HIV-1 induces complement factor C3 synthesis in astrocytes and neurons by modulation of promoter activity

Molecular Immunology 40 (2003) 949–961

HIV-1 induces complement factor C3 synthesis in astrocytes and neurons by modulation of promoter activity Cornelia Bruder a , Magdalena Hagleitner a , Gretchen Darlington b , Iradj Mohsenipour c , Reinhard Würzner a , Isolde Höllmüller a , Heribert Stoiber a , Cornelia Lass-Flörl a , Manfred P. Dierich a , Cornelia Speth a,∗ a

Institute of Hygiene and Social Medicine, University of Innsbruck and Ludwig-Boltzmann-Institute for AIDS Research, Fritz-Pregl-Str. 3, A-6020 Innsbruck, Austria b Department of Pathology, Baylor College of Medicine, Houston, USA c Department of Neurosurgery, Innsbruck, Austria Received 8 July 2003; received in revised form 7 October 2003; accepted 22 October 2003

Abstract Virus-induced complement expression and activation in the brain is hypothesized to contribute to the process of neurodegeneration in AIDS-associated neurological disorders. Previous experiments have shown that the human immunodeficiency virus (HIV) upregulates the low basal production of complement factor C3 in astrocytes and neurons. Since inhibition of complement synthesis and activation in the brain may represent a putative therapeutic goal to prevent virus-induced damage, we analysed the mechanism of the HIV-induced modulation of C3 expression. Detailed studies using different C3 promoter constructs revealed that HIV activates the synthesis of C3 by stimulation of the promoter. This HIV-induced promoter activation could be measured both in different astrocytic cell lines and in neurons. Deletion constructs of the C3 promoter defined the IL-6/IL-1␤ responsive element within the promoter region as a central element for the responsiveness of the C3 promoter towards the influence of HIV. A binding site for the transcription factor C/EBP␦ was identified as important regulatory domain within the IL-6/IL-1␤ responsive element, since a point mutation which eliminates the binding capacity of C/EBP␦ to this site also abolishes the induction by HIV-1. Similarly, the viral proteins Nef and gp41 which had also been shown to stimulate the synthesis of C3, exert their effect via the IL-6/IL-1␤ responsive element with binding of the transcription factor C/EBP␦ representing the critical step. Our experiments clearly define the mechanism for the induction of complement factors in the HIV-infected brain and reveal a decisive role of the regulator protein C/EBP␦ for the HIV-induced increase in C3 expression. © 2003 Elsevier Ltd. All rights reserved. Keywords: HIV; Complement; AIDS; C3, promoter; C/EBP

1. Introduction The human immunodeficiency virus (HIV-1) penetrates the blood–brain barrier in the majority of AIDS patients and induces neurological manifestations in 20–30% of HIV-l-infected individuals (Gray et al., 1996; Johnson et al., 1996; Sinclair et al., 1994). The AIDS dementia complex (ADC) is the most prominent of these neurological compli∗ Corresponding author. Tel.: +43-512-507-3405; fax: +43-512-507-2870. E-mail address: [email protected] (C. Speth).

0161-5890/$ – see front matter © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.molimm.2003.10.016

cations with cognitive, motor and behavioural dysfunctions. The pathogenesis of ADC is unclear since only a limited number of brain cells is infected by HIV. Current hypotheses indicate that host-derived mediators are involved in the induction of neurological lesions. One such potential mediator is the complement system (Speth et al., 2001, 2002a). Complement is an antimicrobial defence mechanism which recognizes a large variety of pathogens and targets them for destruction (reviewed in: Speth et al., 1999). In the brain the complement system is of special importance for the antiviral defence since the blood–brain barrier restricts

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the access of the adaptive immune elements. But beside the protective role of complement there is also evidence from other neurological disorders like stroke, Alzheimer’s disease and multiple sclerosis that chronic complement activation in the brain is associated with inflammation and neurodegeneration (Huang et al., 1999; Veerhuis et al., 1996; Yasojima et al., 1999; reviewed in: Gasque et al., 2000; Speth et al., 2002b). C3 is a central protein of the cascade and participates in the activation of all three pathways of the complement cascade. Its fragments affect many cellular processes in the brain like activation of signalling pathways in astrocytes and neurons (Moller et al., 1997; Osaka et al., 1999a,b) and modulation of cytokine synthesis (Heese et al., 1998; Sayah et al., 1999). C3 is an acute-phase protein (APP), i.e. a protein whose plasma concentration increases following tissue injury and inflammation. C3 can be categorized as class I APP, the synthesis of which is regulated by IL-1-type cytokines (IL-1␣, IL-1␤, TNF-␣) and IL-6 (Baumann et al., 1989; Volanakis, 1993). The gene of C3, which is located on chromosome 19, is well characterized and consists of 41 exons spanning about 42 kb of DNA (Barnum et al., 1989; Fong et al., 1990; Vik et al., 1991). Approximately, 1 kb of DNA upstream of the transcription start site is sequenced and analysed for the presence of responsive elements (Fan et al., 1996; Wilson et al., 1990). One responsive element within the promoter is a 58 bp region that confers interleukin 1 and 6 (IL-1, IL-6) responsiveness to this gene. A sequence analysis reveals two CCAAT/enhancer-binding protein (C/EBP) consensus sequences (bZIP1 and bZIP2) within this region which participate in the acute-phase regulation of C3 (Wilson et al., 1990; Juan et al., 1993). In the brain the normal synthesis of C3 is low but can be stimulated by various stimuli (Barnum and Jones, 1995; Haga et al., 1996; reviewed in: Morgan and Gasque, 1996). Enhanced mRNA levels of complement factor C3 were measured in lesion areas of Alzheimer brains (Yasojima et al., 1999). In addition, viral infection with HIV induced an increase of C3 production in astrocytes and neurons (Speth et al., 2001, 2002a), a finding that is reflected by increased levels of C3 and C4 in the cerebrospinal fluid (CSF) of HIV-infected patients with neurological symptoms and signs of CNS dysfunction (Jongen et al., 2000). In addition to the complete virions purified viral proteins Nef and gp41 also harboured the capacity to induce C3 synthesis in different brain cells (Speth et al., 2002a). Since inhibition of complement synthesis and activation may be an interesting therapeutic approach to prevent neurological damage in the virus-infected brain, we analysed the molecular mechanism of HIV-induced C3 upregulation in astrocytes and neurons. Promoter studies revealed that enhancement of the C3 promoter activity is the relevant mechanism by which HIV increases the production of C3. An IL-6/IL-1␤ responsive element within the promoter region with a binding site for the transcription factor C/EBP␦ seems

to be the central mediator for the effect of HIV-1 on the C3 synthesis induction. 2. Materials and methods 2.1. Cell lines and culture The human glioblastoma/astrocytoma cell line U373MG, the glioblastoma cell lines U138MG and U251MG and the astroglioma cell line U87MG were purchased from the MRC (Centralised Facility for AIDS Reagents, Hertfordshire, UK) and cultured in either Ham’s F12 medium or DMEM medium (Life Technologies, Vienna, Austria) supplemented with 10% fetal calf serum (FCS; BioWhittaker, Verviers, Belgium), penicillin/streptomycin (100 U/ml and 100 ␮g/ml, respectively), 0.1 mM non-essential amino acids and 2 mM l-glutamine (Life Technologies). The neuroblastoma cell line SK-N-SH, the glioblastoma/astrocytoma cell line U118 and the glioblastoma multiforme cell line T98 were from the American Type Culture Collection ATCC (clones HTB-11, HTB-15 and CRL1690) and grown in DMEM medium with the same supplements. The T-lymphoblastoid cell line M8166 used for virus propagation was cultivated in RPMI 1640 medium (Life Technologies) supplemented with 10% FCS, penicillin/streptomycin and 2 mM l-glutamine. 2.2. Virus propagation and purification The HIV-1 IIIB strain was from the MRC (Popovic et al., 1984; Cheng-Mayer and Levy, 1988) and grown for propagation in the cell line M8166. Virus-containing cell culture supernatants were harvested 3 days after infection and frozen at −70 ◦ C. For mock-treatment of cells culture supernatant of non-infected M8166 was collected at the same time points. Viral yield was determined by p24 antigen ELISA developed at the Institute of Applied Microbiology (Vienna, Austria). For that assay the monoclonal antibodies 37G12 and Mo1 were used which were a kind gift from H. Katinger (Institute for Applied Microbiology). Briefly, microplates were coated overnight with the first monoclonal antibody and washed three times with PBS/0.1% Tween 20 (Serva, Heidelberg, Germany). Dilution series of M8166 culture supernatants or purified viral stocks were applied on the plate, together with the second antibody conjugated with biotin, for 1 h. The plates were consequently incubated with streptavidin-␤-galactosidase (Roche, Wien, Austria) for 30 mm. The amount of bound p24 was quantified by adding of resorufin-␤-d-galactopyranoside substrate solution (Sigma) and measuring the optical density at 550 nm using an ELISA reader. The p24 assay was calibrated using baculovirus-derived recombinant p24 as a standard.

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(B) Fig. 1. C3 Promoter structure and constructs derived thereof. (A) Scheme of the 5 , region of the C3 gene and of the different C3 promoter constructs used in this study. For detailed description see Section 2. (B) Nucleotide sequence of the 5 region of the C3 gene. The IL-6/IL-1␤ responsive element is boxed and the binding sites bZIP1 and bZIP2 for the transcription factor C/EBP␦ therein are underlined in bold. The mutation site of the construct C3-199mut which abolishes the binding of C/EBP␦ as well as the putative CAAT and TATA boxes are indicated. The transcription start site is labeled with an arrow.

2.3. Purified proteins Purified viral proteins nef IIIB (deposit by Dr. V. Erfle, Munich, Germany), was purchased from Medical Research Council. Since gp41 is rather hydrophobic, a recombinant fusion protein of maltose-binding protein (mbp) with an 82-amino acid-long extracellular region (aa565–647) of gp41 was used for the experiments (Intracel, Cambridge, USA). To

rule out possible side effects of mbp we used recombinant mbp (New England Biolabs, Beverly, USA) as a negative control. 2.4. Plasmids and constructs The promoter of the C3 gene comprises more than 1000 bp upstream of the transcription start site (Fig. 1A and B). For our experiments several promoter constructs were used

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which had been fused to the firefly luciferase gene as reporter gene. The most extended construct is C3T1luc (a kind gift of D.P. McDonnell, Department of Pharmacology, Durham, USA) which comprised the part −1030 to +58 of the C3 promoter (Fan et al., 1996). The generation of the constructs C3-320 (fragment −320 to −1 bp), C3-199 (fragment −199 to −1 bp), C3-199 mut (fragment −199 to −1 bp; with a six-base substitution of TGTCGA for AGAAAT at position −107 to −102; Fig. 1B), C3-156 (fragment −156 to −1 bp), C3-127 (fragment −127 to −1 bp), C3-114 (fragment −114 to −1 bp), C3-76 (fragment −76 to −1 bp) was described elsewhere (Wilson et al., 1990). All these fragments have been cloned into the vector pXP1. The construction of the expression vector pCMV-hC/EBP␦ is described elsewhere (Juan et al., 1993). In this construct the gene for the transcription factor C/EBP␦ is cloned 3 of the CMV promoter. The plasmids were transfected into the bacterial strain XL1-blue and amplified. For plasmid preparation a kit (Qiagen, Hilden Germany) was used according to manufacturer’s instruction. 2.5. Cell transfections and quantification of luciferase activity 7×104 cells/ml were seeded in a 12-well plate and grown until 80% confluence. The cells were transfected with 2 ␮g purified plasmid DNA per well using the superfect reagent from Qiagen according to the manufacturer’s instruction. The incubation time with DNA and superfect was optimised with 2.5 h. Control cells were incubated with superfect alone without any plasmid DNA. The cells were washed subsequently and either incubated with medium or infected with HIV (20 ␮g/ml p24 if not otherwise indicated). To quantity luciferase activity the luciferase assay kit from Promega (Madison, USA) was used. Briefly, the cell supernatant was removed and the cells were lysed by addition of cell lysis buffer for 15 min. The lysate was transferred in a luminometer vial, substrate was added and the light reaction was quantified by a luminometer (Lumat LB, Berthold, Bundoora, Australia) with an integration interval of 5–30 s. 2.6. Quantification of C3 by ELISA Cell culture supernatants from different experiments were harvested at indicated time points. The amount of C3 protein therein was quantified by a specific sandwich ELISA as described previously (Speth et al., 2001). Briefly, ELISA microplates were coated overnight at 4 ◦ C with the monoclonal C3d antibody BB5 (a kind gift of W. Prodinger, Innsbruck, Austria) as catching antibody in 0.1 M NaHCO3 , pH 9.6. Unspecific binding was inhibited by saturation with 1% BSA (Sigma) in PBS. Cell culture supernatants were added to the coated wells and incubated for 1 h at room temperature. To calculate the exact amount of C3 in the

supernatants purified C3 protein (Sigma) was used as a standard. After washing procedure, bound C3 was detected with polyclonal C3c antibody and a second peroxidase-labeled anti-rabbit IgG antibody (Dako, Glostrup, Denmark). 2.7. Statistical analysis Statistical analysis (Student’s t-test) was performed using the Origin 6.0 software (serial no.: 6014906).

3. Results 3.1. Induction of C3 expression by HIV-1 is mediated by promoter activation To differentiate between C3 mRNA stabilization by HIV and virus-induced activation of the C3 promoter we used the promoter construct C3T1luc, which contains the complete known C3 promoter sequence fused to firefly luciferase as reporter gene (Fig. 1A). This construct was transfected into U373 astrocytes followed by mock-treatment or infection with HIV. Luciferase activity was measured after 48 h to quantify promoter activity. In parallel, production and secretion of C3 protein in the cell culture supernatant was measured by ELISA at different time points. Whereas C3 protein level in cell culture supernatant of U373 astrocytes was low or even absent in uninfected cells, the level increased up to 300 ng/ml in HIV-infected cells (Fig. 2A). In parallel the C3 promoter activity increased eight-fold in HIV-infected cells compared to mock-infected control cells, indicating that HIV upregulated the production of C3 by enhancing the promoter activity (Fig. 2B). The level of promoter induction by HIV varied between different experiments, ranging between 4 and 10-fold. Since the induction of C3 protein synthesis depends on the dose of HIV (Speth et al., 2001), we also tested the dose-dependence of the C3 promoter induction. HIV concentrations between 2 and 30 ng/ml p24 continuously increased the promoter activity, higher amounts of virus had no additional effect (data not shown). Previous experiments had shown that the induction of C3 synthesis by HIV-1 on protein level was not restricted to U373 astrocytes but also found in general for different astrocytic cell lines (Speth et al., 2001). Therefore we evaluated, whether the induction of promoter activity is the appropriate mechanism for C3 induction in all astrocytes. For this purpose the cell lines U373, U251, U138, U118 and T98 were transfected in parallel with the promoter construct C3T1luc and either mock-treated or infected by HIV. Luciferase activity was quantified with a luminometer after 48 h. The basal promoter activity of the different astrocytic cell lines differed widely, ranging between 1 × 103 RLU/s for U373 and 500×103 RLU/s for U251 (Fig. 3). All cell lines upregulated the activity of the C3 promoter construct significantly as a consequence of HIV infection. The increase in promoter ac-

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Fig. 2. Induction of C3 synthesis by HIV-1 via promoter activation (A) U373 astrocytes were either mock-treated with medium or incubated with 20 ng/ml p24 HIV IIIB and cell culture supernatants were harvested at different time points. The amount of C3 protein was quantified by a specific ELISA assay. Results are the mean ± S.D. of triplicate determinations; statistical significance of HIV-induced C3 production versus C3 levels in mock-treated cells was evaluated by Student’s t-test. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.005. (B) U373 astrocytes were transfected with the C3 promoter construct C3T1luc and subsequently either incubated with medium or infected with 20 ng/ml p24 HIV IIIB. Luciferase activity was quantified 48 h after transfection and expressed as relative luminescence units (RLU). Results are the mean ± S.D. of six parallel samples; statistical significance of luciferase activity of HIV-infected cells versus that of non-infected cells was evaluated by Student’s t-test. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.005.

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Fig. 3. Comparison of C3 promoter activity in different astrocytes and neurons. The astrocytic cell lines U373, U251, U118, U138 and T98 as well as the neuronal cell line SK-N-SH were transfected with the C3 promoter construct C3T1luc and subsequently either infected by HIV-1 (20 ng/ml p24) (+) or mock-treated with medium (−). Luciferase activity was quantified 48 h after transfection and expressed as relative luminescence units (RLU). Results are the mean ± S.D. of six parallel samples. Statistical significance of luciferase activity in HIV-infected cells versus that in non-infected cells was evaluated by Student’s t-test. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.005.

tivity was 6 to 7-fold for U373 and U251, 10-fold for U118 and T98 and 30-fold for U138, indicating that C3 promoter activation by HIV is a general mechanism for all astrocytes. Since not only astrocytes but also neurons had been described to enhance C3 synthesis after stimulation by HIV,

we also tested the C3 promoter activity in infected and uninfected SK-N-SH neurons after transfection of the C3T1luc construct. Similar to the astrocytes the SK-N-SH revealed a 10-fold increase in luciferase activity after stimulation with HIV compared to uninfected control cells (Fig. 3).

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Fig. 4. Effect of HIV on the activity of different C3 promoter constructs in U373 astrocytes. Different promoter constructs were transfected in U373 astrocytes followed either by infection with HIV-1 (20 ng/ml p24) or by mock-treatment with medium. Luciferase activity was quantified 48 h after transfection and expressed as relative luminescence units (RLU). Results are the mean ± S.D. of six parallel samples. Statistical significance of luciferase activity in HIV-infected cells versus that in non-infected cells was evaluated by Student’s t-test. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.005.

3.2. Identification of the HIV-responsive element within the C3 promoter To identify the region which mediates the responsiveness of the C3 promoter towards HIV, different constructs were used with C-terminal subfragments of the C3 promoter region fused to the luciferase reporter gene (Fig. 1A). The constructs were transfected into U373 astrocytes and luciferase activity in HIV-infected and uninfected cells was quantified after 48 h. Luciferase activity after infection with HIV was highest in the construct C3T1luc which comprises the complete known C3 promoter. The levels of luciferase were lower in those cells transfected with shorter constructs, however a clear and significant induction by HIV was seen for the constructs C3-320, C3-199, C3-156, C3-127 and C3-114. The inducibility was completely abolished in the construct C3-76 which comprises only the most C-terminal 76 bp of the promoter. The capacity of the promoter to be stimulated by HIV was also abolished by introduction of a point mutation within the IL-6/IL-1␤ responsive element of the promoter

(construct C3-199mut). This mutation affects the binding site bZIP1 for the transcription factor C/EBP␦ and inhibits the binding of this factor (Fig. 1B). No induction of luciferase activity by HIV was possible in cells transfected with this construct C3-199mut, whereas the corresponding construct C3-199 without that mutation was clearly activated by HIV (Fig. 4), indicating the central role of C/EBP␦ for the HIV-induced upregulation of C3 production. To further underline the role of C/EBP␦ for promoter induction by HIV, we co-transfected the cells with the construct C3T1luc and an expression plasmid for C/EBP␦ under control of the strong promoter of CMV. The quantification of luciferase activity revealed that the HIV-induced increase of promoter activity can be amplified by co-expression of large amounts of the transcription factor C/EBP␦ (Fig. 5). Whereas the luciferase activity was about 4 to 5-fold higher in the HIV-infected versus uninfected cells transfected with C3T1luc only, the activity was increased about 14-fold when cells were transfected with both C3T1luc and the plasmid pCMV-CEBP␦ which enables a high expression of the transcription factor.

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C3T1luc HIV C/EBPδ Fig. 5. Impact of transcription factor C/EBP␦ on the HIV-induced C3 promoter activity. U373 astrocytes were either mock-treated or transfected with C3T1luc, pCMV-C/EBP␦, or C3T1luc + pCMV-C/EBP␦, followed by incubation with medium or with HIV (20 ng/ml p24). Luciferase activity was quantified 48 h after transfection and expressed as relative luminescence units (RLU). Results are the mean ± S.D. of five parallel samples. Statistical significance of luciferase activity in HIV-infected cells versus that in non-infected cells and of luciferase activity in infected cells transfected with C3T1luc versus that in cells transfected with C3T1luc + pCMV-C/EBP␦ was evaluated by Student’s t-test. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.005.

3.3. Stimulation of promoter activity by the viral proteins nef and gp4l Previous experiments had shown that not only complete virions are able to stimulate C3 synthesis in astrocytes but also the isolated viral proteins Nef and gp41 (Speth et al., 2002a). Furthermore, Nef but not gp41 is also active in neurons regarding C3 synthesis. Since those two proteins are expressed in the HIV-infected brain and have been correlated to the degree of neuronal damage, we analysed their mechanism to upregulate C3 synthesis in astrocytes and neurons. The promoter construct C3T1luc was transfected into U373 or SK-N-SH, respectively, followed by incubation with Nef. The control cells were incubated with maltose-binding protein which had been shown to exert no effect on C3 synthesis (Speth et al., 2002a). Nef upregulated the production and secretion of C3 protein in both cell types with astrocytes being the more potent system (Fig. 6A and C). A similar upregulation was visible on the C3 promoter level with an 3 to 4-fold higher luciferase level in Nef-treated U373 and a 2 to 2.5-fold higher amount in SK-N-SH neurons compared to the control cells (Fig. 6B and D). Gp4l harboured the capacity to stimulate C3 protein synthesis in U373 astrocytes, whereas no effect was revealed for SK-N-SH neurons (Fig. 7A and C). Parallel quantification of C3 promoter activity showed a correlation between protein synthesis and promoter activation: whereas the luciferase activity was increased 2.5-fold in U373 astrocytes by gp41 compared to the mbp-treated control cells, gp41 did not alter the luciferase activity in SK-N-SH (Fig. 7B and D).

The next step was to examine whether the same responsive promoter element which can be stimulated by HIV is also responsible for the stimulation of C3 promoter activity by Nef and gp41. For this purpose U373 astrocytes were transfected with the constructs C3T1luc, C3-199 or C3-199mut, respectively, followed by incubation with mbp, gp41 or Nef. In those cells transfected with C3T1luc or C3-199 the incubation with gp41 or Nef resulted in a significant increase in luciferase activity compared to the mbp-treated control cells (Fig. 8). The stimulation of promoter activity by gp41 and Nef was completely abolished by the mutation in bZIP1, as shown by transfection with C3-199mut. These results indicate that the gp41 and Nef act via a similar mechanism on the C3 promoter by stimulation of the binding of transcription factor C/EBP to its corresponding binding site within the IL-6/IL-1 responsive element.

4. Discussion The activation of the cerebral complement system during HIV infection can implicate both positive (protective) and negative (degenerative) aspects. Whereas complement can limit the tissue infection and induce the production of neurotrophins, chronic complement activation was associated with neurodegeneration and its products can enhance cerebral inflammation (reviewed in: Speth et al., 2002b). Normal complement synthesis in the brain is low, but can be upmodulated by infection with HIV-1 as shown by our in vitro experiments (Speth et al., 2001, 2002a) and by quantification

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Fig. 6. Induction of C3 synthesis by Nef via promoter activation. U373 astrocytes (A) or SK-N-SH neurons (C) were incubated with 250 ng/ml maltose-binding protein (mbp) or with 250 ng/ml Nef and cell culture supernatants were harvested at different time points. The amount of C3 protein was quantified by a specific ELISA assay. Results are the mean ± S.D. of triplicate determinations; statistical significance of C3 production by cells incubated with Nef versus C3 levels in mbp-treated cells was evaluated by Student’s t-test. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.005. U373 astrocytes (B) or SK-N-SH neurons (D) were transfected with the C3 promoter construct C3T1luc and subsequently incubated either 250 ng/ml maltose-binding protein (mbp) or with 250 ng/ml Nef. Luciferase activity was quantified 48 h after transfection and expressed as relative luminescence units (RLU). Results are the mean ± S.D. of six parallel samples; statistical significance of luciferase activity in cells incubated with Nef versus that in mbp-treated cells was evaluated by Student’s t-test. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.005.

of complement factor C3 in the CSF of HIV-infected patients (Jongen et al., 2000). Since a correlation is implied for the pathogenesis of HIV-induced neurological dysfunctions and the level of complement synthesis/activation the mechanism of C3 induction was studied in detail. Our experiments described here implicate that stimulation of C3 promoter activity rather than enhancement of mRNA stability or translation is the relevant mechanism for the virus-induced increase of C3 production. This fits well to experiments using other stimuli showing that C3 promoter induction is in general the most important mechanism to regulate C3 synthesis. Nuclear run-on experiments had demonstrated that the cytokines IL-1, IFN-␥ and IL-6 all enhanced C3 expression by induction of C3 gene transcription, and glucocorticoid-induced downregulation of C3 production by

macrophages was due to decreased transcription (Darlington et al., 1993; Lappin et al., 1992; Lappin and Whaley, 1991). The C3 gene promoter contains a large array of potentially important regulatory sequences, including consensus responsive elements for NF-kB, AP-1, estrogen and glucocorticoid receptors (Fong et al., 1990; Vik et al., 1991). The responsiveness of C3 gene expression for the cytokines IL-1 and IL-6 was conferred by a 58 bp segment at position −127 to −70. More detailed analysis identified a binding site for the transcription factor CCAAT/enhancer-binding protein C/EBP␦ as the central mediator for the induction of C3 by cytokines (Wilson et al., 1990; Juan et al., 1993). Further experiments with neutralizing antibodies against IL-1␤ and IL-6 however showed that the effect of HIV is not an indirect one involving the upregulation of cytokine expression.

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(A)

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Fig. 7. Induction of C3 synthesis by gp41 via promoter activation. U373 astrocytes (A) or SK-N-SH neurons (C) were incubated with 20 nM maltose-binding protein (mbp) or with 20 nM gp41 and cell culture supernatants were harvested at different time points. The amount of C3 protein was quantified by a specific ELISA assay. Results are the mean ± S.D. of triplicate determinations; statistical significance of C3 production by cells incubated with gp41 versus C3 levels in mbp-treated cells was evaluated by Student’s t-test. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.005. U373 astrocytes (B) or SK-N-SH neurons (D) were transfected with the C3 promoter construct C3T1luc and subsequently incubated with either 20 nM maltose-binding protein (mbp) or with 20 nM gp41. Luciferase activity was quantified 48 h after transfection and expressed as relative luminescence units (RLU). Results are the mean ± S.D. of six parallel samples; statistical significance of luciferase activity in cells incubated with gp41 versus that in mbp-treated cells was evaluated by Student’s t-test. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.005.

Kinetic studies showed that the promoter is activated 48 h after addition of the virus, whereas the C3 protein level increased only 3 days after infection. This time difference might be attributed to the time needed for transcription of the mRNA, translation of the protein and secretion of C3 into the cell culture medium. Further time is needed until the level of C3 in the culture supernatant reaches the detection level of the C3 ELISA. Our experiments show that the same element also mediates the induction of C3 by HIV both in astrocytes and neurons. Whereas the activity of the complete C3 promoter was highly upregulated in HIV-infected cells, shorter promoter constructs, which did not contain the IL-6/IL-1␤ responsive element any more were no longer inducible by HIV. Furthermore, a point mutation within the C3 promoter construct, which abolishes the binding capacity for C/EBP␦ to its binding site bZIP1, also completely prevented the enhancement of promoter activity by HIV, underlining the im-

portance of transcription factor C/EBP␦ for the regulation of the acute-phase protein C3 by various stimuli. The mechanism of the induction of complement factor C3 by the viral proteins Nef and gp41 was investigated in a similar fashion. Nef is the predominant viral protein produced in HIV-infected astrocytes. It modulates the intracellular signalling of astrocytes, reversibly increases the potassium ion current in neurons, and stimulates a productive HIV-1 infection from latency (Brack-Werner, 1999; Fujinaga et al., 1995). Since the Nef protein is released extracellularly in vivo (Cheingsong-Popov et al., 1990), it can exert its effects also in non-infected distant brain regions. The transmembrane glycoprotein gp41 is readily detected in the brains of HIV-infected patients and plays an important role in the HIV pathogenesis of this organ (Achim et al., 1994; Kure et al., 1990, 1991; Soontornniyomkij et al., 1998). Its function in the HIV-infected brain includes induction of nitric oxide synthesis, inhibition of excitatory amino acid transport

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Fig. 8. Effect of gp41 and Nef on the activity of different C3 promoter constructs in U373 astrocytes. The promoter constructs C3T1luc, C3-199 or C3-199mut were transfected in U373 astrocytes followed by treatment with either 20 nM mbp, 20 nM gp41 or 250 ng/ml Nef. Luciferase activity was quantified 48 h after transfection and expressed as relative luminescence units (RLU). Results are the mean ± S.D. of five parallel samples. Statistical significance of luciferase activity in cells incubated with Nef or gp41 versus that in mbp-treated cells was evaluated by Student’s t-test. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.005.

and up-regulation of the cytokines TNF-␣, IL-1 and IL-10 (Adamson et al., 1996; Hori et al., 1999; Kort, 1998; Koka et al., 1995; Speth et al., 2000). Both Nef and gp41 have been shown to increase the synthesis of C3 in the brain. However, whereas Nef is active both in astrocytes and neurons, gp41 can act only in astrocytes (Speth et al., 2002a). Our experiments showed that the regulation of C3 expression by gp41 and Nef in astrocytes is also mediated by promoter activation. The promoter constructs C3T1luc and C3-199 were both activated by stimulation of the transfected astrocytes with Nef or gp41. The inability of gp41 to induce C3 synthesis in neurons coincides with the unresponsiveness of the promoter constructs in neurons. Similar to the mechanism of C3 induction by HIV, the promoter induction by gp41 and Nef in astrocytes was abolished when the construct C3-199mut, where C/EBP␦ can not bind any more, was used for transfection, underlining again the central role of this transcription factor for the regulation of C3 expression. C/EBP␦ is a member of a family of transcription factors that contain typical basic region/leucine zipper domains. The basic region interacts sequence-specifically with the major groove of DNA, whereas the leucine zipper domain can induce formation of homodimers or heterodimers. The family of C/EBPs is critical for normal cellular differentiation and function and they vary from strong activators to dominant negative repressors. Phosphorylation of C/EBP␦ caused

increases in its DNA-binding activity and nuclear translocation (reviewed in; Takiguchi, 1998; Lekstrom-Himes and Yanthopoulos, 1998). Activation of C/EBP␦ by HIV is favourable for viral replication since C/EBP␦ binding activates the HIV-LTR (Ross et al., 2001; Ruocco et al., 1996). Furthermore, the transcription factor has been described to dimerize with NF-kB, the main activator of the viral promoter (Ray et al., 1995). There is indeed a report that heterodimers between C/EBP␦ and NF-kB subunits strongly activate the HIV-1-LTR (Ruocco et al., 1996). The activation of C/EBP␦ by HIV also explains the dependence of promoter regulation on adenylate cyclase and cAMP level as shown by former experiments with specific inhibitors (Speth et al., 2002a). Cyclic AMP has been described to stimulate the expression of C/EBP␦ both on protein level and on mRNA level (Cardinaux and Magistretti, 1996). We can therefore speculate that HIV or its proteins gp41 and Nef activate the production of cAMP by the adenylate cyclase. Cyclic AMP enhances the expression of C/EBP␦ which can bind together with NF-kB to the viral promoter LTR to induce the viral replication. As a bystander effect, C/EBP␦ can also bind to the promoter domain of complement factor C3 and thus strongly activates its synthesis. The effect might be very prominent in HIV-infected cells, since NF-kB is highly upregulated by HIV infection and

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enhances DNA binding of C/EBP to its site without being part of the DNA-protein complex formed (Wedel and Ziegler-Heitbrock, 1995). In conclusion, our results have clarified the mechanism of complement induction by HIV virions as well as by the purified proteins Nef and gp41. The activation of transcription factor C/EBP␦ and its binding to the bZIP1 site within the IL-6/IL-1␤ responsive element of the C3 promoter have been shown to be central steps of the regulation process. Whereas the increased activity of C/EBP␦ might favour the viral replication and transcription, it can also contribute to the process of HIV-induced neurodegeneration via enhancement of C3 expression and therefore stimulation of neuroinflammation.

Acknowledgements We gratefully acknowledge the generous gift of the monoclonal anti-C3 antibody BB5 from W. Prodinger (Institute of Hygiene and Social Medicine, Innsbruck, Austria). D.P. McDonnell (Duke University Medical School, Durham, USA) kindly provided the construct C3T1luc for the studies. This study was supported by the Ludwig-Boltzmann-Society, FWF (project P15375), the Österreichische Nationalbank (project 9374), the BMSG and the State of Tyrol. H.S. was supported by the fifth frame work of the EU (QLK2-CT-1999-01215, QLK2-CT2002-00882). We are grateful to the MRC for providing various reagents. We thank H. Katinger of the Institute of Applied Microbiology (Vienna, Austria) for the p24 antibodies. References Achim, C.L., Wang, R., Miners, D.K., Wiley, C.A., 1994. Brain viral burden in HIV infection. J. Neuropathol. Exp. Neurol. 53, 284–294. Adamson, D.C., Wildemann, B., Sasaki, M., Glass, J.D., McArthur, J.C., Christov, V.I., Dawson, T.M., Dawson, V.L., 1996. Immunologic NO synthase: elevation in severe AIDS dementia and induction by HIV-1 gp41. Science 274, 1917–1921. Barnum, S.R., Amiguet, P., Amiguet-Barras, F., Fey, G., Tack, B.F., 1989. Complete intron/exon organization of DNA encoding the a’ chain of human C3. J. Biol. Chem. 264, 8471–8474. Barnum, S.R., Jones, J.L., 1995. Differential regulation of C3 gene expression in human astroglioma cells by interferon-␥ and interleukin-1␤. Neurosci. Lett. 197, 121–124. Baumann, H., Prowse, K.R., Marinkovic, S., Won, K.A., Jahreis, G.P., 1989. Stimulation of hepatic acute phase response by cytokines and glucocorticoids. Ann. N.Y. Acad. Sci. 557, 280–296. Brack-Werner, R., 1999. Astrocytes: HIV cellular reservoirs and important participants in neuropathogenesis. AIDS 13, 1–22. Cardinaux, J.R., Magistretti, P.J., 1996. Vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, and noradrenaline induce the transcription factors CCAAT/enhancer binding protein (C/BBP)-beta and C/EBP delta in mouse cortical astrocytes: involvement in cAMP-regulated glycogen metabolism. J. Neurosci. 16, 919–929. Cheingsong-Popov, R., Panagiotidi, C., Ali, M., Bowcock, S., Watkins, P., Aronstam, A., Wassef, M., Weber, J., 1990. Antibodies to HIV-1

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