JOURNAL OF VIROLOGY,
Copyright © 1999, American Society for Microbiology. All Rights Reserved.
May 1999, p. 3893–3903Vol. 73, No. 5
I?? Mediates NF-?B Activation in Human Immunodeficiency
SUSANA ASIN,1JULIE A. TAYLOR,1SERGEY TRUSHIN,1GARY BREN,1
AND CARLOS V. PAYA1,2*
Department of Immunology1and Division of Infectious Diseases,2
Mayo Clinic, Rochester, Minnesota 55905
Received 16 September 1998/Accepted 27 January 1999
Human monocytes and macrophages are persistent reservoirs of human immunodeficiency virus (HIV)
type-1. Persistent HIV infection of these cells results in increased levels of NF-?B in the nucleus secondary to
increased I?B?, I?B?, and I?B? degradation, a mechanism postulated to regulate viral persistence. To
characterize the molecular mechanisms regulating HIV-mediated degradation of I?B, we have sought to
identify the regulatory domains of I?B? targeted by HIV infection. Using monocytic cells stably expressing
different transdominant molecules of I?B?, we determined that persistent HIV infection of these cells targets
the NH2but not the COOH terminus of I?B?. Further analysis demonstrated that phosphorylation at S32and
S36is necessary for HIV-dependent I?B? degradation and NF-?B activation. Of the putative N-terminal I?B?
kinases, we demonstrated that the I?? complex, but not p90rsk, is activated by HIV infection and mediates
HIV-dependent NF-?B activation. Analysis of viral replication in cells that constitutively express I?B?
negative transdominant molecules demonstrated a lack of correlation between virus-induced NF-?B (p65/p50)
nuclear translocation and degree of viral persistence in human monocytes.
The Rel family of transcription factors plays an important
role in the transactivation of several viral genes, including
those of human immunodeficiency virus (HIV) type 1 (HIV-1)
(25, 38). HIV-1 replication is regulated, in part, at the tran-
scriptional level through the interplay of viral regulatory pro-
teins with cellular transcription factors interacting with the
viral long terminal repeat (LTR) (39). Since the identification
and functional characterization of NF-?B cis-acting sequences
within the HIV LTR (38), multiple studies have addressed the
essential or dispensable role that this transcription factor plays
in the reactivation of HIV from a true latent state and in the
control of viral persistence (1, 10, 27, 31, 54, 55). Unfortu-
nately, these studies have yielded conflicting results as to the
role of NF-?B in these two steps of the viral life cycle in
infected host cells. Differences in the type of host cells studied,
HIV strain or genetic constructs used, and methodological
approaches may explain these conflicting results.
Understanding the potential impact of NF-?B on the regu-
lation of HIV latency has again become a priority, as recent
studies suggest that NF-?B controls the reactivation of latent
HIV in T cells from HIV-infected patients undergoing highly
active antiretroviral therapy (19). An additional reservoir of
HIV, separate from that of T cells harboring latent HIV, are
cells of the monocyte lineage in which persistent viral replica-
tion is observed (36). During all stages of HIV infection, tissue
macrophages provide a unique viral reservoir. In these cells,
HIV persistently replicates in the absence of cytopathicity,
escapes immune surveillance, and spreads via cell-to-cell con-
tact (reviewed in reference 36). The important role of macro-
phages in AIDS pathogenesis has prompted the investigation
of the molecular mechanisms which regulate HIV-1 persis-
tence in these immune cells; one of these mechanisms is
thought to be NF-?B dependent. Human macrophages express
a constitutive level of NF-?B in the nuclei in the absence of
exogenous cellular activation (25). This constitutive pool of
nuclear NF-?B may be sufficient to allow for the initiation of
HIV transcription immediately following infection. In addi-
tion, NF-?B may be required to further sustain persistent HIV
replication, as multiple studies have demonstrated that persis-
tent HIV replication in human macrophages or monocytes
further upregulates NF-?B activity (2, 34, 40, 43, 48). However,
the mechanisms by which HIV infection induces the activation
of NF-?B in cells of the monocyte lineage remains unknown.
Their identification would greatly enhance the understanding
of this process and allow future testing of whether inhibition of
the virus-induced activation of NF-?B may decrease viral per-
sistence in cells of the monocyte lineage, hence eliminating an
important reservoir of HIV replication in infected patients.
NF-?B is a heterodimeric protein composed of different
combinations of members of the Rel family of transcription
factors. A well-characterized form of NF-?B is a heterodimer
of p50 and Rel-A (p65) (reviewed in references 3 and 4). In the
majority of cells studied, NF-?B is anchored in the cytosol by
an inhibitory protein, I?B. An extensively studied I?B mole-
cule, I?B?, has previously been shown to physically interact
with NF-?B and to mask the nuclear localization signal of p50
and Rel-A (6). Following cell activation by one of an array of
extracellular stimuli, I?B? undergoes a hyperphosphorylation
event that renders the inhibitory molecule susceptible to deg-
radation (7, 13, 47). This process results in the release of
NF-?B, which undergoes nuclear translocation and drives gene
transcription. Significant advances in the understanding of the
molecular mechanisms and the structure-function of the phos-
phorylation and degradation of I?B? have recently been made.
I?B? is constitutively phosphorylated at its COOH terminus by
protein kinase-casein kinase II (PK-CK2) (5, 33, 35, 45). While
the exact function of this phosphorylation is poorly under-
stood, it appears that phosphorylation at the COOH terminus
may play a role in the constitutively rapid protein turnover of
I?B? in resting cells, thus potentially favoring a low degree of
continuous NF-?B translocation. On the contrary, the N ter-
* Corresponding author. Mailing address: Mayo Clinic, 200 First St.
SW, Guggenheim 501, Rochester, MN 55905. Phone: (507) 284-3747.
Fax: (507) 284-3757. E-mail: email@example.com.
minus contains two series (S32and S36) which are required for
stimulus-dependent phosphorylation (8, 9, 11, 15, 46, 49, 50,
52) by specific kinases, such as the ones present in the I??
complex (I??? and I???) or p90rsk(12, 16, 20, 24, 37, 42, 44,
53, 57). Phosphorylation at these sites primes I?B? to undergo
ubiquitination and subsequent degradation by the proteosome.
Our group has previously determined that a mechanism where-
by HIV infection results in an increase in the nuclear translo-
cation of NF-?B involves modification and enhancement of
I?B? turnover (34). The half-life of I?B? in HIV-infected cells
is reduced by at least 50% compared to that in uninfected cells,
and this fact directly correlates with increased levels of the
nuclear pool of NF-?B in HIV-infected cells. That I?B? is the
target of persistent HIV infection in monocytic cells has been
further confirmed by other groups (14, 27); one of those groups
further demonstrated that inhibition of I?B? degradation with
proteosome inhibitors decreases HIV-induced NF-?B activa-
tion (27). What remain to be elucidated are the molecular mech-
anisms whereby HIV infection targets I?B?. Potential mecha-
nisms regulated by HIV infection could target the COOH
terminus of I?B?, favoring an enhanced “basal” turnover of
this inhibitor molecule by activating PK-CK2 or the proteolytic
machinery. Alternatively, HIV infection could result in the
activation of other I?B? kinases that target S32and S36, thus
continuously priming I?B? to be degraded via the proteosome.
Lastly, HIV could target other regulatory sites of I?B? or even
other molecules, such as Rel-A, that could result in the disso-
ciation of NF-?B from I?B?, thus rendering I?B? less stable.
To investigate these possibilities, we have used a cell model
of monocytic cells in which persistent HIV replication results
in NF-?B activation and a variety of genetically modified
tagged I?B? molecules can be constitutively overexpressed.
Our results indicate that HIV infection targets the NH2ter-
minus of I?B?, specifically S32and S36, causing the enhanced
degradation of I?B? and hence increased NF-?B nuclear
translocation. The I?? complex kinase activity is selectively
activated and is shown to mediate increased NF-?B activation
in HIV-infected cells. In addition, we demonstrate that HIV-
mediated NF-?B activation is not necessary to maintain viral
persistence in monocytic cells.
MATERIALS AND METHODS
Reagents and antibodies. Tumor necrosis factor (TNF) was purchased from
Genzyme (Cambridge, Mass.) and stored in aliquots at ?70°C. Cycloheximide
was purchased from Sigma (St. Louis, Mo.) and stored at ?20°C. Calpain
inhibitor I (N-acetyl-Leu-Leu-norleucinal or ALLN) was purchased from Boehr-
inger Mannheim Biochemicals (Indianapolis, Ind.), solubilized in ethyl alcohol,
and stored in aliquots at ?20°C. Bay 11-7082 (41) was purchased from Biomol
(Plymouth Meeting, Pa.), solubilized in ethyl alcohol, and stored at ?20°C. G418
was purchased from Calbiochem-Novabiochem Corporation (La Jolla, Calif.),
solubilized in RPMI medium, and stored in aliquots at ?20°C.
The expression of the Flag-tagged I?B constructs was monitored with an
anti-Flag monoclonal antibody (Kodak, New Haven, Conn.). To control for
equal loading of proteins in sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis (PAGE) analysis, an anti–?-actin polyclonal antibody (Sigma)
and an anti-p90rskantibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) were
used. Polyclonal anti-I?B? serum was generated with a glutathione S-transferase
(GST)–MAD3 fusion protein (34). The viral envelope protein gp120 was de-
tected with an anti-gp120 polyclonal antibody (Center for Biologics Evaluation
and Research, Food and Drug Administration, Bethesda, Md.). The identity of
the complexes binding DNA in the gel shift assays was determined with poly-
clonal antibodies against the different members of the Rel family (Santa Cruz
Biotechnology). Antibodies against p90rsk, I???, I???, Raf-1, and NF-?B were
purchased from Santa Cruz Biotechnology.
DNA constructs. pCMV2-FLAG-I?B?-wt consisted of the full-length “wild-
type” sequence of human I?B? (26) cloned into the SmaI-HindIII sites of
pCMV2FLAG (Kodak) to generate N-terminally Flag-tagged I?B?-wt (Flag-
I?B?-wt). Flag-I?B?-wt was used as a template for subsequent mutations and
deletions by PCR-based techniques. Flag-I?B?-?N consisted of an N-terminal
deletion lacking the first 37 amino acids. This construct was generated with the
sense primer wt-FLAG (5?CGGAATTCATGGACTACAAAGACGAT3?) and
the antisense primer wt-B (5?GGAATTCCTCATAACGTCAGACGCTG3?).
EcoRI sites were created upstream and downstream of the coding sequence.
Flag-I?B?-?C consisted of a C-terminal deletion lacking the last 40 amino acids
and was generated with the sense primer wt-FLAG and the antisense primer ?C
(5?GCGAATTCTCAAAGGTTTTCTAGTGTC3?). This construct contained
an EcoRI site downstream of the coding sequence. Flag-I?B?-2N consisted of
the full-length sequence of I?B?-wt in which S32and S36were mutated to alanine
residues. To generate these mutations, a sense primer with the sequence 5?GA
CGCAGGCCTGGACGCAATG3? and an antisense primer with the sequence
5?CATTGCGTCCAGGCCTGCGTC3? were used. Flag-I?B?-4C was created by
mutation of S283, S288, S293, and T291to alanine residues in the PEST sequence
(35), cloning into the HindIII-EcoRI site of pCMV2FLAG, and then PCR
amplifying with the sense primer wt-FLAG and the antisense primer wt-B.
Flag-I?B?-wt, Flag-I?B?-?N, Flag-I?B?-?C, Flag-I?B?-2N, and Flag-I?B?-4C
were then digested and cloned into the EcoRI site of SFFV-Neo under the
transcriptional regulation of the Friend spleen focus-forming virus (SFFV) 5?
LTR (22). All of the cloning was verified by DNA sequencing.
Plasmid ?B-luc contains three tandem copies of the ?B motif of the HIV LTR
cloned upstream of the minimal conalbumin-luciferase (con-luc) promoter re-
porter gene. Plasmid pBLCAT 2 is a mammalian reporter vector designed for the
expression of chloramphenicol acetyltransferase (CAT) in mammalian cells tran-
scribed by the minimal thymidine kinase (TK) promoter (Promega, Madison,
Wis.). Plasmids I??? wt and kinase dead were kind gifts from Alain Israel,
Institute Pasteur, Paris, France. Plasmids I??? wt and kinase dead were obtained
from M. Roth (Tularik, San Jose, Calif.). I??? kinase dead was generated by
mutation of aspartic acid 144 to asparagine. I??? kinase dead was created by
mutation of lysine 44 to alanine. pcDNA3-I?? expression vectors were generated
by cloning the cDNA of wild-type I??? or I??? or its respective mutant into the
cytomegalovirus expression vector pcDNA3 (Invitrogen).
Gene transfection and generation of cell lines. The U937 promonocytic cell
line was purchased from the American Type Culture Collection and grown in
RPMI 1640 supplemented with 5% heat-inactivated fetal bovine serum (Inter-
gen), 1% glutamine, and 1% penicillin-streptomycin. To generate I?B?-express-
ing cell lines, 107freshly thawed and exponentially growing U937 cells were
resuspended in RPMI 1640 and electroporated with 20 ?g of previously linear-
ized DNA by use of a BTX cell electroporator at 250 V for 10 ms. U937 cells
electroporated without DNA were used as controls. At 24 h after transfection,
cells were resuspended in selection medium containing 5% fetal bovine serum
and 700 ?g of G418 per ml. After 3 to 4 weeks, upon the incipient growth of
neomycin-resistant bulk cultures, cells were cloned by limiting dilution (30).
Stable integration and expression of the transfected genes within each monoclo-
nal population were verified by serial passages of the cultures in the absence of
the selective antibiotic and by immunoblotting with anti-Flag antibodies.
Separate clones expressing equal levels of Flag-I?B? constructs were selected,
and their CD4 surface expression was verified by flow cytometry analysis. There-
after, three clones expressing each of the Flag-I?B? constructs were pooled, and
exponentially growing cells were mock or HIV infected. The level of expression
of each of the tagged I?B? constructs was confirmed before and during the
period of HIV infection by immunoblotting of cytosolic extracts with anti-Flag
Transient transfection of U937 cells was performed as follows. A total of 107
exponentially growing U937 cells were incubated with 4 ?g of the con-luc or
?B-con-luc reporter construct, 6 ?g of the pDNA3-I?? construct, 4 ?g of the
pBLCAT2 reporter, and 300 ?g of DEAE-dextran (Pharmacia, Piscataway, N.J.)
per ml for 90 min at room temperature. Dimethyl sulfoxide (10%) was then
added for 3 min, followed by extensive washing and plating at 0.5 ? 107/cells/ml.
Two days later, cells were harvested. Luciferase levels were measured with the
Promega luciferase assay system, and CAT activity was measured with a CAT
enzyme-linked immunosorbent assay kit (Boehringer).
HIV infection and measurement of HIV replication. U937 cells expressing
SFFV, Flag-I?B?-wt, Flag-I?B?-?N, Flag-I?B?-?C, Flag-I?B?-2N, and Flag-
I?B?-4C were infected with the HIV LAV-Bru strain as previously described (2,
34, 40). Briefly, 107exponentially growing U937 cells were sedimented by low-
speed centrifugation and resuspended overnight in 10 ml of infective supernatant
containing 360 ng of p24 per ml. Mock-infected cells were used as a control.
After 24 h, cells were extensively washed and resuspended in culture medium.
Cells were passaged twice a week at 0.25 ? 106cells/ml and used from day 30
through day 90 postinfection. During this period, cell supernatants were col-
lected, precleared by centrifugation at 1,500 rpm for 5 min at 4°C, and stored for
future analysis of HIV p24 antigen content by an enzyme-linked immunosorbent
assay (Coulter-Immunotech Immunology, Westbrook, Maine). At least eight
consecutive infections were used for each of these experiments. All the cell lines
studied maintained HIV persistence and 100% viability during the study period,
except for the U937 clones expressing Flag-I?B?-?C, which maintained viability
and normal growth while uninfected which underwent immediate and massive
cytopathicity upon HIV infection in four consecutive attempts. Therefore, a
U937 cell line expressing Flag-I?B?-?C could not support a persistent HIV
infection. In addition, in some experiments, HIV gp120 expression was deter-
mined by immunoblotting with anti-gp120 antibodies.
Nuclear and cytosolic extracts, electrophoretic mobility shift assays, and im-
munoblotting. Nuclear and cytosolic extracts were prepared by a modification of
the method of Dignam et al. (17). A total of 107cells were washed with ice-cold
3894 ASIN ET AL.J. VIROL.
phosphate-buffered saline and then with buffer A (10 mM HEPES [pH 7.9], 1.5
mM MgCl2, 10 mM KCl). Cells were then lysed for 10 min on ice in the same
buffer containing 0.1% Nonidet P-40, 0.5 mM dithiothreitol (DTT), 0.5 mM
phenylmethylsulfonyl fluoride (PMSF), 2 ?g of aprotinin per ml, 2 ?g of leu-
peptin per ml, and 2 ?g of pepstatin per ml. After centrifugation, cells were
washed twice with buffer A. Nuclei were pelleted by centrifugation, lysed by
resuspension in 25 ?l of buffer C (20 mM HEPES, 25% glycerol, 0.42 M NaCl,
1.5 mM MgCl2, 0.2 mM EDTA, DTT, PMSF, aprotinin, leupeptin, pepstatin)
and rotated at 4°C for 30 min. After centrifugation, the supernatants were diluted
in 50 ?l of buffer D (20 mM HEPES, 20% glycerol, 0.05 M KCl, 0.2 M EDTA,
DTT, PMSF, aprotinin, leupeptin, pepstatin) and stored at ?70°C.
For electrophoretic mobility shift assays, 6 ?g of nuclear extract was incubated
with a [?-32P]ATP-labeled double-stranded NF-?B oligonucleotide probe in 15
?l of DNA binding buffer for 15 min at room temperature as previously de-
scribed (2, 34, 40). Components of the HIV-induced DNA binding protein
complexes were identified by incubation of the extract with specific polyclonal
antibodies against p50 and Rel-A prior to addition of the labeled probe. The
resulting protein-DNA complexes were resolved on a 5% polyacrylamide gel and
visualized by autoradiography.
To characterize the level of expression of the Flag-I?B? constructs in unin-
fected and infected cells, 40 ?g of cytosolic protein was analyzed by SDS–10%
PAGE. Proteins were transferred to Immobilon-P membranes (Millipore) by
standard procedures and blotted with an anti-Flag monoclonal antibody, fol-
lowed by incubation with rabbit anti-mouse immunoglobulin G (Pierce) and then
horseradish peroxidase (Amersham, Buckinghamshire, England). Immunoreac-
tive proteins were detected with an ECL Western blotting detection kit (Amer-
sham). ?-Actin and p90rskwere used as internal controls for equal loading in all
Preparation of recombinant I?B?. The I?B?-MAD3 cDNA (26) plasmid was
obtained from Cetus Corporation and was used as a template for subsequent
The amino-terminal I?B?-MAD3 (positions 1 to 54) sequence was amplified
with wild-type primer A (5?CGGGATCCATGTTCCAGGCGGCCGAG3?) as
the sense primer, creating a BamHI site upstream of the coding sequence, and
wild-type primer B (5?GGAATTCCTCAGCGGATCTCCTGCAGCT3?) as the
antisense primer, creating an EcoRI site downstream of the coding sequence. An
S32/36Adouble mutant was amplified from the full-length cDNA by use of prim-
ers to create alanines at S32and S36. Following digestion with BamHI-EcoRI,
these sequences were ligated into pGEX-KG (derived from pGEX-2T, from
Pharmacia, Piscataway, N.J.). These constructs were transformed into Esche-
richia coli DH5? cells, which were grown exponentially. After 60 min of stimu-
lation with isopropylthiogalactopyranoside (Sigma), cells were lysed. Proteins
were isolated by affinity chromatography on glutathione-bonded 4% cross-linked
agarose (Sigma). The purity of GST-I?B? (positions 1 to 54) containing the first
54 amino acids of I?B? and GST-I?B? (positions 1 to 54) containing S32/36Awas
analyzed by SDS–10% PAGE and subsequent Coomassie blue staining. The
purity of both proteins was greater than 90%.
Immunoprecipitation of I?B? kinases and in vitro kinase assays. Whole-cell
extracts were prepared for immunoprecipitation and in vitro kinase assays as
follows. Aliquots of 107exponentially growing U937 cells were washed twice with
cold phosphate-buffered saline, resuspended in lysis buffer containing 40 mM
Tris-HCl (pH 8), 0.3 M NaCl, 0.1% Nonidet P-40, 6 mM EDTA, 6 mM EGTA,
10 mM NaF, 10 mM p-nitrophenyl phosphate (PNPP), 10 mM ?-glycerolphos-
phate, 300 ?M sodium orthovanadate, 1 mM DDT, 2 ?M PMSF, 10 ?g of
aprotinin per ml, 1 ?g of leupeptin per ml, and 1 ?g of pepstatin per ml, and
incubated on ice. Cells were then centrifuged at 12,000 ? g for 15 min at 4°C. The
resultant supernatant contained total cellular proteins, which were quantitated
with a Bio-Rad protein assay.
For immunoprecipitation of the I?? complex, p90rsk, or Raf-1, 100 ?g of cell
extract was incubated with anti-I???, anti-I???, anti-p90rsk, or anti–Raf-1 anti-
bodies for 1 h at 4°C, after which protein A-agarose beads (Life Technologies,
Gaithersburg, Md.) were added for 1 h. The beads were then washed three times
with 0.5 M NaCl-based lysis buffer, followed by one wash with a buffer containing
50 mM Tris-HCl (pH 7.4) and 40 mM NaCl. The washed beads were then
incubated in 15 ?l of kinase buffer (20 mM HEPES [pH 7.4], 2 mM MgCl, 2 mM
MnCl, 10 ?M ATP, 10 mM NaF, 10 mM PNPP, 10 mM ?-glycerolphosphate, 300
?M sodium orthovanadate, 2 ?M PMSF, 10 ?g of aprotinin per ml, 1 ?g of
leupeptin per ml, 1 ?g of pepstatin per ml, 1 mM DTT) with 2 ?g of GST-I?B?
(positions 1 to 54) or GST-I?B? (positions 1 to 54) containing S32/36Aand 0.1
?Ci of [?-32P]ATP. The kinase reaction was performed for 30 min at 30°C, and
samples were resolved by SDS-PAGE, transferred to Immobilon-P membranes,
and exposed to film.
Increased degradation of Flag-I?B?-wt in HIV-infected cells.
To confirm the expression and functionality of the Flag-I?B?
constructs, pooled clones expressing equal levels of Flag-I?B
constructs were treated or not treated with TNF, followed by
the analysis of the cytosolic extracts by SDS-PAGE and immu-
noblotting with anti-Flag antibodies. As shown in Fig. 1A, TNF
stimulation led to the rapid hyperphosphorylation and subse-
quent degradation of Flag-I?B?-wt. In contrast, Flag-I?B?-?N
and Flag-I?B?-2N were refractory to TNF-induced hyperphos-
phorylation and subsequent degradation. Flag-I?B?-4C be-
haved similarly to Flag-I?B?-wt in that it was susceptible to
TNF-mediated hyperphosphorylation and degradation. These
results confirm that the constitutively overexpressed Flag-I?B?
molecules are regulated as previously described for native
I?B? and highlight the functional relevance of the N terminus
containing S32and S36in TNF-induced I?B? hyperphosphor-
ylation and degradation.
As expected (34), persistent HIV infection of U937 cells re-
sulted in decreased cytosolic levels of native I?B?. Moreover,
I?B? and I?Bε protein levels were also significantly decreased
in HIV-infected cells compared to uninfected cells (Fig. 1B).
Having determined that overexpressed Flag-I?B? constructs
function similarly to native I?B? upon stimulation with known
inducers of NF-?B and that HIV infection of U937 cells results
in decreased steady-state levels of endogenous I?B, we next
investigated whether Flag-I?B?-wt is also a target of HIV in-
fection. Immunoblotting of cytosolic fractions from mock- and
HIV-infected cells expressing Flag-I?B?-wt was performed
with anti-Flag antibodies. U937 cells transfected with the pa-
rental empty retrovirus vector (SFFV) were also mock or HIV
infected and used as controls. As shown in Fig. 1C, the steady-
state protein levels of Flag-I?B?-wt were decreased in the
cytosolic fractions of HIV-infected cells compared to mock-
infected cells, confirming that HIV infection decreases the
cytosolic levels of Flag-I?B? and indicating that tagged I?B?
constructs can be used to study the regulatory domain(s) tar-
geted by persistent HIV infection in monocytes.
Having previously demonstrated that the decreased level of
native I?B? is a result of the enhanced rate of I?B? degrada-
tion in persistently HIV-infected monocytes, we investigated
whether this process also accounted for the decreased level of
Flag-I?B?-wt in infected cells. The half-life of Flag-I?B?-wt
was estimated by immunoblotting Flag-I?B?-wt from cytosolic
fractions from mock- and HIV-infected cells treated for dif-
ferent time periods with cycloheximide. As shown in Fig. 1D,
the turnover of Flag-I?B?-wt was increased in HIV-infected
cells compared to mock-infected cells. The half-lives of Flag-
I?B?-wt calculated from Fig. 1D were found to be approxi-
mately 60 min in HIV-infected cells and 128 min in uninfected
cells (Fig. 1E).
The NH2terminus but not the PEST sequence present in the
COOH terminus of I?B? is necessary for I?B? degradation by
HIV infection. To characterize which of the regulatory do-
mains of I?B? is targeted by HIV infection, we first focused on
the NH2-terminal domain of I?B?. We analyzed the turnover
and half-life of Flag-I?B?-?N. U937 cells stably transfected
with the empty retrovirus vector (SFFV), Flag-I?B?-wt, or
Flag-I?B?-?N were mock or HIV infected. The half-lives of
these constructs were measured by analyzing the levels of the
Flag-I?B? constructs in cytosolic extracts from cell cultures
treated for different time periods with cycloheximide (as for
Fig. 1). As shown in Fig. 2A, Flag-I?B?-?N was very stable not
only in mock-infected but also in HIV-infected U937 cells, with
the resulting half-lives being estimated at greater than 4 h (Fig.
2B). The enhanced stability of Flag-I?B?-?N in both mock-
and HIV-infected cells contrasts with the more rapid turnover
of Flag-I?B?-wt in mock-infected cells and even more rapid
turnover in HIV-infected cells (Fig. 1D and Fig. 2A). These
results indicate that the increased degradation of I?B? that
ensues in HIV-infected cells appears to be dependent on the
NH2-terminal domain of the molecule. In addition, these re-
VOL. 73, 1999I?? MEDIATES NF-?B ACTIVATION IN HIV-INFECTED CELLS3895
sults highlight the potential relevance of this I?B? domain in
the regulation of the basal turnover of I?B? in unstimulated
Previous studies have demonstrated that mutation of S283,
S288, T291, and S293to alanines eliminates the constitutive
phosphorylation of I?B? mediated by PK-CK2 and may influ-
ence the turnover of I?B? (5, 33, 35, 45). Based on this infor-
mation, we analyzed the turnover and half-life of Flag-I?B?-
4C in mock- or HIV-infected U937 cells and compared them to
those of Flag-I?B?-wt. In mock-infected U937 cells, the basal
turnover of Flag-I?B?-4C was slightly longer than that of Flag-
I?B?-wt (Fig. 3), suggesting a potential role of the C-terminal
amino acids S283, S288, T291, and S293in the basal turnover of
I?B? in unstimulated monocytic cells. The half-life of Flag-
I?B?-4C was shorter in HIV-infected cells than in mock-in-
fected cells but was similar to the half-life of Flag-I?B?-wt in
HIV-infected cells (Fig. 3). These results demonstrate that the
amino acids present in the PEST sequence of I?B? are not
involved in the HIV-mediated degradation and turnover of
FIG. 1. Functional characterization of Flag-I?B? molecules in U937 cells. (A) Pooled clones of U937 cells expressing the different Flag-I?B? constructs were
stimulated with TNF for different time periods, and the cell lysates were analyzed by immunoblotting with anti-Flag antibodies. The hyperphosphorylated form of I?B?
is indicated by a small circle. (B) Immunoblotting of cell lysates from mock-infected (NI) or HIV-infected (HIV), SFFV-expressing U937 cells with anti-I?B?,
anti-I?B?, anti-I?Bε, and antiactin antibodies. The hyperphosphorylated form of I?Bε is indicated by a small circle. (C) Immunoblotting of cell lysates from
mock-infected (NI) or HIV-infected (HIV), SFFV- or Flag-I?B?-wt-expressing U937 cells with anti-Flag and antiactin antibodies. (D) The half-life of Flag-I?B?-wt
was estimated by immunoblotting of cell lysates from mock-infected (NI) or HIV-infected (HIV), Flag-I?B?-wt-expressing U937 cells treated with cycloheximide
(CHX) for different periods of time with anti-Flag antibodies. Equal protein loading was calculated by immunoblotting the same membrane with anti-p90rskantibody.
(E) The half-life of Flag-I?B?-wt was calculated by measuring with a densitometer the disintegrations per minute of Flag-I?B?-wt and normalizing them to those for
p90rskfrom each experimental time point shown in panel D.
3896 ASIN ET AL.J. VIROL.
HIV-induced degradation of Flag-I?B? requires phosphor-
ylation at the NH2-terminal residues S32and S36. Several
studies have identified S32and S36as targets of inducible I?B?
kinases (8, 9, 11, 15, 46, 49, 50, 52). As shown in Fig. 1A,
mutation of S32and S36to alanines yields an I?B? construct
that is refractory to the hyperphosphorylation and subsequent
degradation triggered by TNF in U937 cells. Having identified
the NH2-terminal domain of I?B? as a target of HIV-induced
degradation, we next questioned whether S32and S36could be
the amino acids that are targeted by HIV infection. For this,
we investigated the half-life and turnover of Flag-I?B?-2N in
mock- and HIV-infected U937 cells and compared them to the
half-life and turnover of Flag-I?B?-wt. Following the same
experimental design as that used for Fig. 2 and 3, we observed
that in mock-infected cells, mutation of S32and S36to alanines
significantly prolonged the half-life of I?B? (greater than 5 h)
compared to the more rapid turnover of Flag-I?B?-wt (Fig. 4).
This very low rate of basal degradation of Flag-I?B?-2N is
similar to that observed for Flag-I?B?-?N (Fig. 2). Relevant to
the focus of this study, we demonstrate that the half-life of
Flag-I?B?-wt is significantly reduced in HIV-infected cells
compared to mock-infected cells and that Flag-I?B?-2N was
refractory to HIV-mediated I?B? degradation (Fig. 4). These
results confirm that S32and S36are the I?B? amino acids
targeted by persistent HIV infection to result in enhanced deg-
radation of I?B?.
S32and S36are the targets of I?B? kinases that are activated
by a variety of stimuli, such as inflammatory cytokines, and
phosphorylation at these amino acids renders I?B? susceptible
to degradation by the proteosome (8, 9, 11, 12, 15, 16, 24, 29,
37, 42, 43, 46, 49, 50, 52, 53, 57). To investigate whether HIV
infection results in the hyperphosphorylation of I?B?, mock-
or HIV-infected U937 cells expressing Flag-I?B?-wt were
treated with the proteosome inhibitor ALLN for 3 h, after
which cytosolic extracts were separated by SDS-PAGE and
immunoblotted with anti-Flag antibodies. To control for the
accurate detection of hyperphosphorylated I?B?, mock-in-
fected Flag-I?B?-wt-expressing U937 cells were treated or not
treated with TNF and/or a pharmacological inhibitor previ-
ously shown to inhibit the TNF-induced hyperphosphorylation
of I?B? (Bay 11-7082) (41). As shown in Fig. 5 (upper panel),
a more slowly migrating form of Flag-I?B?-wt was observed in
mock-infected, TNF-treated cells, specifically in the presence
of ALLN (lanes 3 and 4). In HIV-infected Flag-I?B?-wt-ex-
pressing U937 cells, a more slowly migrating form of Flag-
I?B?-wt was observed only when ALLN was used (compare
lanes 6 with lane 5). These effects are dependent on the pres-
ence of S32and S36in the Flag-I?B? construct, as their mu-
tation to alanines abrogated both TNF-induced and HIV-
dependent I?B? hyperphosphorylation (Fig. 5, lower panel).
Altogether, these results indicate that the enhanced degrada-
tion of I?B? that is observed in HIV-infected monocytes is a
result of specific hyperphosphorylation of I?B? at S32and S36.
Whether the differences in the kinetics of I?B? hyperphos-
phorylation at S32and S36between transient stimuli, such as
TNF, and chronic stimuli, such as persistent HIV infection, are
due to the use of different I?B? kinases or simply different
upstream control mechanisms is currently unknown.
The I?? complex mediates HIV-dependent I?B? degrada-
tion and NF-?B activation. Two kinases in the I?? complex
(I??? and I???) have recently been shown to phosphorylate
S32and S36of I?B? and to be the targets of inflammatory
cytokines, such as TNF and interleukin 1 (12, 16, 29, 37, 42, 53,
57). Having identified S32and S36as the regulatory amino acids
of I?B? which are targeted by HIV, we questioned whether the
I?? complex is activated by HIV infection and mediates the
increased levels of nuclear NF-?B activation in infected cells.
Mock-infected and persistently HIV-infected U937 cells were
lysed, followed by immunoprecipitation of the I?? complex,
p90rsk, or Raf-1. The kinase activities of these immunoprecipi-
tates were analyzed in an in vitro kinase reaction with GST-
I?B? (positions 1 to 54) or GST-I?B? (positions 1 to 54) con-
taining S32/36Aas a substrate. In HIV-infected samples,
increased I?B? kinase activity was present in the I?? complex
immunoprecipitate but not in the p90rsk(Fig. 6A) or the Raf-1
(data not shown) immunoprecipitate. This kinase activity was
specific for S32and S36, as their mutation eliminated the basal
and HIV-induced I?? complex activity. Also, as shown in Fig.
6A, there was no difference in the amounts of I?? complex
immunoprecipitated with anti-I??? antibodies in mock- and
HIV-infected U937 cells, thus eliminating the possibility that
HIV infection simply increases the pool of I?? kinases. These
data indicate that HIV infection activates the I?? complex,
resulting in phosphorylation at S32and S36.
The potential relevance of the I?? complex in mediating the
HIV-dependent activation of NF-?B was further analyzed in
transient transfection experiments. Transcription from an NF-
?B-dependent luciferase reporter gene was analyzed with both
mock- and HIV-infected U937 cells in the presence or absence
of wild-type or dominant negative forms of I??? and I???. A
minimal TK promoter driving the expression of CAT was used
to normalize for transfection efficiency differences that might
be present between mock- and HIV-infected cells. The results
of these experiments demonstrated that the increased NF-?B
activity that is observed in HIV-infected cells is reduced by an
I??? dominant negative expression vector but not by a domi-
nant negative form of I??? or the wild-type form of either
kinase (Fig. 6B). Altogether, these studies demonstrate that
FIG. 2. Deletion of the first 37 amino acids of I?B? conveys resistance to
HIV-mediated degradation. (A) Mock-infected (NI) or HIV-infected (HIV),
Flag-I?B?-?N-expressing U937 cells were treated with cycloheximide (CHX) for
different time periods, after which cell lysates were analyzed by immunoblotting
with anti-Flag or antiactin antibodies. (B) The half-life of Flag-I?B?-?N was
calculated as described in the legend to Fig. 1E.
VOL. 73, 1999I?? MEDIATES NF-?B ACTIVATION IN HIV-INFECTED CELLS 3897
the I?? complex is activated by HIV infection and mediates
virus-induced I?B? hyperphosphorylation and NF-?B activa-
HIV-1 replication in U937 cells expressing different trans-
dominant mutants of I?B?. Having determined that S32and
S36of I?B? are required for HIV-mediated I?B? degradation,
we next questioned whether U937 cells expressing Flag-I?B?-
?N or Flag-I?B?-2N (constructs that are refractory to HIV-
dependent degradation) would inhibit HIV-mediated NF-?B
activation, and if so, whether this inhibition would result in
decreased viral replication. Nuclear extracts and cell-free su-
pernatants were obtained from mock- or HIV-infected cells
stably transfected with the SFFV vector, Flag-I?B?-?N or
Flag-I?B?-2N at the same time as the cytosolic fractions were
analyzed to determine the half-lives of the respective I?B?
constructs (Fig. 2 and 4). Nuclear extracts were analyzed by a
gel shift assay with an oligonucleotide containing NF-?B DNA
binding motifs, and viral replication was monitored by measur-
ing p24 levels in culture supernatants. As shown in Fig. 7A and
B, left panels, HIV infection of SFFV-expressing U937 cells
led to nuclear translocation of a DNA binding protein complex
composed of p50 and Rel-A (p65); this finding was not ob-
served in HIV-infected cells expressing either Flag-I?B?-?N
or Flag-I?B?-2N (Fig. 7A and B, right panels). These obser-
vations directly correlate with the inability of these two Flag-
I?B? constructs to undergo HIV-mediated degradation, as
demonstrated in Fig. 2 and 4.
The levels of HIV replication in the cells expressing SFFV,
Flag-I?B?-?N, or Flag-I?B?-2N were then analyzed by mea-
suring HIV p24 levels in supernatants from the same cultures
as those used to study the I?B? half-life (Fig. 2 and 4) and
NF-?B nuclear translocation (Fig. 7A and B). The results of
these experiments indicated that there was no significant re-
duction in the levels of p24 in supernatants of U937 cells
expressing Flag-I?B?-?N or Flag-I?B?-2N compared to su-
pernatants of control cultures (SFFV expressing) (Fig. 7C).
Using an HIV-susceptible promonocytic cell line which can
support persistent viral replication, we have determined that
the I?B? residues S32and S36and the I?? complex (12, 16, 29,
37, 42, 53, 57) are required to mediate HIV-dependent I?B?
degradation and, hence, NF-?B activation. The identification
of a transdominant negative I?B? molecule which is refractory
to HIV-dependent degradation and thus is capable of blocking
the HIV-mediated activation of NF-?B extends and confirms
previous studies from our group indicating that I?B? is a target
molecule and that persistent HIV infection leads to increased
NF-?B activation (34). In addition, it provides supporting data
that HIV-mediated NF-?B (p50/p65) activation is not neces-
sary to support viral persistence in the U937 monocytic cell
The use of pooled clones of monocytic cells that constitu-
FIG. 3. Mutation of the phosphoamino acids present in the PEST sequence does not alter the HIV-mediated degradation of I?B?. (A) Mock-infected (NI) or
HIV-infected (HIV), Flag-I?B?-4C- or Flag-I?B?-wt-expressing U937 cells were treated for different time periods with cycloheximide (CHX), and cell lysates were
analyzed by immunoblotting with anti-Flag and anti-p90rskantibodies. The lysates used for detecting p90rsklevels were the same as those from Flag-I?B?-4C-expressing
U937 cells. Similar results were obtained with Flag-I?B?-wt-expressing U937 cell lysates. (B) The half-lives of Flag-I?B?-4C (left panel) and Flag-I?B?-wt (right panel)
were calculated as described in the legend to Fig. 1E.
3898ASIN ET AL. J. VIROL.
tively express genetically modified I?B? constructs has proven
to be a valuable tool with which to study the role of NF-?B
replication in HIV persistence. Different from punctual stimuli
(inflammatory cytokines or transient expression of human T-
cell leukemia virus type 1 tax), the activation of NF-?B by HIV
infection is dependent on the establishment of viral persis-
tence, achieved only after 6 to 10 days of viral infection (2, 34,
40). Due to this unique virus-host cell interaction, the experi-
mental approaches which can be utilized to address the mech-
anisms by which HIV activates NF-?B have been significantly
limited. Previous attempts have used nonmonocytic cell lines
which are highly susceptible to gene transfection, such as 293
or COS-7 cells (28, 54, 55). However, such studies, rather than
focusing on HIV persistence in stimulating NF-?B activation,
have addressed the role of I?B? in controlling the reactivation
of HIV from latency or in inhibiting the initiation of viral rep-
The use of our genetically modified monocytic cells has al-
lowed us to address the mechanism(s) by which HIV leads to
NF-?B activation and then to study the role of I?B? in con-
trolling viral persistence. To ensure the relevance of this mod-
el, significant efforts were made to verify the maintenance of
stable I?B? expression and CD4 expression and the function-
ality of the tagged overexpressed I?B? clones throughout the
infections (months). In addition, as was the case for the Flag-
I?B?-2N construct, experiments were repeated with each in-
dividual clone separately to verify that the results obtained
with a pool of three clones were not due to the overgrowth of
a single clone. The level of expression of Flag-I?B?-?N and
Flag-I?B?-2N was significantly higher in HIV-infected cells
than in uninfected cells. This result may be due to increased
transcription from the SFFV retrovirus promoter in HIV-in-
fected U937 cells. As both the I?B? N-terminal deletion and
FIG. 4. Mutation of S32and S36of I?B? abrogates HIV-mediated I?B? degradation. (A) Mock-infected (NI) or HIV-infected (HIV), Flag-I?B?-2N- or
Flag-I?B?-wt-expressing U937 cells were treated with cycloheximide (CHX) for different time periods, after which cell lysates were analyzed by SDS-PAGE and
immunoblotted with anti-Flag and anti-p90rskantibodies. The p90rsklysates were the same as those from Flag-I?B?-2N-expressing U937 cells. Similar results were
obtained with Flag-I?B?-wt-expressing U937 cell lysates. (B) The half-lives of Flag-I?B?-2N and Flag-I?B?-wt were calculated as described in the legend to Fig. 1E.
FIG. 5. HIV infection of U937 cells induces hyperphosphorylation of I?B?
which is dependent on S32and S36. Mock-infected (NI) or HIV-infected (HIV),
Flag-I?B?-wt- or Flag-I?B?-2N-expressing U937 cells were treated (?) or not
treated (?) with Bay 11-7082 (Bay 11), TNF, or ALLN, after which the cell
lysates were analyzed by SDS-PAGE and immunoblotted with anti-Flag anti-
bodies. The supershifted hyperphosphorylated I?B? form of Flag-I?B?-wt is
indicated by a bullet.
VOL. 73, 1999I?? MEDIATES NF-?B ACTIVATION IN HIV-INFECTED CELLS3899
the I?B? N-terminal mutation are refractory to HIV-induced
degradation, over time the steady-state Flag-I?B?-?N and
Flag-I?B?-2N protein levels may lead to an increase in the
levels of the transgene.
The reduced half-life of I?B? observed in HIV-infected cells
differs significantly from the very short half-life of I?B? ob-
served following TNF stimulation. This observation initially led
to the hypothesis that sites other than S32and S36are targeted
by HIV infection. However, the finding that S32and S36are
required for enhanced I?B? turnover in HIV-infected cells,
together with the observation that the I?? complex (12, 16, 29,
37, 42, 53) is activated and mediates NF-?B activation in HIV-
infected cells, demonstrates a shared utilization of this kinase
complex and this I?B? domain in mediating NF-?B activation
by unrelated stimuli. What accounts for the significant differ-
ence in I?B? half-lives with two separate stimuli, i.e., TNF (5
to 10 min) and HIV (50 to 60 min), which share the same I?B?
regulatory domain and kinase complex, is unknown. We have
previously demonstrated that within an HIV-infected U937
cell culture, ?90% of cells express intracytoplasmic HIV p24,
thus excluding the possibility that a small subpopulation that is
actively HIV infected results in a dilution effect (34).
From the available data, we conclude that it is a lower
degree of I?? complex activation by HIV infection that corre-
lates with the smaller amount of hyperphosphorylated I?B?
and the slower I?B? turnover in HIV-infected cells than in
TNF-treated cells. Whether the lower degree of I?? activation
is secondary to the utilization of different secondary messen-
gers that lie upstream of I?? is unknown. It is also plausible
that HIV infection targets regulatory processes that control the
basal level of I?? activity rather than its “inducible” activity.
Recent data indicate that protein phosphatase 2A (PP2A)
dephosphorylates I???, resulting in a decrease in its kinase
activity (16) and explaining the NF-?B-activating function of
the PP2A inhibitor okadaic acid (16). It is theoretically possi-
ble that HIV infection inhibits PP2A, resulting in a higher
“basal” I?? activity which is separate from the TNF-inducible
I?? activity. Previous studies from our group have identified
p21ras(21) and the atypical protein kinase C isoforms ? and ?
(20) as essential components of NF-?B activation mediated by
HIV infection. Whether these secondary messengers target the
I?? complex is unknown, but recent advances in the charac-
terization of I?? complex regulation will now enable the study
of the role of these secondary messengers in HIV-induced
FIG. 6. The I?? complex but not p90rskmediates the HIV-induced activation of NF-?B. (A) In vitro kinase assay of I?? and p90rsk. Immunoprecipitates (IP) from
mock-infected (NI), mock-infected and TNF treated (TNF), and HIV-infected (HIV), SFFV-expressing U937 cells were lysed, and the I?? complex and p90rskwere
immunoprecipitated with anti-I??? and anti-p90rskantibodies, respectively. Immunoprecipitates were analyzed in an in vitro kinase (IVK) assay with recombinant
protein I?B?-wt (positions 1 to 54) or I?B? S32/36A(positions 1 to 54) as a substrate (32P-I?B?). The membrane was subsequently stained with Coomassie blue (stained
I?B?) or immunoblotted (Immunoblot) with anti-I???, anti-I???, or anti-p90rskantibodies. (B) SFFV-expressing, HIV-infected U937 cells were transiently transfected
with con-luc (?) or ?B-con-luc (I) together with expression vectors for wild-type (WT) or negative dominant (nd) forms of I??? or I??? and a TK CAT reporter gene.
Luciferase units were normalized to CAT units. The NF-?B luciferase activity of uninfected, SFFV-expressing cells was similar to that of con-luc in HIV-infected cells,
and none of the I??? or I??? (wt or nd) expression vectors modified the basal level of plasmid ?B-luc activity in uninfected cells (data not shown). This experiment
is representative of three additional ones. Each transfection point was determined in duplicate, and error bars indicate ? standard deviations.
3900 ASIN ET AL.J. VIROL.
NF-?B activation and their linkage to the activation of the I??
The apparent lack of dependence of viral persistence on
HIV-mediated NF-?B (p50/p56) activation is a significant con-
clusion from this study. While several groups, including ours,
have consistently demonstrated that persistent HIV infection
of monocytic cells results in the selective activation of NF-?B
(p50/p65) (2, 34, 40, 43), it has not been possible to clearly
demonstrate that the HIV-dependent activation of the p50/p65
heterodimer is necessary to maintain viral persistence in such
cells. Attempts to test this question have been made with
proteosome inhibitors (14, 27). These compounds were shown
to inhibit HIV-dependent I?B? degradation and, hence, p50/
p65 heterodimer nuclear translocation, which correlated with a
reduction in HIV replication. Because proteosome inhibitors
may inhibit a variety of additional cell functions and, poten-
tially, specific steps of the HIV cycle, the role of HIV-depen-
dent activation of NF-?B in regulating viral persistence in
monocytic cells remains to be fully clarified. The use of genetic
approaches such as the one described in this study allows for
more specific inhibition of NF-?B. Interestingly, overexpres-
sion of transdominant mutants of Flag-I?B? is sufficient to
inhibit the nuclear translocation of additional NF-?B (p50/
p65) complexes that may result from the enhanced HIV-de-
pendent degradation of I?B? and/or I?Bε, indicating that an
I?B? negative dominant molecule overrides the functional im-
pact of the two other I?B molecules, at least in HIV-infected
Our results indicate an apparent dispensable role of HIV-
triggered NF-?B (p50/p65) activation in maintaining viral per-
sistence in U937 cells. Whereas it is still possible that HIV-
induced NF-?B (p50/p65) activation is indeed involved in
controlling viral persistence, inhibition of this mechanism may
have allowed for the utilization of complementary mechanisms
to maintain viral persistence in the absence of constitutive or
HIV-induced nuclear translocation of the p50/p65 hetero-
dimer. Other members of the NF-?B family or alternative
transcription factors, such as Sp1, may be constitutively present
in the nuclei of host cells or selectively activated by HIV
infection (51) and thus may compensate for the lack of nuclear
translocation of p50/p65 dimers observed in the clones express-
ing I?B? negative transdominant molecules. Rel-B nuclear
translocation is thought to be refractory to I?B? inhibition (18,
32). Therefore, either the constitutive presence of Rel-B in
nuclei or its potential nuclear translocation following HIV
infection might serve to compensate for a lack of HIV-induced
p50/p65 in the nuclei of infected cells expressing I?B? negative
transdominant molecules. While our gel shift assay experi-
ments with NF-?B DNA concatemers did not demonstrate any
NF-?B DNA binding activity in HIV-infected cells expressing
I?B? S32and S36mutants, the detection of DNA binding
activity of Rel-B may have been elusive, as previously sug-
gested. Infection of U937 cells which express an I?B? S32/36A
mutant with HIV provirus lacking the NF-?B cis-acting motifs
could help clarify the potential role of nuclear proteins which
could bind and regulate transcription through the NF-?B cis-
acting sequences (23).
While the above hypothesis can be adequately tested with
U937 cells, it is ultimately necessary to test the role of the
HIV-mediated activation of NF-?B in regulating viral persis-
tence within true physiological host cells, such as human mac-
rophages. In these cells, persistent HIV infection results in the
continuous activation of NF-?B (34); thus, it is mandatory to
test whether its inhibition alters viral persistence in these host
cells. Unfortunately, the current lack of specific inhibitors of
I?B? phosphorylation at S32and S36and the limitation of
applying genetic approaches, such as those used here with
U937 cells, to primary human macrophages preclude the con-
clusion that the observations derived from promonocytic cells
apply to human macrophages.
With the identification of the I?? complex and I?B? S32and
S36as targets of persistent HIV infection in monocytes, HIV
infection can now be added to the growing list of NF-?B
activators that utilize this recently identified complex of N-
terminal I?B? kinases. In addition, differences in the degree of
I?? activation and hence in I?B? turnover between a “chronic”
stimulus, such as persistent HIV infection, and other, more
“punctual” ones suggest that there may be different means of
activating the I?? complex within the same cell. Lastly, using
genetically modified I?B? constructs, we have been able to
demonstrate that the HIV-mediated activation of NF-?B is not
necessary to maintain viral persistence. Thus, future efforts
should be directed at exploring the complementary role of
other NF-?B family members or additional transcription fac-
tors in regulating viral persistence in human macrophages as
an important cell reservoir of HIV infection.
FIG. 7. Genetic interference with HIV-mediated NF-?B activation does not
result in reduced HIV replication. (A) Gel shift assays of nuclear extracts from
mock-infected (NI) or HIV-infected (HIV), SFFV- or Flag-I?B?-?N-expressing
U937 cells. Antibodies against p50 (p50) or p65 (p65) were added to the gel shift
assay. The corresponding molecular complex is indicated. (B) Same panel A,
except that nuclear extracts from SFFV-expressing U937 cells were compared in
parallel to those from Flag-I?B?-?-2N-expressing U937 cells. (C) The HIV p24
content in supernatants of HIV-infected, SFFV-expressing U937 cells (?) or
cells expressing Flag-I?B?-?N (I) and Flag-I?B?-2N (I) was calculated in
duplicate. This experiment is representative of two additional ones. Error bars
indicate standard deviations.
VOL. 73, 1999I?? MEDIATES NF-?B ACTIVATION IN HIV-INFECTED CELLS 3901
1. Alcamı ´, J., T. Laı ´n de Lera, L. Folgueira, M.-A. Pedraza, J.-M. Jacque ´, F.
Bachelerie, A. R. Noriega, R. T. Hay, D. Harrich, R. B. Gaynor, J.-L. Vire-
lizier, and F. Arenzana-Seisdedos. 1995. Absolute dependence on ?B re-
sponsive elements for initiation and TAT-mediated amplification of HIV
transcription in blood CD4 T lymphocytes. EMBO J. 14:1552–1560.
2. Bachelerie, F., J. Alcami, F. Arenzana-Seisdedos, and J.-L. Virelizier. 1991.
HIV enhancer activity perpetuated by NF-?B induction on infection of
monocytes. Nature (London) 350:709–712.
3. Baeuerle, P. A., and D. Baltimore. 1996. NF-?B ten years after. Cell 87:
4. Baeuerle, P. A., and T. Henkel. 1994. Function and activation of NF-?B in
the immune system. Annu. Rev. Immunol. 12:141–179.
5. Barroga, C. F., J. K. Stevenson, E. M. Schwarz, and I. M. Verma. 1995.
Constitutive phosphorylation of I?B? by casein kinase II. Proc. Natl. Acad.
Sci. USA 92:7637–7641.
6. Beg, A. A., S. M. Ruben, R. I. Scheinman, S. Haskill, C. A. Rosen, and A. S.
Baldwin. 1992. I?B interacts with the nuclear localization sequences of the
subunits of NF-?B: a mechanism for cytoplasmic retention. Genes Dev. 6:
7. Beg, A. A., T. S. Finco, P. V. Nantermet, and A. S. Baldwin. 1993. Tumor
necrosis factor and interleukin-1 lead to phosphorylation and loss of I?B?:
a mechanism of NF-?B activation. Mol. Cell. Biol. 13:3301–3310.
8. Brockman, J. A., D. C. Scherer, T. A. McKinsey, S. M. Hall, X. Qi, W. Y. Lee,
and D. W. Ballard. 1995. Coupling of a signal response domain in I?B? to
multiple pathways for NF-?B activation. Mol. Cell. Biol. 15:2809–2818.
9. Brown, K., S. Gerstberger, L. Carlson, G. Franzoso, and U. Siebenlist. 1995.
Control of I?B? proteolysis by site-specific, signal-induced phosphorylation.
10. Chen, B. K., M. B. Feinberg, and D. Baltimore. 1997. The ?B sites in the
human immunodeficiency virus type 1 long terminal repeat enhance virus
replication yet are not absolutely required for viral growth. J. Virol. 71:
11. Chen, Z., J. Hagler, V. J. Palombella, F. Melandri, D. Scherer, D. Ballard,
and T. Maniatis. 1995. Signal-induced site-specific phosphorylation targets
I?B? to the ubiquitin-proteosome pathway. Genes Dev. 9:1586–1587.
12. Chen, Z. J., L. Parent, and T. Maniatis. 1996. Site-specific phosphorylation
of I?B? by a novel ubiquitination-dependent protein kinase activity. Cell 84:
13. Cordle, S. R., R. Donald, M. A. Reed, and J. Hawiger. 1993. Lipopolysac-
charide induces phosphorylation of MAD3 and activation of c-rel and re-
lated NF-?B proteins in human monocytic THP-1 cells. J. Biol. Chem. 268:
14. DeLuca, C., A. Roulston, A. Koromilas, M. A. Wainberg, and J. Hiscott.
1996. Chronic human immunodeficiency virus type 1 infection of myeloid
cells disrupts the autoregulatory control of the NF-?B/Rel pathway via en-
hanced I?B? degradation. J. Virol. 70:5183–5193.
15. DiDonato, J., F. Mercurio, C. Rosette, J. Wu-Li, H. Suyang, S. Ghosh, and
M. Karin. 1996. Mapping of the inducible I?B phosphorylation sites that
signal its ubiquitination and degradation. Mol. Cell. Biol. 16:1295–1304.
16. DiDonato, J. A., M. Hayakawa, D. M. Rothwarf, E. Zandi, and M. Karin.
1997. A cytokine-responsive I?B kinase that activates the transcription factor
NF-?B. Nature 388:548–554.
17. Dignam, J. D., R. M. Lebovitz, and R. G. Roeder. 1983. Accurate transcrip-
tion initiation by RNA polymerase II in a soluble extract from isolated
mammalian nuclei. Nucleic Acids Res. 11:1475–1489.
18. Ferreira, V., N. Tarantino, and M. Ko ¨rner. 1998. Discrimination between
RelA and RelB transcriptional regulation by a dominant negative mutant of
I?B?. J. Biol. Chem. 273:592–599.
19. Finzl, D., M. Hermankova, T. Pierson, L. M. Carruth, C. Buck, R. E.
Chaisson, T. C. Quinn, K. Chadwick, J. Margolick, R. Brookmeyer, J. Gal-
lant, M. Markowitz, D. D. Ho, D. D. Richman, and R. F. Siliciano. 1997.
Identification of a reservoir for HIV-1 in patients on highly active antiret-
roviral therapy. Science 278:1295–1300.
20. Folgueira, L., J. A. McElhinny, G. D. Bren, W. S. MacMorran, M. T. Diaz-
Meco, J. Moscat, and C. V. Paya. 1996. Protein kinase C-? mediates NF-?B
activation in human immunodeficiency virus-infected monocytes. J. Virol.
21. Folgueira, L., A. Algeciras, W. S. MacMorran, G. D. Bren, and C. V. Paya.
1996. The ras-raf pathway is activated in human immunodeficiency virus-
infected monocytes and participates in the activation of NF-?B. J. Virol. 70:
22. Fuhlbrigge, R. C., S. M. Fine, E. R. Unanue, and D. D. Chaplin. 1988.
Expression of membrane interleukin 1 by fibroblasts transfected with murine
pro-interleukin 1? cDNA. Proc. Natl. Acad. Sci. USA 85:5649–5653.
23. Fuminori, H., H. Tanaka, Y. Hirano, M. Hiramoto, H. Handa, I. Makino,
and C. Scheidereit. 1998. Functional interference of Sp1 and NF-?B through
the same DNA binding site. Mol. Cell. Biol. 18:1266–1274.
24. Ghoda, L., X. Lin, and W. C. Green. 1997. The 90-kDa ribosomal S6 kinase
(pp90rsk) phosphorylates the N-terminal regulatory domain of I?B? and
stimulates its degradation in vitro. J. Biol. Chem. 272:21281–21288.
25. Griffin, G. E., K. Leung, T. M. Folks, S. Kunkel, and G. J. Nabel. 1989.
Activation of HIV gene expression during monocyte differentiation by in-
duction of NF-?B. Nature 339:70–73.
26. Haskill, D., A. A. Beg, S. M. Tompkins, J. S. Morris, A. D. Yurochko, A.
Sampson-Johannes, K. Mondal, P. Ralph, and A. S. Baldwin, Jr. 1991.
Characterization of an immediate-early gene induced in adherent monocytes
that encodes I?B-like activity. Cell 65:1281–1289.
27. Jacque ´, J.-M., B. Ferna ´ndez, F. Arenzana-Seisdedos, D. Thomas, F. Baleux,
J. L. Virelizier, and F. Bachelerie. 1996. Permanent occupancy of the human
immunodeficiency virus type 1 enhancer by NF-?B is needed for persistent
viral replication in monocytes. J. Virol. 70:2930–2938.
28. Kwon, H., N. Pelletier, C. DeLuca, P. Genin, S. Cisternas, R. Lin, M. A.
Wainberg, and J. Hiscott. 1998. Inducible expression of I?B? repressor
mutants interferes with NF-?B activity and HIV-1 replication in Jurkat T
cells. J. Biol. Chem. 273:7431–7440.
29. Lee, F. S., J. Hagler, Z. J. Chen, and T. Maniatis. 1997. Activation of the
I?B? kinase complex by MEKK1, a kinase of the JNK pathway. Cell 88:
30. Lefkovits, I., and H. Waldmann. 1979. Limiting dilution analysis of cells in
the immune system. Cambridge University Press, Cambridge, England.
31. Leonard, J., C. Parrott, A. J. Buckler-White, W. Turner, E. K. Ross, M. A.
Martin, and A. B. Rabson. 1989. The NF-?B binding sites in the human
immunodeficiency virus type 1 long terminal repeat are not required for virus
infectivity. J. Virol. 63:4919–4924.
32. Lernbecher, T., B. Kistler, and T. Wirth. 1994. Two distinct mechanisms
contribute to the constitutive activation of relB in lymphoid cells. EMBO J.
33. Lin, R., P. Beauparlant, C. Makris, S. Meloche, and J. Hiscott. 1996. Phos-
phorylation of I?B? in the C-terminal PEST domain by casein kinase II
affects intrinsic protein stability. Mol. Cell. Biol. 16:1401–1409.
34. McElhinny, J. A., W. S. MacMorran, G. D. Bren, R. M. Ten, A. Israel, and
C. V. Paya. 1995. Regulation of I?B? and p105 in monocytes and macro-
phages persistently infected with human immunodeficiency virus. J. Virol.
35. McElhinny, J. A., S. A. Trushin, G. D. Bren, N. Chester, and C. V. Paya.
1996. Casein kinase II phosphorylates I?B? at S-283, S-288, S-293, and T-291
and is required for its degradation. Mol. Cell. Biol. 16:899–906.
36. Meltzer, M. S., D. R. Skillman, D. L. Hoover, B. D. Hanson, J. A. Turpin, D.
Chester Kalter, and H. E. Gendelman. 1990. Macrophages and the human
immunodeficiency virus. Immunol. Today 11:217–223.
37. Mercurio, F., H. Zhu, B. W. Murray, A. Shevchenko, B. L. Bennett, J. Wu Li,
D. B. Young, M. Barbosa, M. Mann, A. Manning, and A. Rao. 1997. I??-1
and I??-2: cytokine-activated I?B kinases essential for NF?B activation.
38. Nabel, G., and D. Baltimore. 1987. An inducible transcription factor acti-
vates expression of human immunodeficiency virus in T cells. Nature (Lon-
39. Oh, S. H. I., and R. B. Gaynor. 1995. Intracellular factors involved in gene
expression of human retroviruses, p. 97–187. In J. A. Levy (ed.), The Ret-
roviridae, 4th ed. Plenum Press, New York, N.Y.
40. Paya, C. V., R. M. Ten, C. Bessia, J. Alcami, and R. T. Hay. 1992. NF-?B-
dependent induction of the NF-?B p50 subunit gene promoter underlies
self-perpetuation of human immunodeficiency virus transcription in mono-
cytic cells. Proc. Natl. Acad. Sci. USA 89:7826–7830.
41. Pierce, J. W., R. Schoenleber, G. Jesmok, J. Best, S. A. Moore, T. Collins,
and M. E. Gerritsen. 1997. Novel inhibitors of cytokine-induced I?B? phos-
phorylation and endothelial cell adhesion molecule expression show anti-
inflammatory effect in vivo. J. Biol. Chem. 272:21096–21103.
42. Re ´gnier, C. H., H. Y. Song, X. Gao, D. V. Goeddel, Z. Cao, and M. Rothe.
1997. Identification and characterization of an I?B kinase. Cell 90:373–383.
43. Roulston, A., M. D’Addario, F. Boulerice, S. Caplan, M. A. Wainberg, and J.
Hiscott. 1992. Induction of monocyte differentiation and NF-?B-like activi-
ties by human immunodeficiency virus 1 infection of myelomonoblastic cells.
J. Exp. Med. 175:751–752.
44. Schouten, G. J., A. C. O. Vertegaal, S. T. Whiteside, A. Israel, M. Toebes,
J. C. Dorsman, A. J. van der Eb, and A. Zantema. 1997. I?B? is a target for
the mitogen-activated 90 kDa ribosomal S6 kinase. EMBO J. 16:3133–3144.
45. Schwarz, E. M., D. V. Antwerp, and I. M. Verma. 1996. Constitutive phos-
phorylation of I?B? by casein kinase II occurs preferentially at serine 293:
requirement for degradation of free I?B?. Mol. Cell. Biol. 16:3554–3559.
46. Sun, S.-C., J. Elwood, and W. C. Greene. 1996. Both amino- and carboxyl-
terminal sequences within I?B? regulate its inducible degradation. Mol.
Cell. Biol. 16:1058–1065.
47. Sun, S.-C., J. Elwood, C. Beraud, and W. C. Greene. 1994. Human T-cell
leukemia type 1 tax activation of NF-?B/Rel involves phosphorylation and
degradation of I?B?- and RelA (p65)-mediated induction of the c-rel gene.
Mol. Cell. Biol. 14:7377–7384.
48. Suzan, M., D. Salaun, C. Neuveut, B. Spire, I. Hirsch, P. de Bouteiller, G.
Querat, and J. Sire. Induction of NF-?B during monocyte differentiation by
HIV type 1 infection. J. Immunol. 146:377–383.
49. Traenckner, E. B.-M., H. L. Pahl, T. Henkel, K. N. Schmidt, S. Wilk, and
P. A. Baeuerle. 1995. Phosphorylation of human I?B? on serines 32 and 36
3902 ASIN ET AL.J. VIROL.
controls I?B? proteolysis and NF?B activation in response to diverse stimuli. Download full-text
EMBO J. 14:2876–2883.
50. Van Antwerp, D. J., and I. M. Verma. 1996. Signal-induced degradation of
I?B?: association with NF-?B and the PEST sequence in I?B? are not
required. Mol. Cell. Biol. 16:6037–6045.
51. Wang, L., S. Mukherjee, F. Jia, O. Narayan, and L.-J. Zhao. 1995. Interac-
tion of virion protein Vpr of human immunodeficiency virus type 1 with
cellular transcription factor Sp1 and transactivation of viral long terminal
repeat. J. Biol. Chem. 270:25564–25569.
52. Whiteside, S. T., M. K. Ernst, O. LeBail, C. Laurent-Winter, N. Rice, and A.
Israel. 1995. N- and C-terminal sequences control degradation of MAD3/
I?B? in response to inducers of NF-?B activity. Mol. Cell. Biol. 15:5339–
53. Woronicz, J. D., X. Gao, Z. Cao, M. Rothe, and D. V. Goeddel. 1997. I?B
kinase-?: NF?B activation and complex formation with I?B kinase-? and
NIK. Science 278:866–869.
54. Wu, B.-Y., C. Woffendin, C. S. Duckett, T. Ohno, and G. J. Nabel. 1995.
Regulation of human retroviral latency by the NF-?B/I?B? family: inhibition
of human immunodeficiency virus replication by I?B through a Rev-depen-
dent mechanism. Proc. Natl. Acad. Sci. USA 92:1480–1484.
55. Wu, B.-Y., C. Woffendin, I. MacLachlan, and G. J. Nabel. 1997. Distinct
domains of I?B? inhibit human immunodeficiency virus type 1 replication
through NF-?B and Rev. J. Virol. 71:3161–3167.
56. Yin, M.-J., L. B. Christerson, Y. Yamamoto, Y.-T. Kwak, S. Xu, F. Mercurio,
M. Barbosa, M. H. Cobb, and R. B. Gaynor. 1998. HTLV-1 Tax protein binds
to Mekk1 to stimulate I?B kinase activity and NF-?B activation. Cell 93:
57. Zandi, E., D. M. Torhwarf, M. Delhase, M. Hayakawa, and M. Karin. 1997.
The I?B kinase complex (I??) contains two kinase subunits, I??? and I???,
necessary for I?B phosphorylation and NF-?B activation. Cell 91:243–252.
VOL. 73, 1999I?? MEDIATES NF-?B ACTIVATION IN HIV-INFECTED CELLS 3903