The core promoters of HIV-1, HIV-2, and SIV all contain a TATA box and multiple binding sites for the Sp family of transcription factors, and their enhancers all contain at least one binding site for NF-κB. The Sp and NF-κB factor binding sites in the core promoter play important cell type-specific roles in regulating transcription and replication. The promoter of HIV-1 contains three binding sites for Sp factors at -46 to -78 relative to the transcriptional start site (Fig. 1) 58 . Sp factors also regulate transcription by binding to positions +271 to +289 59 60 and -421 to -451 61 relative to the transcriptional start site. Sp family members include Sp1-4, as well as M1 and M2, truncated Sp3 proteins that result from alternative translational start sites within the transactivation domain 62 63 64 65 . All of the Sp proteins contain zinc finger DNA binding domains, and Sp1, 3, and 4 have similar, though not identical, affinities and specificities for GC-rich (GGGGCGGGGC) DNA 62 66 67 . Sp2 binds to GT-rich sequences (GGTGTGGGG) rather than to the GC-rich sequences that constitute the classical Sp binding sites 65 . Sp1 and Sp4 are transcriptional activators, whereas Sp3 has been classified as a repressor of HIV-1 transcription. By itself, Sp3 can weakly activate HIV-1 transcription; however, in the presence of the strong activator Sp1, it competes for binding to the LTR and inhibits activation by Sp1 66 68 69 . In contrast, M1 and M2 have the Sp3 DNA binding domain but lack the transactivation domain and are true repressors of transcription in the absence or presence of other Sp family members 69 . In addition to repressing Sp-mediated transactivation, Sp3 represses LTR activation by the viral protein Tat 66 . Sp4 is expressed predominantly in the brain 62 70 71 , providing an additional HIV-1 LTR transactivator to drive replication in this compartment. Unlike Sp1, Sp4 does not synergistically activate transcription in the presence of multiple Sp binding sites 71 . Consequently, the loss of one binding site due to genetic variation may have less of an effect in the brain than it would in other tissues, because the loss of function would not synergistically disrupt binding.

Genetic variation within the Sp sites is likely to play a role in HIV-1-associated disease progression. The NF-κB-proximal Sp site (site III) is much less conserved during the course of disease than Sp sites I and II 41 and Kilareski and Wigdahl, unpublished results). A C-to-T change at position 5 of Sp site III has been shown to correlate positively with HIV-1-associated disease progression, both in the periphery and in the brain 41 . This variant greatly reduces the affinity of this site for Sp factors, but greatly increases the response of viral replication to tumor necrosis factor α (TNFα) stimulation in peripheral blood mononuclear cells (Kilareski, Pirrone, and Wigdahl, unpublished observation). This finding is likely due to a loss of steric hindrance leading to an increase in NF-κB binding to its adjacent binding sites (Liu, Banerjee, and Wigdahl, unpublished observations). In the presence of Sp4 in the brain, one could speculate that this effect may be magnified, because Sp4 binding to sites I and II is not affected by the loss of Sp binding to site III, and the resulting stimulated LTR may have high levels of both Sp and NF-κB factors bound to their cognate sites.

Sp factors bound to the HIV-1 core promoter cooperate with the TATA binding protein and TATA binding protein-associated factors 110 and 55 to drive basal transcription 72 73 74 75 . They can also recruit P-TEFb to promote phosphorylation of RNA Pol II 76 and play an important role in remodeling chromatin to facilitate or inhibit transcription 77 78 . Histone deacetylases (HDACs) 1 and 2 are regulated through phosphorylation by protein kinase CK2. Sp1 and Sp3 can bind and recruit the phosphorylated HDACs to the LTR to repress LTR activity 79 80 81 . The repressor activity of Sp1 and Sp3 is regulated by the expression of CK2 77 .

The three Sp sites in the HIV-1 promoter have different affinities for Sp factors 39 40 58 82 , and the affinity of Sp for LTR binding sites correlates with replication kinetics; faster viral replication is achieved when a higher affinity Sp binding site is in the NF-κB proximal site 39 . Interestingly, this might, at first glance, seem to contradict the fact presented above that Sp site III has increased genetic variation with the 5T variant (a low binding affinity site) correlating with disease progression, given traditionally low binding affinity correlates with decreased viral production. However, given that a decreased binding affinity has been shown to promote higher levels of NF-κB binding, this variation may actually provide an opportunity for increased replication (Kilareski and Wigdahl, unpublished observations). This suggests that genetic variations within these sites could have significant effects on the overall viral replication kinetics 41 .

Expression patterns of the different Sp isoforms can modulate HIV-1 transcription in different cell types. As cells of the monocyte lineage differentiate, the ratio of Sp1 to Sp3 increases, resulting in increased HIV-1 transcription (McAllister and Wigdahl, unpublished observations). This process allows HIV to replicate at low levels, if at all, in circulating monocytes, and to evade the immune system until the cells are differentiated in peripheral tissues. The importance of the Sp sites also varies depending on the differentiation stage of the cell; in unstimulated monocytes, mutation of the Sp sites reduces LTR activity, whereas in MDMs, transcription of HIV and replication of SIVmac are abolished when these critical binding sites are knocked out 83 84 85 86 .

DNA binding and transactivation activity of Sp factors are regulated both positively and negatively by phosphorylation and other post-translational modifications (reviewed in 87 88 and Fig. 2). Phosphorylation at Sp1 Ser131 by DNA-dependent protein kinase increases the affinity of the protein for DNA and also increases the ability of the protein to cooperate with the viral protein Tat to transactivate the LTR 89 90 91 92 . In contrast, O-linked N-acetylglucosaminylation (O-GLcNAc) of Sp1 inhibits HIV-1 replication 93 . Therefore, modulating O-GLcNAc of transcription factors may play a role in regulation of HIV-1 latency and activation, and may link glucose metabolism to HIV-1 replication.

NF-κB proteins have been shown to be one of the main modulators of the HIV-1 LTR in all cell types and a potential pathway for anti-HIV-1 therapies 94 . NF-κB proteins bind the enhancer at two sites located at nucleotide positions -81 to -91 and -95 to -104 relative to the transcriptional start site 95 96 97 . NF-κB is composed of heterodimers of five c-rel protein family members: p65/RelA, NF-κB1/p50, c-Rel, RelB, and NF-κB2/p52. Functional NF-κB in T cells is predominantly composed of p65 or c-Rel bound to p50 or p52, whereas in MDMs, Rel B replaces p65 97 98 99 100 . In T cells and immature monocytes, NF-κB shuttles between the cytoplasm and the nucleus in response to cellular stimuli. In the cytoplasm, NF-κB is bound to inhibitor (IκB) proteins 101 . As a result of specific stimuli, IκB is phosphorylated and released from NF-κB; after release from the inhibitory complex, NF-κB translocates to the nucleus where it activates many host and viral genes through the initial recruitment of P-TEFb (Fig. 3) 101 102 103 . Interestingly, one of the IκB's, IκBα has been shown to play a role in shuttling of NF-κB from the nucleus and cytosol and in the binding NF-κB in the nucleus of T cells, potentially contributing to the lower activation levels of the HIV-1 LTR and possibly promoting viral latency 104 . However, this mechanism has not been explored in cells of the monocyte-macrophage lineage. NF-κB can also function as a repressor of transcription through the recruitment of HDAC1 (Fig. 3) 78 105 .

NF-κB DNA binding activity first occurs in monocytes as they progress from promonocytes to monocytes; however, in mature monocytes and MDMs, NF-κB is constitutively active in the nucleus, and its DNA binding activity is not increased further in response to cellular activation or differentiation 106 . This constitutive pool of NF-κB allows a low level of basal HIV transcription in the absence of cellular stimuli. Binding of NF-κB to the enhancer of the HIV-1 LTR plays a critical role in the response of the LTR to cellular stimuli in both T cells and maturing monocytes 36 94 97 106 107 108 109 . Deletion or mutation of the NF-κB sites abolishes LTR activity 97 109 110 111 112 and results in reduced production of infectious virus 98 . Activation of monocytes by LPS, IL-6, or TNF-α (Fig. 3) results in enhanced HIV replication, a process that correlates with activation of NF-κB 27 49 50 51 113 . LPS activation of monocytes leads to the induction of the NF-κB pathway through TNF-α 27 50 . In contrast, in differentiated primary MDMs, stimulation by LPS results in the downregulation of LTR activity and viral replication 48 . This activity was not affected by mutation of the NF-κB sites, but did map to the enhancer element (position -156 to -121); thus, this effect may involve NFAT proteins (see below) 48 . While this may seem counter-intuitive, one might speculate that stimulation of cells through the NF-κB pathway would enhance LTR activity and viral replication, it should be noted that LPS stimulation of differentiated macrophages could also induce transcription factors that negatively regulate the LTR, however this has not been explored. This would be very interesting as this might provide another reason for macrophages serving as a latent reservoir for HIV-1. In addition to activating transcription by binding the enhancer region, NF-κB activates transcription by binding to sites -1 to +9 and +31 to +40 relative to the transcriptional start site 114 115 .

The NF-κB site(s) located immediately upstream of the Sp sites in the enhancer in HIV and SIV result in Sp-NF-κB protein-protein interactions that further modulate the LTR activity. Sp1 and NF-κB proteins bind the LTR cooperatively and activate transcription synergistically in response to cellular stimulation 66 82 109 . This activation is mediated by the binding of the DNA-binding domain of p65 to the DNA-binding domain of Sp1 108 (Fig. 4). Sp3 and Sp4 are unable to activate transcription cooperatively with NF-κB 66 . In the absence of functional Sp sites (or in the presence of genetic alterations that inactivate the Sp binding sites), binding of NF-κB to the enhancer can restore replication of the virus in T cells 116 117 118 , perhaps by recruiting Sp to the variant sites.

NFAT proteins are part of a family of Rel-related transcription factors that become active early after T cell activation and are constitutively in monocytes. NFAT exists as several isoforms (NFAT1, NFAT2/NFATc, and NFAT3-5) that activate a variety of genes in immune and non-immune cell populations 119 120 121 . Like NF-κB, NFAT contains a DNA-binding domain that is homologous to rel and shuttles between the cytoplasm and nucleus in response to cellular stimuli 122 123 . In the cytoplasm, NFAT is dephosphorylated and translocates to the nucleus where it activates transcription of many genes 124 125 126 (Fig. 4). NFAT can bind DNA as a high affinity dimer or as a lower affinity monomer 127 128 129 . NFAT proteins frequently cooperate with other transcription factor families when bound to adjacent sites within a promoter.

An NFAT binding site was identified in the HIV-1 LTR at positions -216 and -254, with a footprint extending from -253 to -215 relative to the transcriptional start site 122 130 . Although this site can bind NFAT in vitro, this site was later shown not to be necessary for NFAT-mediated activation of the HIV-1 LTR 131 132 . Instead, NFAT binds the NF-κB binding sites in the enhancer in response to cellular activation in T cells and constitutively in monocytes 110 112 127 130 133 . NFAT activation of genes from κB-like sequences has been documented with a number of host and viral promoters 134 135 (Fig. 4). In addition to binding to the enhancer, NFAT binding at positions +169 to +181 has been reported to activate transcription 59 60 136 .

NFAT proteins activate HIV-1 transcription and replication in a variety of cell types. Whereas NFAT1 and NFAT2/NFATc are responsible for the activation of HIV in T cells 110 133 137 reviewed in 138 139 ), NFAT5, the most evolutionarily divergent NFAT member, regulates HIV replication in monocyte-MDMs 130 . Terminally differentiated MDMs constitutively express high levels of NFAT5, which is able to bind and activate the enhancer of HIV-1 subtypes B, C, and E, HIV-2, and SIV from multiple primate species 130 . Targeting NFAT5 with siRNAs in primary MDMs modestly reduces viral replication 130 ; however, NFAT derived from MDM nuclear extract was unable to compete with NF-κB for binding to the HIV enhancer in vitro 98 . This finding suggests that in vivo, although constitutively expressed NFAT is able to bind the LTR, it is unable to do so in the presence of high levels of NF-κB.

The HIV-1 LTR contains three C/EBP binding sites upstream of the transcriptional start site 156 157 and one binding site downstream of the transcriptional start site, at the 3'-most end of U5 (Liu and Wigdahl, unpublished observations). C/EBPs play a critical role in HIV-1 replication. It has been shown that at least one upstream C/EBP binding site and the presence of C/EBP proteins are necessary for replication in cells of the monocyte-macrophage lineage 157 158 159 160 161 . The two C/EBP binding sites located in the U3 region of the LTR have differing affinities for C/EBP factors, with the upstream site (site II), having a much higher relative affinity than the downstream site (site I) 43 . In addition to activating HIV-1 transcription through direct binding to the LTR, C/EBP factors may inhibit the host cellular antiviral protein APOBEC3G (Fig. 5), allowing more efficient reverse transcription to occur in the cytoplasm 162 .

The C/EBP family of transcription factors consists of six members, including C/EBP α, β, γ, δ, ε, and ζ 163 164 165 166 167 168 169 . C/EBPβ itself has three isoforms that result from the use of internal start codons within a single mRNA 170 171 . C/EBP-1, the full-length isoform, and C/EBP-2, an isoform that lacks the N-terminal 23 amino acids, both contain three transcriptional activation domains and function as activators of HIV-1 transcription. C/EBP-3, which lacks the N-terminal 198 amino acids that include the activation domains, serves as a repressor of HIV-1 transcription, because it retains the C-terminal DNA-binding domain and competes for binding with the activator isoforms of C/EBP.

C/EBP isoform expression depends on the differentiation and activation state of cells in the monocyte-macrophage lineage. C/EBPα levels are high early in monocyte differentiation and then decrease as cells mature, whereas C/EBPβ and C/EBPδ levels are low early in development and increase as cells mature 172 173 . C/EBP isoform expression is also regulated by extracellular stimuli. C/EBPβ expression increases upon cellular activation, whereas expression of the other C/EBP isoforms remains constant 172 174 . Exposure of macrophages to interleukin-1 (IL-1), tumor necrosis factor α (TNFα), or interferon-γ, all of which have been shown to be present at elevated levels during the course of HIV-1 infection, has been shown to induce a reduction in C/EBPα mRNA levels while the levels of C/EBPβ and C/EBPδ expression increase 174 . This results in C/EBPβ and C/EBPδ playing a key role in the regulation of HIV-1 transcription as disease progresses and inflammatory cytokine levels increase (Fig. 4).

An additional level of regulation of C/EBPβ activity resides in two regulatory domains that lie between the activation domains and the DNA binding domain. These domains inhibit C/EBP activity, until phosphorylation results in an increase in DNA binding affinity and transcriptional activation activity 175 176 . Several signaling cascades regulate the phosphorylation state of C/EBP. Phosphorylation of threonine 235 by a ras-dependent mitogen-activated protein kinase increases transcriptional activation 177 ; phosphorylation of serine 288 by cAMP-dependent protein kinase A results in nuclear translocation and subsequent transactivation 178 ; and cyclin-dependent kinase 9 (cdk9) phosphorylates C/EBPβ and leads to an increase in HIV-1 gene expression 179 (Fig. 4).

C/EBPs interact with many nuclear proteins to activate transcription. In addition to binding other bZIP proteins, C/EBP recruits chromatin remodeling complexes such as SWI/SNF 180 , cAMP response element-binding protein/p300 181 182 , and p300/CREB-binding protein-associated factor 183 to the HIV-1 LTR. These proteins remodel the chromatin structure and increase transcription of the HIV-1 genome. C/EBP increases the phosphorylation of p300, which in turn alters its nuclear localization and increases its activity 184 . C/EBP can also act synergistically with Sp proteins to activate transcription of the HIV-1 LTR 185 .

The importance of C/EBP factors in the regulation of HIV-1 gene expression is underscored by the discovery that a 6G configuration (a T-to-G change at nucleotide position 6) in C/EBP site I increases C/EBP binding, increases LTR activity, and is preferentially encountered in proviral LTRs derived from the brain of HIV-1-infected patients 42 186 . C/EBP site II was also found to be preferentially conserved in the consensus subtype B configuration or to contain a 6G variation of this site, which are both high affinity sites for C/EBP factors in LTRs present in proviral DNA in cells located in the mid-frontal gyrus of the brain of infected individuals. A high rate of viral replication occurs in this region of the brain. Interestingly, the presence of the 6G configuration of this binding site also correlates with the presence of HIV-1-associated dementia 42 44 . In contrast, the presence of a 4C C/EBP site II, which is a low-affinity C/EBP site, has been found preferentially in the cerebellum, a region of low viral replication 44 . This observation suggests that high affinity for C/EBP factors may contribute to the maintenance and/or pathogenesis of HIV-1 in the central nervous system, whereas low affinity sites such as 4C may contribute to lower levels of transcription required to maintain a latent reservoir of provirus. We have also identified a 3T configuration (a C-to-T change at position 3) of C/EBP site I that exhibits a low affinity for C/EBP within LTRs in the peripheral blood and brain and has also been shown to correlate with both late stage HIV disease and HIV-1-associated dementia 43 , respectively.

ATF/CREB binds the HIV-1 LTR at a site immediately upstream of the C/EBP binding site I 38 187 and at two sites downstream of the transcriptional start site (sites +160 to +167 and +92 to +102) to regulate the LTR 59 60 188 189 . ATF/CREB and C/EBP factors can bind their adjacent upstream sites individually as homodimers, or C/EBP and ATF/CREB can heterodimerize with each other to regulate HIV-1 expression. This heterodimerization results in the recognition of a site composed of the 3' half of the ATF/CREB site and the 5' half of the C/EBP site 146 . As a result, in the presence of genetic variation that results in a low affinity C/EBP site, ATF/CREB is able to recruit C/EBP factors to the site and vice versa 146 . In addition to activating transcription, ATF/CREB can inhibit transcription by binding to Swi6, a component of the remodeling complex SWI/SNF, to promote the formation of heterochromatin 190 .

AP-1 proteins exist as homodimers of Jun family members (c-Jun, JunB, and JunD) or as heterodimers of Jun and Fos family members (c-Fos, FosB, Fra-1, and Fra-2) (reviewed in 191 ). They bind a palindromic DNA sequence known as the TPA-responsive elements (TRE) at positions -306 to -285 and -242 to -222 of the LTR 59 as well as at positions +95 and +160, downstream of the transcriptional start site 59 60 188 189 . The sequence of these sites has been shown to evolve in a manner that facilitates efficient cell type-specific binding of AP-1 59 192 . AP-1 acts as either an activator or repressor of transcription, depending on the components of the dimer 191 193 . Once bound to the promoter, cFos/cJun heterodimers can recruit the SWI/SNF chromatin remodeling complex to activate transcription, whereas homodimers or heterodimers consisting of other family members lack this ability 194 .

AP-1 mRNA is typically absent in quiescent cells; however, it is significantly up-regulated upon cellular stimulation 195 . Jun levels increase during monocytic maturation and become constitutively expressed in MDMs 196 197 198 199 200 . Despite being expressed, AP-1 in MDMs of some tissues, such as the lung, lacks the ability to bind DNA because of the lack of expression of Ref-1, a protein that modulates the oxidation state of Fos 201 202 . In addition to being regulated by oxidation 201 202 203 , AP-1 protein activity is further controlled post-transcriptionally by sumoylation, which inhibits protein activity 204 205 , and by phosphorylation, which increases activity in response to cellular stimulation 206 .

In addition to directly regulating HIV-1 gene expression, AP-1 proteins can modulate the activity of other transcription factors. C/EBPβ dimerization with c-Fos or c-Jun results in C/EBP being unable to bind DNA thus a reduction in C/EBP-mediated transactivation 153 154 . In contrast, C/EBPα dimerization with c-Jun or c-Fos forms a potent activator of transcription 207 . In response to mitogen or cytokine stimulation, the mitogen-activated protein kinases ERK1/ERK2 phosphorylate AP-1 (reviewed in 208 and 209 ). This phosphorylation promotes the interaction of AP-1 with NF-κB and the enhancer element, which leads to the synergistic activation of the LTR 210 211 212 213 . This cascade of events is one mechanism by which HIV emerges from latency 210 214 .

Tat is a virus-encoded transcriptional transactivator that binds to the RNA secondary structure encoded by the transactivation region (TAR) in the repeat segment of the LTR (+1 to +59) 215 216 . Once bound to the elongating transcript, Tat helps assemble the pre-initiation complex and recruits cdk9 to promote phosphorylation of RNA Pol II 217 218 and P-TEFb to increase processivity of RNA Pol II 219 220 221 222 223 . Interestingly, mechanistic studies of this complex suggest that one of the functions of Tat is to increase the duration of P-TEFb occupancy at the HIV-1 LTR 224 . Tat also significantly remodels chromatin by recruiting the histone acyltransferases Tip60 225 226 , human Nucleosome Assembly Protein-1 (hNAP-1) 227 , p300/cAMP response element-binding protein 228 229 , and p/CAF 230 , as well as the chromatin-remodeling complex SWI/SNF 231 . Tat activity is limited in monocytes due to the lack of sufficient levels of cyclin T1, a component of P-TEFb 54 . Differentiation into macrophages increases Cyclin T1 expression and results in strong Tat activity 54 .

Tat regulates the activity of many other transcription factors through direct protein-protein interactions and the modulation of kinase activities. Tat promotes the phosphorylation of Sp1, which in turn increases binding of Sp to the LTR 92 . Conversely, Sp is also necessary to recruit Tat to the LTR 76 , and deletion or mutation of the Sp binding sites in the promoter abolishes Tat activity 232 233 . It is currently unclear whether direct interaction occurs between Sp factors and Tat 234 235 236 . In addition to regulating Sp1 activity, Tat increases the cooperation between NFAT and AP-1 proteins without altering independent binding of these transcription factors to DNA 137 237 . It also promotes the interaction of NF-κB and AP-1 factors to synergistically activate transcription 238 239 240 .

Vpr is another virus-encoded protein that plays a direct role in the regulation of HIV-1 transcription 241 242 243 . Vpr is found in the viral particle and plays an important role in early transcriptional activation of the LTR before Tat can be expressed 244 245 246 247 248 . Its importance is highlighted by a recent study that describes alterations in Vpr that provide a significant reduction in Vpr nuclear import and virion incorporation uniquely in a long term non-progressor patient 249 . Vpr also causes cell cycle arrest in the G2 phase, the phase of the cell cycle when the LTR is most active, which results in apoptosis. 250 . It is necessary for viral replication in cells of the monocyte-macrophage lineage 251 252 253 254 255 256 . Interestingly, Vpr has been shown to interact with the nuclear form of uracil DNA glycosylase (UNG2), a cellular DNA repair enzyme, which helps incorporate this protein into virus particles leading to a decrease in viral mutation rate. Specifically, the lack of UNG in virions during virus replication in primary monocyte-derived macrophages further increases virus mutant frequencies by 18-fold compared with the 4-fold increase measured in actively dividing cells 257 . In addition, Vpr has been shown to concentrate at the nuclear envelope (NE) shortly after infection (4-6 hrs) as part of the pre-integration complex (PIC), supporting an interaction between Vpr and components of the nuclear pore complex 258 259 260 261 , including the nucleoporin hCG1 262 . Single-point Vpr mutants within the first α-helix of the protein such as Vpr-L23F and Vpr-K27M fail to associate with hCG1, but are still able to interact with other known relevant host partners of Vpr. In primary human monocyte-derived macrophages, these mutants fail to localize at the NE resulting in a diffuse nucleocytoplasmic distribution, impaired the Vpr-mediated G2-arrest of the cell cycle, and subsequently induced cell death. These observations were obtained in primary macrophages from some but not all donors indicating that the targeting of Vpr to the nuclear pore complex may constitute an early step toward Vpr-induced G2-arrest and subsequent apoptosis. These results also suggest that Vpr targeting to the nuclear pore complex is not absolutely required, but can enhance HIV-1 replication in macrophages 263 . Extracellular Vpr is found in the plasma and the CSF 254 264 and can enter monocytes and macrophages and behave as if the protein was endogenously expressed 265 266 267 . Vpr binds the LTR in a sequence-specific manner to activate transcription directly 45 46 and also interacts with Sp1 268 , TFIIB 269 270 , NF-κB 271 , C/EBP 272 , and Tat 244 273 to enhance transcription of the HIV-1 genome. Vpr activates the DNA binding activity of AP-1 by promoting the phosphorylation of cFos and cJun in monocytes and macrophages 267 . It also promotes the translocation of NF-κB p50/p65 to the nucleus by promoting the phosphorylation of IκB 267 , which allows an NF-κB- and AP-1-mediated increase in LTR activity.

C/EBP and Vpr interact at the HIV-1 LTR in two ways. Vpr has been shown to increase C/EBPβ DNA binding activity 272 . It has also been shown that Vpr has a high affinity for LTR C/EBP binding site I variants that exhibit a decreased affinity of the site for C/EBP. The presence of these LTR variants correlates with late-stage HIV-associated disease 45 46 . Thus, as HIV-1-associated disease progresses, viral variants containing this type of LTR C/EBP site I may become more prevalent and function to facilitate a transition from C/EBP-mediated LTR activation to Vpr-mediated transactivation from that site. Alternatively, Vpr and C/EBP may form a complex at that site (Burdo and Wigdahl, unpublished observations). In addition to interacting with cellular proteins, Vpr interacts with Tat and activates transcription in an additive manner 244 274 .

HIV proviral DNA that has integrated into the host genome also becomes subject to host factors that regulate chromatin organization and gene transcription. These mechanisms include histone modification, RNA interference/silencing, and DNA methylation. The mechanisms play a role in the control of gene expression, viral activation, and/or latency. DNA methylation of CpG islands within the HIV-1 LTR is one process that results in the downregulation/silencing of the integrated proviral genome 275 276 277 278 . This form of transcriptional silencing occurs by specific methyltransferases that are directed to the target DNA by methylation of lysine 9 of histone H3 through histone methyltransferases 279 . In cells of the monocyte-macrophage lineage, methylation of the LTR has been found to result in the transcriptional silencing of the promoter which contributes to limited access of transcription factors to the target DNA 280 . In addition, in the CD4⁺ T cell line ACH-2, the transcriptional silencing brought about by DNA methylation of the LTR can be reversed through TNF-α treatment of the cells which leads to demethylation of the 5' LTR and the induction of viral gene expression 281 showing that although this modification is inheritable, it is not permanent. The reduction of LTR expression is possibly explained by the binding of methyl-CpG-binding protein 1 complex and methyl-CpG-binding protein 2 to methylated Sp1 transcription factor binding sites, thereby inhibiting the binding of Sp1 transcription factors 282 283 . In addition, the transcription factors USF and NF-κB lose affinity for their methylated LTR transcription factor binding sites as well 284 . Unfortunately, to date all of these studies have been performed in T cell lines and primary T cells, but not in cells of the monocyte-macrophage lineage.

Cytokines play a critical role in the pathogenesis of HIV-1. IL-6, TNFα, IL-1β, and other proinflammatory cytokine levels are elevated in the blood, bone marrow, and cerebrospinal fluid of HIV-infected patients 285 286 . IL-6 and TNF-α are induced early after HIV monocytic infection, followed by their continued increased expression 52 53 287 . IL-6 is a potent activator of C/EBP, and exposure of monocytes to IL-6 results in increased HIV-1 replication. The increase in C/EBP activity then forms a positive feedback loop for IL-6 expression, because C/EBPβ binds to and activates the IL-6 promoter 288 . C/EBPs can also activate the genes encoding other proinflammatory cytokines such as IL-1β 289 and TNFα 290 291 . TNFα is one of the most potent activators of NF-κB activity known. It acts by causing a signaling cascade that activates the IκB kinase complex, which then phosphorylates IκB, releasing NF-κB. The free NF-κB translocates to the nucleus and induces the activation of the HIV-1 LTR (Fig. 3 and 4).

In addition to being regulated by cytokines, chemokines contribute to HIV-1 infection and pathogenesis. The HIV-1 Nef protein induces HIV-infected macrophages to secrete at least two chemokines, MIP1α and MIP1β, which recruit and activate resting CD4+ T lymphocytes 292 . These T cells can then become infected and produce high levels of virus.