable knockdown
of the integrase cofactor LEDGF/p75 reveals its role in the replication cycle
of human immunodeficiency virus. J. Virol. 80:1886–1896.
69. Va ´zquez, N., T. Greenwell-Wild, N. J. Marinos, W. D. Swaim, S. Nares, D. E.
Ott, U. Schubert, P. Henklein, J. M. Orenstein, M. B. Sporn, and S. M.
Wahl. 2005. Human immunodeficiency virus type 1-induced macrophage
gene expression includes the p21 gene, a target for viral regulation. J. Virol.
70. Verani, A., G. Gras, and G. Pancino. 2005. Macrophages and HIV-1: dan-
gerous liaisons. Mol. Immunol. 42:195–212.
71. Wang, X., W. Hou, Y. Zhou, Y. J. Wang, D. S. Metzger, and W. Z. Ho. 2009.
Cellular microRNA expression correlates with susceptibility of monocytes/
macrophages to HIV-1 infection. Blood 113:671–674.
72. Warbrick, E., D. M. Glover, and L. S. Cox. 1995. A small peptide inhibitor
of DNA replication defines the site of interaction between the cyclin-depen-
dent kinase inhibitor p21WAF1and proliferating cell nuclear antigen. Curr.
73. Wei, B. L., P. W. Denton, E. O’Neill, T. Luo, J. L. Foster, and J. V. Garcia.
2005. Inhibition of lysosome and proteasome function enhances human
immunodeficiency virus type 1 infection. J. Virol. 79:5705–5712.
74. Weiden, M., Y. Qiao, B. Y. Zhao, Y. Honda, K. Nakata, A. Canova, D. E.
Levy, W. N. Rom, and R. Pine. 2000. Differentiation of monocytes to mac-
rophages switches the Mycobacterium tuberculosis effect on HIV-1 replica-
tion from stimulation to inhibition: modulation of interferon response and
CCAAT/enhancer binding protein beta expression. J. Immunol. 165:2028–
75. Wu, W., K. Kehn-Hall, C. Pedati, L. Zweier, I. Castro, Z. Klase, C. S. Dowd,
L. Dubrovsky, M. Bukrinsky, and F. Kashanchi. 2008. Drug 9AA reactivates
p21/Waf1 and Inhibits HIV-1 progeny formation. Virol. J. 5:41.
76. Xaus, J., A. F. Valledor, C. Soler, J. Lloberas, and A. Celada. 1999. Inter-
feron gamma induces the expression of p21waf-1 and arrests macrophage
cell cycle, preventing induction of apoptosis. Immunity 11:103–113.
77. Xiong, Y., H. Zhang, D. Casso, R. Kobayashi, and D. Beach. 1993. p21 is a
universal inhibitor of cyclin kinases. Nature 366:701–704.
78. Yamamoto, N., Y. Wu, M. O. Chang, Y. Inagaki, Y. Saito, T. Naito, H.
Ogasawara, I. Sekigawa, and Y. Hayashida. 2006. Analysis of human immu-
nodeficiency virus type 1 integration by using a specific, sensitive and quan-
titative assay based on real-time polymerase chain reaction. Virus Genes
79. Yoder, K., A. Sarasin, K. Kraemer, M. McIlhatton, F. Bushman, and R.
Fishel. 2006. The DNA repair genes XPB and XPD defend cells from
retroviral infection. Proc. Natl. Acad. Sci. USA 103:4622–4627.
80. Zhang, J., E. Attar, K. Cohen, C. Crumpacker, and D. Scadden. 2005.
Silencing p21Waf1/Cip1/Sdi1expression increases gene transduction efficiency
in primitive human hematopoietic cells. Gene Ther. 12:1444–1452.
81. Zhang, J., D. T. Scadden, and C. S. Crumpaker. 2007. Primitive hema-
topoietic cells resist HIV-1 infection via p21Waf1/Cip1/Sdi1. J. Clin. Investig.
82. Zhao, Y., L. Wu, G. Chai, H. Wang, Y. Chen, J. Sun, Y. Yu, W. Zhou, Q.
Zheng, M. Wu, G. A. Otterson, and W. G. Zhu. 2006. Acetylation of p53 at
lysine 373/382 by the histone deacetylase inhibitor depsipeptide induces
expression of p21Waf1/Cip1. Mol. Cell. Biol. 26:2782–2790.
83. Zybarth, G., H. Schmidtmayerova, B. Sherry, and M. Bukrinsky. 1999.
Activation-induced resistance of human macrophages to HIV-1 infection in
vitro. J. Immunol. 162:400–406.
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