Divergent Transcriptional Regulation among ... - Journal of Virology

JOURNAL OF VIROLOGY, Nov. 1997, p. 8657–8665 0022-538X/97/$04.0010 Copyright © 1997, American Society for Microbiology

Vol. 71, No. 11

Divergent Transcriptional Regulation among Expanding Human Immunodeficiency Virus Type 1 Subtypes MONTY A. MONTANO,1 VLADIMIR A. NOVITSKY,1 JASON T. BLACKARD,1 NANCY L. CHO,1 DAVID A. KATZENSTEIN,2 AND MAX ESSEX1* Harvard AIDS Institute, Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts 02115,1 and Division of Infectious Diseases, Department of Medicine, Stanford University School of Medicine, Stanford, California 943052 Received 1 May 1997/Accepted 6 August 1997

The current AIDS pandemic represents the uneven spread of multiple genetically related subtypes (A to J) of human immunodeficiency virus type 1 (HIV-1). Notably, HIV-1 E in southeast Asia and HIV-1 C in subSaharan Africa are expanding faster and are likely of greater global significance than the HIV-1 B subtype prevalent in the United States and Europe. While many studies have focused on genetic variation among structural genes, we chose to conduct a comparative analysis of the long terminal repeats of HIV-1 E and HIV-1 C isolates and report subtype-specific differences in enhancer copy numbers and sequences, as well as divergent activation in response to the cellular transcriptional activators Rel-p65 and NFATc and viral Tat. This study is the first to identify functional distinctions in promoter architecture between HIV-1 subtypes and raises the possibility that regulatory divergence among the subtypes of HIV-1 has occurred. Divergent transcriptional regulation may explain some of the epidemiologically observed differences in transmission and pathogenesis and underscores the need for further comparative analysis of HIV-1 regulation. HIV-1 B (8, 17, 21, 31, 33, 35, 37), but have not yet been addressed among distinct subtypes of HIV-1. Transcription studies have previously identified the important immunoregulatory determinants NF-kB p50:p65 and NF-AT and their cognate cis-acting enhancer sequences within the HIV-1 LTR (8). Importantly, however, the identification of these cis-acting sequences, and that of a variety of others, has been based on the architecture of the first described viral HIV-1 B sequence isolated from T cells (23, 33, 35, 38). Subsequent studies have revealed that regulators such as NF-kB p50:p65 and NF-AT represent members of large gene families with complex functions in a broad range of cell types (17, 24). Given that HIV-1 has an unusual propensity for genetic diversity and complex expression in a variety of cell types, there emerges a rich potential for alternative regulatory modes to arise, if advantageous (10, 21, 31). Analysis of the current worldwide distribution of HIV-1 subtypes concluded that HIV-1 E and HIV-1 C are the most prevalent HIVs in the world (3). HIV-1 B although prevalent in the West and present in Africa and Asia accounts for only a small number of infections in the latter sites. For this reason, we decided to compare potential LTR and/or regulatory differences between subtypes E and C by characterizing LTRs from infected individuals representing distinct areas experiencing expanding epidemics, namely, southeast Asia (Thailand, HIV-1 E) and sub-Saharan Africa (Zimbabwe, HIV-1 C). Therefore, LTR-specific primers were designed based on identified consensus regions across A, B, D, and O subtypes in the Los Alamos database (22) (no E or C LTR sequences were available) and were used to generate primary PCR products from proviral DNA that were then sequenced directly or cloned and sequenced by using an ABI automated DNA sequencer and dye terminator chemistry.

Although we know that human immunodeficiency virus type 2 (HIV-2) is different from HIV-1 in both virulence and transmission efficiency (12, 19), the extent to which such differences occur between subtypes of HIV-1 remains unclear. Epidemiologic analysis of the spread of these viruses indicates that while HIV-2 is largely restricted to west Africa, HIV-1 has a global and expanding distribution (3). Several subtypes of HIV-1 (A to J) have been described based on phylogenetic analysis of gag and env gene sequences (16). The HIV-1 subtypes have spread unevenly, such that HIV-1 B is predominantly associated with the epidemics linked to injection drug users and homosexual contact in the West while subtypes such as HIV-1 E and HIV-1 C have spread by heterosexual contact in Thailand, India, and sub-Saharan Africa (3, 15, 39). A possible explanation for this may be that Langerhans’ cells, a likely target in heterosexual transmission, provide for more efficient replication of HIV-1 E than HIV-1 B (36). Recent studies of HIV-1 B isolates indicate that selection among genetic variants of HIV-1 env can occur during sexual transmission (34, 42) and that such variation may underlie the differential utilization of chemokine receptors by T-cell tropic (6) and monocyte/macrophage tropic (1) strains of HIV-1 within and, perhaps, between subtypes. However, we reasoned that successful HIV replication also includes postentry events, such as viral transcription, replication, and viral turnover, such that extant genetic sequences in the regulatory region of HIV-1, the long terminal repeat (LTR), may also impact the ability of different HIVs to replicate efficiently through differential recruitment of cellular transcription factors. Differential replication efficiency and activation have been partially addressed for HIV-2 compared to HIV-1 B, and have been examined in longitudinal and cross-sectional studies within

MATERIALS AND METHODS

* Corresponding author. Mailing address: Harvard AIDS Institute, Harvard School of Public Health, 651 Huntington Ave., Boston, MA 02115.

DNA sequencing and analysis. For PCR analysis, DNA was extracted with a Qiagen Blood Kit (Chatsworth, Calif.) from either uncultured or minimally (less

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than three passages) cocultured peripheral blood mononuclear cells from donors. PCRs were carried out in a total volume of 100 ml, containing 100 ng to 1 mg of genomic DNA, by using GeneAmp Kits (Perkin-Elmer Cetus, Foster City, Calif.). Samples were overlaid with 50 ml of mineral oil and then subjected to 30 to 39 amplification cycles consisting of a denaturing step (94°C, 30 s), a primerannealing step (50°C, 30 s), and a primer extension step (72°C, 45 s). Oligonucleotide primers were designed based on an analysis of conserved regions within the LTR across the subtypes represented in the Los Alamos National Laboratory HIV Database (A, B, D, and O) (22). The upstream primer for the LTR was PH59KPN. Downstream primers for the LTR were PH39LTR and PH56. PCR products were sequenced directly or were isolated from agarose gels by using Spin-X columns (Costar, Cambridge, Mass.) and were cloned with the PCR2.1 TA cloning system (Invitrogen, San Diego, Calif.). Sequencing was conducted with fluorescent dye terminators and a 373A Applied Biosystems automated sequencer. Sequences were aligned by using the neighbor-joining algorithm with Clustal W(1.6) (9), gap-stripped with corrections for multiple substitutions, and bootstrap analyzed with 1,000 bootstrap resamplings. Phylograms were generated with NJ-plot (9). Similar analyses and results were obtained using PHYLIP and PAUP (data not shown). A computer algorithm, MatInspector, was used in some cases to identify degenerate enhancer and promoter elements (28). The sequences for the DNA oligonucleotides were as follows: PH59KPN, 59-CTC AGG TAC CTT TAA GAC C-39; PH39LTR, 59-CTG AGG GAT CTC TAG TTA CCA GAG-39; PH56, 59-ATTGAGGCTTAAGCAGTGGG-39; TFIID consensus TATA box (Santa Cruz Biotechnology, Santa Cruz, Calif.), 59-GCAG AGCATATAAAATGAGGTAGGA-39; E-TATA1, 59-CAG ATG CTG CAT AAA AGC AGC CGC T-39; E-TATA2, 59-AGC GGC TGC TTT TAT GCA GCA TCT G-39; B-TATA1, 59-CAG ATC CTG CAT ATA AGC AGC CGC T-39; B-TATA2, 59-AGC GGC TGC TTA TAT GCA GGA TCT G-39; ETATAUp1, 59-CCA GAG TAC TAT AAA GAC TGC TGA C-39; E-TATA Up2, 59-GTC AGC AGT CTT TAT AGT ACT CTG G-39; B-TATAUp1, 59-CCG GAG TAC TTC AAG AAC TGC TGA C-39; B-TATAUp2, 59-GTC AGC AGT TCT TGA AGT ACT CCG G-39; KB1, 59-TAC AAG GGA CTT TCC GCT GG-39; KB2, 59-CCA GCG GAA AGT CCC TTG TA-39; E-KB1, 59-CTA ACT AGG ACT TCC GCT GG-39; E-KB2, 59-CCA GCG GAA GTC CTA GTT AG-39; C-KB1, 59-CAA CTG GGG CGT TCC AGG AG-39; and C-KB2, 59-CTC CTG GAA CGC CCC AGT GG-39. The sequences for the RNA oligonucleotides were as follows: Tar-B, GAC CAG AUC UGA GCC UGG GAG CUC UCU GGC-39; Tar-E, GAC CAG GUC GAG CCC GGG AGG UCC CUG GC-39; and Tar-del, GAC CAG AUC UGA GCC CGG GCU CUG GC-39. Electrophoretic mobility shift assays. DNA binding assays using purified protein or nuclear extracts from unstimulated Jurkat T cells (used in TATA box oligonucleotide competitions) or phorbol-myristate-acetate (PMA)-stimulated Jurkat T cells (used in NF-kB oligonucleotide competitions) were carried out by standard methods and as previously described (20). Briefly, 5 mg of nuclear extract or 100 ng of purified Tat protein was incubated with radiolabeled oligonucleotide (or preincubated for 15 min with cold competitor oligonucleotides) followed by an additional 15-min incubation. DNA-protein or RNA-protein complexes were resolved by native polyacrylamide (6%) gel electrophoresis and exposed from 2 h to overnight by using Bio-MAX AR film. Reporter gene construction and transient-transfection assays. PCR products were digested with KpnI and HindIII and directly cloned into pbgal-Basic (Clontech, Palo Alto, Calif.). The pHIV-lacZ plasmids contain the KpnI-HindIII fragment of the HIV-1 LTR (2450 to 160) from representative clones of HIV-1 subtypes B, C, and E. Plasmid transfection of target cells was conducted by either calcium phosphate transfection (293 cells) or electroporation (Jurkat T cells). Target plasmids were transfected with 100 ng of genomic DNA/105 293 cells or 10 mg of genomic DNA/107 Jurkat T cells. Expression vectors Rel-p65, Rel-p52, and NFATc were transfected with 1 mg in 293 cells or 10 mg in Jurkat cells. Tat was transfected with 100 ng in 293 cells or 10 mg in Jurkat cells. NFATc stimulation of Jurkat T cells required PMA (10 ng/ml) and Ionomycin (10 nM). All transfections were done in duplicate at least three times, and average values (6 standard errors of the mean) from representative transfections are shown. Cells were assayed for b-galactosidase activity by using the methylumbelliferylb-glucuronide assay (20). Fluorescence was determined with a Fluoroskan plate reader (Flow Laboratories, Helsinki, Finland). Heteroduplex mobility assays. PCR products (approximately 550 bp) from proviral DNA, or relevant clones, were used in a modified heteroduplex assay (5, 25) as follows. Briefly, 5 ml of second-round PCR products (;100 to 250 ng of DNA) of target DNA was mixed with 5 ml of subtype reference (or H2O) and 0.5 ml of 103 heteroduplex annealing buffer for a final concentration of 50 mM NaCl-5 mM Tris (pH 7.8)-1 mM EDTA in MicroAmp reaction tubes (PerkinElmer). Heteroduplexes were formed by heating the mixture to 94°C for 2 min and cooling to 4°C for 10 min by using an MJR cycling machine. A total of 0.3 ml from each mixture was applied directly onto an upturned sample applicator which was placed in the PhastSystem (Pharmacia LKB Biotechnology AB, Uppsala, Sweden) and electrophoresis was carried out. Separation of homoduplexes and heteroduplexes was carried out by using PhastGel DNA buffer strips with the following conditions: loading, 100 V; 7 mA; and 1 W at 15°C for 5 V z h; and running, 300 V; 10 mA; and 2 W at 15°C for a total of 150 to 200 V z h. Staining was conducted with the PhastSystem DNA Silver Kit.

J. VIROL. Nucleotide sequence accession numbers. Nucleotide sequences were submitted to GenBank (accession no. AF023426 to AF023441).

RESULTS Phylogenetic and heteroduplex analyses indicate subtype divergence within the LTR. Phylogenetic analysis of proviral DNA obtained from individuals with HIV-1 infections in the United States (subtype B), Thailand (subtype E), and Zimbabwe (subtype C) was conducted (Fig. 1a). This analysis indicated that LTR sequences clustered by subtype (note bootstrap values) and were congruent with subtype classifications based on the env or tat genes (data not shown). The LTR has therefore diverged between these subtypes and may be useful as an additional marker for subtype assignment and for the detection of intergenotypic recombinants. Intrasubtype pairwise distance comparisons were performed and indicated that, consistent with a recent expansion, subtype E displayed a relatively modest distribution (range of 3 to 6%) relative to subtype C isolates, which were broadly distributed (range of 5 to 12%). The B-subtype isolates displayed an intermediate distribution. To investigate LTR quasispecies complexity, heteroduplex mobility assays were conducted. The primary PCR products obtained for direct sequencing (quasispecies) were tested against a panel of clones subsequently established for each subtype. Quasispecies obtained from individuals infected with the same subtype of virus displayed heteroduplex bands with faster mobility, while concomitantly displaying slower-mobility heteroduplex band patterns with different subtypes (Fig. 1b). Consistent with Fig. 1a, heteroduplex analysis confirmed LTR variation between subtypes and supports the potential for functional divergence. HIV-1 subtypes E and C contain divergent promoter architectures. Subtype-related variation within and between HIV-1 B, E, and C was observed in many regulatory sequences, notably NF-kB and NF-AT, but also in USF, TCF-1a, the TATA box, and the TAR region (Fig. 2a and b). The NF-kB and NF-AT sites have been correlated with activation of HIV-1 by cytokines and mitogens and are likely to play a critical role in the recruitment of Rel-related transcription factors that augment HIV-1 expression (14, 23, 24, 33). The USF and TATA box influence basal HIV expression and assembly of the preinitiation complex, respectively (8). The HIV-1 B regulatory sites were highly conserved, with the single exception of a duplicated region upstream of the NF-kB site in one individual. This duplication has been previously observed (41). Direct sequence products from 44 independent sequence reactions (seven individuals) confirmed that the C-subtype isolates consistently contained a minimum of three NF-kB enhancer sites (four sites in one individual) (Fig. 3a). All E-subtype LTRs, representing direct sequence products from 38 independent sequence reactions (six individuals), contained one canonical NF-kB site and one variant NF-kB site. All B-subtype LTRs contained two invariant NF-kB sites in 34 independent sequence reactions (seven individuals). All HIV-1 E isolates also contained consistent changes in the TAR region, including a nucleotide deletion creating a 2-nucleotide bulge which may influence Tat activity (4, 7). Also observed within HIV-1 E subtypes, and potentially relevant to Tat function (2, 26) and NF-kB function (13, 40), were sequence changes in the highly conserved TATA box as well as the presence of an additional putative upstream TATA box. Interestingly, analysis of sequences surrounding the upstream TATA box (2140) indicated a homology with the canonical TATA (230)-initiator region of both HIV-1 E and B (Fig. 3c), as well as with the flanking regions. However, clari-

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FIG. 1. Phylogenetic and heteroduplex analyses indicating LTR divergence among HIV-1 subtypes based on LTR DNA sequences (500 nucleotides, entire U3 through TAR [2455 to 146, relative to transcription start]). (a) Unrooted tree based on neighbor-joining algorithm with 1,000 bootstrap resamplings. Included are the consensus sequences from subtype B primary isolates obtained from homosexual men at the Fenway Community Health Center in Boston, Mass. (B-2, B-19, B-3, B-32, B-B017, and B-56) and from subtype E primary isolates obtained from heterosexual men in northern Thailand (Mahidol University) (E-17, E-18, E-10, E-11, and E-15). The U.S. and Thai samples have been described in detail previously (36, 38). Subtype C primary isolates (C-A, C-B, C-D, C-E, C-G, and C-H) were obtained from heterosexual men in Zimbabwe without a history of travel outside of the region. Also included are the reference Los Alamos database subtype consensus sequences for the LTR (B, A, D, and O), two subtype A sequences from Senegal, and molecular clones cm240 (4) and cm2220 (32). (b) Heteroduplex panel. Selected quasispecies were obtained from individuals infected with either HIV-1 subtype B (isolates B017, 19, and 56), subtype E (isolates 10, 11, and 18), or subtype C (isolates D, G, and A). Subtype viruses were duplexed with heterologous clones from an LTR panel containing B-subtype viral clones (HXB2, B-2.1, and B-3.3), E-subtype viral clones (E-15.2, E-16.2, and E-17.1), and C-subtype viral clones (C-F.1, C-H.1, and C-G.3ii). These clones are identified at the top of the panel as B (1, 2, 3), E (1, 2, 3), and C (1, 2, 3), respectively. Quasispecies displayed relatively homoduplex band patterns (faster mobility) with clones from the same subtype (boxed in figure) while concomitantly displaying heteroduplex band patterns (slower mobility) from the alternate subtypes. Homoduplexes with self are indicated with an asterisk.

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FIG. 2. (a) Alignment of LTR sequences and comparative LTR regulatory landscape in subtypes B, E, and C. Enhancer and promoter elements reported to influence viral gene expression are boxed. Deviations from the B-subtype consensus are indicated. Sequence variations within NF-AT, USF, TCF1a, TATA, and TAR as well as copy number changes in the NF-kB enhancer are evident. Also included are the recently reported E clone, cm240 (4), and the recently reported C clone, c2220 (32). (b) Diagram summarizing sequence data shown in panel a. Solid circles indicate conserved mutations.

fication of the functional significance of this upstream TATA box awaits genetic analysis. In addition, analysis of the NF-AT sites revealed that 9 of 11 mutations were shared among subtype E and C isolates, relative to HIV-1 B (Fig. 3d). DNA and RNA binding assays reveal a divergent phenotype associated with enhancer and promoter elements within sub-

type E and C isolates. To functionally assess the in vitro binding activity of altered NF-kB, TATA, and TAR sequences, gel shift assays were performed with nuclear extracts from Jurkat T cells stimulated with PMA, a potent inducer of both Rel heterodimer NF-kB p50:p65 nuclear translocation and HIV-1 transcriptional activation. Oligonucleotides were synthesized

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FIG. 3. Sequence and biochemical analyses of divergent HIV-1 E and HIV-1 C LTR enhancer and promoter elements. (a) Computer identification of kB sequences within HIV-1 E and HIV-1 C isolates. MatInspector (28) was used with a consensus similarity cutoff score of 0.9 to identify NF-kB enhancer sites. All HIV-1 E isolates contained a single copy, while all HIV-1 C isolates contained a third site (duplicated in one individual). The sequence position, relative to the start of U3, is shown as are the core and matrix similarity (simil.) scores. (1), analysis of plus strand. (b) Gel shift analysis of variant kB, TATA, and TAR sequences. NF-kB enhancer binding activity is shown in lanes 1 to 3 and 4 to 7. The upstream kB site in the E subtype, TAGGACTTCC (lanes 2 and 3), failed to compete for binding to a radiolabeled consensus kB site (lane 1). The third kB site in all C subtype isolates, GGGGCGTTCC, (lanes 4 and 5) competed efficiently, and a polymorphic site resembling a kB motif, GGGGTTTTAC, failed to compete (lanes 6 and 7). NS, non-specific complexes (20). TATA box binding activity is shown in lanes 8 to 12. Both HIV-1 B (lane 9) and HIV-1 E (lane 10) TATA boxes (230) competed for binding to a radiolabeled consensus TATA oligonucleotide (lane 8). The putative upstream TATA box in subtype E (2140) also competed, with reduced affinity, for binding to the TATA box (lane 12), while the cognate upstream site in subtype B (lane 11) displayed no competitive binding (compare lanes 11 and 12 with lane 8). TAR binding activity is shown in lanes 13 to 16. HIV-1 B Tat binding activity to radiolabeled Tar-B (lane 13) was competed for by both Tar-B (lane 14) and the variant Tar-E (lane 15) but not by a Tar-del control (lane 16). Arrows indicate specific complexes. (c) Alignment of HIV-1 TATA sequences. The region surrounding the HIV-1 E putative upstream TATA box (2140) with binding activity for TBP (panel B) is aligned with the canonical TATA boxes (230) in HIV-1 B and HIV-1 E. (d) Alignment of NF-AT sequences. Deviations from the HIV-1 B consensus in the region overlapping the upstream NF-AT site (2298 to 2258) in C- and E-subtype isolates are indicated. Shared nucleotide changes (9 of 11) among HIV-1 E and HIV-1 C (in comparison with HIV-1 B) are marked with an asterisk.

based on the NF-kB consensus within each subtype. Competition assays with oligonucleotides derived from the altered upstream E-subtype kB site, TAGGACTTCC, indicated a dramatically reduced capacity to compete for NF-kB p50:p65 binding (Fig. 3b, lanes 2 and 3); this result is consistent with the existence of a single functional kB site within the HIV-1 E subtype. Alternatively, all isolates of HIV-1 C contained a third functional kB site, GGGGCGTTCC, in addition to two consensus kB sites, which efficiently competed with a consensus oligonucleotide for NF-kB p50:p65 binding (Fig. 3b, lanes 4 and 5). By contrast, an additional polymorphic site resembling a kB motif in one HIV-1 C isolate (C-G), GGGGTTTTAC, failed to compete (Fig. 3b, lanes 6 and 7). Within the core promoter, nucleotide changes were also observed in the highly conserved TATA box, which functions to position RNA synthesis through the recruitment of the preinitiation factor TATA binding protein (TBP), an integral part of the TFIID complex. The altered TATA motif CATAAAA (T3A) was observed in five of the six HIV-1 E isolates. Although unusual for HIV-1, this TATA polymorphism can be observed in many simian immunodeficiency virus isolates obtained from naturally infected African green monkeys (11) and resembles the loss of function in murine leukemia virus TATA previously observed in mutagenesis studies of a laboratory-adapted molecular clone of HIV-1 B (26). Nevertheless, this altered TATA sequence competed with an apparently equivalent efficiency for TBP-TFIID binding as did the wild-type TATA sequence from HIV-1 B (Fig. 3b, compare lanes 9 and 10 with lane 8). Computer analysis predicted the presence of an upstream additional TATA box (2140), ACTATAAA, within the HIV-1 LTR of all E-subtype isolates. In DNA binding assays with extracts from unstimulated Jurkat T cells, oligonucleotides derived from the putative additional TATA box in HIV-1 E (2140) competed for TBP-TFIID binding over a corresponding region in HIV-1 B (Fig. 3b, compare lane 12 with 11), albeit with a reduced efficiency compared to the primary TATA box (230) of subtypes E and B (Fig. 3b, compare lane 12 with lanes 9 and 10). The observed binding efficiencies of the respective (230) and (2140) TATA sequences perhaps indicate a role for flanking sequences surrounding the TATA box in influencing the overall binding activity of TBP-TFIID. In fact, mutagenized TATA and surrounding sequences have been correlated with altered HIV-1 activation by viral TAT (2, 26). Binding assays were conducted to test the potential altered capacity of Tat protein to recognize HIV-1 E TAR (Tar-E) compared to the reference HIV-1 B TAR (Tar-B). RNA was synthesized, 118 to 144, corresponding to Tar-B, Tar-E, or a control deletion construct (Tar-del). Competition assays indicated that both Tar-B (Fig. 3b, lane 14) and Tar-E (lane 15) were efficient at competing for binding to HIV-1 B Tat, whereas the control Tar-del (lane 16) did not effectively com-

pete. Therefore, no obvious defect in HIV-1 B Tat binding was observed. Determination of whether homologous Tat from HIV-1 E displays a similar phenotype or, perhaps, has undergone compensatory changes altering Tat binding activity to homologous TAR sequences awaits cloning and functional characterization (research in preparation). Differential transcriptional activation of HIV-1 E and HIV-1 C in response to Rel-p65, NFATc, and viral Tat. We speculated that NF-kB, NF-AT, and TAR might have altered functions in HIV-1 E and HIV-1 C and chose to assess potential functional changes directly by cotransfection assays. Proviral amplicons of the LTR from each subtype were cloned into the KpnI-HindIII site of pBgal-Basic, allowing for lacZ reporter gene measurements in cotransfection assays with 293 cells or in Jurkat T cells with expression vectors for the NF-kB binding factors Rel-p65 and Rel-p52, as well as NFATc, and HIV-1 B Tat. Transfections with an expression vector for Rel-p65 indicated a correlated activity of NF-kB enhancer copy number with Rel-p65 transcriptional induction such that the HIV-1 E subtype weakly transactivated in comparison with HIV-1 C subtype LTRs, which contained three or four kB sites and were activated more efficiently than either HIV-1 E or HIV-1 B (Fig. 4a and d). Though each subtype consistently differed in response to Rel-p65, we did not observe a similar trend in response to Rel-p52 (Fig. 4a). This may indicate different criteria for p52 function or may represent compensation by other low-affinity p52 sites which are not efficiently utilized by p65 (18, 20). Transfections with an expression vector for NFATc indicated a consistently reduced activity (Fig. 4b and d) by both the C- and E-subtype LTRs compared to HIV-1 B. The activation by NFATc in 293 cells did not require exogenous stimuli, whereas NFATc activation in Jurkat T cells required the use of both PMA and Ionomycin (17, 33). Since PMA influences both NFATc and NF-kB activation, a suboptimal concentration of PMA (10 ng/ml), which had no detectable effect on LTR activation, was chosen to minimize the contribution of PMAinduced NF-kB activity. At higher concentrations of PMA (100 ng/ml) HIV-1 LTR activation was correlated with kB copy number in Jurkat T cells (data not shown). NF-AT binding includes a large family of regulatory proteins that have different cell type distributions (17). Therefore, differential recognition among members of the NF-AT family that partition HIV-1 E and C from HIV-1 B may have occurred, and this possibility awaits further study. However, the reduced NFATc activity may not be due to lack of binding activity, since cotransfections with a Tat expression vector resulted in NFATcspecific transcriptional interference (data not shown). Additionally, since NFATc activity is associated with AP-1 (24), the confounding role of these factors in NF-AT activity remains to be determined. Recently, NFATc activation of an HIV-1 B molecular clone was reported which indicated a critical role for the core kB enhancer (14). Since the core enhancer, as well as

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an HIV-1 B control in 293 cells (Fig. 4b), whereas HIV-1 E activation was clearly reduced in Jurkat T cells (Fig. 4d) with a molecular expression clone of HIV-1 B Tat. The reduced activity of HIV-1 E in Jurkat T cells is consistent with previous reports showing that mutagenized TATA motifs influence TAT function (2, 26). By contrast, Tat activation of HIV-1 C was reproducibly lower than that of either HIV-1 B or HIV-1 E in 293 cells (Fig. 4b) but was comparable to that of the HIV-1 B control in Jurkat T cells (Fig. 4d). The observed subtype differences in activation between 293 cells and Jurkat T cells may be due in part to the presence of E1a in 293 cells. Nevertheless, these results suggest that Tat regulation is sensitive to cell type and promoter context, and further clarification awaits a comparative analysis using representative clones of Tat from HIV-1 E and HIV-1 C subtypes (research in preparation). Cotransfection assays with Rel-p65 augmented Tat activation of HIV-1 B and HIV-1 C LTRs but not that of HIV-1 E (Fig. 4c), consistent with the reduced p65 activity in HIV-1E (Fig. 4a and d).

FIG. 4. Differential transactivation of HIV-1 E and C LTR clones by cotransfection of Rel-p65, Rel-p52, NFATc, and Tat in 293 cells (a, b, and c) and Jurkat T cells (d). (a) Transcriptional activity with Rel-p65 and Rel-p52. Upregulated expression of HIV-1 C-subtype LTRs was observed, but minimal induction was observed in the HIV-1 E-subtype LTRs relative to an HIV-1 B control, consistent with binding data shown in Fig. 3b. (b) NFATc activity was reduced in both HIV-1 E- and HIV-1 C-subtype LTRs relative to the HIV-1 B-subtype LTR, consistent with the sequence changes shown in Fig. 3d. Tat (HIV-1 B) activity was essentially at the wild-type level with HIV-1 E LTR targets; HIV-1 C LTR activation was consistently lower relative to an HIV-1 B LTR reference. (c) Rel-p65 augmented Tat activation of HIV-1 B and HIV-1 C LTRs, but HIV-1 E displayed minimal increased activity, consistent with results shown in panel a. (d) In Jurkat T cells, transcriptional activity with p65 was upregulated in HIV-1 C and reduced in HIV-1 E relative to an HIV-1 B control, consistent with results shown in panel a. NFATc activity in the presence of PMA and Ionomycin (1 P/I) was reduced in both HIV-1 E and HIV-1 C relative to an HIV-1 B control, consistent with results obtained with 293 cells shown in panel b. Tat activation of HIV-1 E was consistently reduced compared to that of HIV-1 B and HIV-1 C, in contrast with results obtained with 293 cells (see text). Target LTRlacZ plasmids E-cl.1, E-cl.2, E-cl.3, C-cl.1, C-cl.2, and C-cl.3 are representative consensus clones from HIV-1 E-subtype isolates E-17, E-15, and E-18 and HIV-1 C-subtype isolates C-G, C-H, and C-E, respectively. The reference clone used (B-ref) is a consensus clone from isolate B-19 and is similar to the laboratory-adapted molecular clone ARV-2 lacZ (20).

the NF-AT sites and others, have changed in distinct ways both in HIV-1 E and HIV-1 C, there are likely to be complex enhancer interactions which underlie HIV-1 transcriptional activity. The HIV-1 E TAR stem loop structure is predicted to contain a 2-nucleotide bulge and an energetically weakened hairpin (4, 7). We observed similar Tat activation of HIV-1 E and

DISCUSSION We have described distinct regulatory landscapes in subtypes HIV-1 E and HIV-1 C that differ with subtype HIV-1 B in both copy number and exact nucleotide sequence of key enhancer and promoter elements. Notably, functional changes implicating the activities of the NF-kB enhancer, NF-AT, USF, the TATA box, and the TAR region were observed. The observed natural variation in response to Rel-p65, NFATc, and TAT supports accumulating evidence from epidemiological and transmission studies that a model for HIV-1 gene expression based only on the HIV-1 B subtype may not encompass the range of regulatory expression and pathogenic potential of expanding subtypes such as HIV-1 E and C. Recent studies suggest that recombinant viruses may account for up to 10% of all HIV-1 genomes, including HIV-1 E which dominates in Asia (29, 30). While emphasis has been placed on identifying and characterizing such recombinants with respect to structural genes, regulatory elements could play an equally important role in the genetic divergence and pathogenic potential of HIV-1 subtypes. The E genotype has been recently described as being an intergenotypic recombinant between A and E, with most of the genome being derived from subtype A but with gp120, an external portion of gp41, and a segment of the LTR being derived from subtype E (4, 7, 29). Therefore, an intriguing hypothesis is that coselection for both structural and regulatory regions may have occurred. HIV-1 promoter variation is qualitatively different from sequence variation in structural genes in that there is no protein product and presumably no direct immune selection pressure. Therefore, dissociating the relative roles of drift and selection is complicated. Nevertheless, whether the observed LTR variation is due to founder effects within subtypes or regions or stochastic effects on virus replication or is the result of selection remains to be determined by further genetic and functional analysis. There is no compelling reason to suspect that regulatory gene expression will necessarily remain linked to subtype or be confined to a given geographic region. Selective pressures related to transmission or pathogenesis may result in the emergence of recombinant viruses with altered regulatory regions, in addition to the observed intersubtype recombination in env and gag genes. Taken together, these results underscore the need to develop an appropriate model for the control of HIV-1 gene expression that is relevant to those subtypes such as HIV-1 E and HIV-1 C that are spreading most rapidly (3). As signaling cascades are better delineated, particularly in cell types rele-

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ACKNOWLEDGMENTS This study was supported in part by grants CA 39805 and AI 07387 from the National Institutes of Health and by training grant 5 D43 TW0004 from the Fogarty International Center, National Institutes of Health. V. Novitsky was sponsored in part by the Educational Commission for Foreign Medical Graduates. We acknowledge discussions with P. Kanki, T. H. Lee, and G. P. Nolan and thank K. MacArthur for editorial assistance.

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