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    Topic review

    Retroviral Latency and Transcription Balance

    Subjects: Pathology
    View times: 19
    Submitted by: Aneta Pluta

    Definition

    The representative of the Lentivirus genus is the human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS). To date, there is no cure for AIDS because of the existence of the HIV-1 reservoir. HIV-1 infection can persist for decades de-spite effective antiretroviral therapy (ART), due to the persistence of infectious latent viruses in long-lived resting memory CD4+ T cells, macrophages, monocytes, microglial cells, and other cell types. However, the biology of HIV-1 latency remains incompletely understood. Retroviral long terminal repeat region (LTR) plays an indispensable role in controlling viral gene expression. Reg-ulation of the transcription initiation plays a crucial role in establishing and maintaining a retro-virus latency. Whether and how retroviruses establish latency and reactivate remains unclear.

    1. Introduction

    The human immunodeficiency virus type 1 (HIV-1) belongs to the family of Retro-viridae, subfamily Orthoretrovirinae, and genus Lentivirus. HIV¬-1 is firmly associated with the acquired immunodeficiency syndrome (AIDS) [1]. Highly pathogenic lentivi-ruses, after integration of double-stranded viral DNA into cellular genome, activate transcription of the viral genome. After synthesis of viral nucleic acid and formation of several viral proteins, to complete the viral life cycle, progeny virions are produced [2]. The efficiency of the initial transcription of integrated DNA from 5′ long terminal re-peat (LTR) region promoter determines the level of viral RNA in an infected cell. Pro-viral 5′ LTR promoter contains numerous cis-regulatory elements, which modulate the rate of viral transcription initiation. However, certain cell types and the cell differen-tiation processes with respect to diversity of cell activation signals may contribute to substantial variations in transcriptional activity of LTR [3]. All these variables gener-ate a remarkably broad range in HIV-1 gene expression level. Contrary to simple ret-roviruses (avian leukemia virus and murine leukemia virus), regulation of lentivirus gene expression involves both cellular and virally encoded regulatory factors. Conse-quently, RNA production in HIV-1 infection is highly variable.

    The latently infected cells are a source of viral reactivation and lead to marked increase of the viral load after a pause of highly active antiretroviral therapy (HAART). In this context, a better understanding of the molecular mechanisms re-sponsible for the regulation of proviral latency and reactivation would define rational strategies aimed at purging the HIV-1 reservoirs in treated patients. The regulation of gene expression in HIV-1 is complex and requires multiple steps, including chromatin organization, allowance of transcription machinery, mRNA processing and its transport to the cytoplasm, translation and posttranslational processes.

    2. LTR Regulatory Elements

    Retroviruses integrate into host DNA as proviruses that are flanked by LTRs at each end of the viral DNA. Transcription of proviral DNA is catalyzed by cellular RNA polymerase II (RNAPII) and initiated at the U3 end of 5ʹ LTR. Each LTR is composed of three regions: unique 3ʹ (U3), repeated (R), and unique 5ʹ (U5). U3 occupies most of the LTR and plays an important role in the induction of retroviral transcription, since it contains the viral promoters and other cis-active elements required for the modulation of promoter activity. The TATA box, located within the LTR promoter element, provides the binding site for RNAPII, determining the site of initiation and also affecting the efficiency of the initiation of transcription [4].

    The U3 region of HIV-1 LTR contains the crucial regulatory elements for the core promoter region: three specific protein 1 (Sp1) sites and TATA box; for the enhancer region: two nuclear factor-κB sites (NF-κB) and one nuclear factor of activated T-cells (NF-AT) site; for the modulatory region: three CCAAT/enhancer binding protein (C/EBP) sites, the activating transcription factor/cyclic AMP response element binding (ATF/CREB) region, two NF-AT sites, two activator protein 1 (AP-1) sites, one upstream stimulatory factor Ets/PU.1, and one T-cell specific transcription factor/lymphoid enhancer binding factor (TCF/LEF-1) [5][6][7][8].

    In HIV-1, the following regulatory sequences downstream of the transcription start site are as follows: the initiator (Inr), the inducer of short transcripts (ITS), and trans-activation responsive element (TAR). TAR forms an RNA stem-loop structure, which recruits the virally encoded transactivator protein (Tat) to the LTR to modulate the activity of the viral promoter [1]. In addition, HIV-1 LTR consists of several substantial transcription factor (TF) binding sites including AP-1 sites, an AP-3-like (AP-3L) sequence, C/EBP/NFAT (nuclear factor for activated T cells) downstream binding site (DS3), two downstream sequence element (DSE) sites, one downstream binding factor (DBF-1) in R region, and two Sp1 binding sites and gag leader sequence (GLS) in the U5 [9]. Enhancer functions have been also mapped to the gagpol regions of simian immunodeficiency virus (SIV) and HIV, but their role in the virus replication has yet to be established.

    The transcription of Lentiviruses is regulated by the interactions between numerous and different viral proteins and transcription factors with binding sites located in the 5′ LTR. Most of regulatory elements encompass the U3 region. Regulatory elements situated in R and U5 regions may improve the promoter and enhancer strengths and provide a broad viral response for stimulating factors and control transcription in cell-type-dependent manner.

    3. A Variety of Enhancers with Regulatory Functions

    The HIV-1 mainly infects CD4+ T cells, monocytes, and macrophages, and in a lower proportion also dendritic cells (DCs) and microglial cells. HIV-1 enhancer sequence consists of two NF-ĸB binding sites and three adjacent Sp1 binding sites that are required for viral transcription [5]. Other factors shown to bind the enhancer include Ets, PU.1, NF-AT, C/EBP, AP-1, cAMP response element-binding protein/ activating transcription factor (CREB/ATF), upstream stimulatory factor (USF), Sp1, Sp3 and chicken ovalbumin upstream promoter transcription factor (COUP-TF) and they play role in enhancing the transcription (Table 1).

    Table 1. Key transcription factors involved in regulation of human immunodeficiency virus type 1 (HIV-1) transcription in different cell types.

    Transcription Factor

    Cell Type

    NF-κB

     T cells*, monocytes, macrophages, iDC, microglial cells

    NF-AT

    T cells

    Sp1

    microglial cells, T cells, monocytes, macrophages, iDC

    Sp3

    microglial cells, monocytes, macrophages

    AP-1

    microglial cells, monocytes, T cells

    COUP-TF

    microglial cells, T cells

    Ets-1

    T cells

    USF

    monocytes, macrophages, iDC, T cells, microglial cells

    C/EBP (NF-IL-6)

    monocytes, macrophages, iDC, T cells, microglial cells

    CREB/ATF

    T cells, microglial cells, monocytes, macrophages

    * transcription factors required for transcriptional activation in cell-type-specific expression of HIV-1; NF-κB, nuclear factor kappa-light-chain-enhancer of activated B cells; NF-AT, nuclear factor of activated T-cells; Sp1, 3, specific protein 1, 3; AP-1, activator protein 1; COUP-TF, chicken ovalbumin upstream promoter transcription factor; Ets-1, E26 transformation-specific (ETS) transcription factor; USF, upstream stimulatory factor; C/EBP, CCAAT/enhancer-binding protein; NF-IL-6, transcription factor nuclear factor interleukin 6; CREB/ATF, cAMP response element-binding protein/ activating transcription factor.

    This variety of binding sites may result in maintenance reverse latency in some cells. As an example, NF-κB transcription factor binding to enhancer sites within LTR activate viral transcription in most HIV-1-infected types of cells [10]. The transcriptional activity of the NF-κB and other transcription factors in primary immune cells versus transformed cell lines is listed in Table 2.

    Table 2. Regulation of HIV-1 gene transcription in primary immune cells and transformed cell lines.

    Transcription Factor

    Cell Type

    Primary Cells

    Transformed Cell Line

     

     

     

    NF-κB

    T cells

    • activates transcription in dopamine-stimulated PBMCs [11]

    • activates transcription in CD4+ T cells by direct occupancy of enhancer by NF-κB p50/p65 [12]

    • activates transactivation in TNF-, IL-1-, and IL-7-stimulated TEC co-cultured with thymocytes [13]

    • activates transcription in dopamine-stimulated lymphoid Jurkat T cell line [11]

    • activates transcription in Jurkat T cell line that stably expresses the Tat [14]

    • activates transcription in latently HIV-1-infected established T lymphoid cell line J1.1 promoted by MRPs [15]

    monocytes/macrophages

    • activates transcription in macrophages by direct occupancy of enhancer by NF-κB p50/p65 [16]

    • involved in efficient activation of viral transcription in monocytes isolated from PBMC [17]

    • activates HIV gene transcription in monocytic cell line U937 and promonocytic cell U1 by direct occupancy of enhancer by NF-κB p50/p65 [18][19]

    microglial cells

    nd

    • activates transcription in human microglial MC-3 cell line and embryonic microglial cell line upon stimulation with IFNγ, IL1β, and TNFα [20][21][22]

     

     

    NF-AT

     

     

    T cells

    • enhances activation of transcription in CD4+ T cells 

    • NF-AT1,2 enhances activation of transcription in PMA/ionomycin stimulated CD4+ T cells [23]

    • NFAT1, 2 positive effect on transcription in PMA-, PHA-, bpV-stimulated PBMC [24]

    efficient binding to the HIV-1 LTR enhancer in Jurkat-derived CD4+ T cells isoform CD45(−), stimulated with PMA/PHA/α-CD3 [25]

    • represses Tat-mediated transactivation in PMA/ionomycin-stimulated Jurkat T-cells  [26]

    • NFAT1, 2 enhances transcription in Jurkat T cells stimulated with PMA, PHA and bpV [24]

     

    Sp1, 3

     

    microglial cells

    nd

    • Sp1 interaction with COUP-TF leads to activation of HIV gene transcription in microglial cell line [18]

    • binding CTIP-2 to Sp1 represses Tat-mediated transcriptional activation HIV promoter [27]

    • Sp3 represses Sp1 and COUP-TF-induced activation in human microglial cell line [18]

    T cells

    • Sp1 associated with Tat activates transcription in CD4+ T cells and PBMCs [28]

     

     

     

    • Tat-induced Sp1 activates promoter in MT-2 cell line and Jurkat T cells [28]

    • Sp1 assembly pre-initiation complex at the LTR TATA box and cooperatively interacts with NF-κB to activate transcription in Jurkat T cells stimulated with PMA[29]

    monocytes/macrophages

    • Sp1-to-Sp3 ratio increases during monocyte lineage differentiation, resulting in increased HIV-1 transcription [30]

    • Sp1 activates LTR-driven transcription in U1 monocytic cells [31][35]

    • Sp1 has moderate impact on transcription activation in human monocytic line U-937 [32]

    iDC

    • Sp1 activates HIV gene transcription in DC differentiated from monocytes derived from PBMCs [35]

    nd

     

     

    AP-1

    microglial cells

    nd

    • c-jun and c-fos interact with TRE sequence and enhance HIV-1 gene transcription in glial cells [34]

     

    monocytes/

    macrophages

    • Vpr-activated AP-1 enhances viral transcription in macrophages differentiated from PBMCs [35]

     

    • Vpr-activated AP-1 enhances viral transcription in U937 cells [35]

    • Nuclear complex of c-fos and c-jun binds directly to the HIV LTR and enhances NF-κB activity in human monocytic cell lines U1 and U937 [36]

    • AP-1 activated by Nef stimulates HIV transcription in U1 and U937 cells [37]

    T cells

    • enhances HIV-1 gene expression in CBMCs more than in PBMCs [38]

    • c-jun and c-fos do not interact with TRE sequence and do not enhance HIV-1 transcription in Jurkat T cells [39]

     

     

    COUP-TF

     

     

    microglial cells

    • cooperates with Tat to promote NF-κB- and Sp1-independent transactivation HIV-1 transcription in human fetal microglial cells [40]

     

    • cooperates with Tat and promotes NF-κB and Sp1-independent activation HIV-1 transcription in microglial cell line [40]

    • COUP-TF Sp1 interaction stimulates HIV transcription in microglial cell line 

    • COUP-TF, Sp1, and CTIP2 cooperation suppresses HIV transcription initiation in microglial cells [41]

    T cells

    nd

    • COUP-TF interaction with Sp1 synergistically stimulates viral transcription in Jurkat T cells in response to cAMP and dopamine [42]

    Ets

    T cells

    • Ets in cooperation with NF-kB/NFAT activates HIV-1 enhancer in human peripheral blood T cells [43]

    • Ets in cooperation with USF-1 enhances transcriptional activity of HIV-1 LTR in Jurkat T cells [44]

     

     

    C/EBP

    (NF-IL-6)

     

    monocytes/macrophages

    • regulates HIV transcription by recruiting HATs to the LTR in primary macrophages [45]

    • recruits HATs to LTR and mediates initiation of transcription in promonocytic U937 cells [46]

    T cells

    nd

    • is not required in HIV transcription in Jurkat CD4+ T cell line [45]

    • cooperates with CREB and mediates prostaglandin E2-induced stimulation of LTR-driven transcription Jurkat E6.1 [47]

    microglial cells

    nd

    • in presence of IL-1, IL-6, and TNF- α, activates LTR-driven transcription versus C/EBPγ that acts as inhibitor [48]

     

     

    CREB

    T cells

     

    • phospho-CREB recruits CBP and basal transcription factors, which increases promoter activation in primary lymphocytes [49]

    • phospho-CREB recruits CBP and basal transcription factors, which increases promoter activation in MT-4 human T cell line [50]

    • mediates cAMP and dopamine-induced transcriptional stimulation through indirect interactions with LTR in Jurkat T cells [42]

    • cooperates with COUP-TF in the presence of forskolin, cAMP, and dopamine to activate HIV-1 gene transcription in Jurkat T cells [42]

    monocytes/macrophages

    nd

    • CREB homodimers bind to their DNA site, interact with C/EBPs, and lead to increase HIV promoter activation in U-937 and THP-1 human monocytic cell lines; sequence variations at the CREB site affect LTR activity [51]

    nd, not determined; TEC, human thymic epithelial cell; MRPs, proinflammatory myeloid-related proteins; PBMC, peripheral blood mononuclear cell; NF-AT, nuclear factor of activated T cells; PMA, Phorbol 12-myristate 13-acetate; NF-κB, nuclear factor-kappa B; PHA, phytohemagglutinin; bpV, bis-peroxovana-dium a protein tyrosine phosphatases (PTP) inhibitor; COUP-TF, chicken ovalbumin upstream promoter transcription factor; MT-2, cell line derived from normal human cord leukocytes cocultivated with leukemic cells from an adult T cell leukemia (ATL) patient; CBMCs, umbilical cord blood mononuclear cells; CTIP2, Chicken ovalbumin upstream promoter transcription factor interacting protein 2; cAMP, cyclic AMP, adenosine 3’,5’-cyclic monophosphate; HATs, histone acetylotransferase; Ets, erythroblast transformation specific transcription factor; Sp1, transcription factor specificity protein 1; AP-1, activator protein; C/EBP, CCAAT/enhancer-binding protein; NF-IL-6, transcription factor nuclear factor interleukin 6; CREB, cAMP response element-binding protein; CBP, CREB binding protein.[53]

    In activated CD4+ T lymphocytes, the Sp1 transcription factors are not sufficient to mediate transcription and further binding NF-ĸB and NF-AT cellular factors to the LTR enhancer region is required to activate transcription. In addition, the USF, Ets, NF-IL-6 and CREB proteins facilitate efficient transcription. In long-lived latently infected CD4+ T cells, NF-ĸB and NF-AT, as key factors for initiation of HIV-1 transcription in these cells, are present in very low nuclear concentrations. In addition, Cyclin T1 protein levels are also very low in comparison to activated T cells. For that reason, the above mechanisms have been proposed to be probably involved in CD4+ T cell latency [52].

    In monocyte–macrophage lineage cells, regulation of HIV-1 transcription varies considerably during macrophage differentiation, as numerous transcription factors are expressed in a differentiation-dependent manner. In monocytes, LTR activity may be regulated during their differentiation stages by changes in the Sp1 (activator):Sp3 (repressor) ratio.

    Increased permissiveness of macrophages for HIV-1 replication leads to expression of the cofactors utilized for Tat transactivation of the LTR, and this leads to a high level of HIV-1 transcription. There are numerous studies supporting that microglial cells are susceptible to HIV-1 infection and can be latently infected, constituting a major reservoir in the brain. In contrast to the monocytes, NF-κB, AP-1, and NFAT proteins are constitutively localized in the nucleus of microglial cells, and the Sp1 expression predominates over the Sp3. Interestingly, latently infected microglial cells can be reactivated by cytokine stimulation. In contrast to other reservoirs, the NF-kB and Sp1 binding sites are sufficient for HIV-1 transcription in microglial cells [53]. Contrary to CD4+ T cells, which express only Sp1, microglial cells produce both Sp1 and Sp3; the latter acting as transcriptional repressor. In addition, C/EBPɤ is expressed and acts as repressor by competing with the transcriptional activator C/EBP (Table 2) [54].

    To conclude, the LTRs play a significant role in cell-type-specific expression of the proviral genome. HIV-1 enhancer sequences contain many binding sites providing mechanisms for a broad viral response to extracellular factors and regulate transcription in the cell-type-dependent manner. These observations thus emphasize the differences in mechanisms underlying HIV-1 latency between infected cells.

    4. Transactivation of LTR by Virus-Encoded Tat Protein

    Lentiviruses are capable of promoting the rate of their gene expression through virus-encoded transactivator proteins. Activation occurs by binding of Tat HIV-1 protein to a specific sequence adjacent to 5′ trans-activation response (TAR) element RNA transcript [55][56][57]. Tat protein of HIV-1 (and related Lentiviruses) interacts with the viral RNA transcript, through a unique RNA regulatory segment of the LTR termed transactivation-responsive element (TAR). The TAR secondary RNA structure is formed from transcription of the +19–43 tract in the LTR R region [58]. Various mechanisms of HIV-1 Tat transactivation have been proposed. One model suggests overriding transcription terminations, since in the absence of Tat transcripts that initiate in LTR pause after synthesis of about 70 nucleotides. It has also been proposed that in early steps of viral transcription, the complex of positive transcription elongation factor b (P-TEFb) composed of Cyclin T1 (CycT1) and cyclin-dependent kinase 9 (CDK9) is recruited to the LTR via nuclear factor kappa B (NF-κB). The recruitment of Tat and P-TEFb to the TAR hairpin facilitates phosphorylation of RNAP II, which increases their combined effectiveness and prevents premature termination [58][59]. On the other hand, several investigations revealed that NF-κB can promote both transcription initiation and elongation complex, at a similar level to that of Tat, in a manner independent of Tat. The NF-κB transcription factors induce LTR regulation via interaction with binding sites located within the enhancer region [59]. Deletion of the NF-κB binding sites strongly reduces basal, as well as Tat-transactivated, LTR activity. The Tat proteins activate NF-κB through a IκB kinase (IKK), which accelerates the degradation of IκB, a protein that regulates NF-κB activity by binding NF-κB and translocating to the nucleus [59][60]. In vitro model systems support an alternative hypothesis where Tat initiates transcription through a protein–protein interaction with the Sp1 transcription factor. This paradigm is supported by findings that nucleotide changes within the cis-acting elements recruiting Sp factors to the HIV-1 LTR reduce Tat-mediated LTR activity [60].

    Additionally,viral protein R (Vpr) is another viral accessory protein capable of enhancing the activity of the HIV-1 LTR. Vpr can bind to histone acetyltransferases (HAT)  CREB-binding protein and p300, glucocorticoid receptor, CycT1, and Tat to activate transcription [61][62]. Vpr can also activate NF-κB-directed transcription (reviewed in [1]). HIV-1 LTR C/EBP and NF-κB complex demonstrates a high affinity for Vpr and a low affinity for C/EBPβ during late-stage HIV in brain cells from patients with HIV-associated dementia (HAD) [61][62]. In addition, Kilareski and co-workers identified specific Tat variants derived from HAD brain, which were defective in LTR transactivation, however still were able to activate promoters of the other proinflammatory cytokine genes. Collectively, in the tissues of the brain, Tat may become less transcriptionally competent, however, in this situation, Vpr may facilitate HIV-1 replication by enhancing transcription in the absence of a fully active Tat. On the other hand, Razooky and co-workers suggested that Tat can control a viral reservoir in infected resting and memory CD4+ T cells, even if the Tat level in these cells is low. They found that Tat mutants exaggerated lower levels of HIV-1 expression in the resting cells [63][64]. In addition, Chakraborty and co-workers data indicated that Tat promotes latency by generating a negative feedback loop at later stages of infection, which leads to the silencing of HIV-1 promoter [65].

    The primary function attributed to Tat is the transactivation of HIV-1 promoter. Additionally, it has been demonstrated that Tat enhances HIV-1 virulence by interacting with various cellular proteins in order to induce T cell apoptosis, co-receptor regulation, and cytokine induction in the host cells [66][67][68]. The effect of Tat on many viral activities in the host cell contributes to the pathogenesis of HIV-1, pointing to this molecule as a potential target for HIV-1 therapy, for example, by blocking viral replication by targeting Tat [69][70][71][72]. The Tat naturally occurring polymorphisms are usually caused by viral mutational escape from CD8+ cytotoxic T lymphocyte (CTL) recognition. The host immune responses mediated by CTLs and less by CD4+ T lymphocytes and B lymphocytes may potentially force selective pressure towards Tat diversity and affect its activity [73][74]. It has been proposed that variations in Tat sequence could modulate transactivation and have implications on HIV-1 latency and the reactivation phase. Ronsard and co-workers reported that the Tat variants with a change of S46F were able to significantly enhance LTR transactivation compared with wild-type Tat [75]. Additionally, the change of S46F caused strong Tat interaction with TAR in in vitro and in silico models. In contrast, a naturally occurring change of the C22S in HIV-1 Oyi strain reduced Tat transactivation activity and was linked with long-term nonprogressive infections [76]. Furthermore, other naturally occurring polymorphisms within Tat identified in HIV-infected patients at acute and/or early infection phase (i.e., P10S, W11R, K19R, A42V, and Y47H) have been shown to significantly impair transactivation activity in the infected CD4+ T lymphocytes [77]. These data suggest that certain naturally occurring changes can change Tat transactivation activity.

    The infected lymphocytes rapidly produce great numbers of viral particles, and it is clear that Tat protein triggers this process. Clones with nonsense changes are unable to replicate and thereby disappear from the spectra in vivo. However, as the infection progresses, some naturally occurring changes in Tat can change its immunogenic properties, prevent transactivation, and may influence viral latency. Nevertheless, it remains unclear to what extent CTL escape changes occurring in the Tat epitope may affect the HIV-1 latency kinetic from establishment to reversal stages [78].

    This entry is adapted from 10.3390/pathogens10010016

    References

    1. Kurth, R.; Bannert, N. Retroviruses: Molecular Biology, Genomics and Pathogenesis; Caister Academic Press: Norfolk, UK, 2010; p. 454.
    2. Coffin, J.; Swanstrom, R. HIV pathogenesis: Dynamics and genetics of viral populations and infected cells. Cold Spring Harb. Perspect. Med. 2013, 3, a012526, doi:10.1101/cshperspect.a012526.
    3. Coffin, J.M.; Hughes, S.H.; Varmus, H.E. Retroviruses. In Retroviruses, Coffin, J.M., Hughes, S.H., Varmus, H.E., Eds.; Cold Spring Harbor (New York): New York, NY, USA, 1997.
    4. Diaz, R.M.; Eisen, T.; Hart, I.R.; Vile, R.G. Exchange of viral promoter/enhancer elements with heterologous regulatory se-quences generates targeted hybrid long terminal repeat vectors for gene therapy of melanoma. J. Virol. 1998, 72, 789–795.
    5. Schiralli Lester, G.M.; Henderson, A.J. Mechanisms of HIV Transcriptional Regulation and Their Contribution to Latency. Mol. Biol. Int. 2012, 2012, 614120, doi:10.1155/2012/614120.
    6. Rohr, O.; Marban, C.; Aunis, D.; Schaeffer, E. Regulation of HIV-1 gene transcription: From lymphocytes to microglial cells. J. Leukoc. Biol. 2003, 74, 736–749, doi:10.1189/jlb.0403180.
    7. Pereira, L.A.; Bentley, K.; Peeters, A.; Churchill, M.J.; Deacon, N.J. A compilation of cellular transcription factor interactions with the HIV-1 LTR promoter. Nucleic Acids Res. 2000, 28, 663–668, doi:10.1093/nar/28.3.663.
    8. Wu, Y.; Marsh, J.W. Gene transcription in HIV infection. Microbes Infect. 2003, 5, 1023–1027, doi:10.1016/s1286-4579(03)00187-4.
    9. Dahiya, S.; Liu, Y.; Williams, J.; Pirrone, V.; Nonnemacher, M.R.; Wigdahl, B. Role of Downstream Elements in Transcrip-tional Regulation of the HIV-1 Promoter. J. Hum. Virol. Retrovirol. 2014, 1, 00006, doi:10.15406/jhvrv.2014.01.00006.
    10. Chan, J.K.; Greene, W.C. NF-kappaB/Rel: Agonist and antagonist roles in HIV-1 latency. Curr. Opin. HIV AIDS 2011, 6, 12–18, doi:10.1097/COH.0b013e32834124fd.
    11. Rohr, O.; Sawaya, B.E.; Lecestre, D.; Aunis, D.; Schaeffer, E. Dopamine stimulates expression of the human immunodefi-ciency virus type 1 via NF-kappaB in cells of the immune system. Nucleic Acids Res. 1999, 27, 3291–3299, doi:10.1093/nar/27.16.3291.
    12. Bosque, A.; Planelles, V. Induction of HIV-1 latency and reactivation in primary memory CD4+ T cells. Blood 2009, 113, 58–65, doi:10.1182/blood-2008-07-168393.
    13. Chene, L.; Nugeyre, M.T.; Barre-Sinoussi, F.; Israel, N. High-level replication of human immunodeficiency virus in thymo-cytes requires NF-kappaB activation through interaction with thymic epithelial cells. J. Virol. 1999, 73, 2064–2073, doi:10.1128/JVI.73.3.2064-2073.1999.
    14. Fiume, G.; Vecchio, E.; De Laurentiis, A.; Trimboli, F.; Palmieri, C.; Pisano, A.; Falcone, C.; Pontoriero, M.; Rossi, A.; Scial-done, A.; et al. Human immunodeficiency virus-1 Tat activates NF-kappaB via physical interaction with IkappaB-alpha and p65. Nucleic Acids Res. 2012, 40, 3548–3562, doi:10.1093/nar/gkr1224.
    15. Ryckman, C.; Robichaud, G.A.; Roy, J.; Cantin, R.; Tremblay, M.J.; Tessier, P.A. HIV-1 transcription and virus production are both accentuated by the proinflammatory myeloid-related proteins in human CD4+ T lymphocytes. J. Immunol. 2002, 169, 3307–3313, doi:10.4049/jimmunol.169.6.3307.
    16. McElhinny, J.A.; MacMorran, W.S.; Bren, G.D.; Ten, R.M.; Israel, A.; Paya, C.V. Regulation of I kappa B alpha and p105 in monocytes and macrophages persistently infected with human immunodeficiency virus. J. Virol. 1995, 69, 1500–1509, doi:10.1128/JVI.69.3.1500-1509.1995.
    17. Palmieri, C.; Trimboli, F.; Puca, A.; Fiume, G.; Scala, G.; Quinto, I. Inhibition of HIV-1 replication in primary human mono-cytes by the IkappaB-alphaS32/36A repressor of NF-kappaB. Retrovirology 2004, 1, 45–45, doi:10.1186/1742-4690-1-45.
    18. Jacqué, J.M.; Fernández, B.; Arenzana-Seisdedos, F.; Thomas, D.; Baleux, F.; Virelizier, J.L.; Bachelerie, F. Permanent occu-pancy of the human immunodeficiency virus type 1 enhancer by NF-kappa B is needed for persistent viral replication in monocytes. J. Virol. 1996, 70, 2930–2938, doi:10.1128/jvi.70.5.2930-2938.1996.
    19. Griffin, G.E.; Leung, K.; Folks, T.M.; Kunkel, S.; Nabel, G.J. Activation of HIV gene expression during monocyte differentia-tion by induction of NF-kB. Nature 1989, 339, 70–73, doi:10.1038/339070a0.
    20. Janabi, N.; Peudenier, S.; Héron, B.; Ng, K.H.; Tardieu, M. Establishment of human microglial cell lines after transfection of primary cultures of embryonic microglial cells with the SV40 large T antigen. Neurosci. Lett. 1995, 195, 105–108, doi:10.1016/0304-3940(94)11792-h.
    21. Janabi, N.; Di Stefano, M.; Wallon, C.; Hery, C.; Chiodi, F.; Tardieu, M. Induction of human immunodeficiency virus type 1 replication in human glial cells after proinflammatory cytokines stimulation: Effect of IFNgamma, IL1beta, and TNFalpha on differentiation and chemokine production in glial cells. Glia 1998, 23, 304–315.
    22. Albright, A.V.; Shieh, J.T.; O’Connor, M.J.; González-Scarano, F. Characterization of cultured microglia that can be infected by HIV-1. J. Neurovirol. 2000, 6 (Suppl. S1), S53–S60.
    23. Cron, R.Q.; Bartz, S.R.; Clausell, A.; Bort, S.J.; Klebanoff, S.J.; Lewis, D.B. NFAT1 Enhances HIV-1 Gene Expression in Pri-mary Human CD4 T Cells. Clinical Immunology 2000, 94, 179–191, doi:10.1006/clim.1999.4831.
    24. Fortin, J.F.; Barbeau, B.; Robichaud, G.A.; Pare, M.E.; Lemieux, A.M.; Tremblay, M.J. Regulation of nuclear factor of activated T cells by phosphotyrosyl-specific phosphatase activity: A positive effect on HIV-1 long terminal repeat-driven transcription and a possible implication of SHP-1. Blood 2001, 97, 2390–2400, doi:10.1182/blood.v97.8.2390.
    25. Robichaud, G.A.; Barbeau, B.; Fortin, J.F.; Rothstein, D.M.; Tremblay, M.J. Nuclear factor of activated T cells is a driving force for preferential productive HIV-1 infection of CD45RO-expressing CD4+ T cells. J. Biol. Chem. 2002, 277, 23733–23741, doi:10.1074/jbc.M201563200.
    26. Macián, F.; Rao, A. Reciprocal modulatory interaction between human immunodeficiency virus type 1 Tat and transcription factor NFAT1. Mol. Cell Biol. 1999, 19, 3645–3653, doi:10.1128/mcb.19.5.3645.
    27. Rohr, O.; Lecestre, D.; Chasserot-Golaz, S.; Marban, C.; Avram, D.; Aunis, D.; Leid, M.; Schaeffer, E. Recruitment of Tat to heterochromatin protein HP1 via interaction with CTIP2 inhibits human immunodeficiency virus type 1 replication in micro-glial cells. J. Virol. 2003, 77, 5415–5427, doi:10.1128/jvi.77.9.5415-5427.2003.
    28. Sancho, R.; Márquez, N.; Gómez-Gonzalo, M.; Calzado, M.A.; Bettoni, G.; Coiras, M.T.; Alcamí, J.; López-Cabrera, M.; Ap-pendino, G.; Muñoz, E. Imperatorin inhibits HIV-1 replication through an Sp1-dependent pathway. J. Biol. Chem. 2004, 279, 37349–37359, doi:10.1074/jbc.M401993200.
    29. Perkins, N.D.; Edwards, N.L.; Duckett, C.S.; Agranoff, A.B.; Schmid, R.M.; Nabel, G.J. A cooperative interaction between NF-kappa B and Sp1 is required for HIV-1 enhancer activation. EMBO J. 1993, 12, 3551–3558.
    30. Kilareski, E.M.; Shah, S.; Nonnemacher, M.R.; Wigdahl, B. Regulation of HIV-1 transcription in cells of the mono-cyte-macrophage lineage. Retrovirology 2009, 6, 118, doi:10.1186/1742-4690-6-118.
    31. Demarchi, F.; D’Agaro, P.; Falaschi, A.; Giacca, M. In vivo footprinting analysis of constitutive and inducible protein-DNA interactions at the long terminal repeat of human immunodeficiency virus type 1. J Virol. 1993, 67, 7450–7460, doi:10.1128/jvi.67.12.7450-7460.1993.
    32. McAllister, J.J.; Phillips, D.; Millhouse, S.; Conner, J.; Hogan, T.; Ross, H.L.; Wigdahl, B. Analysis of the HIV-1 LTR NF-kappaB-proximal Sp site III: Evidence for cell type-specific gene regulation and viral replication. Virology 2000, 274, 262–277, doi:10.1006/viro.2000.0476.
    33. Bergamaschi, A.; Pancino, G. Host hindrance to HIV-1 replication in monocytes and macrophages. Retrovirology 2010, 7, 31, doi:10.1186/1742-4690-7-31.
    34. Canonne-Hergaux, F.; Aunis, D.; Schaeffer, E. Interactions of the transcription factor AP-1 with the long terminal repeat of different human immunodeficiency virus type 1 strains in Jurkat, glial, and neuronal cells. J. Virol. 1995, 69, 6634–6642, doi:10.1128/jvi.69.11.6634-6642.1995.
    35. Varin, A.; Decrion, A.Z.; Sabbah, E.; Quivy, V.; Sire, J.; Van Lint, C.; Roques, B.P.; Aggarwal, B.B.; Herbein, G. Synthetic Vpr protein activates activator protein-1, c-Jun N-terminal kinase, and NF-kappaB and stimulates HIV-1 transcription in promonocytic cells and primary macrophages. J. Biol. Chem. 2005, 280, 42557–42567, doi:10.1074/jbc.M502211200.
    36. Yang, X.; Chen, Y.; Gabuzda, D. ERK MAP kinase links cytokine signals to activation of latent HIV-1 infection by stimulating a cooperative interaction of AP-1 and NF-kappaB. J. Biol. Chem. 1999, 274, 27981–27988, doi:10.1074/jbc.274.39.27981.
    37. Varin, A.; Manna, S.K.; Quivy, V.; Decrion, A.Z.; Van Lint, C.; Herbein, G.; Aggarwal, B.B. Exogenous Nef protein activates NF-kappa B, AP-1, and c-Jun N-terminal kinase and stimulates HIV transcription in promonocytic cells. Role in AIDS path-ogenesis. J. Biol. Chem. 2003, 278, 2219–2227, doi:10.1074/jbc.M209622200.
    38. Sundaravaradan, V.; Saxena, S.K.; Ramakrishnan, R.; Yedavalli, V.R.; Harris, D.T.; Ahmad, N. Differential HIV-1 replication in neonatal and adult blood mononuclear cells is influenced at the level of HIV-1 gene expression. Proc. Natl. Acad. Sci. USA 2006, 103, 11701–11706, doi:10.1073/pnas.0602185103.
    39. Li, Y.; Mak, G.; Franza, B.R., Jr. In vitro study of functional involvement of Sp1, NF-kappa B/Rel, and AP1 in phorbol 12-myristate 13-acetate-mediated HIV-1 long terminal repeat activation. J Biol. Chem. 1994, 269, 30616–30619.
    40. Rohr, O.; Schwartz, C.; Hery, C.; Aunis, D.; Tardieu, M.; Schaeffer, E. The nuclear receptor chicken ovalbumin upstream promoter transcription factor interacts with HIV-1 Tat and stimulates viral replication in human microglial cells. J. Biol. Chem. 2000, 275, 2654–2660, doi:10.1074/jbc.275.4.2654.
    41. Marban, C.; Suzanne, S.; Dequiedt, F.; de Walque, S.; Redel, L.; Van Lint, C.; Aunis, D.; Rohr, O. Recruitment of chroma-tin-modifying enzymes by CTIP2 promotes HIV-1 transcriptional silencing. EMBO J. 2007, 26, 412–423, doi:10.1038/sj.emboj.7601516.
    42. Rohr, O.; Schwartz, C.; Aunis, D.; Schaeffer, E. CREB and COUP-TF mediate transcriptional activation of the human im-munodeficiency virus type 1 genome in Jurkat T cells in response to cyclic AMP and dopamine. J. Cell Biochem. 1999, 75, 404–413.
    43. Bassuk, A.G.; Anandappa, R.T.; Leiden, J.M. Physical interactions between Ets and NF-kappaB/NFAT proteins play an im-portant role in their cooperative activation of the human immunodeficiency virus enhancer in T cells. J. Virol. 1997, 71, 3563–3573, doi:10.1128/jvi.71.5.3563-3573.1997.
    44. Sieweke, M.H.; Tekotte, H.; Jarosch, U.; Graf, T. Cooperative interaction of ets-1 with USF-1 required for HIV-1 enhancer activity in T cells. EMBO J. 1998, 17, 1728–1739, doi:10.1093/emboj/17.6.1728.
    45. Henderson, A.J.; Calame, K.L. CCAAT/enhancer binding protein (C/EBP) sites are required for HIV-1 replication in primary macrophages but not CD4(+) T cells. Proc. Natl. Acad. Sci. USA 1997, 94, 8714–8719, doi:10.1073/pnas.94.16.8714.
    46. Henderson, A.J.; Connor, R.I.; Calame, K.L. C/EBP activators are required for HIV-1 replication and proviral induction in monocytic cell lines. Immunity 1996, 5, 91–101, doi:10.1016/s1074-7613(00)80313-1.
    47. Dumais, N.; Bounou, S.; Olivier, M.; Tremblay, M.J. Prostaglandin E2-Mediated Activation of HIV-1 Long Ter-minal Repeat Transcription in Human T Cells Necessitates CCAAT/Enhancer Binding Protein (C/EBP) Binding Sites in Addi-tion to Cooperative Interactions Between C/EBPβ and Cyclic Adenosine 5′-Monophosphate Response Element Binding Pro-tein. J. Immunol. 2002, 168, 274–282, doi:10.4049/jimmunol.168.1.274.
    48. Schwartz, C.; Catez, P.; Rohr, O.; Lecestre, D.; Aunis, D.; Schaeffer, E. Functional interactions between C/EBP, Sp1, and COUP-TF regulate human immunodeficiency virus type 1 gene transcription in human brain cells. J. Virol. 2000, 74, 65–73, doi:10.1128/jvi.74.1.65-73.2000.
    49. Hofmann, B.; Nishanian, P.; Nguyen, T.; Liu, M.; Fahey, J.L. Restoration of T-cell function in HIV infection by reduction of intracellular cAMP levels with adenosine analogues. Aids 1993, 7, 659–664, doi:10.1097/00002030-199305000-00008.
    50. Nokta, M.; Pollard, R. Human immunodeficiency virus infection: Association with altered intracellular levels of cAMP and cGMP in MT-4 cells. Virology 1991, 181, 211–217, doi:10.1016/0042-6822(91)90486-u.
    51. Ross, H.L.; Nonnemacher, M.R.; Hogan, T.H.; Quiterio, S.J.; Henderson, A.; McAllister, J.J.; Krebs, F.C.; Wigdahl, B. Interac-tion between CCAAT/enhancer binding protein and cyclic AMP response element binding protein 1 regulates human im-munodeficiency virus type 1 transcription in cells of the monocyte/macrophage lineage. J. Virol. 2001, 75, 1842–1856, doi:10.1128/jvi.75.4.1842-1856.2001.
    52. Budhiraja, S.; Famiglietti, M.; Bosque, A.; Planelles, V.; Rice, A.P. Cyclin T1 and CDK9 T-loop phosphorylation are downreg-ulated during establishment of HIV-1 latency in primary resting memory CD4+ T cells. J. Virol. 2013, 87, 1211–1220, doi:10.1128/JVI.02413-12.
    53. Ross, H.L.; Gartner, S.; McArthur, J.C.; Corboy, J.R.; McAllister, J.J.; Millhouse, S.; Wigdahl, B. HIV-1 LTR C/EBP binding site sequence configurations preferentially encountered in brain lead to enhanced C/EBP factor binding and increased LTR-specific activity. J. Neurovirol. 2001, 7, 235–249, doi:10.1080/13550280152403281.
    54. Li, Y.; Kappes, J.C.; Conway, J.A.; Price, R.W.; Shaw, G.M.; Hahn, B.H. Molecular characterization of human immunodefi-ciency virus type 1 cloned directly from uncultured human brain tissue: Identification of replication-competent and -defective viral genomes. J. Virol. 1991, 65, 3973–3985.
    55. Liu, Y.; Nonnemacher, M.R.; Alexaki, A.; Pirrone, V.; Banerjee, A.; Li, L.; Kilareski, E.; Wigdahl, B. Functional Studies of CCAAT/Enhancer Binding Protein Site Located Downstream of the Transcriptional Start Site. Clin. Med. Insights Pathol. 2017, 10, 1179555717694556, doi:10.1177/1179555717694556.
    56. Tan, J.; Hao, P.; Jia, R.; Yang, W.; Liu, R.; Wang, J.; Xi, Z.; Geng, Y.; Qiao, W. Identification and functional characterization of BTas transactivator as a DNA-binding protein. Virology 2010, 405, 408–413, doi:10.1016/j.virol.2010.05.037.
    57. Derse, D. Bovine leukemia virus transcription is controlled by a virus-encoded trans-acting factor and by cis-acting response elements. J. Virol. 1987, 61, 2462–2471.
    58. Parada, C.A.; Roeder, R.G. Enhanced processivity of RNA polymerase II triggered by Tat-induced phosphorylation of its carboxy-terminal domain. Nature 1996, 384, 375–378, doi:10.1038/384375a0.
    59. Roebuck, K.A.; Saifuddin, M. Regulation of HIV-1 transcription. Gene Expr. 1999, 8, 67–84.
    60. Kreb, F.C.; Hogan, T.H.; Quiterio, S.; Gartner, S.; Wigdahl, B. Lentiviral LTR-directed Expression, Sequence Variation, and Disease Pathogenesis. Available online: https://www.hiv.lanl.gov/content/sequence/HIV/REVIEWS/WIGDAHL2001/Wigdahl.html (accessed on 1 December 2020).
    61. Siddappa, N.B.; Venkatramanan, M.; Venkatesh, P.; Janki, M.V.; Jayasuryan, N.; Desai, A.; Ravi, V.; Ranga, U. Transactiva-tion and signaling functions of Tat are not correlated: Biological and immunological characterization of HIV-1 subtype-C Tat protein. Retrovirology 2006, 3, 53, doi:10.1186/1742-4690-3-53.
    62. Spector, C.; Mele, A.R.; Wigdahl, B.; Nonnemacher, M.R. Genetic variation and function of the HIV-1 Tat protein. Med. Mi-crobiol. Immunol. 2019, 208, 131–169, doi:10.1007/s00430-019-00583-z.
    63. Razooky, B.S.; Pai, A.; Aull, K.; Rouzine, I.M.; Weinberger, L.S. A hardwired HIV latency program. Cell 2015, 160, 990–1001, doi:10.1016/j.cell.2015.02.009.
    64. DeMaster, L.K.; Liu, X.; VanBelzen, D.J.; Trinite, B.; Zheng, L.; Agosto, L.M.; Migueles, S.A.; Connors, M.; Sambucetti, L.; Levy, D.N.; et al. A Subset of CD4/CD8 Double-Negative T Cells Expresses HIV Proteins in Patients on Antiretroviral Ther-apy. J. Virol. 2015, 90, 2165–2179, doi:10.1128/JVI.01913-15.
    65. Chakraborty, S.; Manisha Kabi, M.; Ranga, U. A stronger transcription regulatory circuit of HIV-1C drives rapid establish-ment of latency with implications for the direct involvement of Tat. bioRxiv 2020, doi:10.1101/2020.02.20.958892.
    66. Bucci, M. Viral mechanisms: Tat modulates DAT. Nat. Chem. Biol. 2015, 11, 240, doi:10.1038/nchembio.1779.
    67. Mediouni, S.; Marcondes, M.C.; Miller, C.; McLaughlin, J.P.; Valente, S.T. The cross-talk of HIV-1 Tat and methamphetamine in HIV-associated neurocognitive disorders. Front. Microbiol. 2015, 6, 1164, doi:10.3389/fmicb.2015.01164.
    68. Yuan, Y.; Huang, X.; Midde, N.M.; Quizon, P.M.; Sun, W.L.; Zhu, J.; Zhan, C.G. Molecular mechanism of HIV-1 Tat inter-acting with human dopamine transporter. ACS Chem. Neurosci. 2015, 6, 658–665, doi:10.1021/acschemneuro.5b00001.
    69. Hamy, F.; Gelus, N.; Zeller, M.; Lazdins, J.L.; Bailly, C.; Klimkait, T. Blocking HIV replication by targeting Tat protein. Chem. Biol. 2000, 7, 669–676, doi:10.1016/s1074-5521(00)00012-0.
    70. Burton, D.R.; Desrosiers, R.C.; Doms, R.W.; Koff, W.C.; Kwong, P.D.; Moore, J.P.; Nabel, G.J.; Sodroski, J.; Wilson, I.A.; Wy-att, R.T. HIV vaccine design and the neutralizing antibody problem. Nat. Immunol. 2004, 5, 233–236, doi:10.1038/ni0304-233.
    71. Sun, G.; Li, H.; Wu, X.; Covarrubias, M.; Scherer, L.; Meinking, K.; Luk, B.; Chomchan, P.; Alluin, J.; Gombart, A.F.; et al. Interplay between HIV-1 infection and host microRNAs. Nucleic Acids Res. 2012, 40, 2181–2196, doi:10.1093/nar/gkr961.
    72. Romani, B.; Engelbrecht, S.; Glashoff, R.H. Functions of Tat: The versatile protein of human immunodeficiency virus type 1. J. Gen. Virol. 2010, 91, 1–12, doi:10.1099/vir.0.016303-0.
    73. Lichterfeld, M.; Gandhi, R.T.; Simmons, R.P.; Flynn, T.; Sbrolla, A.; Yu, X.G.; Basgoz, N.; Mui, S.; Williams, K.; Streeck, H.; et al. Induction of strong HIV-1-specific CD4+ T-cell responses using an HIV-1 gp120/NefTat vaccine adjuvanted with AS02A in antiretroviral-treated HIV-1-infected individuals. J. Acquir. Immune Defic. Syndr. 2012, 59, 1–9, doi:10.1097/QAI.0b013e3182373b77.
    74. Goldstein, G.; Tribbick, G.; Manson, K. Two B cell epitopes of HIV-1 Tat protein have limited antigenic polymorphism in geo-graphically diverse HIV-1 strains. Vaccine 2001, 19, 1738–1746, doi:10.1016/s0264-410x(00)00393-5.
    75. Ronsard, L.; Lata, S.; Singh, J.; Ramachandran, V.G.; Das, S.; Banerjea, A.C. Molecular and genetic characterization of natu-ral HIV-1 Tat Exon-1 variants from North India and their functional implications. PLoS ONE 2014, 9, e85452, doi:10.1371/journal.pone.0085452.
    76. Peloponese, J.M., Jr.; Collette, Y.; Gregoire, C.; Bailly, C.; Campese, D.; Meurs, E.F.; Olive, D.; Loret, E.P. Full peptide synthe-sis, purification, and characterization of six Tat variants. Differences observed between HIV-1 isolates from Africa and other continents. J. Biol. Chem. 1999, 274, 11473–11478, doi:10.1074/jbc.274.17.11473.
    77. Yukl, S.; Pillai, S.; Li, P.; Chang, K.; Pasutti, W.; Ahlgren, C.; Havlir, D.; Strain, M.; Gunthard, H.; Richman, D.; et al. Latent-ly-infected CD4+ T cells are enriched for HIV-1 Tat variants with impaired transactivation activity. Virology 2009, 387, 98–108, doi:10.1016/j.virol.2009.01.013.
    78. Kamori, D.; Ueno, T. HIV-1 Tat and Viral Latency: What We Can Learn from Naturally Occurring Sequence Variations. Front. Microbiol. 2017, 8, 80, doi:10.3389/fmicb.2017.00080.
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