Human Immunodeficiency Virus Promoter Research Articles

Direct and indirect effects of CYTOR lncRNA regulate HIV gene expression.

The implementation of antiretroviral therapy (ART) has effectively restricted the transmission of Human Immunodeficiency Virus (HIV) and improved overall clinical outcomes. However, a complete cure for HIV remains out of reach, as the virus persists in a stable pool of infected cell reservoir that is resistant to therapy and thus a main barrier towards complete elimination of viral infection. While the mechanisms by which host proteins govern viral gene expression and latency are well-studied, the emerging regulatory functions of non-coding RNAs (ncRNA) in the context of T cell activation, HIV gene expression and viral latency have not yet been thoroughly explored. Here, we report the identification of the Cytoskeleton Regulator (CYTOR) long non-coding RNA (lncRNA) as an activator of HIV gene expression that is upregulated following T cell stimulation. Functional studies show that CYTOR suppresses viral latency by directly binding to the HIV promoter and associating with the cellular positive transcription elongation factor (P-TEFb) to activate viral gene expression. CYTOR also plays a global role in regulating cellular gene expression, including those involved in controlling actin dynamics. Depletion of CYTOR expression reduces cytoplasmic actin polymerization in response to T cell activation. In addition, treating HIV-infected cells with pharmacological inhibitors of actin polymerization reduces HIV gene expression. We conclude that both direct and indirect effects of CYTOR regulate HIV gene expression.

Naphthalene Diimide-Tetraazacycloalkane Conjugates Are G-Quadruplex-Based HIV-1 Inhibitors with a Dual Mode of Action.

Human immunodeficiency virus 1 (HIV-1) therapeutic regimens consist of three or more drugs targeting different steps of the viral life cycle to limit the emergence of viral resistance. In line with the multitargeting strategy, here we conjugated a naphthalene diimide (NDI) moiety with a tetraazacycloalkane to obtain novel naphthalene diimide (NDI)-tetraazacycloalkane conjugates. The NDI inhibits the HIV-1 promoter activity by binding to LTR G-quadruplexes, and the tetraazacycloalkane mimics AMD3100, which blocks HIV entry into cells by interfering with the CXCR4 coreceptor. We synthesized, purified, and tested the metal-free NDI-tetraazacycloalkane conjugate and the two derived metal-organic complexes (MOCs) that incorporate Cu2+ and Zn2+. The NDI-MOCs showed enhanced binding to LTR G4s as assessed by FRET and CD assays in vitro. They also showed enhanced activity in cells where they dose-dependently reduced LTR promoter activity and inhibited viral entry only of the HIV-1 strain that exploited the CXCR4 coreceptor. The time of addition assay confirmed the dual targeting at the different HIV-1 steps. Our results indicate that the NDI-MOC conjugates can simultaneously inhibit viral entry, by targeting the CXCR4 coreceptor, and LTR promoter activity, by stabilizing the LTR G-quadruplexes. The approach of combining multiple targets in a single compound may streamline treatment regimens and improve the overall patient outcomes.

Molecular components of the circadian clock regulate HIV-1 replication

Human immunodeficiency virus 1 (HIV-1) causes major health burdens worldwide and still lacks curative therapies and vaccines. Circadian rhythms are endogenous daily oscillations that coordinate an organism's response to its environment and invading pathogens. Peripheral viral loads of HIV-1 infected patients show diurnal variation; however, the underlying mechanisms remain unknown. Here, we demonstrate a role for the cell-intrinsic clock to regulate rhythmic HIV-1 replication in circadian-synchronized systems. Silencing the circadian activator Bmal1 abolishes this phenotype, and we observe BMAL1 binding to the HIV-1 promoter. Importantly, we show differential binding of the nuclear receptors REV-ERB and ROR to the HIV-long terminal repeat at different circadian times, demonstrating a dynamic interplay in time-of-day regulation of HIV-1 transcription. Bioinformatic analysis shows circadian regulation of host factors that control HIV-1 replication, providing an additional mechanism for rhythmic viral replication. This study increases our understanding of the circadian regulation of HIV-1, which can ultimately inform new therapies.

Zipper interacting protein kinase (ZIPK) is a negative regulator of HIV-1 replication that is restricted by viral Nef protein through proteasomal degradation

Human immunodeficiency virus-1 (HIV-1) infection leads to the development of acquired immunodeficiency syndrome (AIDS). To establish a productive infection, HIV-1 hijacks the cellular machinery and modulates various physiological processes to propagate itself. The pathways altered by HIV-1 include cell cycle, autophagy, apoptosis, cell stress pathways, immune response, antiviral response, etc. Zipper interacting protein kinase (ZIPK) is a member of the death-associated protein kinase (DAPK) family of proteins, known to be one of the key regulators of cell death and cell survival pathways. ZIPK is also involved in regulating many cellular processes that are altered during HIV-1 infection; thus, we have explored the functional role of ZIPK in HIV-1 infection. Our results show that ZIPK protein expression is downregulated during HIV-1 infection in Nef dependent manner. Overexpression of ZIPK leads to downregulation in LTR-driven gene expression and virus production, whereas ZIPK knockdown induces viral gene expression and replication. HIV-1 promoter activity is reportedly enhanced by Nef-mediated activation of some transcription factors like NFκB and STAT3. ZIPK is reported to inhibit the STAT3 activity by phosphorylating it at ser-727. Our results show that STAT3 (ser-727) phosphorylation is decreased upon overexpression of Nef with simultaneous downregulation of ZIPK expression. We finally show that HIV-1 Nef interacts with ZIPK and induces its proteasomal degradation. Overall, our data suggests that Nef is involved in downregulation of ZIPK thereby increasing the virus production through rescue of STAT3 activity.

Hydrogen sulfide blocks HIV rebound by maintaining mitochondrial bioenergetics and redox homeostasis.

A fundamental challenge in human immunodeficiency virus (HIV) eradication is to understand how the virus establishes latency, maintains stable cellular reservoirs, and promotes rebound upon interruption of antiretroviral therapy (ART). Here, we discovered an unexpected role of the ubiquitous gasotransmitter hydrogen sulfide (H2S) in HIV latency and reactivation. We show that reactivation of HIV is associated with downregulation of the key H2S producing enzyme cystathionine-γ-lyase (CTH) and reduction in endogenous H2S. Genetic silencing of CTH disrupts redox homeostasis, impairs mitochondrial function, and remodels the transcriptome of latent cells to trigger HIV reactivation. Chemical complementation of CTH activity using a slow-releasing H2S donor, GYY4137, suppressed HIV reactivation and diminished virus replication. Mechanistically, GYY4137 blocked HIV reactivation by inducing the Keap1-Nrf2 pathway, inhibiting NF-κB, and recruiting the epigenetic silencer, YY1, to the HIV promoter. In latently infected CD4+ T cells from ART-suppressed human subjects, GYY4137 in combination with ART prevented viral rebound and improved mitochondrial bioenergetics. Moreover, prolonged exposure to GYY4137 exhibited no adverse influence on proviral content or CD4+ T cell subsets, indicating that diminished viral rebound is due to a loss of transcription rather than a selective loss of infected cells. In summary, this work provides mechanistic insight into H2S-mediated suppression of viral rebound and suggests exploration of H2S donors to maintain HIV in a latent form.

The Block-and-Lock Strategy for Human Immunodeficiency Virus Cure: Lessons Learned from Didehydro–Cortistatin A

Antiretroviral therapy effectively controls human immunodeficiency virus (HIV) infection. However, a reservoir of latently infected cells persists under suppressive therapy, constituting a major barrier to an HIV cure. The block-and-lock approach to a functional cure aims at the transcriptional and epigenetic silencing of proviruses, blocking viral reactivation in the absence of therapy, preventing disease progression and transmission, despite the presence of detectable integrated proviruses. This approach has been put forward for exploration based on the activity of didehydro-cortistatin A, an inhibitor of the HIV transcriptional activator Tat. Here we review the mechanisms by which didehydro-cortistatin A inhibition of Tat's feedback loop transcriptional amplification results in epigenetic silencing of the HIV promoter, and we discuss the benefits and limitations of the block-and-lock approach for an HIV cure.

Genome-wide CRISPR knockout screen identifies ZNF304 as a silencer of HIV transcription that promotes viral latency.

Despite the widespread use of anti-retroviral therapy, human immunodeficiency virus (HIV) still persists in an infected cell reservoir that harbors transcriptionally silent yet replication-competent proviruses. While significant progress has been made in understanding how the HIV reservoir is established, transcription repression mechanisms that are enforced on the integrated viral promoter have not been fully revealed. In this study, we performed a whole-genome CRISPR knockout screen in HIV infected T cells to identify host genes that potentially promote HIV latency. Of several top candidates, the KRAB-containing zinc finger protein, ZNF304, was identified as the top hit. ZNF304 silences HIV gene transcription through associating with TRIM28 and recruiting to the viral promoter heterochromatin-inducing methyltransferases, including the polycomb repression complex (PRC) and SETB1. Depletion of ZNF304 expression reduced levels of H3K9me3, H3K27me3 and H2AK119ub repressive histone marks on the HIV promoter as well as SETB1 and TRIM28, ultimately enhancing HIV gene transcription. Significantly, ZNF304 also promoted HIV latency, as its depletion delayed the entry of HIV infected cells into latency. In primary CD4+ cells, ectopic expression of ZNF304 silenced viral transcription. We conclude that by associating with TRIM28 and recruiting host transcriptional repressive complexes, SETB1 and PRC, to the HIV promoter, ZNF304 silences HIV gene transcription and promotes viral latency.

Pharmacological activation of the circadian component REV-ERB inhibits HIV-1 replication

Human immunodeficiency virus 1 (HIV-1) is a life-threatening pathogen that still lacks a curative therapy or vaccine. Despite the reduction in AIDS-related deaths achieved by current antiretroviral therapies, drawbacks including drug resistance and the failure to eradicate infection highlight the need to identify new pathways to target the infection. Circadian rhythms are endogenous 24-h oscillations which regulate physiological processes including immune responses to infection, and there is an emerging role for the circadian components in regulating viral replication. The molecular clock consists of transcriptional/translational feedback loops that generate rhythms. In mammals, BMAL1 and CLOCK activate rhythmic transcription of genes including the nuclear receptor REV-ERBα, which represses BMAL1 and plays an essential role in sustaining a functional clock. We investigated whether REV-ERB activity regulates HIV-1 replication and found REV-ERB agonists inhibited HIV-1 promoter activity in cell lines, primary human CD4 T cells and macrophages, whilst antagonism or genetic disruption of REV-ERB increased promoter activity. The REV-ERB agonist SR9009 inhibited promoter activity of diverse HIV-subtypes and HIV-1 replication in primary T cells. This study shows a role for REV-ERB synthetic agonists to inhibit HIV-1 LTR promoter activity and viral replication, supporting a role for circadian clock components in regulating HIV-1 replication.

Histone deacetylase inhibitor chidamide promotes reactivation of latent human immunodeficiency virus by introducing histone acetylation

Highly active antiretroviral therapy can reduce the human immunodeficiency virus (HIV) viral load in the plasma to undetectable levels. However, because of the presence of latent HIV reservoirs, it is difficult to completely eradicate HIV in infected patients. Our objective was to assess the potency of chidamide, a novel histone deacetylase inhibitor recently approved for cancer treatment by the China Food and Drug Administration, to reactivate latent HIV-1 via histone acetylation. Viral reactivities of chidamide were accessed in 2 latent HIV pseudotype virus cell reporter systems (J-Lat Tat-green fluorescent protein clone A72 and TZM-bl), a latently infected full-length HIV virus cell system (U1/HIV), and resting CD4+ T cells from 9 HIV-infected patients under highly active antiretroviral therapy with undetectable viral load. Chidamide was able to increase HIV expression in each cell line, as evidenced by green fluorescent protein, luciferase activity, and p24, as well as to reactivate latent HIV-1 in primary CD4+ T cells of HIV-infected patients. Histone acetylation adjacent to the HIV promoter in A72 cells was determined by chromatin immunoprecipitation. Chidamide was able to increase histone H3 and H4 acetylation at the HIV promoter. In brief, chidamide induced the reactivation of latent HIV in pseudotype virus reporter cells, latently infected cells, and primary CD4+ T cells, making this compound an attractive option for future clinical trials.

Super elongation complex promotes early HIV transcription and its function is modulated by P-TEFb

ABSTRACTEarly work on the control of transcription of the human immunodeficiency virus (HIV) laid the foundation for our current knowledge of how RNA Polymerase II is released from promoter-proximal pausing sites and transcription elongation is enhanced. The viral Tat activator recruits Positive Transcription Elongation Factor b (P-TEFb) and Super Elongation Complex (SEC) that jointly drive transcription elongation. While substantial progress in understanding the role of SEC in HIV gene transcription elongation has been obtained, defining of the mechanisms that govern SEC functions is still limited, and the role of SEC in controlling HIV transcription in the absence of Tat is less clear.Here we revisit the contribution of SEC in early steps of HIV gene transcription. In the absence of Tat, the AF4/FMR2 Family member 4 (AFF4) of SEC efficiently activates HIV transcription, while gene activation by its homolog AFF1 is substantially lower. Differential recruitment to the HIV promoter and association with Human Polymerase-Associated Factor complex (PAFc) play key role in this functional distinction between AFF4 and AFF1. Moreover, while depletion of cyclin T1 expression has subtle effects on HIV gene transcription in the absence of Tat, knockout (KO) of AFF1, AFF4, or both proteins slightly repress this early step of viral transcription. Upon Tat expression, HIV transcription reaches optimal levels despite KO of AFF1 or AFF4 expression. However, double AFF1/AFF4 KO completely diminishes Tat trans-activation. Significantly, our results show that P-TEFb phosphorylates AFF4 and modulates SEC assembly, AFF1/4 dimerization and recruitment to the viral promoter.We conclude that SEC promotes both early steps of HIV transcription in the absence of Tat, as well as elongation of transcription, when Tat is expressed. Significantly, SEC functions are modulated by P-TEFb.

Distinct promoter activation mechanisms modulate noise-driven HIV gene expression

Latent human immunodeficiency virus (HIV) infections occur when the virus occupies a transcriptionally silent but reversible state, presenting a major obstacle to cure. There is experimental evidence that random fluctuations in gene expression, when coupled to the strong positive feedback encoded by the HIV genetic circuit, act as a ‘molecular switch’ controlling cell fate, i.e., viral replication versus latency. Here, we implemented a stochastic computational modeling approach to explore how different promoter activation mechanisms in the presence of positive feedback would affect noise-driven activation from latency. We modeled the HIV promoter as existing in one, two, or three states that are representative of increasingly complex mechanisms of promoter repression underlying latency. We demonstrate that two-state and three-state models are associated with greater variability in noisy activation behaviors, and we find that Fano factor (defined as variance over mean) proves to be a useful noise metric to compare variability across model structures and parameter values. Finally, we show how three-state promoter models can be used to qualitatively describe complex reactivation phenotypes in response to therapeutic perturbations that we observe experimentally. Ultimately, our analysis suggests that multi-state models more accurately reflect observed heterogeneous reactivation and may be better suited to evaluate how noise affects viral clearance.

The CD8+ cell non-cytotoxic antiviral response affects RNA polymerase II-mediated human immunodeficiency virus transcription in infected CD4+ cells.

A CD8+ cell non-cytotoxic antiviral response (CNAR), mediated by a CD8+ cell antiviral factor (CAF), is associated with a long-term healthy state in human immunodeficiency virus (HIV) infection. CNAR/CAF reduces viral transcription without a known effect on specific viral sequences in the HIV genome. In studies to define the mechanism involved in the block in viral transcription, we now report that transcription from the HIV-LTR reporter is reduced in infected CD4+ cells upon treatment with CAF. In agreement with this observation, the amount of RNA polymerase II (RNAPII) on the HIV promoter and other viral regions was strongly diminished in HIV-infected CD4+ cells co-cultivated with CNAR-expressing CD8+ cells. These results demonstrate further that CNAR/CAF has a specific role in regulating HIV transcription and a step during the preinitiation complex assembly appears to be sensitive to CNAR/CAF.

Involvement of Sp1 in Butyric Acid-Induced HIV-1 Gene Expression

Background/Aims: The ability of human immunodeficiency virus-1(HIV-1) to establish latent infection and its re-activation is considered critical for progression of HIV-1 infection. We previously reported that a bacterial metabolite butyric acid, acting as a potent inhibitor of histone deacetylases (HDACs), could lead to induction of HIV-1 transcription; however, the molecular mechanism remains unclear. The aim of this study was to investigate the effect of butyric acid on HIV-1 gene expression. Methods: Butyric acid-mediated HIV-1 gene expression was determined by luciferase assay and Chromatin immunoprecipitation assay. Western blot analysis and ELISA were used for the detection of HIV-1. Results: We found that Sp1 binding sites within the HIV-1 promoter are primarily involved in butyric acid-mediated HIV-1 activation. In fact, Sp1 knockdown by small interfering RNA and the Sp1 inhibitor mithramycin A abolished the effect of butyric acid. We also observed that cAMP response element-binding-binding protein (CBP) was required for butyric acid-induced HIV-1 activation. Conclusions: These results suggest that butyric acid stimulates HIV-1 promoter through inhibition of the Sp1-associated HDAC activity and recruitment of CBP to the HIV-1 LTR. Our findings suggest that Sp1 should be considered as one of therapeutic targets in anti-viral therapy against HIV-1 infection aggravated by butyric acid-producing bacteria.

A single point mutation in cyclin T1 eliminates binding to Hexim1, Cdk9 and RNA but not to AFF4 and enforces repression of HIV transcription.

BackgroundHuman immunodeficiency virus (HIV) gene expression is primarily regulated at the step of transcription elongation. The viral Tat protein recruits the Positive Transcription Elongation Factor b (P-TEFb) and the Super Elongation Complex (SEC) to the HIV promoter and enhances transcription by host RNA polymerase II.ResultsTo map residues in the cyclin box of cyclin T1 that mediate the binding of P-TEFb to its interacting host partners and support HIV transcription, a pool of N-terminal cyclin T1 mutants was generated. Binding and functional assays in cells identified specific positions in cyclin T1 that are important for (i) association of P-TEFb with Hexim1, Cdk9 and SEC/AFF4 (ii) supporting Tat-transactivation in murine cells and (iii) inhibition of basal and Tat-dependent HIV transcription in human cells. Significantly, a unique cyclin T1 mutant where a Valine residue at position 107 was mutated to Glutamate (CycT1-V107E) was identified. CycT1-V107E did not bind to Hexim1 or Cdk9, and also could not assemble on HIV TAR or 7SK-snRNA. However, it bound strongly to AFF4 and its association with HIV Tat was slightly impaired. CycT1-V107E efficiently inhibited HIV replication in human T cell lines and in CD4(+) primary cells, and enforced HIV transcription repression in T cell lines that harbor a transcriptionally silenced integrated provirus.ConclusionsThis study outlines the mechanism by which CycT1-V107E mutant inhibits HIV transcription and enforces viral latency. It defines the importance of N-terminal residues of cyclin T1 in mediating contacts of P-TEFb with its transcription partners, and signifies the requirement of a functional P-TEFb and SEC in mediating HIV transcription.

HMGA1 directly interacts with TAR to modulate basal and Tat-dependent HIV transcription

The transactivating response element (TAR) of human immunodeficiency virus 1 (HIV-1) is essential for promoter transactivation by the viral transactivator of transcription (Tat). The Tat-TAR interaction thereby recruits active positive transcription elongation factor b (P-TEFb) from its inactive, 7SK/HEXIM1-bound form, leading to efficient viral transcription. Here, we show that the 7SK RNA-associating chromatin regulator HMGA1 can specifically bind to the HIV-1 TAR element and that 7SK RNA can thereby compete with TAR. The HMGA1-binding interface of TAR is located within the binding site for Tat and other cellular activators, and we further provide evidence for competition between HMGA1 and Tat for TAR-binding. HMGA1 negatively influences the expression of a HIV-1 promoter-driven reporter in a TAR-dependent manner, both in the presence and in the absence of Tat. The overexpression of the HMGA1-binding substructure of 7SK RNA results in a TAR-dependent gain of HIV-1 promoter activity similar to the effect of the shRNA-mediated knockdown of HMGA1. Our results support a model in which the HMGA1/TAR interaction prevents the binding of transcription-activating cellular co-factors and Tat, subsequently leading to reduced HIV-1 transcription.

Controlling Cellular P-TEFb Activity by the HIV-1 Transcriptional Transactivator Tat

The human immunodeficiency virus 1 (HIV-1) transcriptional transactivator (Tat) is essential for synthesis of full-length transcripts from the integrated viral genome by RNA polymerase II (Pol II). Tat recruits the host positive transcription elongation factor b (P-TEFb) to the HIV-1 promoter through binding to the transactivator RNA (TAR) at the 5′-end of the nascent HIV transcript. P-TEFb is a general Pol II transcription factor; its cellular activity is controlled by the 7SK small nuclear RNA (snRNA) and the HEXIM1 protein, which sequester P-TEFb into transcriptionally inactive 7SK/HEXIM/P-TEFb snRNP. Besides targeting P-TEFb to HIV transcription, Tat also increases the nuclear level of active P-TEFb through promoting its dissociation from the 7SK/HEXIM/P-TEFb RNP by an unclear mechanism. In this study, by using in vitro and in vivo RNA-protein binding assays, we demonstrate that HIV-1 Tat binds with high specificity and efficiency to an evolutionarily highly conserved stem-bulge-stem motif of the 5′-hairpin of human 7SK snRNA. The newly discovered Tat-binding motif of 7SK is structurally and functionally indistinguishable from the extensively characterized Tat-binding site of HIV TAR and importantly, it is imbedded in the HEXIM-binding elements of 7SK snRNA. We show that Tat efficiently replaces HEXIM1 on the 7SK snRNA in vivo and therefore, it promotes the disassembly of the 7SK/HEXIM/P-TEFb negative transcriptional regulatory snRNP to augment the nuclear level of active P-TEFb. This is the first demonstration that HIV-1 specifically targets an important cellular regulatory RNA, most probably to promote viral transcription and replication. Demonstration that the human 7SK snRNA carries a TAR RNA-like Tat-binding element that is essential for the normal transcriptional regulatory function of 7SK questions the viability of HIV therapeutic approaches based on small drugs blocking the Tat-binding site of HIV TAR.

Oral bacteria induce a differential activation of human immunodeficiency virus‐1 promoter in T cells, macrophages and dendritic cells

The human immunodeficiency virus (HIV) can integrate into T cells, macrophages and dendritic cells resulting in a latent infection. Reports have also demonstrated that various microbial and host cell factors can trigger HIV reactivation leading to HIV recrudescence, potentially undermining highly active antiretroviral therapies. This study evaluated the capacity of oral bacteria associated with chronic periodontal infections to stimulate HIV promoter activation in various cell models of HIV latency. T cells (1G5) challenged with oral bacteria demonstrated a dose-response of HIV promoter activation with a subset of the bacteria, as well as kinetics that were generally similar irrespective of the stimuli. Direct bacterial challenge of the T cells resulted in increased activation of approximately 1.5- to 7-fold over controls. Challenge of macrophages (BF24) indicated different kinetics for individual bacteria and resulted in consistent increases in promoter activation of five fold to six fold over basal levels for all bacteria except Streptococcus mutans. Dendritic cells showed increases in HIV reactivation of 7- to 34-fold specific for individual species of bacteria. These results suggested that oral bacteria have the capability to reactivate HIV from latently infected cells, showing a relationship of mature dendritic cells > immature dendritic cells > macrophages > or = T cells. Expression of various pattern recognition receptors on these various cell types may provide insight into the primary receptors/signaling pathways used for reactivation by the bacteria.

Correction of Laminin-5 Deficiency in Human Epidermal Stem Cells by Transcriptionally Targeted Lentiviral Vectors

Deficiency of the basement membrane component laminin-5 (LAM5) causes junctional epidermolysis bullosa (JEB), a severe and often fatal skin adhesion defect. Autologous transplantation of epidermal stem cells genetically corrected with a Moloney leukemia virus (MLV)-derived retroviral vector reconstitutes LAM5 synthesis, and corrects the adhesion defect in JEB patients. However, MLV-derived vectors have genotoxic characteristics, and are unable to reproduce the physiological, basal layer-restricted expression of LAM5 chains. We have developed an alternative gene transfer strategy based on self-inactivating (SIN) or long terminal repeat (LTR)-modified lentiviral vectors, in which transgene expression is under the control of different combinations of promoter-enhancer elements derived from the keratin-14 (K14) gene. Analysis in human keratinocyte cultures and in fully differentiated skin regenerated onto immunodeficient mice showed that gene expression directed by K14 enhancers is tissue-specific and restricted to the basal layer of the epidermis. Transcriptionally targeted lentiviral vectors efficiently transduced clonogenic stem/progenitor cells derived from a skin biopsy of a JEB patient, restored normal synthesis of LAM5 in cultured keratinocytes, and reconstituted normal adhesion properties in human skin equivalents transplanted onto immunodeficient mice. These vectors are therefore an effective, and potentially more safe, alternative to MLV-based retroviral vectors in gene therapy of JEB.Molecular Therapy (2008) 16 12, 1977-1985 doi:10.1038/mt.2008.204.

Bromodomain Protein Brd4 Regulates Human Immunodeficiency Virus Transcription through Phosphorylation of CDK9 at Threonine 29

Positive transcription elongation factor b (P-TEFb), composed of cyclin-dependent kinase 9 (CDK9) and cyclin T, is a global transcription factor for eukaryotic gene expression, as well as a key factor for human immunodeficiency virus (HIV) transcription elongation. P-TEFb phosphorylates the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II (RNAP II), facilitating the transition from nonprocessive to processive transcription elongation. Recently, the bromodomain protein Brd4 has been shown to interact with the low-molecular-weight, active P-TEFb complex and recruit P-TEFb to the HIV type 1 long terminal repeat (LTR) promoter. However, the subsequent events through which Brd4 regulates CDK9 kinase activity and RNAP II-dependent transcription are not clearly understood. Here we provide evidence that Brd4 regulates P-TEFb kinase activity by inducing a negative pathway. Moreover, by analyzing stepwise initiation and elongation complexes, we demonstrate that P-TEFb activity is regulated in the transcription complex. Brd4 induces phosphorylation of CDK9 at threonine 29 (T29) in the HIV transcription initiation complex, inhibiting CDK9 kinase activity. P-TEFb inhibition is transient, as Brd4 is released from the transcription complex between positions +14 and +36. Removal of the phosphate group at T29 by an incoming phosphatase released P-TEFb activity, resulting in increased RNAP II CTD phosphorylation and transcription. Finally, we present chromatin immunoprecipitation studies showing that CDK9 with phosphorylated T29 is associated with the HIV promoter region in the integrated and transcriptionally silent HIV genome.

Promoter-targeted siRNAs Induce Gene Silencing of Simian Immunodeficiency Virus (SIV) Infection In Vitro

RNA interference is a conserved process by which sequence-specific double-stranded RNA is converted into small interfering double-stranded RNAs (siRNAs) that can induce gene silencing via two pathways: post-transcriptional gene silencing and transcriptional gene silencing (TGS). We previously reported TGS of human immunodeficiency virus-1 (HIV-1) could be induced by siRNAs targeting regions within its 5'-long-terminal repeat (5'LTR) promoter region. Here we show that promoter-targeted siRNAs can also induce silencing of simian immunodeficiency virus (SIV) replication by similar mechanisms. Suppression of productive infection was achieved in two different cell lines: a CD4, CCR5, CXCR4 expressing HeLa cell line (MAGIC-5) and in a human lymphoid cell line (CEMx174). HpaII digestion demonstrated induction of methylation at a CpG site within the SIV promoter region following siRNA-induced suppression. Both 5-azacytidine (5-AzaC) and trichostatin A (TSA), inhibitors of DNA methyltransferases (DNMTs) and histone deacetylation, respectively, partially reversed the silencing effect. Furthermore, using chromatin immunoprecipitation (ChIP) assays we found enrichment in the region of the LTR of heterochromatin markers dimethylated histone 3 lysine 9 (H3K9) and trimethylated histone 3 lysine 27 (H3K27) in the siRNA silenced cultures. Together, these results strongly suggest certain siRNAs targeting the promoter region of SIV can effect viral silencing through the induction of epigenetic changes.