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Originally published In Press as doi:10.1074/jbc.M604220200 on August 21, 2006

J. Biol. Chem., Vol. 281, Issue 42, 31348-31358, October 20, 2006
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SerpinB2 Is an Inducible Host Factor Involved in Enhancing HIV-1 Transcription and Replication*Formula

Grant A. Darnell{ddagger}, Wayne A. Schroder{ddagger}, Joy Gardner{ddagger}, David Harrich{ddagger}, Hong Yu§, Robert L. Medcalf§, David Warrilow{ddagger}, Toni M. Antalis, Secondo Sonza||, and Andreas Suhrbier{ddagger}1

From the {ddagger}Queensland Institute of Medical Research, Brisbane, Queensland 4029, Australia, the §Australian Centre for Blood Diseases, Monash University, Melbourne, Victoria 3800, Australia, the Department of Physiology, University of Maryland School of Medicine, Rockville, Maryland 20854, and the ||AIDS Pathogenesis Research, Macfarlane Burnet Centre for Medical Research, Melbourne, Victoria 3001, Australia

Received for publication, May 3, 2006 , and in revised form, August 7, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The serine protease inhibitor SerpinB2 (plasminogen activator inhibitor-2) is a major product of activated monocytes and macrophages and is substantially induced during most inflammatory processes. Distinct from its widely described extracellular role as an inhibitor of urokinase plasminogen activator, SerpinB2 has recently been shown to have an intracellular role as a retinoblastoma protein (Rb)-binding protein that inhibits Rb degradation. Here we show that HIV-1 infection and gp120 treatment of human peripheral blood mononuclear cells resulted in induction of SerpinB2. Furthermore, SerpinB2 expression in THP-1 monocyte/macrophage, Jurkat T, and HeLa cell lines increased replication of HIV-1 and enhanced transcription from the HIV-1 long terminal repeat (LTR) promoter by 3–10-fold. Increased HIV-1 gene expression and transcription was also observed in activated macrophages from SerpinB2+/+ mice compared with macrophages from SerpinB2–/– mice. The SerpinB2-mediated elevation of Rb protein levels appeared to be responsible for enhancing transcription from the core promoter region of the LTR by relieving HDM2-mediated inhibition of Sp1 and/or by increasing the Sp1/Sp3 expression ratios. This is the first report associating HIV-1 replication with SerpinB2 expression and illustrates that SerpinB2 is a potentially important inducible host factor that significantly promotes HIV-1 replication.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
SerpinB2, also known as plasminogen activator inhibitor type-2 (PAI-2),2 is one of the most abundant proteins of activated monocytes and macrophages, whose expression is substantially up-regulated during most inflammatory processes (1, 2). SerpinB2 is also induced following infection with a range of parasitic (3, 4), viral (5–8), and bacterial (9–12) pathogens. Although SerpinB2 is widely described as an inhibitor of the extracellular urokinase plasminogen activator, the evidence that SerpinB2 has a major physiological role in the regulation of plasminogen activation is not compelling (13). SerpinB2 is a member of the Clade B or ovalbumin-like serine protease inhibitor (ov-serpin) subgroup of serpins, which lack a classical secretory signal peptide and are frequently found to reside intracellularly with a cytoplasmic or nucleocytoplasmic distribution (14). SerpinB2 has recently been shown to have an intranuclear activity as a retinoblastoma protein (Rb)-binding protein (15–18). SerpinB2 expression inhibited Rb degradation, which resulted in increased Rb protein levels. This activity required both (i) the C-D interhelical region of SerpinB2, which mediates Rb binding, and (ii) the reactive site loop (RSL), which mediates the protease inhibitory activity of SerpinB2. The SerpinB2-mediated increase in Rb protein levels was found to enhance Rb-mediated activities, such as repression of E2F-1-dependent gene expression and promotion of G1 cell cycle arrest (15). SerpinB2 expression has also been shown to influence transcription of mammalian (15, 19) and viral genes (16), with the latter two studies illustrating that this activity was mediated via the elevation of Rb protein levels.

Cells of the monocyte/macrophage lineage are important targets for infection by the human immunodeficiency virus type-1 (HIV-1). Infected monocytes/macrophages are significant reservoirs for persistence of HIV-1, serve as vehicles for dissemination of HIV-1, and can be a major source of virus throughout the course of infection (20, 21). Under certain conditions HIV-1 replication in vivo is likely to occur in monocytes/macrophages expressing SerpinB2. Certain inflammatory conditions (22), TNF{alpha} (23) and bacterial infections (21, 24) enhance the replication of HIV-1 in monocytes/macrophages and potently induce SerpinB2 expression in these cells (2). These considerations and the reported influence of SerpinB2 expression on transcription (15, 16, 19), prompted an investigation of the role of SerpinB2 expression in HIV-1 infection.

HIV-1 infection initially involves binding of viral gp120 to CD4 and chemokine receptors. Following cell entry, HIV-1 RNA is reverse transcribed, viral DNA is integrated into the genome, and transcription from this DNA leads to generation of viral progeny. Transcription from integrated HIV-1 DNA is controlled by the HIV-1 long terminal repeat promoter (HIV-1 LTR), which contains (i) the modulatory region, (ii) the enhancer region, which usually contains tandem NF-{kappa}b sites, (iii) the core promoter region, which usually contains three Sp1 sites, and (iv) the leader region, which encodes a short leader RNA called TAR that binds the HIV-1-encoded transactivator Tat. Initially transcription is Tat-independent, however after Tat is synthesized, transcription is substantially amplified as Tat and TAR recruit host proteins that promote transcription (25).

Here we show that SerpinB2 is a potentially important host factor involved in enhancing HIV-1 transcription and replication. HIV-1 and gp120 induced the expression of SerpinB2 in human PBMC, and virus transcription and replication was increased 3–10-fold in several cell lines expressing SerpinB2. In addition, HIV-1 gene expression and transcription was significantly reduced in primary macrophages from SerpinB2–/– mice. Using a series of truncated HIV-1 LTR reporter constructs, the SerpinB2 expression-associated enhancement of both Tat-independent and Tat-dependent transcription was localized to the core promoter region, which contains three Sp1 sites that are critical for transcription in the presence and absence of Tat (25). The SerpinB2-mediated increase in Rb protein levels was shown to be responsible for the enhanced transcription, with the activity localizing to the C-pocket region of Rb, which binds both SerpinB2 (15) and HDM2 (26). SerpinB2-expressing cells showed increases in Sp1/Sp3 protein ratios and increased binding of Sp1 and/or decreased binding of the inhibitory Sp3 (27) to the core promoter region. Thus SerpinB2 expression was shown to enhance HIV-1 transcription and replication by stimulating an Rb-dependent increase in Sp1-mediated transcription from the core promoter region of the HIV-1 LTR.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Lines—HeLa and Jurkat cell lines and C33A cells (ATCC HTB-31) were maintained as described previously (15). The THP-1 cells lines were generated as described (29) and were maintained in complete medium comprising RPMI 1640 supplemented with antibiotics and 10% low endotoxin fetal calf serum (Invitrogen, Carlsbad, CA). The complete medium contained endotoxin levels below those detectable by the RAW264-HIV-LTR-LUC assay (68).

SerpinB2 protein expression levels were determined for each cell line by ELISA. The levels found in the SerpinB2-transfected cells were at the lower end of those found for endogenous expression (supplemental Table S1). Western analysis of protein expression for the HeLa and Jurkat cell lines is shown in supplemental Fig. S1.

HIV-1 Infection and gp120 Treatment of PBMC—The M-tropic HIV-1AD8 (69) was prepared in 293 cells (70) and purified using a sucrose cushion (71). PBMC (6 x 106 in 1 ml of complete medium) were infected with 40 µl of HIV-1AD8 virus or heat-inactivated virus (72 °C for 25 min) (1 µg of p24/ml) for 2 h at 37 °C. The cells were washed three times in RPMI 1640 supplemented with 1% low endotoxin fetal calf serum and seeded at 106 cells per well of a 24-well plate in complete medium. Fresh medium (0.5 ml) was added on day 7. Supernatants were analyzed for p24 by ELISA (HIV-1 P24 ELISA kit; PerkinElmer Life Sciences). Levels of SerpinB2 mRNA were determined by quantitative real time RT-PCR as described (15) and normalized to c-FMS mRNA (72). SerpinB2 protein levels were determined by Western analysis of whole cell lysates (15) using the MAI21 monoclonal antibody (Biopool, Umeä, Sweden). Recombinant gp120 (see "Acknowledgments") and heat-denatured gp120 (28) (100 nM) were added to PBMC and SerpinB2 protein levels determined as above.

Western Analysis—Western analyses were performed as described (15) using antibodies specific for SerpinB2 (MAI21 or polyclonal from American Diagnostica, Victoria, Australia), Rb (G3–245) (PharMingen/BD Biosciences), Sp1 (E3) Sp3 (D-20) and actin (C-11) (Santa Cruz Biotechnology, Santa Cruz, CA).

Infection/Transfection of Cell Lines with HIV-1—The T-tropic HIV-1NL4.3 was prepared as described (70). Jurkat lines (2 x 106 cells in 1 ml of medium) were incubated with 50–100 µl HIV-1NL4.3 virus (200 ng of p24/ml) for 2 h at 37 °C. THP-1 cells (2 x 106 cell/ml) were infected with 40 µl of HIV-1AD8 virus (1 µg p24/ml) for 2 h at 37 °C. The cells were washed three times and resuspended in 1 ml of complete medium and seeded at 106 cells per well of a 24-well plate in duplicate. Supernatants were assayed for p24 antigen by ELISA. Viable cell counts were performed by trypan blue exclusion and cells re-seeded at 106 viable cells/well.

HeLa cell lines were seeded (2 x 104 cells/well) in a 24-well plate and cultured overnight. Cells were transfected using GeneJammer transfection reagent (3 µl per well) according to manufacturer's instructions (Stratagene, La Jolla, CA) with a total of 1 µg of plasmid DNA comprising a proviral plasmid encoding HIV-1NL4.3 virus (70) and a pCMV beta-galactosidase reporter plasmid (0.5 µg) (Promega). At the indicated times cell lysates were assayed using the beta-galactosidase assay system (Promega) and supernatants assayed using the p24 ELISA.

Pseudotyped HIV-1 and Adenovirus HIV-1 LTR Reporter Infection of Macrophages from SerpinB2–/– Mice—SerpinB2–/– mice, backcrossed 6 times onto the C57/BL6 background, were kindly supplied by Dr. D. Ginsburg (13). The mice were backcrossed a further six times onto the same background and a SerpinB2+/+ littermate control line was also generated. Peritoneal macrophages were isolated by peritoneal lavage from both SerpinB2–/– and SerpinB2+/+ littermate control mice 5 days after 2 ml of intraperitoneal injection of sterile 3% thioglycolate (Sigma-Aldrich). Pseudotyped HIV-1VSV/NL4.3 virus (31) (200 µl of 200 ng of p24/ml) was used to infect 2 x 106 macrophages for 2 h at 37°C. The cells were washed and cultured as described in the previous section. At the indicated times parallel cultures were lysed and assayed in duplicate for p24 antigen by ELISA per the manufacturer's instructions (RETROtec, ZeptoMetrix Corporation, Buffalo, New York), and nuclear lysates (15) analyzed by Western blotting. An adenovirus vector encoding luciferase under the control of the HIV-1 LTR (73) and an adenovirus vector encoding beta-galactosidase (74) were used to coinfect thiogycolate-elicited macrophages from SerpinB2–/– and SerpinB2+/+ mice (2 x 106 macrophages; multiplicity of infection 50 for each vector). After 72 h, LUC activity was assayed in triplicate using the Steady Glo Luciferase Assay System (Promega) and the 1450 microbeta luminometer (PerkinElmer Life Sciences and Analytical Sciences). LUC was normalized to beta-galactosidase levels, which were measured as above.


Figure 1
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  		FIGURE 1.
Induction of SerpinB2 expression in PBMC by HIV-1 and gp120. A, unstimulated human PBMC were infected with the M-tropic HIV-1AD8 or with the same inoculum of heat-inactivated virus. Supernatants were analyzed in triplicate for p24 antigen levels by ELISA. B, cells from the same cultures were analyzed for SerpinB2 mRNA by real-time RT-PCR and for SerpinB2 protein expression by Western analysis. The amount of SerpinB2 mRNA (analyzed in duplicate) is shown as a fold increase over the levels seen in freshly isolated PBMC (Time 0) (left hand y-axis, bars). Cell lysates (15 µg of protein) were analyzed by immunoblot using an anti-SerpinB2 antibody and expressed (right hand y-axis, lines) as a fold increase as determined by densitometry over the levels seen in freshly isolated PBMC (Time 0). C, unstimulated human PBMC were treated with soluble gp120 (100 nM) or the same amount of heat-denatured gp120. At the indicated times SerpinB2 protein levels were determined as for B.

 
Generation of HIV-1 LTR Reporter Constructs—The HIV-LTR reporter plasmids were derived from the LTR of the SF2 B clade strain of HIV-1, which was cloned into pJGFCAT18 (75, 76). pHIV-LTR(–309 to +83)-CAT was constructed by cutting with Mae1 and HindIII and cloning into pJGFCAT18. pHIV-LTR(–118 to +83)-CAT was constructed by cutting with TaqI and HindIII and cloning into pJGFCAT18. To generate the pHIV-LTR(–177 to +83)-LUC construct, a DNA fragment containing nucleotides –177 to +83 was cut from pHIV-LTR(–177 to +83)-CAT (75) using BamHI and HindIII and was inserted into the BamHI and BglII sites of the pGL3-basic vector (Promega). QuikChange Site-directed Mutagenesis kit (Stratagene) was used to delete –177 to –82 from pHIV-LTR (–177 to +83)-LUC to generate pHIV-LTR(-81 to +83)-LUC and to delete –171 to –46 from pHIV-LTR (-177 to +83)-LUC to generate pHIV-LTR(–45 to +83)-LUC. All LTR regions were confirmed by DNA sequencing.

Reporter Assays—Cells were seeded (2 x 104 cells/well) in a 24-well plate and cultured overnight. Cells were then transfected as described above with a total of 1 µg of plasmid DNA comprising (i) a plasmid encoding HIV-1 Tat or a beta-globin control (77) (0.2 µg), (ii) a pCMV beta-galactosidase reporter plasmid (0.4 µg), and (iii) the pHIV-LTR-CAT reporter plasmids (0.4 µg). At 72-h post-transfection, cells were harvested and assayed for beta-galactosidase activity using beta-galactosidase enzyme assay system, and CAT activity using CAT ELISA kits (Roche Applied Sciences, Penzberg, Germany). LUC assays were performed at 24 h and normalized to beta-galactosidase levels as described above.

C33A cells were seeded (2 x 104 cells/well) in a 24-well plate and cultured overnight, then transfected with pHIV-LTR(–81 to +83)-LUC (0.4 µg), and the indicated combinations of plasmids encoding p130414–1135, Rb379–928, Rb SE, Rb SE{Delta} (0.4 µg) (15, 35), and/or HDM2 (0.2 µg) (cDNA for HDM2 was cloned into pEGFP-C3, Clontech/BD Biosciences, Franklin Lakes, NJ), plus pCMV beta-galactosidase (0.5 µg).

The Sp1 (2)-luciferase reporter construct was purchased from Panomics (Fremont, CA). LUC assays were performed 24 h after transfection and LUC activity normalized to beta-galactosidase levels as described above.

Rb siRNA—Cells were seeded into 24-well plates (5 x 104 cells per well in duplicate) and were treated with Rb siRNA or control siRNA as described previously (15). After 48 h, the cells were transfected with the indicated plasmids and LUC activity determined after a further 24 h.

EMSA Experiments—Protein-DNA complexes were analyzed as described (15). For antibody supershifting, 2 µl of 200 µg/ml anti-Sp1 antibody (E-3) or anti-Sp3 antibody (D-20) was added to nuclear lysates containing the above reaction mixture for 30 min 4 °C prior to addition of EMSA probes. The biotinylated dsDNA oligonucleotides were; consensus Sp1, forward 5'-ATTCGATCGGGGCGGGGCGAG-3' and reverse 5'-CTCGCCCCGCCCCGATCGAAT-3'; and LTR Sp1 region (–78 to –43), forward 5'-AGGGAGGCGTGGCCTGGGCGGACTGGGAGTGGCGT-3' and reverse, 5'-ACGCCACTCCCAGTCCGCCCAGGCCACGCCTCCCT-3'.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
HIV-1 and gp120-induced SerpinB2 Expression in PBMC—SerpinB2 expression is induced in monocyte/macrophage cells following alphavirus infection and treatment with double-stranded RNA (7). To determine whether HIV-1 infection of PBMC induces SerpinB2 expression, unstimulated PBMC were infected with M-tropic HIV-1AD8. Capsid p24 was detectable by day 4 postinfection and rose steadily thereafter, whereas no p24 was detected following inoculation of PBMC with the same amount of heat-inactivated HIV-1 (Fig. 1A). Real time RT-PCR analysis illustrated that SerpinB2 mRNA levels were detectable 2 h post-infection and were induced ~16-fold by day 4, dropping to 2–3-fold over controls by days 10 and 14 (Fig. 1B, right hand y-axis, black bars). SerpinB2 protein expression followed a similar pattern (Fig. 1B, left hand y-axis, black squares). SerpinB2 mRNA (Fig. 1B, right hand y-axis, white bars) and protein levels (Fig. 1B, left hand y-axis, dashed line) were not significantly induced by heat-inactivated HIV-1. These data illustrated that M-tropic HIV-1 infection rapidly induced SerpinB2 mRNA and protein expression in human PBMC.


Figure 2
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  		FIGURE 2.
HIV-1 replication is increased in SerpinB2-expressing cells. A, THP-1 cell lines infected with M-tropic HIV-1. THP-1 cell lines expressing SerpinB2 (THP-1/PAI-2), parental THP-1 cells (THP-1), THP-1 cells expressing an empty vector (THP-1/neo), were infected with the M-tropic HIV-1AD8 virus. At the indicated times supernatants were assayed in triplicate for p24 levels using ELISA. B, Jurkat T cell lines infected with T-tropic HIV-1. Jurkat lines stably expressing SerpinB2 (J.PAI-2a/b), parental Jurkat cells (Jurkat), Jurkat cells expressing vector only (Vector), Jurkat cells stably expressing the C-D interhelical mutant of SerpinB2 (J.C-D PAI-2), or the RSL mutant of SerpinB2 (J.PAI-2 Ala380) were infected with HIV-1NL4.3. p24 levels were determined in triplicate as for A. C, HeLa cell lines transfected with an infectious DNA clone of HIV-1NL4.3. HeLa cell lines stably expressing SerpinB2 (S1a/b), parental HeLa cells (HeLa), HeLa cells stable expressing antisense SerpinB2 (A2/7), the C-D interhelical mutant of SerpinB2 (C-D PAI-2a), or the RSL mutant of SerpinB2 (PAI-2Ala380) were transfected with the proviral DNA construct, pHIV-1NL4.3 and a plasmid encoding beta-galactosidase. p24 levels were determined as for A and normalized to beta-galactosidase activity. For A–D, p24 levels from all SerpinB2-expressing cells were significantly higher than control cells at the final time points (Student's t test p < 0.05).

 
To determine whether binding of HIV-1 gp120 to cellular receptors is sufficient for induction of SerpinB2, PBMC were incubated with recombinant gp120 derived from an M-tropic virus. As a control, PBMC were treated with heat denatured gp120 (28). Native gp120, but not heat denatured gp120, induced SerpinB2 protein expression (Fig. 1C) with similar levels and kinetics to that seen when using whole HIV-1 virus (Fig. 1B), indicating that gp120 receptor binding is responsible for SerpinB2 induction. Of the cells present in PBMC-activated monocytes and macrophages represent the only cell types for which significant SerpinB2 expression has been reported (1).

SerpinB2 Expression Increased HIV-1 Replication in THP-1, Jurkat, and HeLa Cells—Unlike most monocyte/macrophage cell lines, the THP-1 cell line is defective for endogenous SerpinB2 expression. THP-1 cell lines that stably expressed SerpinB2 (THP-1/PAI-2) and control transfected cells expressed only the neomycin resistance gene (THP-1/neo) were generated by Yu et al. (29). Following infection with M-tropic HIV-1, significantly greater virus replication occurred in THP-1/PAI-2 cells compared with the control cells, comprising parental THP-1 and THP-1/neo cells (Fig. 2A). This increased replication of M-tropic virus may in part be because of the 3-fold greater expression of CCR5 (a co-receptor for M-tropic HIV-1) in THP-1/PAI-2 cells compared with control cells (supplemental Fig. S2). However, T-tropic virus showed a similar increase in replication in these cells (supplemental Fig. S3), and expression of CD4 (the major HIV-1 receptor) and CXCR4 (a co-receptor for T tropic HIV-1) were not significantly altered (supplemental Fig. S2).

Significant increases in virus replication compared with control cells were also observed following infection with T tropic HIV-1 of two Jurkat T cell lines stably expressing SerpinB2 (Fig. 2B, J.PAI-2a and J.PAI-2b). Control cells included parental Jurkat T cells, and lines stably expressing the neomycin resistance gene (Vector), the C-D interhelical mutant (J.CD PAI-2) or the RSL mutant of SerpinB2 (J.PAI-2Ala380) (Fig. 2B). No significant differences in CD4 or CXCR4 expression (supplemental Fig. S2), or viability postinfection (data not shown) were observed between any of the Jurkat cell lines.


Figure 3
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  		FIGURE 3.
HIV-1 gene expression, Rb protein, and transcription from the HIV LTR is reduced in macrophages from SerpinB2–/– mice. A, thioglycolate-elicited peritoneal macrophages from SerpinB2–/– (white squares) and SerpinB2+/+ littermate control mice (black squares) were infected with pseudotyped HIV-1VSV/NL4.3. At the indicated times cell lysates were assayed in duplicate for p24 levels by ELISA. B, Western analysis of Rb and SerpinB2 from nuclear lysates of the macrophages described in A at day 0 and 5 days postinfection with the pseudotyped HIV-1VSV/NL4.3. C, macrophages from SerpinB2–/– (white bar) or SerpinB2+/+ littermate control mice (black bar) (prepared as above) were coinfected with two adenovirus vectors, one encoding LUC under the control of the HIV-1 LTR promoter and the other beta-galactosidase. After 72 h, LUC activity was determined in triplicate and normalized to beta-galactosidase expression.

 
Significant increases in virus production over control cells were also observed following transfection with an HIV-1 proviral DNA construct of two HeLa cells lines stably expressing SerpinB2 (S1a and S1b) (Fig. 2C). (HeLa cells do not express CD4 and thus cannot be readily infected with HIV-1 virus). No significant differences in virus production were seen for the different control cell lines; parental HeLa cells, and HeLa cells expressing antisense SerpinB2 (A2/7), the C-D interhelical mutant (CD PAI-2a), or the RSL mutant of SerpinB2 (PAI-2Ala380a) (Fig. 2C). Because the cells were transfected with DNA rather than infected with virus, this data also indicates that the increased HIV-1 replication seen in S1a and S1b cells, was unlikely to be due to (i) changes in HIV-1 receptor or co-receptor expression or (ii) SerpinB2 inhibiting extracellular urokinase plasminogen activator (30).

In summary these data illustrated (i) that SerpinB2 expression in three different cell lines increased HIV-1 replication, indicating a robust and consistent effect, (ii) that enhanced replication required intact wild-type SerpinB2, implicating Rb in the activity (15), and (iii) that enhanced virus replication/production was not dependent on expression of viral receptors (Fig. 2C).

HIV-1 Gene Expression Is Reduced in Macrophages from SerpinB2–/– Mice—Fig. 2 illustrated that SerpinB2 expression enhanced HIV-1 replication. To determine whether loss of SerpinB2 expression was associated with reduced HIV-1 gene expression in primary cells, thioglycate-elicited macrophages from SerpinB2–/– mice were infected with HIV-1 virus pseudotyped with vesicular stomatitis virus G envelope glycoprotein (31). Nitkiewicz et al. (32) have recently demonstrated that, in contrast to murine 3T3 cells, there is actually no intrinsic intracellular block to HIV-1 gene expression in primary mouse macrophages once infection has been accomplished with pseudotyped virus. Following infection with the pseudotyped virus, the macrophages from SerpinB2–/– mice (Fig. 3A, white squares) expressed significantly less HIV-1 p24 than macrophages from SerpinB2+/+ mice (Fig. 3A, black squares). SerpinB2 expression in thioglycate-elicited macrophages from SerpinB2+/+ mice was confirmed by Western blot analysis, but did not increase following infection with the pseudotyped virus (Fig. 3B, SerpinB2), supporting the view that gp120 binding rather than HIV replication is responsible for SerpinB2 induction. Importantly, macrophages from SerpinB2–/– mice showed a 3.6 ± 0.9 (S.D.) and 3.0 ± 0.2-fold decrease in nuclear Rb protein levels at day 0 and day 5 postinfection, respectively, when compared with macrophages from SerpinB2+/+ mice (Fig. 3B). Taken together with the known influence of SerpinB2 on Rb-mediated transcription (15, 16, 19), these data suggest that SerpinB2 may be involved in enhancing HIV-1 transcription via its effect on Rb levels (15).

The experiments using pseudotyped virus (Fig. 3, A and B), taken together with the enhanced HIV-1 gene expression following transfection of SerpinB2-expressing cells with proviral HIV-1 DNA (Fig. 2C), also indicate that SerpinB2 is unlikely to be influencing HIV-1 entry.

Transcription from the HIV-1 LTR Is Reduced in Macrophages from SerpinB2–/– Mice—Transcription of HIV-1 genes is controlled by the HIV-1 LTR promoter. To determine whether SerpinB2 expression influences HIV-1 LTR activity in primary cells, macrophages from SerpinB2–/– mice and SerpinB2+/+ mice were infected with an adenovirus vector encoding an HIV-1 LTR luciferase reporter construct. Macrophages from SerpinB2+/+ mice showed {approx}3-fold greater normalized LUC activity compared with macrophages from SerpinB2–/– mice (Fig. 3C). These data illustrated that SerpinB2 expression increases transcription from the HIV-1 LTR in primary mouse macrophages.

SerpinB2 Expression Increased Transcriptional Activity of the HIV-1 LTR—The HIV-1 LTR promoter has been divided into three regions that can influence LTR activity, the modulatory region, the enhancer region, and the core promoter region (25) (Fig. 4A). To investigate the role of SerpinB2 expression on HIV-1 transcription, a CAT reporter construct containing these three promoter regions (pHIV-LTR(–309 to +83)-CAT)) was transiently transfected (together with plasmids coding for HIV Tat and beta-galactosidase) into a panel of human SerpinB2-expressing and control cell lines. In each of the three cell types, wild-type SerpinB2 expression was associated with a significant increase in CAT activity (Fig. 4A). The control lines for each cell type gave similar CAT activity (Fig. 4A). The lack of increased CAT activity in cell lines stably expressing the C-D interhelical mutant (J.C-D PAI-2a and C-D PAI-2a) or the RSL (Ala380) mutant of SerpinB2 (THP-1/PAI-2 Ala380, J.PAI-2Ala380a, PAI-2 Ala380a) suggested a role for Rb, since both the C-D loop and an intact RSL are required for the SerpinB2-mediated protection of Rb from degradation (15, 16).

The Modulatory Region Was Not the Target of SerpinB2 Activity—When using a reporter plasmid from which the modulatory region was removed (pHIV-LTR(–118 to +83)-CAT) the SerpinB2 expression-associated increase in transcriptional activity was retained (Fig. 4B), illustrating that this region was not the target of SerpinB2 activity. This observation is consistent with data showing that NFAT and AP-1 activity was not significantly increased in SerpinB2-expressing cells (supplemental Fig. S4).


Figure 4
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  		FIGURE 4.
SerpinB2 expression enhances transcription from the HIV-1 LTR promoter. A, reporter assays using a plasmid containing the modulatory, enhancer and core promoter regions. The panel of SerpinB2-expressing and control THP-1, Jurkat, and HeLa cell lines were cotransfected with the HIV-LTR(–309 to +83)-CAT reporter plasmid, and plasmids expressing HIV-Tat and beta-galactosidase. Cells were harvested at 72-h post-transfection and CAT protein levels determined by ELISA with optical density (OD) normalized to beta-galactosidase activity. B, reporter assays using a plasmid containing the enhancer and core promoter regions. Cell lines were analyzed as in A using pHIV-LTR(–118 to +83)-CAT. C, reporter assays using a plasmid containing the core promoter region. SerpinB2 expressing and parental cell lines were transfected with the pHIV-LTR(–81 to +83)-LUC reporter plasmid and a plasmid encoding beta-galactosidase. Cells were tested in the presence of a cotransfected plasmid encoding Tat (+Tat)(left hand x-axis) or with a cotransfected control plasmid encoding beta-globin (-Tat) (right hand x-axis). Relative LUC units (RLU) were determined after 24 h by luminometer and normalized to beta-galactosidase activity. All graphs represent the mean of at least three independent experiments performed in duplicate ± S.D. In A–C SerpinB2-expressing cells gave significantly more reporter activity than control cells (Student's t test p < 0.05). D, reporter assays using a plasmid containing only the TATA box and TAR region. The experiment described in C was repeated using pHIV-LTR(–45 to +83)-LUC).

 
CAT reporter plasmids were insufficiently sensitive in plasmids with larger deletions in the LTR (see below), necessitating a switch to a LUC reporter system. The experiments shown in Fig. 4B were repeated using a luciferase (LUC) reporter construct, pHIV-LTR(–177 to +83)-LUC, and showed the same fold up-regulation in SerpinB2-expressing cells (data not shown). A similar pattern of results was also obtained using this reporter plasmid in the presence and absence of Tat, although the LUC activities were {approx}10-fold lower without Tat (data not shown).

The Enhancer Region Was Not the Target of SerpinB2 Activity—The enhancer region contains two important NF-{kappa}B sites, however, when the enhancer region was deleted to leave a reporter construct containing only the core region of the promoter (pHIV-LTR(–81 to +83)-LUC), the SerpinB2-mediated increase in transcriptional activity was retained (Fig. 4C). Experiments using NF-{kappa}B-secreted alkaline phosphatase reporter assays and EMSA assays also both failed to show increased NF-{kappa}B activity in SerpinB2-expressing cells (supplemental Figs. S4 and S5). The enhancement of transcription mediated by SerpinB2 expression seen in Fig. 4B was also retained when NF-{kappa}B activity was maximally stimulated in THP-1 and Jurkat cells with phorbol ester (12-O-tetradecanoylphorbol-13-acetate) in the presence or absence of Tat (data not shown). Taken together these data suggest that the enhancer region was not the primary target of SerpinB2 activity.

Kundu et al. (33) reported that in U-87MG astrocytic glioma cells E2F-1 were able to bind an E2F site embedded between the two NF-{kappa}B sites and mediate transcriptional repression that could be alleviated by Rb expression. A mutation in this site (–98TTTCCGC–92 to –98TTTCCTA–92 in pHIV-LTR(–177 to +83)-LUC) enhanced transcription 2–3-fold in parental THP-1, Jurkat, and HeLa lines (data not shown), supporting the view that the E2F site is able to mediate transcriptional repression (33). However, the ability of SerpinB2 expression to enhance transcription was not altered by this mutation (data not shown), illustrating that this site was not the target of SerpinB2 activity.


Figure 5
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  		FIGURE 5.
The role of Rb. A, Western analysis of Rb, SerpinB2 and actin in THP-1 cell lines. B, role of Rb in the SerpinB2 expression-associated enhancement of transcription. The HIV-LTR(–81 to +83)-LUC reporter plasmid containing the core promoter region was transfected into parental cell lines (gray bars) or SerpinB2-expressing cells (black bars). The latter were untreated or treated with siRNA specific for Rb (Rb siRNA), or control siRNA (Cont siRNA) 48 h prior to transfection with pHIV-LTR(–81 to +83)-LUC and a plasmid encoding beta-galactosidase, or had a plasmid encoding HDM2 co-transfected with the latter two plasmids (HDM2). RLU activity was determined after 24 h as for Fig. 4C. C, Western analysis of siRNA-treated SerpinB2-expressing cells. Cells were untreated, or treated with Rb siRNA or control siRNA (Cont siRNA) for 72 h and were then analyzed by Western blotting using anti-Rb, anti-SerpinB2, and anti-actin antibodies. D, role of the C-pocket of Rb. C33A cells were transfected pHIV-LTR(–81 to +83)-LUC and a plasmid encoding beta-galactosidase, together with plasmids encoding (i) beta-globin (Control), (ii) Rb379–928, comprising the A, B, and C pockets of Rb (Rb), (iii) p130414–1135 comprising the A, B, and C pockets of p130 (p130), (iv) SerpinB2 (SerpinB2), (v) Rb768–928 comprising the C pocket of Rb (Rb SE), (vi) Rb SE with a deletion in the region 785–806 (RbSE{Delta}), and/or (vii) HDM2 (HDM2). LUC activity was determined as for B.

 
SerpinB2 Enhances Transcription through the Core Region of the HIV-1 LTR—The SerpinB2 expression-associated increase in transcriptional activity was retained when the reporter construct containing only the core promoter region (Fig. 4C, pHIV-LTR(–81 to +83)-LUC) was tested. The increase in transcriptional activity again occurred in the presence and absence of Tat, although as expected Tat increased transcriptional activity by {approx}10-fold in each cell line (Fig. 4C). SerpinB2 ability to enhance transcription was lost when a reporter plasmid was used where the Sp1 sites had been deleted (pHIV-LTR(–45 to +83)-LUC) (Fig. 4D). Thus the enhancement of transcription associated with SerpinB2 expression localized to the three Sp1 sites within the core promoter region, suggesting that SerpinB2 expression changes Sp1 and/or Sp3 activity (27).

The Role of Rb in the SerpinB2 Expression-associated Increase in Transcription—SerpinB2 expression in both Jurkat and HeLa cells resulted in significant increases in Rb protein levels (15, 16). Rb protein levels were similarly increased in THP-1 cells expressing SerpinB2, with this increase also requiring the RSL of SerpinB2 to be intact (Fig. 5A). These increases in Rb levels were associated with increased HIV-1 transcription (Fig. 4). Furthermore, loss of SerpinB2 was associated with reduced Rb levels and reduced transcription (Fig. 3). To establish directly the role of increased Rb protein levels in the enhancement of transcription from the core promoter, the SerpinB2-expressing cells were treated with small interfering RNA (siRNA) specific for Rb and reporter activity was measured using pHIV-LTR(–81 to +83)-LUC). Treatment of SerpinB2-expressing cells with Rb siRNA, but not control siRNA, reversed the enhancing effect of SerpinB2 on transcription (Fig. 5B). Western analysis confirmed that Rb siRNA treatment significantly reduced Rb, but not SerpinB2, protein levels (Fig. 5C). (Rb siRNA is particularly effective in S1a cells as loss of Rb results in reappearance of E7, which accelerates Rb degradation, Ref. 16). These results illustrated that the SerpinB2-mediated increase in Rb protein levels were required for the enhancement of transcription from the HIV-1 core promoter region. This observation is consistent with the previously reported central role of Rb in the transcriptional regulation seen in SerpinB2-expressing cells (15, 16).

MDM2, and its human homologue, HDM2, are known as important mediators of p53 degradation. However, overexpression of MDM2 also increases Rb degradation (34). Transient overexpression of HDM2 in the SerpinB2-expressing cell lines resulted in significant reductions in Rb protein levels (data not shown), illustrating that SerpinB2 is not able to inhibit the ubiquitin-dependent degradation of Rb mediated by HDM2. Importantly, overexpression of HDM2 resulted in reversal of SerpinB2 activity in the presence (data not shown) and absence of Tat (Fig. 5B, HDM2), further supporting the role of Rb in the SerpinB2 mechanism of action. p53 cannot be the target of HDM2-mediated reversal of SerpinB2 activity in THP-1 and Jurkat cells as these cells are p53-defective.

SerpinB2 and the C-pocket Region of Rb—To further characterize the ability of Rb to enhance transcription from the HIV core promoter region, transcription from pHIV-LTR(–81 to +83)-LUC was tested in the Rb and p53 defective, human papillomavirus-negative, cervical carcinoma cell line, C33A, a cell line frequently used for analysis of Rb activities (35). Expression of Rb, but not the p130 pocket protein (36), was able to enhance LUC activity in C33A cells (Fig. 5D, Rb, p130). Expression of SerpinB2 in these Rb negative cells had no effect on transcription (Fig. 5D, SerpinB2), whereas co-expression of Rb and SerpinB2 increased transcription over 3-fold (Fig. 5D, SerpinB2+Rb) compared with expression of Rb alone. These data again support the view that SerpinB2 requires the expression of Rb to enhance transcription. This Rb dependence and the lack of enhanced transcription following expression of SerpinB2 alone also argue against a role for urokinase plasminogen activator inhibition (30).

A series of experiments were undertaken to determine which region of Rb was responsible for enhancing transcription from the core promoter region. It emerged that a fragment comprising amino acids 768–928 of Rb (Fig. 5D, Rb SE), which contains the C-pocket region of Rb (35), was as active as Rb379–928 (which contains the A, B, and C pockets of Rb) (Fig. 5D, Rb). The C pocket region of Rb has recently been shown to bind MDM2, with amino acids 785–803 of Rb shown to be critical for MDM2 binding (26). A Rb C-pocket mutant lacking residues 785–806 (Rb SE{Delta}) (35) would thus be defective for MDM2/HDM2 binding. Expression of Rb SE{Delta} was unable to enhance transcription (Fig. 5D, Rb SE{Delta}), implicating the HDM2-binding region within the C pocket region of Rb in the Rb-mediated enhancement of transcription. These observations are consistent with increased Rb levels leading to increased transcription from the core promoter region, since Rb is reported to increase Sp1 activity by releasing Sp1 from MDM2/Sp1 complexes allowing Sp1 to bind DNA (37).

We have previously shown that SerpinB2 protects Rb from degradation by binding the C-pocket region of Rb (15), although SerpinB2 and HDM2 have distinct binding sites (15, 26, 35). Co-expression of SerpinB2 with Rb SE increased transcription (Fig. 5D, SerpinB2+Rb SE) over that seen following Rb SE expression, and reached levels similar to those seen following co-expression of SerpinB2 with Rb379–928. These observations (i) confirm that the C-pocket of Rb is the target of SerpinB2 activity, and (ii) suggest that the C-pocket of Rb is involved in enhancing transcription from the HIV-1 core promoter in SerpinB2-expressing cells.

As expected, the increased LUC activity seen in cells co-expressing SerpinB2 and Rb, or SerpinB2 and Rb SE could be reversed by over-expressing HDM2 (Fig. 5D, SerpinB2+ Rb+HDM2, SerpinB2+Rb SE+HDM2), consistent with HDM2-mediated degradation of Rb (34). Overexpression of HDM2 alone had little effect on transcription (Fig. 5D, HDM2).

SerpinB2 Expression Resulted in Increased Sp1 Expression, Binding, and Activity—Figs. 4C, 4D, 5B, and 5D suggest that changes in Sp1 and/or Sp3 activity (27) are involved in the enhancement of transcription seen in SerpinB2-expressing THP-1, Jurkat, and HeLa cells. Western blot and scanning densitometry analysis illustrated that SerpinB2 expression in the three cell lines was associated with an average 2.7 ± 0.6 S.D. fold increase in Sp1 protein levels, and a 0.35 ± 0.05-fold reduction in Sp3 levels, with little evidence of major differences in phosphorylation states (Fig. 6A).

EMSA assays and densitometry analysis showed an average 2.8 ± 0.14-fold increase in Sp1 binding, and a 0.5 ± 0.14-fold decrease in Sp3 binding to a consensus Sp1 oligonucleotide probe in SerpinB2-expressing cells (Fig. 6B). Using an oligonucleotide probe containing the three Sp1 sites in the HIV-1 LTR (nucleotides –78 to –43), an average 2.3 ± 0.5 increase in Sp1 binding and a 0.6 ± 0.02-fold reduction in Sp3 binding was observed in SerpinB2-expressing cells (Fig. 6C). The identity of the Sp1 and Sp3 bands was verified by supershifting with Sp1- and Sp3-specific antibodies (Fig. 6C). Although Rb is often, and SerpinB2 has recently been found associated with DNA (17), EMSA supershifting experiments illustrated that neither SerpinB2 or Rb was associated with this core promoter region DNA (supplemental Fig. S6).

A Sp1-Luciferase reporter construct containing 3 consensus Sp1 binding elements was used to directly analyze Sp1 activity in SerpinB2-expressing cells. In all three SerpinB2-expressing cell lines Sp1-mediated transcription was increased when compared with parental cells (Fig. 6D).

Both in the presence and absence of Tat, Sp1 activates and Sp3 represses transcription from the core promoter region (27). Thus the increased transcription from the HIV-1 LTR seen in SerpinB2 expressing cells appears to be due to increased Sp1 and decreased Sp3 binding to the core promoter region resulting in increased Sp1-mediated transcription.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Here we show that infection of human PBMC with M-tropic HIV-1 or treatment with gp120-induced expression of SerpinB2, with SerpinB2 mRNA and protein levels increasing up to 15–20-fold. SerpinB2 expression in the THP-1 monocyte/macrophage line was associated with increased HIV-1 transcription, and a greater than 10-fold increase in the replication of M-tropic HIV-1 (Fig. 2A). In addition, primary macrophages from SerpinB2–/– mice showed reduced HIV-1 gene expression and transcription (Fig. 3). Apart from a study showing increased SerpinB2/PAI-2 activity in lungs of AIDS patients with Pneumocystis carinii infection (38), to our knowledge the current study represents the first association of SerpinB2 and HIV-1. The study illustrates that HIV-1-induced SerpinB2 expression represents a potentially important inducible host factor that promotes HIV-1 replication by enhancing transcription from the core promoter region of the HIV-1 LTR.


Figure 6
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  		FIGURE 6.
Sp1 and Sp3. A, left hand panel shows a representative Western blot analysis of Sp1 and Sp3 expression in parental and SerpinB2-expressing cell lines. The right hand bar charts show the fold change in Sp1 and Sp3 levels in SerpinB2-expressing cells (black bars) compared with parental cell lines (white bars). Scanning densitometry was used to quantify bands from three independent experiments. For HeLa, Jurkat, and THP-1 cells significant differences (p < 0.05 by paired Student's t test) in both Sp1 and Sp3 expression levels were observed between parental and SerpinB2-expressing cells. B, left hand panel shows a representative EMSA analysis which used a biotin-labeled Sp1 dsDNA oligonucleotide probe and nuclear lysates from SerpinB2-expressing and parental cell lines. Probe in the absence of nuclear lysate is shown (no lysate), as is lysate and labeled probe with a 100-fold excess of unlabeled probe (100x unlabeled). The right hand bar charts show the fold change in Sp1 and Sp3 complexes in SerpinB2-expressing cells (black bars) compared with parental cell lines (white bars). Scanning densitometry was used to quantify bands from three independent experiments and in all cases (except Sp3 and J.PAI-2a and Jurkat) parental and SerpinB2 showed significant differences (p < 0.05 by paired Student's t test) in Sp1 and Sp3 binding. C, left hand panel shows a representative EMSA analysis, which used an oligonucleotide probe containing the three Sp1 sites in the HIV-1 LTR (–78 to –43) and the same protocol as in B. Supershift experiments were performed using THP-1/PAI-2 cells and anti-Sp1 and anti-Sp3 antibodies to confirm the positions of Sp1 and Sp3; the supershifted bands are indicated (*). The right hand bar charts show the fold change in Sp1 and Sp3 complexes in SerpinB2 expressing cells (black bars) compared with parental cell lines (white bars). Scanning densitometry and statistics as for B. D, LUC reporter assay using a plasmid containing 3 consensus Sp1 binding sites. SerpinB2 expressing and parental cell lines were transfected with the Sp1-LUC reporter plasmid and a plasmid encoding beta-galactosidase. Relative LUC units (RLU) were determined after 24 h and normalized to beta-galactosidase activity. In all cases, parental and SerpinB2 showed significant differences (p < 0.05 by paired Student's t test) in Sp1 activity.

 
The SerpinB2-associated enhancement of HIV-1 transcription and HIV-1 replication occurred in three different human cell lines and in primary mouse macrophages and thus appears to represent a robust effect independent of cell type. The SerpinB2 activity is particularly relevant in monocyte/macrophage cells, which are a major source of virus throughout the course of HIV-1 infection (20) and the major source of inducible SerpinB2 in PBMC. SerpinB2 expression may play a role in amplifying virus replication in monocyte/macrophages at several stages of HIV-1 infection. SerpinB2 expression was shown to enhance transcription from the core promoter in the absence of Tat, indicating that HIV-1-induced SerpinB2 expression may be critical for promoting Sp1-dependent and Tat-independent gene transcription in monocytes/macrophages early in infection (39, 40). SerpinB2 activity was also retained after NF-{kappa}B stimulation, consistent with the known cooperative interaction between NF-{kappa}B and Sp1 (41). SerpinB2 expression may therefore also contribute to increased HIV-1 replication under inflammatory conditions, which stimulate NF-{kappa}B (21, 22) and also induce high levels of SerpinB2 (2). SerpinB2 expression may play a similar role in latently infected macrophages in which HIV replication is activated by inflammatory mediators (42). Latently infected U1 cells, when stimulated with phorbol esters to induce HIV-1 replication also produce large amounts of SerpinB2 (data not shown).

The SerpinB2 activity described herein may also be relevant in microglial cells, which are infected by HIV-1 and express SerpinB2 (43, 44). Although SerpinB2 is a major product of differentiating keratinocytes (2) and HIV-1 replication is increased in SerpinB2-expressing HeLa cells, the physiological relevance of HIV-1 infection of epithelial cells remains to be established (45). No reports of primary T cell expression of SerpinB2 have been published, and in our hands PBMC-derived and PHA-stimulated T cell blasts express little or no SerpinB2 (data not shown). Nevertheless, SerpinB2 mRNA expressed sequence tags have been detected in activated T cells (GenBankTM accession numbers: aa381739 and aa382051). T cell lymphoblastic leukemia lines often express SerpinB2 (46), and CEM-T4 cells, a human T cell lymphoblastoid line frequently used in HIV research, also expresses high levels of SerpinB2 (data not shown). Whether certain activation states and/or subsets of T cells (47) express physiologically relevant levels of SerpinB2 remains to be established.

The ability of gp120 to induce SerpinB2 in PBMC (Fig. 1C) is consistent with the recent observation that NF-{kappa}B and p38 are involved in activating SerpinB2 expression (48), since these transcription factors are activated by gp120 binding to CD4 and/or chemokine receptors (28, 49, 50). Binding of gp120 to CD4 also rapidly induces TNF{alpha} (28) and IL-1 secretion (51), inflammatory mediators known to induce SerpinB2 (2). Stabilization of SerpinB2 mRNA may also be involved (2). The reduction in SerpinB2 expression apparent by day 10 after infection (Fig. 1, B and C) may be caused by induction of IL-10 by HIV-1 and/or gp120 (52–54) because IL-10 has recently been shown to down-regulate SerpinB2 expression (55).

The increased transcription from the HIV-1 LTR, both in the presence and absence of Tat, was localized to the core promoter region, a region containing three Sp1 sites that have a critical role in HIV-1 transcription (25). SerpinB2 expression was associated with an increase in Sp1/Sp3 ratios, and with increased binding of Sp1 and decreased binding of the repressive Sp3 (27) to the core promoter region. The enhanced transcription from the core promoter was caused by the SerpinB2-mediated increase in Rb protein levels, consistent with previous reports of transcriptional regulation associated with SerpinB2 expression (15, 16). Increased Rb expression appeared able to regulate Sp1/Sp3 activity in at least two ways. Rb is known to activate Sp1 by relieving HDM2-mediated repression of Sp1 activity (37). That this mechanism can operate for increasing transcription from the HIV-1 core promoter is indicated in Fig. 5C, where the HDM2-binding domain of the C-pocket of Rb was implicated in the increased transcription. SerpinB2 expression was also associated with an increase in Sp1/Sp3 protein expression ratios (Fig. 6A), which would again lead to enhanced transcription from the core promoter (27). Sp1 and Sp3 are regulated by a complex series of transcriptional and post-transcriptional events (56–58). How elevated Rb expression might increase Sp1/Sp3 ratios remains unclear, although Sp1 has a tissue-specific distribution resembling that of Rb (59) and E2F-1 is known to bind Sp1 (60). Interestingly, an increase in Sp1/Sp3 ratios is also seen in differentiating keratinocytes (61), and differentiation in these cells is associated with potent induction of SerpinB2.

There has been considerable interest in host factors that influence HIV-1-replication, particularly where polymorphisms influence disease outcomes (62, 63). At least two sequence variants of SerpinB2 are known which differ by three amino acids and these polymorphisms have been associated with increased risk of stroke and prostate cancer (64, 65). Two other non-synonymous single nucleotide polymorphisms (SNPs) have been identified in the coding sequence of SerpinB2 (rs17850949, rs6100), indicating that additional variants exist. A number of SNPs have also been found in the non coding regions (66, 67) including in the SerpinB2 promoter and the 3'-untranslated region region. Whether these polymorphisms affect SerpinB2 activity, SerpinB2 induction by HIV-1 and/or correlate with the clinical outcome of HIV-1 infection remains to be established.


   FOOTNOTES
 
* This work was supported in part by the National Health and Medical Research Council of Australia and the National Institutes of Health (R01-CA098369 [GenBank] ). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S6 and Table S1. Back

1 To whom correspondence should be addressed: Queensland Institute of Medical Research, Post Office Royal Brisbane Hospital, Queensland 4029, Australia. Tel.: 61-7-33620415; Fax: 61-7-33620107; E-mail: Andreas.Suhrbier{at}qimr.edu.au.

2 The abbreviations used are: PAI-2, plasminogen activator inhibitor-2 (SerpinB2); Rb, retinoblastoma protein; RSL, the reactive site loop; SEAP, secreted alkaline phosphatase; M-tropic, macrophage tropic; T-tropic, T cell tropic; LUC, luciferase; CAT, chloramphenicol acetyltransferase; ov-serpin, ovalbumin-like serine protease inhibitor; PBMC, human peripheral blood mononuclear cells; siRNA, small inhibitory RNA; MDM2, mouse double minute 2; HDM2, human double minute 2; EMSA, electrophoretic mobility shift assay; HIV-1 LTR, HIV-1 long terminal repeat promoter; IL, interleukin; SNP, single nucleotide polymorphism. Back


   ACKNOWLEDGMENTS
 
We thank Dr. T. Ny (Umea University, Sweden) for the gift of MAI21 antibody, and Dr. J. Y. Wang (University of California) for the C-pocket Rb constructs. The following reagent was obtained through the National Institutes of Health AIDS Research & Reference Reagent Program, Division of AIDS (DAIDS), NIAID, National Institutes of Health: HIV-1BaL gp120 from DAIDS, NIAID.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
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 DISCUSSION
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