Activation Of Long Terminal Repeat Research Articles

LTRs activated by Epstein-Barr virus-induced transformation of B cells alter the transcriptome.

Endogenous retroviruses (ERVs) are ancient viral elements that have accumulated in the genome through retrotransposition events. Although they have lost their ability to transpose, many of the long terminal repeats (LTRs) that originally flanked full-length ERVs maintain the ability to regulate transcription. While these elements are typically repressed in somatic cells, they can function as transcriptional enhancers and promoters when this repression is lost. Epstein-Barr virus (EBV), which transforms primary B cells into continuously proliferating cells, is a tumor virus associated with lymphomas. We report here that transformation of primary B cells by EBV leads to genome-wide activation of LTR enhancers and promoters. The activation of LTRs coincides with local DNA hypomethylation and binding by transcription factors such as RUNX3, EBF1, and EBNA2. The set of activated LTRs is unique to transformed B cells compared with other cell lines known to have activated LTRs. Furthermore, we found that LTR activation impacts the B cell transcriptome by up-regulating transcripts driven by cryptic LTR promoters. These transcripts include genes important to oncogenesis of Hodgkin lymphoma and other cancers, such as HUWE1/HECTH9. These data suggest that the activation of LTRs by EBV-induced transformation is important to the pathology of EBV-associated cancers. Altogether, our results indicate that EBV-induced transformation of B cells alters endogenous retroviral element activity, thereby impacting host gene regulatory networks and oncogenic potential.

Lysine-specific demethylase 1 cooperates with BRAF-histone deacetylase complex 80 to enhance HIV-1 Tat-mediated transactivation.

Despite the notable success of combination antiretroviral therapy, how to eradicate latent HIV-1 from reservoirs poses a challenge. The Tat protein plays an indispensable role in HIV reactivation and histone demethylase LSD1 promotes Tat-mediated long terminal repeats (LTR) activation. However, the role of LSD1 in remodeling chromatin and the role of its component BHC80 in activation of latent HIV-1 in T cells are unknown. Our findings indicate that LSD1 could decrease the level of histone H3 lysine 4 trimethylation (H3K4me3) at the HIV-1 promoter by recruiting histone lysine demethylase 5A (KDM5A) and preventing histone methyltransferase Set1A and WD-40 repeat protein 5 (WDR5) from binding to LTR. Moreover, BHC80 is necessary for LSD1-triggered LTR activation and assists LSD1 in activating LTR by binding to nucleotides 305-631 of LTR. In activated J-Lat-A2 cells, BHC80 expression was elevated and its isoform BHC80-6 promoted the association of BHC80 with LSD1. These results suggest that the LSD1-BHC80 complex enhances HIV-1 transcription by a decrease of H3K4me3 level at the viral promoter. Therefore, it might be used as a new drug target to reactivate latent HIV-1.

Scaffold attachment factor B suppresses HIV-1 infection of CD4+ T cells by preventing binding of RNA polymerase II to HIV-1's long terminal repeat

The 5' end of the HIV, type 1 (HIV-1) long terminal repeat (LTR) promoter plays an essential role in driving viral transcription and productive infection. Multiple host and viral factors regulate LTR activity and modulate HIV-1 latency. Manipulation of the HIV-1 LTR provides a potential therapeutic strategy for combating HIV-1 persistence. In this study, we identified an RNA/DNA-binding protein, scaffold attachment factor B (SAFB1), as a host cell factor that represses HIV-1 transcription. We found that SAFB1 bound to the HIV-1 5' LTR and significantly repressed 5' LTR-driven viral transcription and HIV-1 infection of CD4+ T cells. Mechanistically, SAFB1-mediated repression of HIV-1 transcription and infection was independent of its RNA- and DNA-binding capacities. Instead, by binding to phosphorylated RNA polymerase II, SAFB1 blocked its recruitment to the HIV-1 LTR. Of note, SAFB1-mediated repression of HIV-1 transcription from proviral DNA maintained HIV-1 latency in CD4+ T cells. In summary, our findings reveal that SAFB1 binds to the HIV-1 LTR and physically interacts with phosphorylated RNA polymerase II, repressing HIV-1 transcription initiation and elongation. Our findings improve our understanding of host modulation of HIV-1 transcription and latency and provide a new host cell target for improved anti-HIV-1 therapies.

Investigation of functional roles of transcription termination factor-1 (TTF-I) in HIV-1 replication.

Transcription termination factor-1 (TTF-I) is an RNA polymerase 1-mediated transcription terminator and consisting of a C-terminal DNA-binding domain, central domain, and N-terminal regulatory domain. This protein binds to a so-called ‘Sal box’ composed of an 11-base pair motif. The interaction of TTF-I with the ‘Sal box’ is important for many cellular events, including efficient termination of RNA polymerase-1 activity involved in pre-rRNA synthesis and formation of a chromatin loop. To further understand the role of TTF-I in human immunodeficiency virus (HIV)-I virus production, we generated various TTF-I mutant forms. Through a series of studies of the over-expression of TTF-I and its derivatives along with co-transfection with either proviral DNA or HIV-I long terminal repeat (LTR)-driven reporter vectors, we determined that wild-type TTF-I downregulates HIV-I LTR activity and virus production, while the TTF-I Myb-like domain alone upregulated virus production, suggesting that wild-type TTF-I inhibits virus production and trans-activation of the LTR sequence; the Myb-like domain of TTF-I increased virus production and trans-activated LTR activity.

SAMHD1 Impairs HIV-1 Gene Expression and Negatively Modulates Reactivation of Viral Latency in CD4+ T Cells.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) restricts human immunodeficiency virus type 1 (HIV-1) replication in nondividing cells by degrading intracellular deoxynucleoside triphosphates (dNTPs). SAMHD1 is highly expressed in resting CD4+ T cells, which are important for the HIV-1 reservoir and viral latency; however, whether SAMHD1 affects HIV-1 latency is unknown. Recombinant SAMHD1 binds HIV-1 DNA or RNA fragments in vitro, but the function of this binding remains unclear. Here we investigate the effect of SAMHD1 on HIV-1 gene expression and reactivation of viral latency. We found that endogenous SAMHD1 impaired HIV-1 long terminal repeat (LTR) activity in monocytic THP-1 cells and HIV-1 reactivation in latently infected primary CD4+ T cells. Overexpression of wild-type (WT) SAMHD1 suppressed HIV-1 LTR-driven gene expression at a transcriptional level. Tat coexpression abrogated SAMHD1-mediated suppression of HIV-1 LTR-driven luciferase expression. SAMHD1 overexpression also suppressed the LTR activity of human T-cell leukemia virus type 1 (HTLV-1), but not that of murine leukemia virus (MLV), suggesting specific suppression of retroviral LTR-driven gene expression. WT SAMHD1 bound to proviral DNA and impaired reactivation of HIV-1 gene expression in latently infected J-Lat cells. In contrast, a nonphosphorylated mutant (T592A) and a dNTP triphosphohydrolase (dNTPase) inactive mutant (H206D R207N [HD/RN]) of SAMHD1 failed to efficiently suppress HIV-1 LTR-driven gene expression and reactivation of latent virus. Purified recombinant WT SAMHD1, but not the T592A and HD/RN mutants, bound to fragments of the HIV-1 LTR in vitro These findings suggest that SAMHD1-mediated suppression of HIV-1 LTR-driven gene expression potentially regulates viral latency in CD4+ T cells.IMPORTANCE A critical barrier to developing a cure for HIV-1 infection is the long-lived viral reservoir that exists in resting CD4+ T cells, the main targets of HIV-1. The viral reservoir is maintained through a variety of mechanisms, including regulation of the HIV-1 LTR promoter. The host protein SAMHD1 restricts HIV-1 replication in nondividing cells, but its role in HIV-1 latency remains unknown. Here we report a new function of SAMHD1 in regulating HIV-1 latency. We found that SAMHD1 suppressed HIV-1 LTR promoter-driven gene expression and reactivation of viral latency in cell lines and primary CD4+ T cells. Furthermore, SAMHD1 bound to the HIV-1 LTR in vitro and in a latently infected CD4+ T-cell line, suggesting that the binding may negatively modulate reactivation of HIV-1 latency. Our findings indicate a novel role for SAMHD1 in regulating HIV-1 latency, which enhances our understanding of the mechanisms regulating proviral gene expression in CD4+ T cells.

Transcriptionally active LTR retroelement-related sequences and their relationship with small RNA in moso bamboo (Phyllostachys edulis)

Many different cultivars or forms with diverse phenotypes of moso bamboo have been produced during its long cultivation history. The diverse phenotypes of moso bamboo are mainly reversible and unpredictable during cultivation, which lead to the hypothesis of their epigenetic origin. Earlier studies have shown that transposable elements might be involved in the different expression patterns of moso bamboo genes. LTR (long terminal repeat) retroelement populations are the main components of moso bamboo genomes. In the present study, a genome-wide analysis addressing their potential impact on host gene expression and the regulatory network was carried out. The results show that LTR retroelements are usually inserted far away from the gene regions. Transcriptional activity appears to be common in some moso bamboo retroelement-related sequences. The expression level and number of retroelement-related sequences tend to decrease with increasing distance from the closest genes, indicating that the interaction between retroelement-related sequences and near genes might play a role in the expression pattern of retroelement-related sequences. Retroelement-related sequences generate more than 30% of the siRNAs in moso bamboo. Both 21-nt siRNA and 24-nt siRNA mainly target within LTR regions of the bamboo LTR retroelements. Given the high copy number of LTR retroelements, the transcriptional activity of LTR retroelements, and the high number of siRNAs derived from the LTR retroelements, moso bamboo LTR retroelements might be involved in the transcriptional regulation of host genes, and this may be responsible for the diverse phenotypes of moso bamboo.

Virion-Associated Vpr Alleviates a Postintegration Block to HIV-1 Infection of Dendritic Cells.

Viral protein R (Vpr) is an HIV-1 accessory protein whose function remains poorly understood. In this report, we sought to determine the requirement of Vpr for facilitating HIV-1 infection of monocyte-derived dendritic cells (MDDCs), one of the first cell types to encounter virus in the peripheral mucosal tissues. In this report, we characterize a significant restriction of Vpr-deficient virus replication and spread in MDDCs alone and in cell-to-cell spread in MDDC-CD4+ T cell cocultures. This restriction of HIV-1 replication in MDDCs was observed in a single round of virus replication and was rescued by the expression of Vpr in trans in the incoming virion. Interestingly, infections of MDDCs with viruses that encode Vpr mutants unable to interact with either the DCAF1/DDB1 E3 ubiquitin ligase complex or a host factor hypothesized to be targeted for degradation by Vpr also displayed a significant replication defect. While the extent of proviral integration in HIV-1-infected MDDCs was unaffected by the absence of Vpr, the transcriptional activity of the viral long terminal repeat (LTR) from Vpr-deficient proviruses was significantly reduced. Together, these results characterize a novel postintegration restriction of HIV-1 replication in MDDCs and show that the interaction of Vpr with the DCAF1/DDB1 E3 ubiquitin ligase complex and the yet-to-be-identified host factor might alleviate this restriction by inducing transcription from the viral LTR. Taken together, these findings identify a robust in vitro cell culture system that is amenable to addressing mechanisms underlying Vpr-mediated enhancement of HIV-1 replication.IMPORTANCE Despite decades of work, the function of the HIV-1 protein Vpr remains poorly understood, primarily due to the lack of an in vitro cell culture system that demonstrates a deficit in replication upon infection with viruses in the absence of Vpr. In this report, we describe a novel cell infection system that utilizes primary human dendritic cells, which display a robust decrease in viral replication upon infection with Vpr-deficient HIV-1. We show that this replication difference occurs in a single round of infection and is due to decreased transcriptional output from the integrated viral genome. Viral transcription could be rescued by virion-associated Vpr. Using mutational analysis, we show that domains of Vpr involved in binding to the DCAF1/DDB1/E3 ubiquitin ligase complex and prevention of cell cycle progression into mitosis are required for LTR-mediated viral expression, suggesting that the evolutionarily conserved G2 cell cycle arrest function of Vpr is essential for HIV-1 replication.

Flow Cytometric Analysis of Drug-induced HIV-1 Transcriptional Activity in A2 and A72 J-Lat Cell Lines.

The main obstacle to eradicating HIV-1 from patients is post-integration latency (Finzi et al., 1999). Antiretroviral treatments target only actively replicating virus, while latent infections that have low or no transcriptional activity remain untreated (Sedaghat et al., 2007). A combination of antiretroviral treatments with latency-purging strategies may accelerate the depletion of latent reservoirs and lead to a cure (Geeraert et al., 2008). Current strategies to reactivate HIV-1 from latency include use of prostratin, a non-tumor-promoting phorbol ester (Williams et al., 2004), BET inhibitors (Filippakopoulos et al., 2010; Delmore et al., 2011), and histone deacetylase (HDAC) inhibitors, such as suberoylanilidehydroxamic acid (i.e., SAHA or Vorinostat) (Kelly et al., 2003; Archin et al., 2009; Contreras et al., 2009; Edelstein et al., 2009). As the mechanisms of HIV-1 latency are diverse, effective reactivation may require combinatorial strategies (Quivy et al., 2002). The following protocol describes a flow cytometry-based method to quantify transcriptional activation of the HIV-1 long terminal repeat (LTR) upon drug treatment. This protocol is optimized for studying latently HIV-1-infected Jurkat (J-Lat) cell lines that contain a GFP cassette. J-Lats that contain a different reporter, for example Luciferase, can be treated with drugs as described but have to be analyzed differently.

Naf1 Regulates HIV-1 Latency by Suppressing Viral Promoter-Driven Gene Expression in Primary CD4+ T Cells.

HIV-1 latency is characterized mainly by a reversible silencing of LTR promoter-driven transcription of an integrated provirus. Cellular and viral proteins regulating LTR activity contribute to the modulation of HIV-1 latency. In this study, we found that the host protein Naf1 inhibited HIV-1 LTR-driven transcription of HIV genes and contributed to the maintenance of HIV-1 latency. Our findings provide new insights into the effects of host modulation on HIV-1 latency, which may lead to a potential therapeutic strategy for HIV persistence by targeting the Naf1 protein.

A novel long intergenic noncoding RNA indispensable for the cleavage of mouse two-cell embryos.

Endogenous retroviruses (ERVs) are transcriptionally active in cleavage stage embryos, yet their functions are unknown. ERV sequences are present in the majority of long intergenic noncoding RNAs (lincRNAs) in mouse and humans, playing key roles in many cellular processes and diseases. Here, we identify LincGET as a nuclear lincRNA that is GLN-, MERVL-, and ERVK-associated and essential for mouse embryonic development beyond the two-cell stage. LincGET is expressed in late two- to four-cell mouse embryos. Its depletion leads to developmental arrest at the late G2 phase of the two-cell stage and to MAPK signaling pathway inhibition. LincGET forms an RNA-protein complex with hnRNP U, FUBP1, and ILF2, promoting the cis-regulatory activity of long terminal repeats (LTRs) in GLN, MERVL, and ERVK (GLKLTRs), and inhibiting RNA alternative splicing, partially by downregulating hnRNP U, FUBP1, and ILF2 protein levels. Hnrnpu or Ilf2 mRNA injection at the pronuclear stage also decreases the preimplantation developmental rate, and Fubp1 mRNA injection at the pronuclear stage causes a block at the two-cell stage. Thus, as the first functional ERV-associated lincRNA, LincGET provides clues for ERV functions in cleavage stage embryonic development.

Sequence Instability in the Proviral Long Terminal Repeat and gag Regions from Peripheral Blood and Tissue-Derived Leukocytes of FIV-Infected Cats during the Late Asymptomatic Phase.

Feline immunodeficiency virus (FIV) infection results in viral persistence, a prolonged asymptomatic phase, and progressive immunopathology. During the asymptomatic phase, a cohort of experimentally FIV-infected cats exhibits features of viral latency in blood suggestive of inactive viral replication. We sought to investigate viral replication activity and genomic stability of the FIV proviral long terminal repeat (LTR) and the 5′ aspect of gag over time. FIV-infected cats during the asymptomatic phase demonstrated undetectable plasma FIV gag RNA transcripts and intermittent to undetectable blood-derived cell-associated FIV gag RNA. The LTR sequence demonstrated instability in blood-derived cells over time, in spite of low to undetectable viral replication. Sequence variation in the LTR was identified in CD4+ and CD21+ leukocytes from blood and surgically removed lymph nodes. Three single nucleotide polymorphisms (SNPs) in the LTR were commonly identified. Promoter functionality of a common LTR SNP and rare U3 mutation were examined by reporter gene assays and demonstrated either no change or increased basal FIV promoter function, respectively. In conclusion, this cohort of asymptomatic FIV-infected cats demonstrated instability of the LTR and 5’ gag sequences during the study period, in spite of undetectable plasma and rare to undetectable viral gag RNA, which suggests that blood may not accurately represent viral activity in asymptomatic FIV-infected cats.

Plant active LTR retrotransposons: a review

Long terminal repeat (LTR) retrotransposons are mobile DNA sequences that ubiquitously exist in eukaryotic genomes. They replicate themselves in the genome by copy-paste mechanism with RNA as medium. In higher plants, many active LTR retrotransposons have been applied to analyze molecular marker technology, genetic tagging, insertion mutation and gene function. Here, we systematically review the characteristics of plant active LTR retrotransposons, including their structures, copy numbers and distributions. We further analyzed the gag (group-specific antigen) and pol (polymerase) sequence features of different plants active LTR retrotransposons and the distribution patterns of the cis-acting elements in LTR regions. The results show that autonomous active LTR retrotransposons must contain LTR regions and code Gag, Pr, Int, Rt, Rh proteins. Both LTR regions are highly homologous with each other and contain many cis-regulatory elements; RVT and RNase_H1_RT domain are essential for Rt and Rh protein respectively. These results provide the basis for subsequent identification of plant active LTR retrotransposons and their functional analysis.

HIV-1 transcriptional silencing caused by TRIM22 inhibition of Sp1 binding to the viral promoter.

BackgroundIntracellular defense proteins, also referred to as restriction factors, are capable of interfering with different steps of the viral life cycle. Among these, we have shown that Tripartite motif 22 (TRIM22) suppresses basal as well as phorbol ester-induced HIV-1 long terminal repeat (LTR)-mediated transcription, independently of its E3 ubiquitin ligase activity, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) binding to the U3 region and Tat interaction with the TAR region of the HIV-1 LTR. As basal HIV-1 transcription is driven by the transcription factor specificity protein 1 (Sp1), we have investigated whether TRIM22 could interfere with Sp1-driven transcriptional activation of the HIV-1 LTR.Findings293T cells, devoid of endogenous TRIM22 expression, were transfected with a TRIM22-expressing plasmid together with reporter plasmids driven by the HIV-1 LTR promoter either containing or lacking Sp1 binding sites or with reporter plasmids driven by non-viral promoter sequences either containing or lacking the three Sp1 binding sites from the HIV-1 LTR. These reporter assays showed that TRIM22 efficiently inhibited Sp1-driven transcription. Knocking down TRIM22 expression in the CD4+ SupT1 T cell line increased the replication of Sp1-dependent HIV-1 variants. TRIM22 did not interact with Sp1, but prevented binding of Sp1 to the HIV-1 promoter, as demonstrated in protein-DNA pull down and chromatin immunoprecipitation assays.ConclusionTRIM22 acts as a suppressor of basal HIV-1 LTR-driven transcription by preventing Sp1 binding to the HIV-1 promoter.

Suppression of HTLV-1 transcription by SIRT1 deacetylase

Infection with HTLV-1 causes adult T-cell leukemia and tropical spastic paraparesis in different subsets of infected people. Treatments for HTLV-1-associated diseases are unspecific and unsatisfactory. Prophylactic measures have not been developed. Although HTLV-1 pathogenesis involves multiple stages and factors, high proviral load has been singled out as a major risk factor which predicts disease. HTLV-1 encodes Tax transactivator that potently activates transcription from viral long terminal repeats (LTR) and cellular promoters harbouring cAMP-responsive or KB element. Blocking Tax activity in infected cells should provide prophylactic and therapeutic benefits. In this study, we characterize the negative regulatory role of SIRT1 deacetylase in Tax-induced activation of HTLV-1 LTR. SIRT1 is a sirtuin with anti-cancer and anti-aging activity. Whereas over expression of SIRT1 abolished the activity of Tax to activate the LTR, compromising SIRT1 by RNA interference augmented Tax activation of the LTR. Resveratrol is a natural inhibitor of SIRT1 widely sold as a nutritional supplement and extensively tested for beneficial effects in various diseases. A SIRT1-dependent inhibition of the transcriptional activity of Tax was observed in cells treated with resveratrol. Consistently, treatment of HTLV-1-transformed T cells with resveratrol led to the activation of SIRT1 and the suppression of HTLV-1 proviral transcription. On the contrary, specific inhibition of SIRT1 by Sirtinol or Ex527 promoted HTLV-1 mRNA expression. The amount of HTLV-1 virion collected from culture supernatant was decreased in MT2 cells treated with resveratrol. Tax was found to interact with SIRT1 in HTLV-1-transformed T cells. Administration of resveratrol blocked the interaction of Tax with CREB and suppressed the recruitment of CREB and CRTC1 to the LTR. Taken together, our findings document the suppression of Tax activation of HTLV-1 transcription by SIRT1 and reveal potential benefits of small-molecule activators of SIRT1 such as resveratrol in the prevention and intervention of HTLV-1-associated diseases. Supported by SKY-MRF (2011), HK-RGC (HKU7674/12M, HKU7686/13M and HKU1/CRF/11G) and HK-HMRF (13121052) grants.

The dual action of poly(ADP-ribose) polymerase -1 (PARP-1) inhibition in HIV-1 infection: HIV-1 LTR inhibition and diminution in Rho GTPase activity.

Multifactorial mechanisms comprising countless cellular factors and virus-encoded transactivators regulate the transcription of HIV-1 (HIV). Since poly(ADP-ribose) polymerase 1 (PARP-1) regulates numerous genes through its interaction with various transcription factors, inhibition of PARP-1 has surfaced recently as a powerful anti-inflammatory tool. We suggest a novel tactic to diminish HIV replication via PARP-1 inhibition in an in vitro model system, exploiting human primary monocyte-derived macrophages (MDM). PARP-1 inhibition was capable to lessen HIV replication in MDM by 60–80% after 7 days infection. Tat, tumor necrosis factor α (TNFα), and phorbol 12-myristate 13-acetate (PMA) are known triggers of the Long Terminal Repeat (LTR), which can switch virus replication. Tat overexpression in MDM transfected with an LTR reporter plasmid resulted in a 4.2-fold increase in LTR activation; PARP inhibition caused 70% reduction of LTR activity. LTR activity, which increased 3-fold after PMA or TNFα treatment, was reduced by PARP inhibition (by 85–95%). PARP inhibition in MDM exhibited 90% diminution in NFκB activity (known to mediate TNFα- and PMA-induced HIV LTR activation). Cytoskeleton rearrangements are important in effective HIV-1 infection. PARP inactivation reduced actin cytoskeleton rearrangements by affecting Rho GTPase machinery. These discoveries suggest that inactivation of PARP suppresses HIV replication in MDM by via attenuation of LTR activation, NFκB suppression and its effects on the cytoskeleton. PARP appears to be essential for HIV replication and its inhibition may provide an effective approach to management of HIV infection.

SIRT1 Suppresses Human T-Cell Leukemia Virus Type 1 Transcription.

Human T-cell leukemia virus type 1 (HTLV-1)-associated diseases are poorly treatable, and HTLV-1 vaccines are not available. High proviral load is one major risk factor for disease development. HTLV-1 encodes Tax oncoprotein, which activates transcription from viral long terminal repeats (LTR) and various types of cellular promoters. Counteracting Tax function might have prophylactic and therapeutic benefits. In this work, we report on the suppression of Tax activation of HTLV-1 LTR by SIRT1 deacetylase. The transcriptional activity of Tax on the LTR was largely ablated when SIRT1 was overexpressed, but Tax activation of NF-κB was unaffected. On the contrary, the activation of the LTR by Tax was boosted when SIRT1 was depleted. Treatment of cells with resveratrol shunted Tax activity in a SIRT1-dependent manner. The activation of SIRT1 in HTLV-1-transformed T cells by resveratrol potently inhibited HTLV-1 proviral transcription and Tax expression, whereas compromising SIRT1 by specific inhibitors augmented HTLV-1 mRNA expression. The administration of resveratrol also decreased the production of cell-free HTLV-1 virions from MT2 cells and the transmission of HTLV-1 from MT2 cells to uninfected Jurkat cells in coculture. SIRT1 associated with Tax in HTLV-1-transformed T cells. Treatment with resveratrol prevented the interaction of Tax with CREB and the recruitment of CREB, CRTC1, and p300 to Tax-responsive elements in the LTR. Our work demonstrates the negative regulatory function of SIRT1 in Tax activation of HTLV-1 transcription. Small-molecule activators of SIRT1 such as resveratrol might be considered new prophylactic and therapeutic agents in HTLV-1-associated diseases. Human T-cell leukemia virus type 1 (HTLV-1) causes a highly lethal blood cancer or a chronic debilitating disease of the spinal cord. Treatments are unsatisfactory, and vaccines are not available. Disease progression is associated with robust expression of HTLV-1 genes. Suppressing HTLV-1 gene expression might have preventive and therapeutic benefits. It is therefore critical that host factors controlling HTLV-1 gene expression be identified and characterized. This work reveals a new host factor that suppresses HTLV-1 gene expression and a natural compound that activates this suppression. Our findings not only provide new knowledge of the host control of HTLV-1 gene expression but also suggest a new strategy of using natural compounds for prevention and treatment of HTLV-1-associated diseases.

Impact of Naturally Occurring Genetic Variation in the HIV-1 LTR TAR Region and Sp Binding Sites on Tat-Mediated Transcription

HIV-1 Tat-mediated transactivation of the long terminal repeat (LTR) requires an interaction with the transactivation response element (TAR) and is facilitated by NF-κB and Sp factors binding to sequences upstream of the transcriptional start site. Studies conducted pre- and post antiretroviral therapy identified HIV- 1-infected patients harboring a C-to-T change at position 5 of the consensus B sequence of Sp binding site III (a knockout configuration with respect to binding of Sp1). HIV-1 LTRs and Tat were amplified and cloned from patients in the Drexel Medicine CNS AIDS Research and Eradication Study (CARES) Cohort. As one simple approach to examine the impact of sequence variation on LTR fitness, LTR clones from a single patient (patient 19) were used in transient expression studies in both T-cell and monocyte-macrophage cell lines. These results demonstrated that one of 4 clones could not be transactivated by Tat derived from laboratory strain IIIB or from the patient. After inspection of the sequence of the patient-derived LTR clone defective with respect to Tat-mediated transactivation, additional alterations within the LTR were identified in a number of transcription factor binding sites in the LTR core region as well as in the TAR element that suggested that these sites may also contribute to the Tat non responsiveness. In this regard, site-directed mutagenesis indicated that optimal Tat-mediated transactivation depended on both position 32 of the TAR element and binding of Sp to all three Sp binding sites within the LTR. These studies indicate that a vast majority of LTRs derived from the integrated provirus in the peripheral blood compartment of a number of infected patients maintained the ability to drive basal and Tat- mediated transcription but that just a small number of nucleotides were shown to have a detrimental impact on LTR activation in both T cells and cells of the monocyte-macrophage lineage.

Human Pirh2 is a novel inhibitor of prototype foamy virus replication.

Prototype foamy virus (PFV) is a member of the unconventional and nonpathogenic retroviruses. PFV causes lifelong chronic infections, which are partially attributable to a number of host cell factors that restrict viral replication. Herein, we identified human p53-induced RING-H2 protein (Pirh2) as a novel inhibitor of prototype foamy virus. Overexpression of Pirh2 decreased the replication of PFV, whereas knockdown of Pirh2 with specific siRNA increased PFV replication. Dual-luciferase assays and coimmunoprecipitation demonstrated that Pirh2 negatively influences the Tas-dependent transcriptional activation of the PFV long terminal repeat (LTR) and internal promoter (IP) by interacting with the transactivator Tas and down-regulating its expression. In addition, the viral inhibitory function of Pirh2 is N-terminal and RING domain dependent. Together, these results indicated that Pirh2 suppresses PFV replication by negatively impacting its transactivator Tas and the transcription of two viral promoters, which may contribute to the latency of PFV infection.

Differential expression of HERV-K (HML-2) proviruses in cells and virions of the teratocarcinoma cell line Tera-1.

Human endogenous retrovirus (HERV-K (HML-2)) proviruses are among the few endogenous retroviral elements in the human genome that retain coding sequence. HML-2 expression has been widely associated with human disease states, including different types of cancers as well as with HIV-1 infection. Understanding of the potential impact of this expression requires that it be annotated at the proviral level. Here, we utilized the high throughput capabilities of next-generation sequencing to profile HML-2 expression at the level of individual proviruses and secreted virions in the teratocarcinoma cell line Tera-1. We identified well-defined expression patterns, with transcripts emanating primarily from two proviruses located on chromosome 22, only one of which was efficiently packaged. Interestingly, there was a preference for transcripts of recently integrated proviruses, over those from other highly expressed but older elements, to be packaged into virions. We also assessed the promoter competence of the 5’ long terminal repeats (LTRs) of expressed proviruses via a luciferase assay following transfection of Tera-1 cells. Consistent with the RNASeq results, we found that the activity of most LTRs corresponded to their transcript levels.

Impact of viral activators and epigenetic regulators on HIV-1 LTRs containing naturally occurring single nucleotide polymorphisms.

Following human immunodeficiency virus type 1 (HIV-1) integration into host cell DNA, the viral promoter can become transcriptionally silent in the absence of appropriate signals and factors. HIV-1 gene expression is dependent on regulatory elements contained within the long terminal repeat (LTR) that drive the synthesis of viral RNAs and proteins through interaction with multiple host and viral factors. Previous studies identified single nucleotide polymorphisms (SNPs) within CCAAT/enhancer binding protein (C/EBP) site I and Sp site III (3T, C-to-T change at position 3, and 5T, C-to-T change at position 5 of the binding site, respectively, when compared to the consensus B sequence) that are low affinity binding sites and correlate with more advanced stages of HIV-1 disease. Stably transfected cell lines containing the wild type, 3T, 5T, and 3T5T LTRs were developed utilizing bone marrow progenitor, T, and monocytic cell lines to explore the LTR phenotypes associated with these genotypic changes from an integrated chromatin-based microenvironment. Results suggest that in nonexpressing cell clones LTR-driven gene expression occurs in a SNP-specific manner in response to LTR activation or treatment with trichostatin A treatment, indicating a possible cell type and SNP-specific mechanism behind the epigenetic control of LTR activation.