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1996, American Society for Microbiology

Transdominant Mutants of I

k

B

a

Block Tat-Tumor Necrosis

Factor Synergistic Activation of Human Immunodeficiency

Virus Type 1 Gene Expression and Virus Multiplication

PIERRE BEAUPARLANT,

1

HAKJU KWON,

1

MICHELLE CLARKE,

1

RONGTUAN LIN,

1

NAHUM SONENBERG,

2

MARK WAINBERG,

1AND

JOHN HISCOTT

1

*

Lady Davis Institute for Medical Research and Departments of Microbiology and Medicine,

McGill AIDS Center, McGill University, Montreal, Canada H3T 1E2,

1

and Department

of Biochemistry and McGill Cancer Center, McGill University,

Montreal, Canada H3G 1Y6

2

Received 13 February 1996/Accepted 16 May 1996

The human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) contains two binding sites

for the NF-

k

B/Rel family of transcription factors which are required for the transcriptional activation of viral

genes by inflammatory cytokines such as tumor necrosis factor alpha (TNF-

a

) and interleukin-1. In the present

study, we examined the effect of transdominant mutants of I

k

B

a

on the synergistic activation of the HIV-1 LTR

by TNF-

a

and the HIV-1 transactivator, Tat, in Jurkat T cells. The synergistic induction of HIV-1 LTR-driven

gene expression represented a 50- to 70-fold stimulation and required both an intact HIV-1 enhancer and

Tat-TAR element interaction, since mutations in Tat protein (R52Q, R53Q) or in the bulge region of the TAR

element that eliminated Tat binding to TAR were unable to stimulate LTR expression. Coexpression of I

k

B

a

inhibited Tat-TNF-

a

activation of HIV LTR in a dose-dependent manner. Transdominant forms of I

k

B

a

,

mutated in critical serine or threonine residues required for inducer-mediated (S32A, S36A) and/or

constitu-tive (S283A, T291A, T299A) phosphorylation of I

k

B

a

were tested for their capacity to block HIV-1 LTR

transactivation. I

k

B

a

molecules mutated in the N-terminal sites were not degraded following inducer-mediated

stimulation (

t

1/2

, >4 h) and were able to efficiently block HIV-1 LTR transactivation. Strikingly, the I

k

B

a

(S32A, S36A) transdominant mutant was at least five times as effective as wild-type I

k

B

a

in inhibiting

synergistic induction of the HIV-1 LTR. This mutant also effectively inhibited HIV-1 multiplication in a

single-cycle infection model in Cos-1 cells, as measured by Northern (RNA) blot analysis of viral mRNA species

and viral protein production. These experiments suggest a strategy that may contribute to inhibition of HIV-1

gene expression by interfering with the NF-

k

B/Rel signaling pathway.

The intracellular efficiency of human immunodeficiency

vi-rus type 1 (HIV-1) gene expression and replication is due in

part to the ability of HIV-1 to utilize host signaling pathways to

mediate its own transcriptional regulation. In this regard, the

NF-

k

B/Rel pathway plays a central role in HIV-1 long terminal

repeat (LTR)-driven transcription. The HIV-1 LTR contains

two adjacent high-affinity

k

B-binding sites in its enhancer

re-gion (

2

109 to

2

79) (55). Transient-transfection studies with

HIV-1 LTR or HIV-1 enhancer reporter constructs

demon-strate that HIV-1 gene expression increases upon induction of

NF-

k

B DNA-binding activity with stimulators such as tumor

necrosis factor alpha (TNF-

a

) and interleukin-1 (IL-1)

(re-viewed in references 4, 64, and 79).

Experiments with HIV-1 LTR reporter constructs in

lym-phoid cells showed that mutation of NF-

k

B motifs reduced

gene expression in the presence and absence of the HIV-1 Tat

protein (1, 3, 10, 11, 28, 35, 38, 51, 55, 76). The 15-kDa Tat

protein enhances LTR-derived gene expression and is required

for high-level expression of all viral genes (reviewed in

refer-ence 24). Tat acts via physical association with a stem-loop

RNA structure, the transactivation response (TAR) sequence

(30, 65, 66), present in the 5

9

end of all nascent viral mRNA

species. TAR RNA is formed by nucleotides

1

1 and

1

59, and

the core secondary stem-loop structure (nucleotides

1

18 to

1

44) is critical for Tat-TAR interactions and for Tat-mediated

transactivation. The bulge region in TAR (

1

22 to

1

24) serves

as the primary binding site for Tat, and the loop sequences

(

1

30 to

1

35) also contribute to this interaction (65, 66, 72).

TAR RNA essentially serves as an anchor for Tat and certain

cellular factors (TRP-185 and p68) (52, 73) to facilitate

inter-actions with promoter elements such as TATA, Sp1, and the

HIV-1 enhancer (11, 43). Deletion of NF-

k

B- and Sp1-binding

sites from the HIV-1 promoter abrogates Tat-mediated

trans-activation, suggesting that once brought into the vicinity of the

promoter, Tat interacts with transcription factors bound at the

NF-

k

B/Sp1 region to stimulate transcription initiation and

sta-bilize elongation complexes (10). Tat may be able to

transac-tivate independently of TAR RNA, since TAR-defective

vi-ruses are replication competent but still require Tat. In each

case, the Tat-responsive element maps to the NF-

k

B sites

within the HIV-1 enhancer (1, 5, 76).

The NF-

k

B/Rel family of transcription factors plays a pivotal

role in the regulation of the immunomodulatory genes and

activates genes including cytokines, cell surface receptors, and

acute-phase proteins, as well as viral genes including the HIV-1

LTR (for reviews, see references 4, 64, and 79). The NF-

k

B/

Rel family members can be subdivided into two subgroups

based on their structure and function: (i) the DNA-binding

proteins NF-

k

B1 (p50) (16, 33, 44), NF-

k

B2 (p52) (15, 57, 71),

RelA (p65) (58, 67), c-Rel (20), and RelB (68, 69), and (ii) the

NF-

k

B1 (p105) and NF-

k

B (p100) precursors, which are

pro-* Corresponding author. Mailing address: Lady Davis Institute for

Medical Research, 3755 Cote Ste. Catherine, Montreal, Quebec,

Canada H3T 1E2. Phone: (514) 8260, ext. 5265. Fax: (514)

340-7576.

5777

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(2)

teolytically cleaved to generate DNA-binding proteins (p50

and p52, respectively) (15, 16, 33, 44, 57, 71). All members of

the family share an N-terminal 300-amino-acid homology

re-gion involved in the formation of a DNA-binding dimer

com-plex which associates with a decameric consensus sequence,

5

9

-GGGPuNNPyPyCC-3

9

, found in the promoters of several

target genes (reviewed in references 4, 64, and 79). The dimer

composition of NF-

k

B contributes to the differential specificity

of gene activation (48, 60).

NF-

k

B activity is regulated in part at the level of subcellular

localization. In unstimulated cells, the NF-

k

B complex is

re-tained in the cytoplasm by inhibitory I

k

B proteins which bind

to and mask the nuclear localization sequence contained

within the Rel homology domain, thus preventing the

translo-cation of the DNA-binding proteins (8, 9). All the members of

I

k

B family, including I

k

B

a

(36), I

k

B

b

(77), I

k

B

g

(32, 41), and

Bcl-3 (37, 56), as well as the precursor proteins p105 (50) and

p100 (54), contain multiple ankyrin repeats. These sequences

represent the minimal requirements for I

k

B function as they

are involved in the physical interaction with NF-

k

B

DNA-binding subunits and in inhibitory activity (40, 42). The most

extensively studied of the I

k

B proteins is I

k

B

a

. Upon

stimu-lation by many activating agents, including cytokines, viruses,

and double-stranded RNA, I

k

B

a

is rapidly phosphorylated

and degraded, resulting in the release of NF-

k

B (8).

Phosphor-ylation of I

k

B

a

does not impair the ability of I

k

B

a

to associate

with NF-

k

B but, rather, represents a signal for degradation of

the inhibitor (2, 26, 49, 78) via the ubiquitin-proteasome

deg-radation pathway (21). Once released, NF-

k

B is able to

acti-vate target genes until new I

k

B

a

is synthesized. Since I

k

B

a

contains NF-

k

B-binding sites in its promoter, NF-

k

B is able to

autoregulate the transcription of its own inhibitor (19, 22, 46,

75). This autoregulatory control of I

k

B

a

/NF-

k

B expression is

in part responsible for the transient nature of the NF-

k

B

ac-tivation of gene expression.

Recent studies demonstrated that mutation of either Ser-32

or Ser-36 blocked signal-induced I

k

B

a

phosphorylation and

degradation (17, 18, 21). Constitutive phosphorylation of I

k

B

a

in the C-terminal PEST (proline, glutamate, serine, threonine)

domain by casein kinase II has also been demonstrated, and

triple point mutation of I

k

B

a

at Ser-283, Thr-291, and Thr-299

eliminated constitutive phosphorylation in vitro and in vivo (6,

47). Together with results demonstrating a role for S32/S36

sites in inducer-mediated phosphorylation and degradation of

I

k

B

a

, these studies define the regulatory phosphorylation sites

in I

k

B

a

.

In this study, we demonstrate that Tat synergizes with

TNF-

a

via NF-

k

B induction to transactivate the HIV-1

LTR-driven gene expression. We also show that I

k

B

a

is able to

inhibit the Tat–TNF-

a

synergistic activation of the HIV-1 LTR

in a dose-dependent manner. I

k

B

a

molecules mutated in

N-terminal sites involved in inducer-mediated phosphorylation

were stable in transfected cells (t

1/2

,

.

4 h) and efficiently

blocked HIV-1 LTR-directed gene expression and reduced

HIV-1 multiplication in a single-cycle infection model. These

experiments suggest a strategy that may contribute to the

in-hibition of HIV-1 gene expression by interfering with the

NF-k

B/Rel signaling pathway.

MATERIALS AND METHODS

Plasmid and cell lines.Plasmids SVK3-IkBaand SVK3-IkBa(3C) encoding wild-type IkBaand IkBa(3C), respectively, are described elsewhere (39, 47). IkBa(3C) is a full-length human IkBain which serine 283, threonine 291, and threonine 299 are substituted by alanine residues. The substitutions S32A and S36A in mutant IkBa(2N) and S32A, S36A, S283A, T291A, and T299A in mutant IkBa(2N13C) were generated by overlap PCR mutagenesis with Pfu DNA polymerase. The resulting mutated IkBacDNAs were inserted in

expres-sion vector SVK3 (Pharmacia) and plasmid pREP-9 CMVt (7). The presence of the mutations was confirmed by sequencing. HIV-1 LTR chloramphenicol acetyltransferase (CAT) plasmids ptzIIICAT, 2109/279, IIID23, and IIIDA were a kind gift from Eric Cohen (for a schematic map, see Fig. 2). The plasmids expressing wild-type and R52Q, R53Q mutant Tat, HIV-1 LTR (DB andD B-ACU, respectively), as well as the plasmid containing the HIV-1 proviral DNA (pSVC21 BH10), were described previously (23, 25, 65). Cell lines inducibly expressing wild-type IkBaor IkBa(3C), tTA-IkBa(wt), and tTA-IkBa(3C), re-spectively, are described elsewhere (7). tTA 3T3 cells were transfected with pREP9-CMVt-IkBa(2N) or pREP9-CMVt-IkBa(2N13C). Cells which inducibly

expressed IkBa(2N) or IkBa(2N13C) were selected and maintained in Dulbec-co’s modified Eagle’s medium containing 10% calf serum, 300mg of hygromycin B per ml, and 400mg of G418 (Gibco BRL) per ml.

Analysis of HIV-1 LTR transcription in reporter gene experiments.The cells were transiently transfected by the DEAE-dextran method (45). The precipitated DNAs (0.5 to 8mg), representing either HIV-1 LTR CAT reporter plasmids or pSVexTat plasmids (wild-type [wt] Tat or the R52Q, R53Q Tat mutant) (25), were resuspended in TS solution (8 mg of NaCl per ml, 0.38 mg of KCl per ml, 0.1 mg of Na2HPO4z7H2O per ml, 3.0 mg of Tris per ml, 0.1 mg of MgCl2per

ml, 0.1 mg of CaCl2per ml [pH 7.4]). After resuspension, 0.05 mg of

DEAE-dextran (Pharmacia) was added. For each transfection, 107

cells in exponential phase were washed once in TS solution, resuspended with the DNA solution, and incubated at room temperature for 20 min. The cells were then incubated at 378C for 30 min in 10 ml with medium containing 10% serum and 0.1 mM chloroquine (Sigma Chemical Co.), after which they were centrifuged and resuspended in fresh medium containing serum. At 32 h after transfection, the cells were in-duced with 5 ng of ml TNF-a(Boehringer Mannheim) per ml. At 16 h after induction, they were harvested and lysed. Extracts (10 to 100 mg) were assayed for CAT activity for 10 to 120 min, depending on the experiment. The percent acetylation was determined by ascending thin-layer chromatography as previ-ously described (31) and quantified with a Bio-Rad Gelscan Phosphoimager and the Molecular Analyst (Bio-Rad) software program.

Immunoblot analysis of IkBaturnover.tTA-IkBa-expressing cells (7) were cultured in tetracycline-free Dulbecco’s modified Eagle’s medium supplemented with 10% calf serum and treated with 5 ng of TNF-a(Gibco-BRL) per ml and 50mg of cycloheximide per ml for 4 h. The cells were washed with phosphate-buffered saline and lysed in 10 mM Tris HCl (pH 8.0)–60 mM KCl–1 mM EDTA–1 mM dithiothreitol–0.5% Nonidet P-40–0.5 mM phenylmethylsulfonyl fluoride–0.01mg of leupeptin perml–0.01mg of pepstatin perml–0.01mg of aprotinin perml (WBL buffer). Equivalent amounts of protein (20mg) were electrophoresed on a sodium dodecyl sulfate–10% polyacrylamide gel. The pro-teins were transferred to a nitrocellulose membrane, and IkBawas detected with IkBamonoclonal antibody MAD 10B (42) as previously described (59).

Analysis of HIV-1 protein and RNA synthesis in a single-cycle infection model.

Cos-1 cells were transfected with 10mg of HIV-1 proviral DNA (pSVC21 BH10) and 1, 5, or 10mg of IkBa-expressing plasmid: either SVK3-IkBa-IkBa(2N), -IkBa(3C), or -IkBa(2N13C). In all experiments, the total amount of DNA transfected was completed to 20mg with unrelated DNA (pUC8). DNA was introduced into cells by lipofection (Lipofectamine) as specified by the manu-facturer (Promega Inc.). At 3 days after transfection, the medium and the cells were collected. The relative amount of virion protein p24 present in the medium was determined by enzyme-linked immunosorbent assay (ELISA) (61). Proteins were extracted from a portion of the collected cells by being resuspended in WLB buffer. The proteins were analyzed by immunoblotting as described above with IkBamonoclonal antibody (42), human serum from an HIV-1-seropositive individual, or actin monoclonal antibody (ICN). Total RNA was extracted from the remaining cells with an RNeasy kit (Qiagen) as specified by the manufac-turer. RNA was denatured, electrophoresed through a 1.2% agarose gel in formaldehyde buffer, and transferred to a nylon membrane. Hybridization was carried out with32

P-labeled, random-primed HIV-1 proviral DNA orb-actin cDNA probes. The HIV-1 proviral cDNA probes were the 2- and 2.2-kbp HindIII fragments derived from pSVC21 BH10. Theb-actin cDNAs used as the probes were the 1-kbp PstI fragments derived from plasmid pb-actin.

RESULTS

Tat–TNF-

a

stimulation of the HIV-1 LTR.

To determine the

specific conditions required for Tat–TNF-

a

synergistic

activa-tion of HIV-1 LTR-driven reporter constructs in Jurkat T cells,

titration of wtTat expression plasmid and/or recombinant

TNF-

a

was initially performed (data not shown). In

subse-quent experiments, Tat plasmid was used at 2

m

g while TNF-

a

was used at 5 ng/ml. Tat stimulated the HIV-1 LTR CAT

reporter plasmid approximately 10-fold, while TNF-

a

treat-ment alone induced reporter gene expression about 5-fold

(Fig. 1A). Addition of both activators produced a true

syner-gistic stimulation of the HIV-1 LTR, resulting in a 50- to

70-fold induction of gene expression (Fig. 1A).

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Tat-TNF-

a

synergism requires Tat binding to the TAR

ele-ment.

Previous studies demonstrated that efficient

transactiva-tion of HIV-1 gene expression by Tat required physical

inter-action between Tat and the TAR element (25). The R52Q,

R53Q point mutations of the HIV-1 Tat protein abrogated Tat

binding to the TAR element and gene transactivation (25). To

test whether the Tat–TNF-

a

synergism defined in the above

model system required Tat-TAR association, the R52Q, R53Q

mutant of Tat (designated TatRQ) was used together with

TNF-

a

to stimulate expression of the HIV-1 LTR-driven CAT

reporter (Fig. 1B). Whereas Tat plus TNF-

a

activated the

HIV-1 LTR up to 70-fold, TNF-

a

alone, wt Tat alone, or

TatRQ alone induced LTR-mediated gene expression 4-, 6-,

and 3-fold, respectively. Significantly, the combination of

TNF-

a

treatment and TatRQ coexpression was only weakly

effective (fivefold induction) in mediating the activation of the

HIV-1 LTR (Fig. 1B). Similarly, mutations within the TAR

element (

D

B and B-ACU) that altered the Tat protein-binding

site (25, 65) also were not activated by Tat-TNF-

a

treatment

(Fig. 1C and D). These results thus reflect a requirement for

Tat-TAR interactions in the synergistic transactivation of the

HIV-1 LTR.

Tat-TNF-

a

synergism requires intact NF-

k

B sites.

To

char-acterize the region of the HIV-1 LTR involved in synergistic

activation, HIV-1 LTR deletion mutants were transfected into

Jurkat cells and stimulated with Tat, TNF-

a

, or Tat plus

TNF-

a

. As expected, the intact LTR-CAT construct (plasmid

ptzIIICAT) was strongly inducible by both activators (Fig. 2),

with an 18-fold induction by Tat–TNF-

a

. In contrast, the

2

109/

2

79 plasmid, a construct lacking the NF-

k

B sites, had

only baseline level of activity and was not transactivated by

TNF-

a

. Surprisingly, this construct was not inducible by wtTat

expression plasmid, even though it contained an intact TAR

element. Nevertheless, the combination of Tat and TNF-

a

was

able to stimulate the

2

109/

2

79 plasmid about fivefold. The

III

D

23 construct, which was deleted for the upstream

modu-latory sequences of the HIV-1 LTR (upstream of

2

167), was

stimulated by both activators as efficiently as was the wt

LTR-CAT plasmid, indicating that the upstream elements in the

2

423 to

2

167 domain of the LTR did not play a

complemen-tary role in Tat-TNF-

a

activation. Plasmid III

D

A was deleted

for all of the LTR sequences upstream of

2

57 and was not

activated significantly by either inducer alone, although a

re-sidual fourfold induction was observed with Tat–TNF-

a

. This

experiment indicates that strong synergistic activation of the

HIV-1 LTR by the combination of Tat and TNF-

a

required

intact NF-

k

B sites. The fact that some activation could occur in

the absence of the enhancer element (in constructs

2

109/79

and IIIA) suggests that TNF-

a

may be able to potentiate Tat

activity at the TAR element independently of the NF-

k

B sites.

Stability of the I

k

B

a

mutants.

Recent experiments have

[image:3.612.101.512.68.334.2]

defined specific sites of inducer-mediated and constitutive

phosphorylation in the I

k

B

a

regulatory protein (summarized

in Fig. 3). In particular, mutation of the N-terminal

phosphor-ylation sites at Ser-32 and Ser-36 in the signal response domain

of I

k

B

a

prevented inducer-mediated phosphorylation and

sub-sequent proteasome-dependent degradation of I

k

B

a

(17, 18,

21). Also, triple-point mutation of I

k

B

a

in the C-terminal

residues S-283, T-291, and T-299 abrogated constitutive

phos-phorylation in vivo by casein kinase II and increased the

in-trinsic stability of I

k

B

a

but did not affect inducer-mediated

degradation of I

k

B

a

(47). I

k

B

a

expression plasmids that

pro-duced I

k

B

a

proteins singly mutated in Ser-32 or Ser-36, in

both Ser-32 and Ser-36 [I

k

B

a

(2N)], in the three C-terminal

FIG. 1. Tat–TNF-aactivation of the HIV-1 LTR requires Tat binding to the TAR element. Jurkat T cells were cotransfected with 5mg of ptzIIICAT (A and B) and 2mg of pSVexTat (A) or Tat(R52Q,R53Q) (B) expression plasmids; they were also transfected with 2mg of LTR-DB (C) or LTR-B-ACU (D) and 2mg of pSVexTat. At 32 h after transfection, the cells were incubated for an additional 16 h in the presence or absence of TNF-a. CAT activities were assayed with whole-cell extracts (50mg for 30 min). Cells transfected only with ptzIIICAT in the absence of activators were used as negative control. The results are the mean of three experiments.

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sites [I

k

B

a

(3C)], and in all five regulatory phosphorylation

sites [I

k

B

a

(2N

1

3C)] were generated. The inducer-mediated

turnover of these proteins was analyzed in stably transfected

NIH 3T3 cells (7, 47) at different times after treatment with

cycloheximide and TNF-

a

(Fig. 4A). Immunoblot analysis with

an I

k

B

a

-specific antibody was able to distinguish between the

[image:4.612.64.552.74.435.2]

endogenous murine I

k

B

a

and the exogenously expressed

hu-man I

k

B

a

(Fig. 4A, lane 1). Cycloheximide was added to

eliminate the complicating effects of de novo synthesis of

I

k

B

a

after induction. The endogenous murine I

k

B

a

, human

wtI

k

B

a

, and I

k

B

a

(3C) all degraded rapidly in response to

TNF-

a

addition, with a t

1/2

of much less than 15 min (lanes 2

FIG. 2. Maximum Tat–TNF-asynergism requires intact NF-kB sites. Jurkat T cells were cotransfected with 5mg of ptzIIICAT,2109/279, IIID23, or IIIDA in the absence or presence of pSVexTat (2mg). TNF-awas added at 32 h after transfection, and the mixture was incubated for an additional 16 h. Whole-cell extracts were prepared, normalized for total protein, and assayed for CAT activity (50mg for 30 min).

FIG. 3. Schematic summary of N- and C-terminal phosphorylation sites in IkBa. The five ankyrin repeats are indicated by hatched boxes. There are two phosphorylation sites at Ser-32 and Ser-36 (triangles) in the N-terminal region of IkBa; these are required for inducer-mediated degradation of IkBa. Within the highly acidic C-terminal region, several potential casein kinase II phosphorylation sites are clustered around S-283, T-291, and T-299 (47). Sites at S-288, T-296 and S-293 are also potential CKII sites.

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[image:4.612.142.473.583.692.2]
(5)

to 6), indicating that triple mutation of S-283, T-291, and T-299

did not affect inducer-mediated degradation of IkBa. In

con-trast, both S36A (Fig. 4A) and S32A mutations (data not

shown) stabilized I

k

B

a

; both point mutations increased the t

1/2

of I

k

B

a

to approximately 90 to 120 min (lanes 2 to 6).

Double-point mutated I

k

B

a

(2N) and I

k

B

a

(2N

1

3C) were extremely

stable in the presence of TNF and cycloheximide, with a t

1/2

of

greater than 4 h (Fig. 4B). This experiment demonstrates the

increased stability of point-mutated I

k

B

a

molecules and

sug-gests that both IkBa(2N) and IkBa(2N13C) should be stable

transdominant mutants of the NF-

k

B response.

Inhibition of Tat–TNF-

a

activation of the HIV-1 LTR by

I

k

B

a

transdominant mutants.

To examine the effect of wt and

mutated forms of I

k

B

a

on Tat–TNF-

a

synergistic activation of

HIV-1 LTR-directed gene expression, the different forms of

I

k

B

a

were cotransfected into Jurkat cells together with the

reporter construct and the wtTat expression plasmid.

Expres-sion of both wtI

k

B

a

and I

k

B

a

(3C) reduced Tat–TNF-

a

acti-vation in a dose-dependent manner from a level of about

70-fold transactivation to 15- to 30-fold induction (Fig. 5).

However, neither wtI

k

B

a

nor I

k

B

a

(3C) completely inhibited

HIV-1 LTR-mediated expression. Expression of the S32A

mu-tant of I

k

B

a

(or S36A) dramatically reduced Tat–TNF-

a

ac-tivation to 10- to 20-fold stimulation. Strikingly, the I

k

B

a

(2N)

and I

k

B

a

(2N

1

3C) mutants were able to eliminate Tat–TNF-

a

transactivation at low concentrations of inhibitory plasmid;

I

k

B

a

(2N) or I

k

B

a

(2N

1

3C) at 1

m

g/ml reduced LTR-directed

reporter gene expression to only fivefold stimulation. At higher

concentrations, Tat–TNF-

a

transactivation of HIV-1

LTR-driven gene expression was completely suppressed by the

transdominant I

k

B

a

mutants (Fig. 5).

Inhibition of HIV-1 protein and RNA synthesis in a

single-cycle infection model.

To examine the ability of the I

k

B

a

[image:5.612.79.535.74.357.2]

mutant proteins to interfere with HIV-1 multiplication, HIV-1

proviral DNA (pSVC21 BH10) was transfected together with

FIG. 4. Inducer-mediated degradation of IkBa. tTA-IkBa(wt), tTA-IkBa(2N), tTA-IkBa(S36A), tTA-IkBa(3C), and tTA-IkBa(2N13C) cells were treated with TNF-a(5 ng/ml) and cycloheximide (50mg/ml) for 0 (lane 1), 15 (lane 2), 60 (lane 3), 120 (lane 4), 180 (lane 5), or 240 (lane 6) min. (A) Endogenous murine and exogenous human IkBawere detected in whole-cell extracts (15mg) by immunoblotting with affinity-purified AR20 antibody. (B) Levels of IkBawere quantified by laser densitometry and presented graphically.

FIG. 5. Transdominant IkBamutants inhibit Tat–TNF-aactivation of the HIV-1 LTR. In Jurkat cells, pTZIIICAT (2mg) was cotransfected with pSVexTat and different amounts (1, 2, and 4mg) of pSVK-wtIkBa, pSVK-S32, pSVK-2N (S32/S36), pSVK-3C (S283, T291, T299), or pSVK-2N13C and treated with TNF-a(5 ng/ml) for 16 h beginning at 32 h posttransfection. The level of HIV-1 LTR-driven reporter gene expression was determined by CAT assay on the whole protein extracts of the cells. The positive control (1Tat/TNF) was ob-tained by cotransfection with HIV-1 LTR CAT (2mg) and pSVexTat (2mg), and induction with TNF-a(5 ng/ml). The negative control (lane2) was obtained by transfection with the HIV-1 LTR reporter construct only. The results represent the mean of three independent experiments.

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increasing amounts of wtI

k

B

a

- or mutant I

k

B

a

-expressing

plasmid into Cos-1 cells. This single-cycle infection model

per-mits a single round of virus multiplication and release of

in-fectious HIV-1, but reinfection does not occur because of the

absence of the CD4 receptor on Cos-1 cells. At 3 days

post-transfection, high levels of transfected I

k

B

a

accumulated in

Cos-1 cells (Fig. 6B, lane 3 to 14) compared with the

endoge-nous level of primate I

k

B

a

(lanes 1 and 2). By using a human

antiserum that recognizes HIV-1 structural proteins, a

dra-matic reduction in the amounts of virus-specific p24 core

an-tigen, p55 precursor for virion core proteins, and gp120

enve-lope glycoprotein was observed in cells expressing I

k

B

a

(2N)

(Fig. 6A, lanes 6 to 8). In other experiments with the

single-cycle infection model, we found that wtI

k

B

a

, I

k

B

a

(3C), and

I

k

B

a

(2N

1

3C) were also effective in blocking a round of

HIV-1 replication but that I

k

B

a

(2N) was consistently more

effective in a dose-dependent manner. Analysis of the

intracel-lular accumulation of viral mRNA also confirmed that I

k

B

a

and the transdominant negative forms of I

k

B

a

differentially

inhibited HIV-1 multiplication (Fig. 7). Expression of I

k

B

a

inhibited HIV-1 proviral transcription in a dose-dependent

manner (Fig. 7A, lanes 3 to 14). In particular, I

k

B

a

(2N) was

the strongest inhibitor of HIV-1 transcript accumulation (lanes

6 to 8). Complementary results were obtained when the level

of p24 antigen release into the supernatant was measured by

an ELISA-based viral antigen capture assay (Fig. 7D).

To-gether, these results indicate that I

k

B

a

(2N) inhibited HIV-1

transcript levels, intracellular viral protein accumulation, and

release of virions into the supernatant. Surprisingly, additional

mutations within the C-terminal phosphorylation sites in I

k

B

a

(2N

1

3C) reduced the inhibitory capacity relative to I

k

B

a

(2N)

(Fig. 6A, lanes 12 to 14; Fig. 7A, lanes 12 to 14), suggesting an

important role for the intact C-terminal PEST domain in the

inhibition of HIV-1 multiplication.

DISCUSSION

[image:6.612.59.293.68.411.2]

In the present study, we examined the ability of different

forms of I

k

B

a

, mutated in distinct regulatory phosphorylation

sites, to inhibit the Tat–TNF-

a

synergistic activation of the

HIV-1 LTR in Jurkat cells. We found in our Jurkat cell model

that transactivation of the HIV-1 LTR was dependent upon

both functional Tat-TAR interaction and the presence of

NF-k

B-binding sites in the

2

100 enhancer region of the HIV-1

LTR. Coexpression of wtI

k

B

a

or mutant I

k

B

a

inhibited

Tat-TNF synergism in a dose-dependent manner. Interestingly, the

transdominant mutants I

k

B

a

(2N) and I

k

B

a

(2N

1

3C) were

each at least five times as effective as wtI

k

B

a

in inhibiting

HIV-1 LTR-directed gene expression. Moreover, I

k

B

a

(2N)

but, surprisingly, not I

k

B

a

(2N

1

3C) was more effective in

blocking HIV-1 protein and RNA synthesis in a single-cycle

infection model than was wtI

k

B

a

or I

k

B

a

(3C). The

observa-tion that mutaobserva-tions in the C-terminal PEST domain of I

k

B

a

decreased the inhibitory potential of I

k

B

a

(2N) is surprising

and indicates that an intact C terminus is required for maximal

inhibition of HIV-1 multiplication by I

k

B

a

(2N). This effect of

the C-terminal domain was not apparent in assays measuring

FIG. 6. Inhibition of viral protein expression by transdominant IkBa

mu-tants. (A to C) Cos-1 cells were transfected with HIV-1 proviral DNA (10mg of pSVC21 BH10), and viral protein expression was inhibited by cotransfecting either 1mg (lanes 3, 6, 9, and 12), 5mg (lanes 4, 7, 10, and 13), or 10mg (lanes 5, 8, 11, and 14) of plasmid expressing wtIkBa(lanes 3 to 5), IkBa(2N) (lanes 6 to 8), IkBa(3C) (lanes 9 to 11) or IkBa(2N13C) (lanes 12 to 14). Three days after transfection, cells were collected and analyzed by immunoblotting for expression of HIV proteins (A), IkBa(B), andb-actin (C). Bands corresponding to the viral envelope glycoprotein gp120, the p55 polyprotein precursor for virion core proteins, and the viral capsid protein p24 are indicated. (D) The release of HIV-1 p24 antigen into the supernatant of infected cells was measured by p24 ELISA; the results represent the mean of two independent experiments.

FIG. 7. Inhibition of viral transcription by transdominant IkBa mutants. Cos-1 cells were transfected with HIV-1 proviral DNA (pSVC21 BH10; 10mg) and either 1mg (lanes 3, 6, 9, and 12), 5mg (lanes 4, 7, 10, and 13), or 10mg (lanes 5, 8, 11, and 14) of plasmids expressing wtIkBa(lanes 3 to 5), IkBa(2N) (lanes 6 to 8), IkBa(3C) (lanes 9 to 11), or IkBa(2N13C) (lanes 12 to 14). Three days after transfection, the cells were collected and RNA was extracted. (A) Viral transcripts were detected by Northern blot analysis; the HIV-1 proviral cDNA probes were the 2- and 2.2-kbp HindIII fragments derived from pSVC21 BH10. The positions of the 9, 4.5, and 2 kb transcripts are indicated. (B) The blot in panel A was stripped and reprobed with ab-actin probe to illustrate the relative amount of RNA present in each lane. Theb-actin cDNA used as probe was the 1-kbp PstI fragment derived from pb-actin plasmid.

5782

BEAUPARLANT ET AL.

J. V

IROL

.

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inhibition of LTR transactivation and may thus reflect a

dis-tinct functional activity for the IkBa

C terminus. These

exper-iments suggest a strategy that may contribute to the inhibition

of HIV-1 gene expression by interfering with the NF-

k

B/Rel

signaling pathway.

Our results are complementary to those of a number of

recent experiments which have addressed the role of NF-

k

B

transcription factors in HIV-1-regulated gene expression.

Wes-tendorp et al. demonstrated that HIV-1 Tat protein amplified

the activity of TNF-

a

with regard to TNF-

a

-induced activation

of NF-

k

B and TNF-

a

-mediated cytotoxicity via the formation

of reactive oxygen intermediates (80). Tat suppressed the

ex-pression of Mn-dependent superoxide dismutase which

nor-mally functions as part of the cellular response to oxidative

stress, thus shifting the cellular redox state toward prooxidative

conditions. Under these conditions, higher levels of NF-

k

B-binding activity contributing to stimulation of HIV-1

LTR-directed gene expression were observed (23a). Under

condi-tions of maximal Tat–TNF-

a

synergism, I

k

B

a

was nonetheless

able to interfere with NF-

k

B induction, by sequestering NF-

k

B

in the cytoplasm in a concentration-dependent manner (6a).

I

k

B

a

molecules mutated in the N-terminal signal response

phosphorylation sites S-32 and/or S-36 did not undergo rapid

inducer-mediated degradation (Fig. 4) and were at least five

times as effective as the wt in blocking LTR-directed gene

expression. Our experiments furthermore extend a recent

study, published during the review of this paper, demonstrating

that I

k

B molecules inhibited Tat-mediated transactivation of

the HIV-1 LTR (34).

Biswas et al. showed that Tat protein provided a low level of

activation of the viral LTR, even in the absence of a functional

TAR element (12, 13), thus confirming the previously

de-scribed TAR-independent mode of Tat action (1, 5, 76). The

TAR-independent mode of Tat action was proposed to occur

through the transcriptional activation of TNF-

a

, which would

in turn stimulate NF-

k

B-binding activity (13, 80). These

obser-vations are reminiscent of a study demonstrating that

interleu-kin-2 (IL-2) secretion was upregulated at the transcriptional

level by the addition of extracellular Tat to activated T cells.

The response element in the IL-2 promoter also mapped to the

NF-

k

B site at positions

2

206 to

2

195 (81). As was observed in

the present study, Biswas et al. found that mutations in the

NF-

k

B motifs decreased Tat activation dramatically, indicating

that maximal stimulation of the LTR-directed gene expression

required Tat–TNF-

a

cooperation (13).

A systematic comparison of HIV-1 LTR activity in human

CD4 primary T cells and a transformed lymphoblastoid cell

line, J-Jhan, was performed, and strikingly different

require-ments for maximal LTR activation were observed (1). In

un-stimulated CD4 T lymphocytes, a low basal level of LTR

ac-tivity was detected, whereas in the lymphoblastoid cell line, a

high spontaneous level of LTR activity that was essentially

independent of the NF-

k

B-responsive elements was found. In

contrast, in primary lymphocytes, there was an absolute

depen-dence upon the NF-

k

B sites for initiation and Tat-mediated

amplification of HIV-1 transcription. These results are in

keeping with differences in the permissiveness to HIV-1

rep-lication of primary and established cell types. In

lymphoblas-toid cell lines, HIV infection resulted in active replication in

the absence of other stimuli, whereas in primary T cells,

rep-lication was undetectable and was dependent upon T-cell

ac-tivation for triggering of viral replication (1). In the present

study, we also found that HIV-1 LTR-directed gene activity

required both Tat-TAR transactivation and NF-

k

B induction,

indicating that our Jurkat cell model may reflect more closely

the quiescent state of primary T cells rather than the activated

state of other lymphoblastoid cell lines.

Phosphorylation, ubiquitination, and degradation of I

k

B

a

represent critical biochemical events required for NF-

k

B

acti-vation (reviewed in references 64 and 79). Recent studies

dem-onstrate that the amino terminus of I

k

B

a

represents a signal

response domain for activation of NF-

k

B. Substitution of

ala-nine for either Ser-32 or Ser-36 completely abolished the

sig-nal-induced phosphorylation and degradation of I

k

B

a

and

blocked the activation of NF-

k

B (17, 18). These mutations also

blocked in vitro ubiquitination of the I

k

B

a

protein (21, 70).

Mutation of C-terminal casein kinase II phosphorylation sites

completely blocked constitutive phosphorylation of I

k

B

a

in

vivo (6, 47, 53). The amino terminus of I

k

B

a

is necessary for

signal-induced degradation, but inducible degradation of IkBa

also requires the C-terminal domain of the protein (7, 17, 18,

27, 42). In this regard, the C-terminal domain may contact the

N-terminal region of the protein in vivo and act as a hinge or

lever to mask the signal response domain (7, 42).

All of the above studies suggest a transcriptional role for

I

k

B

a

in the inhibition of HIV-1 LTR-driven gene expression,

consistent with the sequestration of NF-

k

B subunits in the

cytoplasm. A recent study by Wu et al., however, implicated

I

k

B

a

at a distinct level in the HIV-1 life cycle (82). By using

wtI

k

B

a

, HIV-1 replication was blocked at the

posttranscrip-tional level of Rev function, not at a transcripposttranscrip-tional level.

Because I

k

B

a

did not interact directly with Rev and NF-

k

B

expression vectors potentiated Rev stimulation, it was

con-cluded that NF-

k

B/I

k

B regulated a cellular factor required for

Rev function (82). Recent experiments have demonstrated

that Rev contains an RNA-binding domain, required for

inter-action with HIV-1 RNA, and an effector domain, required for

RNA-bound Rev to function. The Rev effector domain

inter-acted specifically with host proteins with homology to

nucleo-porins, a class of proteins that mediate nucleocytoplasmic

transport (14, 29, 74). It is possible that a novel function of

I

k

B

a

is to interfere with Rev-mediated nuclear export of viral

structural mRNA.

HIV-1 infection causes constitutive activation of NF-

k

B

DNA-binding activity in infected cells (64). A direct temporal

correlation exists between HIV-1 infection and the appearance

of NF-

k

B DNA-binding activity in myeloid cells (62, 63), which

may in turn prime or stimulate cytokine release. Cytokine

release from HIV-1-infected cells may contribute to the

ele-vated levels of TNF-

a

, IL-1, IL-6, transforming growth factor

b

, and gamma interferon, present in the sera of AIDS patients

in late-stage disease (reviewed in reference 64). Elevated

al-pha/beta interferon activity is also present in the sera of AIDS

patients in late-stage disease and serves as a marker for a poor

prognosis (64). Expression of an I

k

B

a

transdominant mutant

may thus interfere with HIV-1 infection at multiple levels: at

the level of NF-

k

B dependent transcription activation of the

LTR; at the level of Rev posttranscriptional activity; and at the

level of expression of HIV-1-induced inflammatory cytokines.

ACKNOWLEDGMENTS

P.B. and H.K. contributed equally to this work.

We thank the AIDS Research and Reference Program for plasmids

and antisera used in this study.

These studies were supported by the Medical Research Council of

Canada, Cancer Research Society, Inc., and by the National Health

Research and Development Program, Health and Welfare, Canada.

P.B. was the recipient of a MRC Studentship, and J.H. was the

recip-ient of a MRC Screcip-ientist Award.

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Figure

FIG. 1. Tat–TNF-�pSVexTat. At 32 h after transfection, the cells were incubated for an additional 16 h in the presence or absence of TNF-extracts (50and 2 activation of the HIV-1 LTR requires Tat binding to the TAR element
FIG. 3. Schematic summary of N- and C-terminal phosphorylation sites in I�phosphorylation sites at Ser-32 and Ser-36 (triangles) in the N-terminal region of Iacidic C-terminal region, several potential casein kinase II phosphorylation sites are clustered a
FIG. 5. Transdominant I�transfection with the HIV-1 LTR reporter construct only. The results representHIV-1 LTR
FIG. 7. Inhibition of viral transcription by transdominant I�(2N) (lanes6 to 8), Iafter transfection, the cells were collected and RNA was extracted

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