Reciprocal Regulation of the Nuclear Factor of Activated T Cells and HIV-1

REVIEW Reciprocal regulation of the nuclear factor of activated T cells and HIV-1

F Pessler1,2 and RQ Cron1,2 1Division of Rheumatology, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA; 2Department of Pediatrics, University of Pennsylvania School of Medicine, Philadelphia, PA, USA

The human immunodeficiency virus type 1 (HIV-1) has evolved to coordinate its replication with the activation state of the host CD4T cell. To this end, it taps into major host cell signaling pathways and their associated transcription factors. Of these, T-cell activation and the transcription factor NF-kB, respectively, have become the best-studied examples. The past several years have revealed compelling evidence that another transcription factor family involved in T-cell activation, the nuclear factor of activated T cells (NFAT), plays an important role in the regulation of HIV-1. Major advances have been made in our understanding of the interaction of HIV-1 with this intriguing transcription factor. The duplicated NF-kB binding sites in the HIV- 1 enhancer surprisingly also bind NFAT proteins and appear to be the most important targets for NFAT transactivation of the HIV-1 long terminal repeat. The crystal structure of NFAT1 bound to one of these duplicated sites was solved recently. Interestingly, it showed that NFAT1 binds to this site as a homodimer and occupies the core of the NF-kB site, suggesting mutually exclusive binding and alternate transactivation by these two factors. NFAT also regulates HIV-1 infection indirectly, as it can relieve a block to reverse transcription in quiescent T cells. In turn, HIV-1, and particularly its Tat and Nef gene products, can upregulate NFAT expression and activity. This reciprocal regulation between virus and transcription factor potentially creates a positive feedback loop, which may facilitate the establishment of early HIV-1 infection and, later, the transition from latent to productive infection. The immunosuppressive drug cyclosporin A (CsA) inhibits NFAT activity and thus represents a potential treatment for HIV-1 infection. Recent small-scale clinical trials have yielded optimistic results, suggesting roles for CsA after organ transplantation in HIV-1 þ individuals and as adjunct treatment in stable early HIV-1 infection. Genes and Immunity (2004) 5, 158–167. doi:10.1038/sj.gene.6364047 Published online 5 February 2004

Keywords: cyclosporin A; nuclear factor of activated T cells; HIV-1; T lymphocytes; transcription; AIDS

HIV-1—a transcriptional parasite activation, the nuclear factor of activated T cells (NFAT or NF-AT, the former abbreviation being used in Like other viruses, the primate immunodeficiency this review), plays an important role in the regulation viruses are intracellular molecular parasites that depend of HIV-1.3 on the host cell biosynthetic machinery to complete their NFAT participates in T-cell activation through signal- life cycle. Intricate mechanisms have evolved by which ing events distinct from NF-kB. Downstream from viruses tap into various steps of host cell nucleic acid and phospholipase C (PLC), NF-kB is activated via the protein synthesis to coordinate viral replication with diacylglycerol/Ca2 þ /protein kinase C (PKC) pathway, specific aspects of the host cell life cycle and to while NFAT regulation follows the IP3/Ca2 þ /calcineurin counteract or circumvent the host immune response. pathway.4 Thus, the interaction with NFAT creates a Indeed, the interaction between nuclear factor kappa-B potentially important additional means for HIV-1 to fine- (NF-kB) and the human immunodeficiency virus type 1 tune its life cycle with the activation state of the host cell. (HIV-1) enhancer has become a prime example of The observation that the well-studied immunosuppres- how a virus usurps a major cellular signaling pathway, sive drug cyclosporin A (CsA) can interfere with NFAT presumably to coordinate viral replication with a state activation of HIV-1 gene expression lends additional of high host cell biosynthetic activity1 (reviewed in clinical relevance to this interaction.3 Thus, the past 10 Hiscott et al2). More recently, several lines of evidence years have afforded major insights into the various have suggested that another factor involved in T-cell interactions between HIV-1 and this intriguing transcription factor. Among the most significant advances have been the identification of the long terminal repeat (LTR) Correspondence: RQ Cron, Division of Rheumatology, The Children’s enhancer sites as the primary target for transcriptional Hospital of Philadelphia, 1102B Abramson Pediatric Research Center, transactivation by NFAT, the findings that NFAT and 3615 Civic Center Boulevard, Philadelphia, PA 19104, USA. E-mail: [email protected] HIV-1 regulate each other in a reciprocal manner, and Received 07 October 2003; revised 03 November 2003; accepted 04 last, but not least, the solution of the crystal structure of November 2003 NFAT1 bound to the HIV-1 enhancer. NFAT and HIV-1 F Pessler and RQ Cron 159 NFAT—a versatile transcriptional regulator cytoplasmic immunophilins, the immunosuppressive drugs CsA and FK506 inhibit the phosphatase activity Since the discovery of a DNA-binding activity that of calcineurin and thus interfere with NFAT depho- correlated with induction of the IL-2 gene during T- sphorylation and nuclear translocation (Figure 1).16 lymphocyte activation,5 five NFAT proteins have been Nuclear localization of NFAT in T cells is maintained isolated.6 Members of the NFAT family play important by a persistent Ca2 þ signal provided by Ca2 þ release roles in cellular differentiation and development in activated channels (CRACs), in the absence of which diverse tissues such as the heart, blood vessels, skeletal NFAT is rephosphorylated by specific kinases and muscle, skin, adipose tissue, and the peripheral immune rapidly returns to the cytoplasm (Figure 1).17,18 Kinases system (for reviews see Hogan et al,6,Crabtree and that oppose calcineurin activity also help maintain NFAT Olson,7 and Horsley and Pavlath8). NFAT1 (also called proteins in their phosphorylated, inactive state in resting NFATp or NFATc2) and NFAT2 (also known as NFATc or cells.6 NFAT proteins recognize purine-rich binding sites NFATc1) are the most abundant family members in the whose primary and adjacent sequences determine the human peripheral immune system, and the best studied composition of NFAT-containing protein/DNA com- in HIV-1 regulation. In primary human T cells, NFAT1 plexes.19 For instance, the binding sites found in the IL- predominates,9–11 and NFAT2 levels are increased upon 2 promoter and GM-CSF enhancer consist of abutting T-cell activation.12 Interestingly, NFAT1 itself is involved NFAT and AP-1 sites and mediate cooperative binding of in the induction of NFAT2 via binding to the NFAT2 both factors to these sites (reviewed in Rao et al15,19). promoter.13 The major steps in NFAT regulation in T Similarly, adjacent NFAT and GATA binding sites are lymphocytes are represented diagrammatically in found in the murine IL-4 and IL-5 enhancers.19,20 The NF- Figure 1. A common property of NFAT proteins, except kB/NFAT binding sites in the HIV-1 enhancer probably NFAT5, is that their inactive, phosphorylated form is bind NFAT monomers or homodimers (see below), sequestered in the cytoplasm. Upon an appropriate although binding of AP-2 to a short spacer between stimulus (T-cell receptor engagement in the case of T these sites has been described,21 and other cofactors may lymphocytes), the Ca2 þ –calmodulin-dependent phos- exist. phatase calcineurin dephosphorylates NFAT proteins and thus exposes their nuclear localization sequences. This leads to their translocation into the nucleus and the NFAT binding sites in the HIV-1 LTR/ subsequent activation of genes carrying NFAT binding sites in their regulatory regions. These genes include T- promoter cell-derived cytokines (eg, IL-2 and IL-4), CD154 (CD40 NFAT protein binding sites are found within three ligand), and others.14,15 Interestingly, in conjunction with regions of the HIV-1 LTR: in the distal LTR (‘upstream

Figure 1 Schematic representation of the major steps of NFAT regulation in T lymphocytes. Some intermediate factors have been omitted for the sake of clarity. T-cell receptor (TCR) stimulation activates phospholipase C-g (PLC-g). Generation of inositol-1,4,5-triphosphate (IP3) and its binding to IP3 receptors (IP3r) on the endoplasmic reticulum (ER) results in the release of calcium ions (Ca þþ) into the cytoplasm. After depletion of Ca þþ stores in the ER, increased free Ca þþ levels are sustained by the entry of extracellular Ca þþ through Ca þþ release activated channels (CRACs). Increased Ca þþ levels activate the calcium–calmodulin-dependent phosphatase calcineurin. The resulting dephosphorylation of NFAT proteins leads to their translocation into the nucleus and the transcription of NFAT-dependent genes. CsA, FK506, and the VIVIT peptide inhibit the activity of calcineurin and the subsequent activation of NFAT proteins. The HIV-1 Tat and Nef proteins induce NFAT activity, most likely by modulating intracellular signaling pathways that activate calcineurin. In addition, Tat may interact with NFAT directly in the nucleus. In the absence of sustained Ca þþ signaling, rephosphorylation of NFAT proteins by kinases leads to their relocation into the cytoplasm. Sustained high intracellular Ca þþ levels inhibit these kinases and thus oppose nuclear export and deactivation of NFAT. Other abbreviations: TATA, TATA-box; arrow, start site of transcription.

Genes and Immunity NFAT and HIV-1 F Pessler and RQ Cron 160 region’), the enhancer region, and in the 50 untranslated strand at the 50 end and those that preferentially interfere region (UTR) (Figure 2). DNase I footprinting analysis with NFAT binding on the noncoding strand at the 30 end has revealed two binding sites in the distal LTR: a of each site (Figure 2).22 Both monomeric and dimeric stronger one5,22 between base pairs (bp) –216 to –254 and NFAT complexes are observed on probes containing both a weaker one5 between –288 and –303. This latter site is sites, and the addition of AP-1 does not result in the not seen in footprinting assays with recombinant NFAT formation of additional complexes. Interestingly, two protein and may represent binding of another factor NFAT-containing bands of different mobilities are seen present in the nuclear extract. Bacterially expressed on a probe containing a single site, suggesting the recombinant NFAT2 strongly protects a region in the possibility of an NFAT dimer bound to a single site.22 enhancer region between –104 and –74,23 while baculo- NFAT2 can be immunoprecipitated with Ets1 from virus-generated recombinant NFAT2 leaves a somewhat normal human T lymphocytes, and this interaction smaller footprint between –99 and –77.22 This region depends on the presence of DNA. However, the overlaps with the duplicated NF-kB enhancer sites. In existence of an Ets/NFAT/DNA protein complex in vivo the H9 cell line, a T-cell line chronically infected with has not been demonstrated.26 By in vitro footprinting and HIV-1, in vivo protein occupancy has been demonstrated gel shift assays, we have recently identified a binding site by DMS-interference footprinting on the stronger one of for the c-Maf transcription factor just upstream of the the two upstream NFAT sites and on the enhancer NF- enhancer sites.27 This factor synergizes with NFAT1 to kB/NFAT sites.24 The monocytic U1 cell line is a model regulate IL-4 production by lymphocytes.28 We are for PMA-inducible cellular and viral transcription. Here, currently exploring whether it may play a similar role constitutive protein occupancy was found on the higher- by cooperating with NFAT on the HIV-1 LTR/promoter. affinity upstream NFAT site and at the 50 end of the 30 The proximity between the NF-kB and NFAT sites in enhancer site. Upon induction, interference extended to the HIV-1 enhancer suggests a physical interaction an additional guanine at the 50 end of the 30 enhancer site between these two factors on DNA. Surprisingly, few outside of the sequences believed to mediate NFAT binding studies have addressed this question directly. binding.25 Thus, additional factors may play a role in Gel shift assays with this region, performed under binding to this region. conditions of probe excess, reveal two protein/DNA Gel shift analysis with mutant oligonucleotide probes complexes, one corresponding to NFAT and the other to suggested that NF-kB and NFAT bind with different NF-kB. A ternary complex, presumably containing both specificities to overlapping sequences in the enhancer factors, is not seen.22 In competition gel shift analysis, sites. The residues that, when mutated, preferentially NFAT1 can displace NF-kB p50 from a probe containing interfere with NF-kB binding are located on the coding a single 30 enhancer half-site and the adjacent SP-1 site,

Figure 2 NFAT binding sites in the HIV-1 LTR. The upper panel depicts an overview of the three regions containing NFAT binding sites. For reasons of clarity, of the more than 50 proteins known to bind the HIV-1 LTR,75 only the NFAT, NF-kB, and SP-1 binding sites are shown. The bracket ‘lower affinity’ refers to the weak additional footprint identified by Shaw et al,5 but not seen in subsequent studies. The arrow and ‘ þ 1’ refer to the start site of transcription. The lower panel shows a magnified view of the NF-kB/NFAT binding sites in the enhancer region. The arrows above the sequence denote the NF-kB sites as described in Berkowitz et al76 and references therein. The arrows below the sequence identify the consensus NFAT binding sequence. The underlined residues designate G–C base pairs which, when mutated, preferentially interfere with NFAT (regular type) or NF-kB(italics) binding.22 The spacer between the NF-kB/NFAT sites is shaded. The box labeled ‘Crystal structure’ depicts the residues that form specific hydrogen bonds with NFAT1 in the recently identified crystal structure (adapted from Giffin et al31). The brackets delineate the DNase I footprints of bacterially expressed (bp À104 to –7423) and baculovirus- expressed recombinant NFAT2 (bp À99 to –7722).

Genes and Immunity NFAT and HIV-1 F Pessler and RQ Cron 161 and, again, a higher order complex containing both Lastly, in addition to the upstream and enhancer factors is not seen.29 Taken together, the results of these binding sites, in vivo and in vitro footprinting and gel and other binding studies were most consistent with a shift analysis in stimulated Jurkat cells revealed an model where an NFAT monomer or homodimer binds to additional, mitogen-inducible NFAT binding site in the 50 each enhancer site. Indeed, the crystal structure of the UTR between positions þ 165 to þ 181 with respect to DNA-binding domains of NFAT1 and AP-1 (fos-jun the transcription start site.32,33 The functional relevance of heterodimer) bound to the distal antigen-response this site, however, remains unclear. receptor element (ARRE) of the IL-2 promoter revealed that the ternary complex consists of one molecule each of NFAT1, AP-1, and the ARRE.30 The most critical NFAT/ Regulation of HIV-1 by NFAT DNA interactions occur by hydrogen bonding with Numerous studies have addressed the potential effects of 1 2 3 1 2 3 4 5 6 residues G G A of the core site G G A A A A , while NFAT proteins on the HIV-1 LTR. These are summarized 3 additional van der Waals interactions occur with T in Table 1. Prior indirect evidence that NFAT may 6 through T on the opposite strand. These results agree regulate HIV-1 transcription came from four unrelated well with in vivo DMS footprinting analysis of the IL-4 observations: (1) putative NFAT1 binding sites were promoter proximal NFAT sites, which has confirmed the identified in the HIV-1 LTR by DNase I footprint 1 2 3 11 importance of the G G A core for NFAT binding. analysis,5 (2) an approximately 110 kDa protein of Recently, the crystal structure of NFAT1 bound to one unknown identity was identified using a DNA affinity 31 HIV-1 enhancer site was solved. Surprisingly, it precipitation assay of the HIV-1 LTR34 (some NFAT revealed that, in contrast to its heterodimeric binding isoforms migrate at approximately 120 kDa), (3) reporter to the ARRE, NFAT1 actually binds to this site as a mice transgenic for the HIV-1 LTR35 exhibited an cooperative homodimer that encircles the DNA like a expression pattern similar to mice expressing the SV40 pair of pliers (Figure 3). Hydrogen bonds are formed T antigen under the control of multimerized NFAT 10 9 8 between one monomer and the G G A of the consensus binding sites,36 and (4) CsA, an inhibitor of NFAT, binding site on the noncoding strand and, in addition, diminished the stimulatory effects of TCR/CD3 1 2 3 between the other monomer and the G G G of a signaling on the HIV-1 LTR.37 nonconsensus site of the coding strand, previously believed to bind NF-kB but not NFAT (Figure 2). The The upstream binding region duplicated HIV-1 enhancer sites could theoretically bind The initial search for effects of NFAT on HIV-1 gene up to two homodimers, that is, a maximum of four NFAT expression focused on the stronger one of the two molecules. This crystal structure also explains why NFAT upstream sites. Surprisingly, mutating or deleting this might outcompete NF-kB for binding to this site,29 since site had no significant effect on HIV LTR transcription in binding of both factors to the same site seems sterically Jurkat cells38 and Jurkat-tat cells, a cell line that expresses impossible. Thus, regulation of the HIV-1 enhancer by Tat constitutively.39 Moreover, removal of this site NFAT and NF-kB may be mutually exclusive. actually caused a modest increase in viral replication in Jurkat cells41 and in HIV-1 LTR transcription in primary human CD4T cells.22 This was consistent with the previous implication of this region as a negative regulatory element.39,40

The 50 UTR binding site Only one study has examined potential functions of this site.32 Mutating the 50 UTR NFAT site alone had no effect on viral replication but decreased basal transcription by 50%. Interestingly, when combined with mutations in other 50 UTR protein binding sites, it reduced both viral replication and transcription.

The enhancer binding sites and effects not localized to a specific site In contrast to the mostly negative results obtained in the studies of the upstream binding region, a seminal report by Kinoshita et al23 has provided evidence that the most important sequences for NFAT regulation are actually located in the HIV-1 enhancer region. Recombinant NFAT2 protects nucleotides –104 to –74 from DNase I digestion, and this binding activity appears in nuclear extracts upon mitogen stimulation. Moreover, in Jurkat Figure 3 Crystal structure of a homodimer of NFAT1 (residues thymoma cells, NFAT2 activates the HIV-1 LTR syner- 396–678) bound to a single NF-kB/NFAT HIV-1 enhancer site. The gistically with Tat, and mutating the 4-bp spacer between monomers (labeled A and B) interact via their C-terminal RHR the NF-kB/NFAT sites (nucleotides –93 to –90) reduces domains (marked with a star, *) and encircle the DNA like a pair of pliers. Monomer A is bound to the consensus GGA site (30 end, this effect substantially. Replication of the HIV-1 strain lower strand, Figure 2) and monomer B to the nonconsensus GGG NL4-3 is about three-fold higher in cells stably over- site (50 end, upper strand, Figure 2). Image provided by courtesy of expressing NFAT2. These findings have been corrobo- L Chen (University of Colorado at Boulder, CO). rated by work from several other laboratories.3,22,26,41,42

Genes and Immunity NFAT and HIV-1 F Pessler and RQ Cron 162 Table 1 Summary of studies about NFAT and HIV-1 transcription

Site examineda Cell type Stimulation NFAT type Assay usedb Remarks Reference

(A) Studies showing stimulatory effects Enhancer (gs) Jurkat, primary CD4+ aCD43, a-CD3 ND tt, vr FK506 + effect 44 Enhancer (gs) Jurkat, CD45(À)Jurkat aCD3/CD28, PMA/ NFAT1 tt, vr FK506, CsA + effect 46 PHA/a-CD3 Enhancer (gs) Jurkat and derivatives, PMA, PHA, bpV NFAT1,2 tt FK506, CsA, aNFAT 42 PBMC, 293 peptide + effect Enhancer Jurkat E6.1 and IG5 aCD3, CD28 ND tt, vr Dominant negative NFAT 45 + effect Enhancer Primary T cells PMA/ionomycin NFAT1 tt Effect + by aNFAT 3 peptide Enhancer (fp,gs), Primary T cells PMA/ionomycin NFAT1,2 tt, vr CsA, FK506, dominant 22 upstream (fp,D) negative NFAT1 + effect, D upstream site * effect Enhancer (gs) Primary T cells, PMA/ aCD3, TNF-a ND tt, vr Rolipram, a 41 ionomycin phosphodiesterase type IV inhibitor + effect Upstream (D), 293, Jurkat PHA, TNF-a NFAT2 tt, vr D upstream region had no 23 enhancer effect (D,fp,gs), spacer (pmut) 50 UTR Jurkat, PBMCs TPA/ionomycin (for gs) ND tt, vr Effect * by mutating 32 additional 50UTR protein binding sites (B) Studies showing inhibition or no effect Upstream (D), Jurkat, CEM PMA/PHA ND tt No effect 48 spacer (pmut) Enhancer (gs) Jurkat PMA/ionomycin NFAT1 tt Inhibition of Tat 29 transactivation Upstream (pmut) Jurkat, EL-4 PMA/PHA, aCD3 ND tt Slight + of basal tx by 38 mutations in upstream site, no effect on Tat Upstream (D) Jurkat-tat — ND tt, vr * in vr by D of upstream 39 site

aMethod by which the effect of this site was examined. gs, a specific NFAT complex formed in a gel shift assay; D, the site was deleted; fp, an NFAT footprint was observed on the site; pmut, D, site was inactivated by point mutations or deletion, respectively; ND, not determined. bAssay used to determine NFAT effect. tt, transient transfection; vr, viral replication; + , decrease; * , increase.

Importantly, activation of the HIV-1 LTR and stimula- TCR signaling molecules, p56lck, ZAP-70, and p21ras, tion of viral replication by both NFAT1 and 2 have also suggested that activities of these factors were increased been verified in primary CD4T lymphocytes.3,22 Notably, by bpV, most likely by inhibiting dephosphorylation of the immunosuppressive drugs CsA and FK506, both of p56lck and ZAP-70 by SHP-1, and by increased p21ras which interfere with calcineurin activity, as well as a phosphorylation by these kinases. Thus, activation of synthetic peptide, VIVIT,43 which competes for NFAT standard CD3/TCR-mediated signaling pathways likely binding to calcineurin and thereby inhibits the depho- forms the backbone of NFAT-driven HIV-1 LTR tran- sphorylation and nuclear translocation of NFAT1, 2, and scription. 4, all reduce LTR transcription in polyclonally stimulated Clearly, other signaling pathways can modulate this (PMA þ ionomycin) primary human CD4T cells.3,22 Ta- activity, and the importance of intracellular signaling ken together, these results add an additional mechanistic mediated by cell surface molecules other than the TCR explanation to the previous observation that CsA inhibits has been demonstrated. Crosslinking of CD43 potenti- HIV-1 replication in culture37 (reviewed in Cron3). ates TCR-dependent activation of HIV-1 transcription In an attempt to identify the signaling events leading and viral replication, and this effect correlates with NFAT to NFAT and HIV-1 LTR activation, Fortin et al42 added and NF-kB binding to the HIV-1 enhancer.44 Antibody- bis-peroxyvanadium (bpV), an inhibitor of protein mediated engagement of the TCR/CD3 complex and tyrosine phosphatases, to Jurkat cells transfected with host-cell attachment by HIV-1 virions ectopically expres- HIV-1 LTR reporter constructs. This compound induced sing CD86 can synergize to activate NFAT-stimulated NFAT translocation to the nucleus and upregulated LTR- HIV-1 LTR transcription.45 Also, consistent with the driven transcription. Addition of CsA or FK506 and previous finding that NFAT levels are upregulated in overexpression of the SHP-1 phosphatase ameliorated memory/effector CD4T cells,11 NFAT1 was recently this effect, suggesting that (1) bpV acts upon signaling identified as a major factor responsible for the prefer- steps upstream of calcineurin and (2) that the SHP-1 ential replication of HIV-1 in CD45RO þ ‘memory’ T phosphatase is a bpV target. Moreover, assays with lymphocytes,46 a potential reservoir of latently infected dominant negative mutants and cell lines deficient in the cells. Interestingly, NFAT-driven HIV-1 LTR transcription

Genes and Immunity NFAT and HIV-1 F Pessler and RQ Cron 163 and NFAT1 binding to the HIV-1 enhancer region are studies of a different virus, the African swine fever virus, potentiated in a CD45-deficient cell line, suggesting that have revealed an intriguing precedent of how a virus can CD45 signaling can inhibit HIV-1 transcription by modulate NFAT activity. The viral A238L protein downregulating NFAT1.47 possesses a 14-amino-acid domain with great similarity In contrast to the above reports, two studies have to an NFAT epitope. This domain inhibits the phospha- shown inhibitory or no effects of NFAT proteins at the tase activity of calcineurin and thus interferes with enhancer sites. In one report, NFAT1 reduced Tat translocation of NFAT2 into the nucleus and the resultant transactivation and had no measurable effect on basal activation of cellular immunomodulatory genes.51,52 LTR transcription in Jurkat thymoma cells.29 Likewise, it reduced Rel A (NF-kB)-mediated activation of the HIV-1 HIV-1 infection and NFAT. TCR/CD3g expression is LTR. Moreover, in a gel shift assay, recombinant NFAT1 downregulated in HIV-1 and -2 infection, possibly displaced recombinant NF-kB p50 from oligonucleotides contributing to the immune dysfunction in HIV infec- containing the 30 enhancer site and the adjacent SP-1 site. tion. Decreased TCR/CD3g expression in HIV-1-infected It was therefore concluded that NFAT1 inhibits tran- cells correlates with the increased binding of NFAT1 and scription by interfering with NF-kB activity at the 2 and NF-kB p50 to regulatory sequences in the CD3g enhancer NF-kB/NFAT sites.29 In agreement with this gene, suggesting that, in this scenario, these factors report, but in stark contrast to the deleterious effect might act as repressors of CD3g transcription. It has been reported by Kinoshita et al23 (see above), mutating the postulated that this induction of NFAT activity and the spacer between the NF-kB sites had no effect on basal or consequent loss of a receptor critical for T-cell signaling Tat-transactivated HIV-1 LTR transcription in phorbol would present a means for the virus to downregulate its myristil acetate (PMA)- and phytohemagglutinin (PHA)- own gene expression and favor a state of latency.53 stimulated Jurkat or CEM cells.48 These divergent results appear difficult to reconcile. They may relate to the Nef and NFAT. Studies into the mechanism of action of differential activities of signaling pathways and/or the HIV-1 Nef protein have shown that in transient NFAT cofactors under the respective experimental con- transfection assays this protein can increase NFAT ditions. Indeed, depending on the presence or absence of translocation into the nucleus and the subsequent cooperative binding with AP-1, NFAT regulates different activation of NFAT-dependent transcription. This effect sets of cellular genes in the same cell type,49 and it has is TCR independent, mediated by influx of extracellular been suggested that some NFAT/DNA complexes con- calcium, and potentiated by the Ras/MAPK pathway.54 tain transcriptional repressors.7 Nevertheless, the bulk of The presence of the HIV-1 Vpr protein further potentiates the available evidence suggests that NFAT stimulates the increase in NFAT-directed transcription, probably via HIV-1 transcription. However, one can postulate that the a regulatory step located further downstream.55 Cell lines overall effect is actually bimodal, such that under certain expressing inducible Nef have also revealed increases in conditions NFAT may act as an inhibitor of HIV-1 gene NFAT-dependent reporter gene transcription and HIV-1 expression. Given the complexity of the HIV-1 life cycle replication.56 in natural infection, both effects are biologically plau- sible, although the positive effects likely predominate. Tat and NFAT. Another HIV-1 gene product, the Tat protein, has been shown to modulate NFAT activity. An Nontranscriptional effects of NFAT on HIV-1. An equally early indication that Tat increases NFAT activity was that intriguing aspect of the interaction between NFAT and Tat enhanced IL-2 promoter activity, and deleting an HIV is that NFAT appears to regulate at least one NFAT binding site between –279 and –263 bp abolished important nontranscriptional aspect of the viral life this effect.57 Subsequently, it was found that Tat cycle. In nondividing primary host cells, blocks to viral enhanced NFAT1-dependent transcription of a reporter replication exist. As a consequence, HIV-1 replicates in construct containing three canonical NFAT binding PHA-stimulated primary CD4T cells but not in quiescent sites.29 This effect correlated with the ability of Tat to ones. Primary T cells stably overexpressing NFAT2 interact with the transactivation domain of NFAT1. permit viral replication in the absence of PHA.50 This Moreover, in gel shift assays, a ternary complex contain- permissiveness is reversible by CsA and FK506 and has ing NFAT1 and Tat formed on NFAT binding sites, been found to be due to relief of a block to reverse suggesting that Tat acts by physically recruiting addi- transcription, possibly by the induction of one or more tional NFAT molecules to the promoter.29 In primary cellular cofactors of reverse transcription regulated by vascular endothelial cells and a Kaposi’s sarcoma cell NFAT2.50 Identification of such cofactors is currently in line, Tat alone had only a small effect, but it potentiated progress (G Nolan, Stanford University, personal NFAT activation by the human herpes virus 8-encoded communication). chemokine homolog vGPCR, an activator of the PI3-K/ Akt signaling pathway.58 Regulation of NFAT by HIV-1 Given the significant effects of NFAT proteins on HIV-1 Other mechanisms of NFAT regulation by HIV-1. The gene expression, one might expect that the virus, in turn, MAPK pathway represents another mechanism by which has evolved strategies to modulate NFAT activity. Viral NFAT activity can be increased. HIV-1-infected CD4 T strains capable of doing so might experience selective lymphocytes express elevated levels of Grb3-3, an advantages in two ways: (1) enhanced NFAT activity adaptor molecule of the MAPK pathway. This molecule would lead to greater rates of viral replication and (2) can stimulate NFAT-driven transcription upon CD3/ reduced NFAT activity would increase the likelihood of CD28 engagement, and this effect is suppressed by the the virus remaining in a quiescent state, thus allowing MEKK1 kinase or CsA, both of which interfere with infected cells to escape immune surveillance. Indeed, NFAT translocation into the nucleus.59 Genomic and

Genes and Immunity NFAT and HIV-1 F Pessler and RQ Cron 164 proteomic methods are becoming widely used tools in NFAT proteins in HIV-2 regulation may exist: (1) CsA the study of virus–host interactions. Along these lines, inhibits replication of HIV-266 and SIV,67 (2) Elf-1 evidence for HIV-1 regulation of NFAT activity has associates with NFAT in Jurkat cells,26 and (3) an NFAT recently been captured by large-scale screens. Since binding site, which is identical to the HIV-1 30 NF-kB/ NFAT activity is regulated primarily at the post-tran- NFAT site and could potentially bind an NFAT dimer, is scriptional level, one does not necessarily expect to present in the HIV-2 and SIV enhancers. detect changes in NFAT mRNA levels by DNA micro- array and other mRNA-based assays. Nonetheless, three NFAT—a target for antiretroviral therapy? studies of host cell gene expression changes induced by Given the predominantly stimulatory effect of NFAT on HIV-1 have revealed upregulation of NFAT proteins. In HIV-1, NFAT proteins represent an attractive pharmaco- one study, NFAT4 was identified among several genes logic target for the treatment of HIV-1 infection. Indeed, that were upregulated in HIV-1 IIIB-infected CEM-SS T there has been considerable interest in the use of the cells that were relatively protected against undergoing immunosuppressive agent CsA for this purpose. At first apoptotic cell death.60 Another study revealed that glance, it would seem counterintuitive to use an ectopic expression of HIV-1 Nef led to global changes immunosuppressive drug for the treatment of an in cellular transcription reminiscent of those following immunodeficiency. However, this interest was motivated TCR stimulation, and NFAT2 was among the upregu- by rational observations such as (1) CsA inhibited HIV-1 lated genes.61 Lastly, the binding of HIV-1 gp120 to replication in culture,68 (2) treatment of HIV-1-infected peripheral blood mononuclear cells and macrophages organ transplant recipients with CsA correlated with a revealed significant upregulation of NFAT1 protein and lower risk of progression to AIDS over 5 years (31 vs NFAT2 mRNA, along with mRNAs and polypeptides 90%),69 and (3) in a nonrandomized pilot study, a subset corresponding to cytokines, chemokines, kinases, and of non-AIDS HIV-1-infected individuals experienced other transcription factors believed to contribute to increased CD4 T-cell numbers while on CsA.70 Unfortu- productive HIV-1 infection.62 Keeping in mind that nately, individuals with clinical AIDS did not show a NFAT2 can relieve a block to reverse transcription in benefit in this study. Consistent with this lack of positive quiescent lymphocytes (see above), induction of NFAT effect in the late stage of HIV-1 infection, eight patients activity during the earliest stages of infection might with clinical AIDS deteriorated while on CsA.71 This led enable the virus to overcome this block. to a lack of interest in treating HIV-1 infection with CsA. Taken together, the above results suggest that the However, in the 10 or more years since these early African swine fever virus and HIV-1 evolved to observations, primary antiretroviral treatment has im- modulate NFAT activity in diametrically opposite ways: proved significantly and more HIV-1-infected indivi- while the former inhibits NFAT and, potentially, the duals are in need of organ transplants. Given the resultant activation of the host cell, HIV-1 has evolved to aforementioned poor outcomes of CsA use in patients primarily upregulate the activity of NFAT proteins. The with advanced HIV-1 infection, the focus on CsA in HIV- virus then utilizes this increase in NFAT activity to 1 infection has thus shifted to its use in early stable optimize viral survival and spread in the host in two infection and in organ transplantation. Along these lines, ways: (1) by creating a host state more permissive to viral the results of three clinical trials were published recently. infection and (2) by directly increasing viral gene In one trial, treatment with CsA (2 mg/kg twice a day) in expression and replication. stable early HIV-1 infection (CD4 cell count 4500 Â 106/ Thus, NFAT and HIV-1 can upregulate each other. This l, plasma HIV-1 RNA 4600 copies/ml, concurrent remarkable reciprocal regulation could potentially create treatment with 0–2 antiretroviral drugs) showed no a positive feedback loop, which may facilitate the benefit, but a small increase in plasma HIV-1 RNA.72 In establishment of early HIV-1 infection and, later, the another study, plasma HIV-1 RNA remained undetect- transition from latent to productive infection. It is able, while CD4 cell counts decreased in four (29%) in a tempting to speculate that negative effects of NFAT cohort of 14 non-AIDS HIV-1 þ liver and kidney proteins on HIV-1 gene expression might serve to transplant patients (plasma HIV-1 RNA undetectable, downregulate this amplification loop under certain CD4 cell count 4100 (liver) or 4200 (kidney)) who were circumstances. treated with CsA, mycophenolate mofetil, and predni- sone.73 Thus, these reports did not detect a therapeutic Is there a role for NFAT proteins in HIV-2 or SIV? benefit but they support the notion that using Surprisingly, there are no published reports about immunosuppressants for organ transplantation in stable, potential effects, or lack thereof, of NFAT proteins on non-AIDS HIV-1 þ individuals may be relatively safe. HIV-2 or simian immunodeficiency virus (SIV) gene The true benefit of CsA may be as an adjunct therapy expression or replication. The reason for this apparent along with highly active antiretroviral therapy (HAART). neglect may be that the initial work on NFAT and HIV-1 In a nonrandomized pilot study, nine patients on focused on the –254 to –216 upstream binding site, which combination therapy of CsA (0.3–0.6 mg/kg for 8 weeks) is not found in HIV-2 or SIV. Moreover, the HIV-2 LTR plus HAART (stavudine, lamivudine, nelfinavir, and transcriptional control elements differ fundamentally saquinavir) had comparable decreases of plasma viral from those found in HIV-1.63,64 In particular, the HIV-2 load as expected from HAART, but, in contrast to 29 enhancer possesses only one functional NF-kB site in the control patients, had significantly increased CD4 cell enhancer region but contains at least four additional cis- counts within 1 week. Surprisingly, this effect persisted inducible elements that bind, among other unidentified until the end of the follow-up period of 64 weeks.74 factors, the Ets family proteins Elf-1 and Pu.1 (see Clark Certainly, larger trials will be needed to explore this et al65 and references therein). In spite of the current lack fascinating phenomenon further. A more extensive trial of experimental evidence, there are hints that a role for about CsA as adjunct treatment with HAART is currently

Genes and Immunity NFAT and HIV-1 F Pessler and RQ Cron 165 in progress (G Rizzardi, University of Lausanne, Switzer- References land, personal communication). The development of an NFAT inhibitory peptide by 1 Nabel G, Baltimore D. An inducible transcription factor combinatorial peptide selection43 and the demonstration activates expression of human immunodeficiency virus in that its expression in primary T cells decreases HIV-1 T cells. Nature 1987; 326: 711–713. transcription3 are first steps toward the design of anti- 2 Hiscott J, Kwon H, Genin P. Hostile takeovers: viral appro- HIV-1 drugs that target NFAT specifically. Such NFAT priation of the NF-kappaB pathway. J Clin Invest 2001; 107: inhibitors promise to be important additions to the 143–151. 3 Cron RQ. HIV-1, NFAT, and cyclosporin: immunosuppresion pharmacologic treatment of HIV-1 infection. There is, for the immunosuppressed? DNA Cell Biol 2001; 20: 761–767. however, a potentially significant caveat. Considering 4 Dolmetsch RE, Lewis RS, Goodnow CC, Healy JI. Differential 22 that CsA inhibits transcription factors other than NFAT activation of transcription factors induced by Ca2+ response and that it interferes with several steps of the HIV-1 life amplitude and duration. Nature 1997; 386: 855–858. cycle other than transcription (reviewed in Cron3), anti- 5 Shaw JP, Utz PJ, Durand DB et al. Identification of a putative NFAT designer drugs may have fewer side effects but regulator of early T cell activation genes. Science 1988; 241: may also be less effective than CsA in interfering with 202–205. HIV-1 infection. 6 Hogan PG, Chen L, Nardone J, Rao A. Transcriptional regulation by calcium, calcineurin, and NFAT. Genes Dev 2003; 17: 2205–2232. Outlook 7 Crabtree GR, Olson EN. NFAT signaling: choreographing the After the initial searches for a role of NFAT through the social lives of cells. Cell 2002; 109: S67–S79. upstream HIV-1 binding site(s), the past 6 years have 8 Horsley V, Pavlath GK. NFAT: ubiquitous regulator of cell redirected the focus onto the duplicated NF-kB/NFAT differentiation and adaptation. J Cell Biol 2002; 156: 771–774. enhancer sites. Significant advances have been made, but 9 Lyakh L, Ghosh P, Rice NR. Expression of NFAT-family many questions remain unanswered. For instance, what, proteins in normal human T cells. Mol Cell Biol 1997; 17: 2475–2484. if any, physical interactions do NFAT proteins undergo 10 Timmerman LA, Healy JI, Ho SN et al. Redundant expression with other factors in this region? Do they really bind but selective utilization of nuclear factor of activated T cells alone to the enhancer sites or are cofactors involved? family members. J Immunol 1997; 159: 2735–2740. How do the various signaling pathways modulate NFAT 11 Cron RQ, Bart SJ, Wang Y et al. T cell priming enhances IL-4 activity at these sites? Could NFAT have a bimodal effect, gene expression by increasing nuclear factor of activated for instance by interacting with repressors vs activators? T cells. J Immunol 1999; 162: 860–870. Which factors mediate the nontranscriptional effects of 12 Northrop JP, Ho SN, Chen L et al. NF-AT components define a NFAT proteins on HIV-1? The effects of HIV-1 on NFAT family of transcription factors targeted in T-cell activation [see activity have thus far been examined almost exclusively comments]. Nature 1994; 369: 497–502. by experiments with individual viral gene products, for 13 Zhou B, Cron RQ, Wu B et al. Regulation of the murine example, Tat, Nef, and gp120. It will now be important to NFATc1 gene by NFATc2. J Biol Chem 2002; 277: 10704–10711. 14 Crabtree GR. Generic signals and specific outcomes: signaling examine better how the virus alters NFAT activity in 2+ natural infection. Moreover, the variation among natural through Ca , calcineurin, and NF-AT. Cell 1999; 96: 611–614. 15 Rao A, Luo C, Hogan PG. Transcription factors of the NFAT viral isolates in their interactions with NFAT proteins has family: regulation and function. Annu Rev Immunol 1997; 15: not been studied systematically. It will also be of great 707–747. interest to define further the role of NFAT proteins in the 16 Ho S, Clipstone N, Timmerman L et al. The mechanism of HIV-1 life cycle in resting host cells. Could they enable action of cyclosporin A and FK506. Clin Immunol Immunopathol the virus to replicate in these cells at a chronic, low level 1996; 80: S40–S45. and thus contribute to the maintenance of the viral 17 Serafini AT, Lewis RS, Clipstone NA et al. Isolation of mutant reservoir? The coming years also promise to yield T lymphocytes with defects in capacitative calcium entry. important results from larger, controlled clinical studies Immunity 1995; 3: 239–250. about the use of CsA in the treatment of HIV-1 infection 18 Beals CR, Sheridan CM, Turek CW et al. Nuclear export of NF- and organ transplantation in HIV-1 þ individuals. ATc enhanced by glycogen synthase kinase-3. Science 1997; Finally, the development of specific NFAT inhibitors 275: 1930–1934. may add to the pharmacologic armamentarium against 19 Macian F, Lopez-Rodriguez C, Rao A. Partners in transcrip- this deadly virus. The intricate interplay between tion: NFAT and AP-1. Oncogene 2001; 20: 2476–2489. 20 Grogan JL, Locksley RM. T helper cell differentiation: on members of the NFAT family and HIV-1 certainly still again, off again. Curr Opin Immunol 2002; 14: 366–372. has some surprises in store for us. 21 Perkins ND, Agranoff AB, Duckett CS, Nabel GJ. Transcrip- tion factor AP-2 regulates human immunodeficiency virus type 1 gene expression. J Virol 1994; 68: 6820–6823. 22 Cron RQ, Bartz SR, Clausell A et al. NFAT1 enhances HIV-1 Acknowledgements gene expression in primary human CD4T cells. Clin Immunol We thank Drs Lin Chen (University of Colorado at 2000; 94: 179–191. Boulder, CO), Terri H Finkel (The Children’s Hospital of 23 Kinoshita S, Su L, Amano M et al. The T cell activation factor NF-ATc positively regulates HIV-1 replication and gene Philadelphia, PA), and H Ralph Schumacher (University expression in T cells. Immunity 1997; 6: 235–244. of Pennsylvania, PA) for critical reading of the manu- 24 Demarchi F, D’Agaro P, Falaschi A, Giacca M. Probing script and helpful comments, and L Chen for providing protein–DNA interactions at the long terminal repeat of the image of the NFAT1/DNA crystal structure. RQ Cron human immunodeficiency virus type 1 by in vivo footprinting. was supported in part by grants from the Elizabeth J Virol 1992; 66: 2514–2518. Glaser Pediatric AIDS Foundation and the Mary L Smith 25 Demarchi F, D’Agaro P, Falaschi A, Giacca M. In vivo Charitable Trust and by NIH Grant P30-AI45008. footprinting analysis of constitutive and inducible protein–

Genes and Immunity NFAT and HIV-1 F Pessler and RQ Cron 166 DNA interactions at the long terminal repeat of human virus production that is induced upon TCR/CD3 stimulation. immunodeficiency virus type 1. J Virol 1993; 67: 7450–7460. J Biol Chem 2002; 277: 28714–28724. 26 Bassuk AG, Anandappa RT, Leiden JM. Physical interactions 45 Bounou S, Dumais N, Tremblay MJ. Attachment of human between Ets and NF-kappaB/NFAT proteins play an impor- immunodeficiency virus-1 (HIV-1) particles bearing host- tant role in their cooperative activation of the human encoded B7-2 proteins leads to nuclear factor-kappa B- and immunodeficiency virus enhancer in T cells. J Virol 1997; 71: nuclear factor of activated T cells-dependent activation of HIV- 3563–3573. 1 long terminal repeat transcription. J Biol Chem 2001; 276: 27 Cron RQ, Genin A, Clausell A. Regulation of HIV-1 transcrip- 6359–6369. tion by c-maf (abstract). FASEB J 2001; 15: A696. 46 Robichaud GA, Barbeau B, Fortin JF et al. Nuclear factor of 28 Ho IC, Hodge MR, Rooney JW, Glimcher LH. The proto- activated T cells is a driving force for preferential productive oncogene c-maf is responsible for tissue-specific expression of HIV-1 infection of CD45RO-expressing CD4+ T cells. J Biol interleukin-4. Cell 1996; 85: 973–983. Chem 2002; 277: 23733–23741. 29 Macian F, Rao A. Reciprocal modulatory interaction between 47 Barbeau B, Robichaud GA, Fortin JF, Tremblay MJ. Negative human immunodeficiency virus type 1 Tat and transcription regulation of the NFAT1 factor by CD45: implication in HIV-1 factor NFAT1. Mol Cell Biol 1999; 19: 3645–3653. long terminal repeat activation. J Immunol 2001; 167: 30 Chen L, Glover JN, Hogan PG et al. Structure of the DNA- 2700–2713. binding domains from NFAT, Fos and Jun bound specifically 48 West MJ, Lowe AD, Karn J. Activation of human immunode- to DNA. Nature 1998; 392: 42–48. ficiency virus transcription in T cells revisited: NF-kappaB 31 Giffin MJ, Stroud JC, Bates DL et al. Structure of NFAT1 bound p65 stimulates transcriptional elongation. J Virol 2001; 75: as a dimer to the HIV-1 LTR kappaB element. Nat Struct Biol 8524–8537. 2003; 10: 800–806. 49 Macian F, Garcia-Rodriguez C, Rao A. Gene expression 32 Van Lint C, Amella CA, Emiliani S et al. Transcription factor elicited by NFAT in the presence or absence of cooperative binding sites downstream of the human immunodeficiency recruitment of Fos and Jun. EMBO J 2000; 19: 4783–4795. virus type 1 transcription start site are important for virus 50 Kinoshita S, Chen BK, Kaneshima H, Nolan GP. Host control infectivity. J Virol 1997; 71: 6113–6127. of HIV-1 parasitism in T cells by the nuclear factor of activated 33 el Kharroubi A, Verdin E. Protein–DNA interactions within T cells. Cell 1998; 95: 595–604. DNase I-hypersensitive sites located downstream of the HIV-1 51 Miskin JE, Abrams CC, Goatley LC, Dixon LK. A viral promoter. J Biol Chem 1994; 269: 19916–19924. mechanism for inhibition of the cellular phosphatase calci- 34 Franza Jr BR, Josephs SF, Gilman MZ et al. Characterization of neurin. Science 1998; 281: 562–565. cellular proteins recognizing the HIV enhancer using a 52 Miskin JE, Abrams CC, Dixon LK. African swine fever virus microscale DNA-affinity precipitation assay. Nature 1987; protein A238L interacts with the cellular phosphatase calci- 330: 391–395. neurin via a binding domain similar to that of NFAT. J Virol 35 Vogel J, Hinrichs SH, Reynolds RK et al. The HIV tat gene 2000; 74: 9412–9420. induces dermal lesions resembling Kaposi’s sarcoma in 53 Badran BM, Wolinsky SM, Burny A, Willard-Gallo KE. transgenic mice. Nature 1988; 335: 606–611. Identification of three NFAT binding motifs in the 50-upstream 36 Verweij CL, Guidos C, Crabtree GR. Cell type specificity and region of the human CD3gamma gene that differentially bind activation requirements for NFAT-1 (nuclear factor of acti- NFATc1, NFATc2, and NF-kappa B p50. J Biol Chem 2002; 277: vated T-cells) transcriptional activity determined by a new 47136–47148. method using transgenic mice to assay transcriptional activity 54 Manninen A, Renkema GH, Saksela K. Synergistic activation of an individual nuclear factor. J Biol Chem 1990; 265: of NFAT by HIV-1 nef and the Ras/MAPK pathway. J Biol 15788–15795. Chem 2000; 275: 16513–16517. 37 Tong-Starkesen SE, Luciw PA, Peterlin BM. Signaling through 55 Lahti AL, Manninen A, Saksela K. Regulation of T cell T lymphocyte surface proteins, TCR/CD3 and CD28, activates activation by HIV-1 accessory proteins: Vpr acts via distinct the HIV-1 long terminal repeat. J Immunol 1989; 142: 702–707. mechanisms to cooperate with Nef in NFAT-directed gene 38 Markovitz DM, Hannibal MC, Smith MJ et al. Activation of the expression and to promote transactivation by CREB. Virology human immunodeficiency virus type 1 enhancer is not 2003; 310: 190–196. dependent on NFAT-1. J Virol 1992; 66: 3961–3965. 56 Wang JK, Kiyokawa E, Verdin E, Trono D. The Nef protein of 39 Lu YC, Touzjian N, Stenzel M et al. Identification of cis-acting HIV-1 associates with rafts and primes T cells for activation. repressive sequences within the negative regulatory element Proc Natl Acad Sci USA 2000; 97: 394–399. of human immunodeficiency virus type 1. J Virol 1990; 64: 57 Vacca A, Farina M, Maroder M et al. Human immunodefi- 5226–5229. ciency virus type-1 tat enhances interleukin-2 promoter 40 Rosen CA, Sodroski JG, Haseltine WA. Location of cis-acting activity through synergism with phorbol ester and calcium- regulatory sequences in the human T-cell leukemia virus type mediated activation of the NF-AT cis-regulatory motif. Biochem I long terminal repeat. Proc Natl Acad Sci USA 1985; 82: 6502– Biophys Res Commun 1994; 205: 467–474. 6506. 58 Pati S, Foulke Jr JS, Barabitskay et al. Human herpesvirus 8- 41 Navarro J, Punzon C, Jimemez JL et al. Inhibition of encoded vGPCR activates nuclear factor of activated T cells phosphodiesterase type IV suppresses human immunodefi- and collaborates with human immunodeficiency virus type 1 ciency virus type 1 replication and cytokine production in Tat. J Virol 2003; 77: 5759–5773. primary T cells: involvement of NF-kappaB and NFAT. J Virol 59 Li X, Multan MC, Henin Y et al. Grb3-3 is up-regulated in HIV- 1998; 72: 4712–4720. 1-infected T-cells and can potentiate cell activation through 42 Fortin JF, Barbeau B, Robichand GG et al. Regulation of NFATc. J Biol Chem 2000; 275: 30925–30933. nuclear factor of activated T cells by phosphotyrosyl-specific 60 Yin J, Chen MF, Finkel TH. Differential gene expression during phosphatase activity: a positive effect on HIV-1 long terminal HIV-1 infection analyzed by suppression subtraction hybridi- repeat-driven transcription and a possible implication of SHP- zation. AIDS in press. 1. Blood 2001; 97: 2390–2400. 61 Simmons A, Aluvihare V, McMichael A. Nef triggers a 43 Aramburu J, Yaffe MB, Lopez-Rodrique et al. Affinity-driven transcriptional program in T cells imitating single-signal T peptide selection of an NFAT inhibitor more selective than cell activation and inducing HIV virulence mediators. cyclosporin A [see comments]. Science 1999; 285: 2129–2133. Immunity 2001; 14: 763–777. 44 Barat C, Tremblay MJ. Engagement of CD43 enhances human 62 Cicala C, Artho SJ, Selig SM et al. HIV envelope induces a immunodeficiency virus type 1 transcriptional activity and cascade of cell signals in non-proliferating target cells that

Genes and Immunity NFAT and HIV-1 F Pessler and RQ Cron 167 favor virus replication. Proc Natl Acad Sci USA 2002; 99: 9380– four cases and review of the literature. Transplantation 1993; 55: 9385. 95–103. 63 Hilfinger JM, Clark N, Smith M et al. Differential regulation of 70 Andrieu JM, Evan P, Venet A et al. Effects of cyclosporin on the human immunodeficiency virus type 2 enhancer in T-cell subsets in human immunodeficiency virus disease. Clin monocytes at various stages of differentiation. J Virol 1993; Immunol Immunopathol 1988; 47: 181–198. 67: 4448–4453. 71 Phillips A, Wainberg MA, Coates R et al. Cyclosporine- 64 Tong-Starksen SE, Welsh TM, Peterlin BM. Differences in induced deterioration in patients with AIDS. CMAJ 1989; transcriptional enhancers of HIV-1 and HIV-2. Response to 140: 1456–1460. T cell activation signals. J Immunol 1990; 145: 4348–4354. 72 Calabrese LH, Lederman MM, Spritzler J et al. Placebo- 65 Clark NM, Hannibal MC, Markovitz DM. The peri-kappa B controlled trial of cyclosporin-A in HIV-1 disease: implications site mediates human immunodeficiency virus type 2 enhancer for solid organ transplantation. J Acquir Immune Defic Syndr activation in monocytes but not in T cells. J Virol 1995; 69: 2002; 29: 356–362. 4854–4862. 73 Stock PG, Roland ME, Carlson L et al. Kidney and liver 66 Karpas A, Lowdell M, Jacobson SK, Hill F. Inhibition of transplantation in human immunodeficiency virus-infected human immunodeficiency virus and growth of infected T cells patients: a pilot safety and efficacy study. Transplantation 2003; by the immunosuppressive drugs cyclosporin A and FK 506. Proc Natl Acad Sci USA 1992; 89: 8351–8355. 76: 370–375. 67 Polacino PS, Liang HA, Firpo EJ, Clark EA. T-cell activation 74 Rizzardi GP, Harari A, Capoluppi B et al. Treatment of primary influences initial DNA synthesis of simian immunodeficiency HIV-1 infection with cyclosporin A coupled with highly active virus in resting T lymphocytes from macaques. J Virol 1993; 67: antiretroviral therapy. J Clin Invest 2002; 109: 681–688. 7008–7016. 75 Pereira LA, Bantley K, Peeters A et al. A compilation of 68 Wainberg MA, Dascal A, Blain N et al. The effect of cellular transcription factor interactions with the HIV-1 LTR cyclosporine A on infection of susceptible cells by human promoter. Nucleic Acids Res 2000; 28: 663–668. immunodeficiency virus type 1. Blood 1988; 72: 1904–1910. 76 Berkowitz B, Huang DB, Chan-Park FE et al. The X-ray crystal 69 Schwarz A, Offermann G, Keller F et al. The effect of structure of the NF-kappa B p50.p65 heterodimer bound to cyclosporine on the progression of human immunodeficiency the interferon beta-kappa B site. J Biol Chem 2002; 277: virus type 1 infection transmitted by transplantation—data on 24694–24700.

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