enhancer binding protein transcription factor family in the simian immunodeficiency virus long terminal repeat

Biomedicine & Pharmacotherapy 57 (2003) 34–40 www.elsevier.com/locate/biopha

Dossier: AIDS

Identification of binding sites for members of the CCAAT/enhancer binding protein transcription factor family in the simian immunodeficiency virus long terminal repeat Michael R. Nonnemacher a, Tricia H. Hogan a, Shane Quiterio a, Brian Wigdahl a, Andrew Henderson b, Fred C. Krebs a,* a

Department of Microbiology and Immunology (H107), The Pennsylvania State University, College of Medicine, 500 University Drive, P.O. Box 850, Hershey, PA 17033, USA b Department of Veterinary Science, The Pennsylvania State University, University Park, PA 16802, USA Received 6 September 2002; accepted 1 October 2002

Abstract Members of the CCAAT/enhancer binding protein (C/EBP) transcription factor family are necessary for human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) activity and viral replication in cells of monocyte/macrophage lineage. The integral roles that HIV-1-infected monocytes and macrophages play in the development and progression of HIV-1-associated disease in the immune and central nervous systems underscore the importance of the C/EBP transcription factor family within the context of regulating HIV-1 gene expression. Although there are considerable similarities between HIV-1 and simian immunodeficiency virus (SIV), including viral-induced immunopathogenesis and neurologic dysfunction, infection of CD4+ T cells and cells of monocyte/macrophage origin, and LTR structure/function, the involvement of C/EBP factors in regulating SIV transcription has not been previously demonstrated. Analyses of the SIVmac239 LTR sequence indicated the presence of five putative C/EBP binding sites within the LTR. Electrophoretic mobility shift (EMS) analyses demonstrated that four of the five sites within the SIV LTR were able to bind C/EBP factors (a and b) and compete for DNA-protein complexes formed by the HIV-1 C/EBP site located adjacent to the promoter–distal NF-jB site. DNase I protection assays indicated that purified C/EBPb specifically was able to occupy each of the four binding sites. These studies suggest that C/EBP factors may also have important roles in the regulation of SIV gene expression and replication, and that these factors and signal transduction pathways that regulate their activity may impact SIV-associated pathogenesis. © 2003 E´ditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: SIV; LTR; C/EBP

1. Introduction Replication of human immunodeficiency virus type 1 (HIV-1), the etiologic agent of the acquired immune deficiency syndrome (AIDS), is regulated, in part, by the long terminal repeat (LTR), which serves as the promoter for the integrated proviral genome [1,13,14]. The LTR functions as a convergence point for transcription factors and elements of the transcriptional machinery from the host cell as well as virus-encoded proteins that enhance or modulate LTR activity and the subsequent expression of viral RNA and proteins. Numerous cis-acting sequences along the length of the LTR * Corresponding author. E-mail address: [email protected] (F.C. Krebs). © 2003 E´ditions scientifiques et médicales Elsevier SAS. All rights reserved. PII: S 0 7 5 3 - 3 3 2 2 ( 0 2 ) 0 0 3 3 4 - 7

facilitate interactions with transcription factors that modulate LTR activity in a manner specific to the host cell, or in response to cellular differentiation, activation, or progression through the cell cycle [18]. In cells of monocyte/macrophage lineage, the ability of the HIV-1 LTR to regulate viral gene expression is dependent on interactions with members of the CCAAT/enhancer binding protein (C/EBP) transcription factor family. Multiple C/EBP binding sites have been identified within the HIV-1 LTR, including several sites in the U3 region (Fig. 1) [25]. One site (site I) is positioned immediately 5’ of the promoter– distal NF-jB binding site and downstream of a binding site for members of the activating transcription factor/cyclic AMP response element binding (ATF/CREB) family [15].

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Fig. 1. Prominent regulatory landmarks in the HIV-1 and SIV LTRs. Locations of the binding sites for TATA binding protein (TBP), Sp factors, NF-jB, and ATF/CREB factors are illustrated relative to the structural divisions of the LTR (not to scale). The locations of the C/EBP sites in both LTRs are also depicted. The SIV LTR structure shown is that of SIVmac239.

A second site (site II) is located further upstream. Studies have shown that C/EBP factors are required for basal LTR activity and necessary for maximal LTR induction by phorbol myristate acetate (PMA) and lipopolysaccharide (LPS) in U-937 human monocytic cells [7]. Deletion of sites I and II resulted in severely diminished LTR-directed basal transcription and viral replication in a human monocytic cell line [6,7] and in primary macrophages [5]. However, replication in unstimulated CD4+ T cells was unaffected by C/EBP site deletions [5]. Recent studies have demonstrated that C/EBP factors that occupy site I interact with factors bound to the neighboring ATF/CREB site, and that naturally occurring site sequence variation at both sites impacted how these factors interact both physically and functionally [24]. The impact of C/EBP factors on HIV-1 LTR function has prompted studies concerning the links between C/EBPregulated HIV-1 gene expression and the development of HIV-1-associated disease. In the central nervous system (CNS), compartmentalization of viral genomes carrying LTRs with C/EBP site I and II sequence variants that alter LTR activity [23] may play a role in the initiation and progression of HIV-1-associated neuropathogenesis and HIV-1 dementia (HAD). The CCAAT/enhancer binding protein factors may also impact opportunistic infections of the lung, as suggested by studies that demonstrated modulation of HIV-1 LTR activity by C/EBP factors in HIV-1-infected macrophages co-infected with Mycobacterium tuberculosis[9]. In CD4+ T cells, the involvement of C/EBP factors in prostaglandin E2 (PGE2)-mediated activation of LTR activity [4] implies that HIV-1 replication in lymphocytes may be linked to modulation of immune responses by the inflammatory mediator PGE2.

Expression of the simian immunodeficiency virus (SIV) is similarly regulated by its 5’ LTR. Several studies have led to the identification of numerous cis-acting binding sites along the length of the SIV LTR and have demonstrated an overall organization similar to the HIV-1 LTR. For example, the LTR from the SIVmac239 strain includes four Sp binding sites and a single NF-jB binding site [21], as well as other cis-acting elements in the U3 region (Fig. 1). Additional LTR configurations have been identified in other SIV strains, including SIVmac142, which contains three Sp sites in the core promoter of the U3 region [22], and SIVsmmPBj1.9 which contains a second NF-jB site in the enhancer region that may contribute to the acutely pathogenic phenotype of this strain [3]. However, no studies to date have documented the regulation of SIV LTR activity by members of the C/EBP transcription factor family, despite the demonstrated replication of SIV in cells of the monocyte/macrophage lineage and the role of infected monocytic cells in SIV-associated pathogenesis in macaques [8]. Simian immunodeficiency virus infection of Asian macaques results in the appearance of immunodeficiency and CNS abnormalities similar to those associated with HIV-1 infection. These traits have made the SIV-infected macaque an attractive system for modeling the pathogenesis associated with HIV-1 infection of humans, for the development and testing of potential vaccines for HIV-1, and for evaluating pharmacotherapeutic approaches to treating HIV-1infected individuals [8]. The utility of an SIV animal model is strengthened by strong similarities between both viruses, including genomic organization, mechanisms of viral gene expression, and tropism for CD4+ T lymphocytes and macrophages. This report describes the identification of binding

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sites for members of the C/EBP transcription factor family in the SIV LTR, which is an additional similarity between HIV-1 and SIV. These sites may have roles in SIV replication similar to those previously associated with analogous binding sites within the HIV-1 LTR.

2. Materials and methods 2.1. Cell maintenance and preparation of nuclear extracts Cells of the U-937 human monocytic cell line (ATCC CRL-1593.2) were maintained in RPMI 1640 media supplemented with heat-inactivated 10% fetal bovine serum, L-glutamine (0.3 mg/ml), antibiotics (penicillin, streptomycin, and kanamycin, each at a concentration of 0.04 mg/ml), and sodium bicarbonate (0.05%). Nuclear extracts were prepared as described [2]. 2.2. Electrophoretic mobility shift (EMS) analyses Electrophoretic mobility shift analyses were conducted using previously described procedures [24]. In brief, complementary single-stranded DNA oligonucleotides were synthesized in the Penn State College of Medicine macromolecular core facility and annealed to form double-stranded probes. The HIV-1 C/EBP site I (3A) probe (100 ng) was radiolabeled with c32P-dATP (0.75 ng, 75 000 cpm/lane) and incubated with 1 µg of non-specific DNA (poly [d(I-C)]) and 20 µg of U-937 nuclear extract at 30 °C for 30 min. Competition studies were conducted using unlabeled competitor oligonucleotides at concentrations up to 100-fold molar excess over the probe concentration. Supershift analyses were initiated by incubation of the complexes for an additional 30 min at 30 °C in the presence of antibodies specific to C/EBPa and C/EBPb (Santa Cruz Biotechnologies). DNAprotein complexes were resolved on 5% non-denaturing polyacrylamide gels at 200 V at 4 °C for approximately 2.5 h. Gels were dried at 80 °C for 1 h and subjected to autoradiography. In the competition EMS analyses, DNA-protein complexes were quantitated by phosphoroimage analysis using the ImageQuant system (Molecular Dynamics). 2.3. C/EBPb synthesis and DNase I protection assays Polyhistidine-tagged C/EBPb was produced and purified as previously described [24]. Briefly, tagged C/EBPb was produced from the C/EBPb-BD-pRSET A expression vector in isopropyl- b-d-thiogalactosidase-induced E. coli [strain BL21(DE3)pLysS] and purified using a nickel-chelating column. DNase I protection analyses were conducted using radiolabeled probes PCR-amplified from the SIVmac239 LTR [12,21]. The probes for US2 (upstream site 2) and US1 (upstream site 1) were synthesized using primer pairs specific to those regions of the LTR (US2, nucleotides (nt) –376 to -352 and –209 to -183 with respect to the U3/R border; US1, nt –131 to -101 and –17 to +14). The probe encompass-

ing sites DS1 (downstream site 1) and DS2 (downstream site 2) was synthesized using an additional primer pair (nt +103 to +133 and +294 to +311). Each gel-purified, 5’radiolabeled probe (2000 cpm) was incubated for 10 min at 4 °C with purified C/EBPb (0, 1, 5, 20, or 50 µl at an approximate concentration 0.35 µg/µl) in the presence of 0.5 µg poly [d(I-C)] and 1.5 µg bovine serum albumin in a total volume of 50 µl. After addition of calcium and magnesium (CaCl2 and MgCl2 at 2 mM each; 1 min), DNase I (3 min), and phenol-chloroform to stop the reactions, the digested DNA was extracted and resolved by electrophoresis on a 6% polyacrylamide denaturing gel. Digestion patterns were visualized by autoradiography and localized using sequencing reactions run in parallel [16]. 3. Results 3.1. Sequence analysis of the SIVmac239 LTR identifies five putative C/EBP sites To identify C/EBP binding sites within the SIV LTR, the LTR sequence from the SIVmac239 strain [12,21] was analyzed using MatInspector v2.2 [19]. Sequence analyses (0.75 core similarity threshold, 0.85 matrix similarity threshold) indicated the presence of five putative binding sites for factors of the C/EBP transcription family (Fig. 1). Three sites were located in the U3 region of the LTR (US2, nt –385 to –372; US1, nt –100 to –87; US0, nt –43 to –30; locations with respect to the U3/R border). Two additional sites were located in the R and U5 regions (DS1, nt +133 to +146; DS2; nt +267 to +281). The location of US1 was particularly notable based on its apparent overlap with the promoter–distal Sp binding site and the NF-jB binding site (Fig. 1). To verify that these sites were not limited to the SIVmac239 LTR, 115 SIV LTRs compiled from the Genbank and Los Alamos HIV sequence databases were analyzed for the presence of these sites. Long terminal repeats were aligned to the SIVmac239 LTR using the MegAlignTM program under the clustal method. Of the 115 LTRs examined, 64 aligned to the SIVmac239 LTR. Within this LTR subset, variants of sites US1, US2, DS1, and DS2 were found in 23, 33, 35, and 10 LTRs, respectively (only 23 of the 64 LTRs examined contained downstream sequence information that extended through the location of DS2). Variant C/EBP binding sites were characterized by a single base pair (bp) substitution or an insertion of a single nucleotide within the binding site sequence. The effects of these changes on C/EBP binding and function will be explored in future studies. 3.2. C/EBP factors bind to four of the five putative C/EBP sites Simian immunodeficiency virus LTR sequences identified as putative C/EBP binding sites were incorporated into double stranded DNA oligonucleotide probes for EMS analyses (Fig. 2A). Electrophoretic mobility shift binding

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Fig. 2. The CCAAT/enhancer binding protein factors bind to SIV LTR oligonucleotide probes. (A) Sequences of the putative C/EBP binding sites in the SIV LTR. (B) The CCAAT/enhancer binding protein site I (3A variant) in the HIV-1 LTR. Double-stranded DNA oligonucleotide probes for each C/EBP binding site were synthesized using the sequences shown. Sequences identified by MatInspector as putative SIV C/EBP sites are highlighted in gray. (C) Supershift EMS analyses using the US2, US1, DS1, and DS2 oligonucleotide probes. Electrophoretic mobility shift analyses were conducted as described in Materials and methods using U-937 nuclear extract and antibodies specific to C/EBPa and C/EBPb. Open triangles indicate positions of the DNA-protein complexes specific to each probe; closed triangles indicate positions of the DNA-protein complexes after antibody supershifting.

studies using U-937 nuclear extract demonstrated that four of the five sites were able to form DNA–protein complexes of similar mobility (Fig. 2C). In contrast, no DNA–protein complexes were formed by the combination of U-937 nuclear extract and the US0 oligonucleotide probe (data not shown). In supershift EMS analyses, complexes formed by the US2 and US1 probes were abrogated by C/EBPa antibodies and supershifted by antibodies specific to C/EBPb (Fig. 2C). Complexes formed by the DS1 and DS2 probes were supershifted by both antibodies (Fig. 2C). These results specifically indicate the presence of C/EBPa and C/EBPb in DNA–protein complexes formed using probes containing sites US2, US1, DS1, and DS2. In competition studies, HIV-1 LTR C/EBP site I variant 3A (Fig. 2B), which has a C-to-A substitution at position 3 of the clade B consensus C/EBP site I, was used as a radiolabeled probe against which the putative SIV sites would compete for C/EBP factors. The CCAAT/enhancer binding protein site I (3A), which was identified in LTRs derived from peripheral blood samples from HIV-1-infected patients [17], has been shown to specifically bind members of the C/EBP transcription factor family (Beyer and Wigdahl, unpublished observations), including C/EBPa and C/EBPb,

with a specificity and relative affinity for C/EBP factors comparable to the 6G site I variant [24] found in the LAI LTR. Increasing amounts of unlabeled SIV competitor oligonucleotides were used to compete for DNA-protein complexes formed by the HIV-1 site I (3A) probe. The site I (3A) probe was also subjected to homologous competition for comparative purposes. Competition EMS analyses indicated that four of the five oligonucleotide probes containing SIV C/EBP binding sites were able to compete for DNA–protein complexes formed using the HIV-1 site I (3A) probe (Fig. 3). Upstream sites 1 and 2 probes effectively competed for complex formation at competitor concentrations as low as 12.5-fold molar excess (Fig. 3A). Similar results were obtained using the DS2 and DS1 probes (Fig. 3B). Competition by each of the four probes was comparable to homologous competition using the HIV-1 site I (3A) probe (Fig. 3A, B). In contrast, the US0 probe (Fig. 2A) containing the fifth putative C/EBP site was unable to compete for the formation of complexes formed by the site I (3A) probe (data not shown). Quantitative phosphoroimage analyses of the competitive EMS assays also demonstrated that the SIV C/EBP sites were effective competitors with respect to formation of

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Fig. 3. Simian immunodeficiency virus LTR C/EBP sites compete for C/EBP complex formation using an HIV-1 C/EBP binding site. Electrophoretic mobility shift analyses were conducted using the radiolabeled HIV-1 C/EBP site I (3A) oligonucleotide probe and U-937 nuclear extract. Probe and extract were incubated in the absence of competitor or in the presence of 12.5-, 25-, 50-, or 100-fold molar excess of unlabeled competitor oligonucleotides. The CCAAT/enhancer binding protein complexes on each gel (indicated by the arrows) were quantitated by fluorometry. Competition for complex formation is expressed as a percentage of the complex formed in the absence of competition. (A) Electrophoretic mobility shift analyses demonstrating competition by SIV US1, SIV US2, and HIV-1 site I (3A). (B) Electrophoretic mobility shift analyses demonstrating competition by SIV DS2, SIV DS1, and HIV-1 site I (3A). (C and D) Quantitation of C/EBP complexes in gels shown in panels A and B, respectively.

DNA–protein complexes containing C/EBP factors. Competition by the US1 and US2 probes (Fig. 3C), which resulted in a 50% reduction in complex formation at 11- and 17-fold molar excess concentrations, respectively, was comparable to the level of competition achieved by the HIV-1 site I (3A) probe (50% reduction at 11-fold molar excess). Similar results (Fig. 3D) were obtained using probes specific to sites DS2 (50% reduction at 10-fold molar excess) and DS1 (50% reduction at 12-fold molar excess), which were also as effective as the site I (3A) oligonucleotide competitor (50% reduction at 11-fold molar excess). 3.3. DNA-protein interactions between C/EBPb and the SIV LTR are detected by DNase I protection Specific occupancy of each SIV C/EBP binding site was also demonstrated using DNase I protection (footprint) analyses. Radiolabeled probes derived by PCR from the SIVmac239 LTR were used in conjunction with purified, recombinant C/EBPb to demonstrate protection of each of the four putative SIV LTR C/EBP sites shown to be effective competitors in the EMS studies. DNase I-digested probes were incubated in the absence or presence of increasing amounts of purified C/EBPb. These analyses indicated that sites US2 and US1 were specifically protected from digestion

by the presence of C/EBPb (Fig. 4). Similarly, SIV LTR sites DS1 and DS2 were also protected by C/EBPb (Fig. 4). 4. Discussion Sequence analyses identified five potential binding sites for C/EBP factors within the SIVmac239 LTR. Electrophoretic mobility shift analyses, using antibodies specific to members of the C/EBP transcription factor family, demonstrated the recruitment of C/EBPa and C/EBPb to four of the five sites. Four of the five sites were able to effectively compete for DNA–protein complexes formed using a relatively high affinity HIV-1 C/EBP site and were specifically protected by recombinant C/EBPb in DNase I footprint analyses. However, studies using oligonucleotides that included both the US1 site and the overlapping Sp binding site indicated that C/EBP and Sp factors may bind competitively to this region of the LTR (data not shown). The overlap between US1 and the upstream NF-jB binding site also suggests that occupancy of these two sites may be competitive. Future studies will address the functional impact of interactions between factors bound to US1 and the overlapping Sp and NF-jB binding sites. The identification of C/EBP binding sites in the R and U5 regions of the SIV LTR also prompted us to reexamine the

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Fig. 4. DNase I protection analyses indicate occupancy of the SIV C/EBP binding sites by C/EBPb. DNase I protection assays were conducted using purified C/EBPb and probes derived from the SIVmac239 LTR (as described in Materials and methods). Each bar indicates the extent of protection; the open portion of the bar indicates the position of the binding site sequence.

HIV-1 LTR for analogous sites. In studies performed by Tesmer et al. [25], DNase I protection experiments were limited to LTR sequences in the U3 and R regions. Analyses of the complete HIV-1 LAI LTR (accession # X01762) [20,26] and downstream sequences (87 nt beyond the end of the LTR) reaffirmed the positions of the C/EBP sites in the U3 region. In addition, they indicated the presence of additional, putative C/EBP sites in the U5 region and sequences in the adjacent untranslated region of the provirus (Fig. 1). Previous studies [5,6], which demonstrated that mutation of C/EBP sites I and II was sufficient to severely curtail HIV-1 replication in U-937 monocytic cells and primary human macrophages, suggest that the downstream sites, unlike sites I and II, provide no redundant mechanisms for C/EBP activation of LTR activity under the conditions examined. However, these studies do not preclude the existence of conditions, possibly related to differentiation or immune responses in cells of monocyte/macrophage or T cell origin, which may modulate LTR activity through these sites. Studies are now underway to document the functional contributions of the SIV LTR C/EBP sites to viral gene expression and replication. In previous SIV structure and function studies performed by Ilyinskii and colleagues [10,11], mutations were placed in the NF-jB and Sp sites of the SIVmac239 LTR to examine their importance in SIV replication and pathogenesis. The results of these studies indicated that, despite extensive changes in this region of the promoter–enhancer, mutant viruses replicated in rhesus monkey peripheral blood mononuclear cells (PBMCs) with kinetics similar to or only slightly delayed compared to parental SIVmac239 [10]. However, in primary rhesus macrophages, viral replication was clearly impaired to varying

degrees by the presence of these mutations, suggesting a greater dependence by SIV on these LTR elements in cells of monocyte/macrophage lineage [10]. While viruses containing a subset of these mutations were all able to cause AIDS in rhesus monkeys, the only virus associated with diminished replication in vivo was the mutant virus containing deletions of the NF-jB site, all of the Sp sites, and the US1 site [11]. Deletion or substitution mutations of the NF-jB and promoter–distal Sp binding sites used in these studies would have also resulted in the elimination or alteration of the US1 C/EBP site. Although the overlap of the NF-jB and Sp sites complicates analyses of the US1 contribution to viral replication, these reports suggest that elimination of the US1 site does not affect replication in rhesus PBMCs [10]. This observation is consistent with the finding that C/EBP sites I and II (Fig. 1) are not necessary for HIV-1 LTR activity or viral replication in CD4+ T cells [5]. Furthermore, the delayed replication kinetics produced in primary rhesus macrophages by mutations in this region of the LTR indicate that the non-overlapping Sp sites have greater importance in these cells than the sites that overlap US1 [10]. These results are also in agreement with previous studies of the HIV-1 LTR which demonstrated that (i) elimination of both sites I and II resulted in a severe reduction in HIV-1 replication in monocytic cells [6] and in primary macrophages [5], and (ii) sites I and II are functionally similar under the conditions examined, since elimination of only one site does not impact basal or LPS/PMA-induced LTR activity [7]. Presuming that sites US1 and US2 are functionally similar, mutation of US1 (as a consequence of the NF-jB/Sp site mutations) would still permit C/EBP induction of LTR activity and viral replication

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through the intact US2 site in cells of monocyte/macrophage lineage. The presence of C/EBP binding sites in the SIV LTR suggests that, like HIV-1, SIV may be regulated by signal transduction pathways and transcription factors that are closely tied to processes integral to cells of monocyte/ macrophage origin. Studies are now underway to determine the impact of these sites on viral gene expression and replication, and to examine the effect that sequence variation at these sites has on their function. These questions will be addressed using numerous in vitro approaches to study both LTR function and virus replication. Subsequent in vivo experiments will utilize SIV-infected primates to examine potential links between C/EBP function and pathogenesis, particularly the development and progression of neuropathogenesis associated with CNS infection. Results from these animal model experiments should provide further insights into HIV-1 infection of cells of monocyte/macrophage lineage and their roles in HIV-1-associated immune system and CNS disease.

Acknowledgements

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[16]

These studies were supported by United States Public Health Service grants NS32092 (BW) and AI46261 (AH). Training support (TH) was provided in part by United States Public Health Services grant CA60395.

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