Regulation of HIV-1 transcription in cells of the monocyte-macrophage lineage

Abstract

Human immunodeficiency virus type 1 (HIV-1) has been shown to replicate productively in cells of the monocyte-macrophage lineage, although replication occurs to a lesser extent than in infected T cells. As cells of the monocyte-macrophage lineage become differentiated and activated and subsequently travel to a variety of end organs, they become a source of infectious virus and secreted viral proteins and cellular products that likely initiate pathological consequences in a number of organ systems. During this process, alterations in a number of signaling pathways, including the level and functional properties of many cellular transcription factors, alter the course of HIV-1 long terminal repeat (LTR)-directed gene expression. This process ultimately results in events that contribute to the pathogenesis of HIV-1 infection. First, increased transcription leads to the upregulation of infectious virus production, and the increased production of viral proteins (gp120, Tat, Nef, and Vpr), which have additional activities as extracellular proteins. Increased viral production and the presence of toxic proteins lead to enhanced deregulation of cellular functions increasing the production of toxic cellular proteins and metabolites and the resulting organ-specific pathologic consequences such as neuroAIDS. This article reviews the structural and functional features of the cis-acting elements upstream and downstream of the transcriptional start site in the retroviral LTR. It also includes a discussion of the regulation of the retroviral LTR in the monocyte-macrophage lineage during virus infection of the bone marrow, the peripheral blood, the lymphoid tissues, and end organs such as the brain. The impact of genetic variation on LTR-directed transcription during the course of retrovirus disease is also reviewed.

Figure 1

Structure of retroviral LTRs. Retroviral LTRs are divided into the U3, R, and U5 regions, and the U3 region is further divided into the Modulatory, Enhancer (E) and Promoter regions (top bars). HIV-1, HIV-2, and SIV all contain highly conserved promoters containing TATA boxes (yellow) and Sp factor binding sites (red) and enhancers (labeled E in light blue bar) containing NF-κB binding sites (blue). The R region of each contains a trans-acting responsive element (TAR) (orange) that forms an RNA stem loop structure upon transcription that binds to the viral protein Tat. A negative regulatory element (NRE, pink) was identified that was subsequently shown to serve as both activator and repressor by binding NFAT proteins (dark blue), AP-1 proteins (purple), and C/EBP factors (green). The modulatory regions of SIVmac and HIV-2 also contain purine box arrays (PuB, gold) and sites that bind members of the Ets family (teal).

Figure 2

Important Sp transcription factor signaling in monocyte-macropahges. (a) Activation of HIV transcription by the interaction of viral protein Tat with DNA-dependent protein kinase (DNA-PK) results in the subsequent phosphorylation at Ser131 of Sp1. Phosphorylated Sp1 results in increased transcription of proviral DNA, resulting in an increase in Tat production, perpetuating the cycle. (b) Inhibition of HIV transcription involves O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) catalyzing the addition of O-GlcNAC to Sp proteins which blocks their interaction with their binding sites on the LTR, resulting in an inhibition/reduction in HIV transcription.

Figure 3

Important NF-κB transcription factor signaling in monocyte-macrophages. (a) Activation of HIV transcription: Translocation of NF-κB from the cytoplasm to the nucleus is controlled by association of IκB with the NF-κB hetero-/homo-dimer. Once IκB is phosphorylated, it relesases NF-κB which then translocates to the nucleus where it can bind the LTR and induce HIV transcription. (b) Inhibition of HIV transcription: In T cells, IκBα has been shown to contribute to lower levels of LTR transcription and potentially contribute to latency. It is postulated that a similar mechanism of action could be in place for cells of the monocyte-macrophage lineage. In addition, NF-κB's association with the histone deacetylase inhibitor HDAC1 results in constriction of the chromatin so that RNA polymerase does not have access to its target DNA.

Figure 4

Important C/EBP transcription factor signaling in monocyte-macrophages: (a) Activation of HIV transcription: C/EBP, located in the cytoplasm of the cell, can become phosphorylated by the MAP kinase, PKA, or cdk9 through a variety of pathways. Once phosphorylated, C/EBP is translocated into the nucleus where it can transactivate the LTR. In addition, C/EBP associates with histone acetyl transferases such as p300, which when bound to the LTR, make the chromosome accessible for RNA polymerases to bind and transcribe the integrated proviral DNA. Finally, association of C/EBP with APOBEC3G allows for better reverse transcription in the cytoplasm. (b) Inhibition of HIV transcription: The binding of IFNβ to its receptor begins a JAK/STAT signaling cascade that results in increased production of C/EBP3 (LIP). C/EBP3, which does not contain the transactivation domain of full-length C/EBPs, does not interact with histone acetyl transferases and when bound to the LTR, blocks the binding of full-length C/EBPs, thereby leading to a repression of LTR activity.

Figure 5

Regulation of HIV-1 transcription in circulating monocytes. Transcription of HIV-1 in circulating monocytes is dependent on the ratio of activator to repressor isoforms of transcription factors, the phosphorylation state of transcription factors, and the inducible translocation of NF-κB and NFAT factors from the cytoplasm. NF-κB can be induced to translocate to the nucleus by TNFα-mediated phosphorylation of IκB. NFAT is dephosphorylated in the cytoplasm by calcineurin, which acts in response to calcium levels within the cell. Once it is dephosphorylated, it translocates to the nucleus where it activates transcription by constitutively binding the NF-κB site in the enhancer. Phosphorylation plays a critical role in regulating the activity of C/EBP factors in monocytes. Phosphorylation of C/EBPα by ras-dependent mitogen-activated protein (MAP) kinase, signaled by IL-6 or by cAMP-dependent protein kinase A, results in its nuclear translocation and subsequent transactivation of the LTR. Cyclin-dependent kinase (cdk) 9 specifically phosphorylates C/EBPβ, which then translocates into the nucleus, binds to the LTR, and leads to an increase in HIV-1 gene expression. Once in the nucleus, C/EBP factors then regulate the activity of AP-1 factors. Relatively high levels of C/EBPα dimerize with AP-1 factors to form potent activators of transcription. Lower levels of C/EBPβ balance this activation by binding AP-1 leading to a loss in DNA binding affinity. Sp1 and Sp3 are constitutively expressed in the nucleus. In the presence of Sp1, which is a strong activator, Sp3 competes for binding to the LTR and inhibits activation by Sp1.

Figure 6

Cytokine-regulation of HIV-1 transcription in monocytes. Cytokines play an integral role in regulating the availability and activity of transcription factors that regulate the LTR. TNFα strongly induces the nuclear localization of NF-κB in monocytes. As a result, the subsequently stimulated LTR interfaces with increased levels of Sp and NF-κB factors. Cellular activation increases the expression of C/EBP, particularly activation by IL-6. TNF-α, IL-1, and interferon-γ reduce the expression of C/EBPα and increase expression of both C/EBPβ and C/EBPδ. Stimulation increases the expression of AP-1 in the cell where its interaction with NF-κB at the enhancer element leads to synergistic activation of the LTR. (Black arrows: translocation to nucleus; red arrows: decrease in expression; green arrows: increase in expression).

Figure 7

Regulation of HIV-1 transcription in differentiated macrophages. In differentiated macrophages, NF-κB and NFAT are constitutively localized in the nucleus; however, in the presence of large amounts of NF-κB, NFAT is unable to bind the LTR. NF-κB-Sp1 protein-protein interactions bind the LTR cooperatively and activate transcription synergistically in response to cellular stimuli. Sp sites are necessary for viral replication, and the ratio of Sp1 proteins to Sp3 proteins increases, thus increasing transcription of the virus. As the cell matures, C/EBPα levels decrease and C/EBPβ and C/EBPδ levels increase. AP-1 is constitutively expressed but loses its ability to bind to the LTR. Tat binds to the transactivation response region (TAR) structure on the viral RNA and recruits (P-TEFb (the Cyclin dependent kinase 9 (Cdk9) and cyclin T1 (CycT1) complex) through binding to cyclin T1. Recruitment of P-TEFb to TAR induces hyperphosphorylation of CTD by Cdk9, thereby enhancing the transcriptional elongation of HIV-1.