Activation Of Long Terminal Repeat Research Articles

Transcriptome analysis of HTLV-1-infected cells in comparison to the normal and Tax-expressing T cells

The transactivator protein of HTLV, Tax, is required for the activity of viral promoter LTR (long terminal repeat) and is capable of regulating both virus and host transcription. Tax is known to interact with several cellular factors including CREB/ATF, NF-κB and those involved in chromatin remodeling, cell cycle and DNA damage/repair. However, the process of Tax-mediated transactivation of chromosomally integrated LTR is not fully understood. Therefore, we have performed an extensive Protein/DNA transcription factor array (PD array I, Panomics) analysis on the HTLV-1 infected cells (MT-2) in comparison to the Tax-expressing cells (C8166) and normal T cells (Jurkat). Besides known factors, significant changes were observed in several new nuclear and cytoplasmic factors such as RxR, AP-1, AP-2, in both MT-2 and C8166 cells as compared to Jurkat indicating an overlap with viral replication and Tax-activity. Interestingly, MT-2 cells demonstrated the down-regulation of majority of the factors analyzed while several nuclear (NF-E1, NF-E2, TFIID) and cytoplasmic (MEF-1, RAR) factors were upregulated in C8166 suggesting Tax activity independent of viral replication. Overall, this data suggest that more scrutiny on nuclear and cytoplasmic factors associated with Tax transactivation process is needed to clearly elucidate the mechanism underlying adult-T-cell leukemia and tropical spastic paraparesis associated with HTLV-1 infection.

Making Sense out of Antisense Transcription in Human T-Cell Lymphotropic Viruses (HTLVs)

Retroviral gene expression generally depends on a full-length transcript that initiates in the 5′ long terminal repeat (LTR), which is either unspliced or alternatively spliced. We and others have demonstrated the existence of an antisense transcript initiating in the 3′ LTR of the Human T-cell Leukemia Virus type 1 (HTLV-1) that is involved in the production of HBZ (HTLV-1 basic leucine zipper (bZIP) factor). HBZ is a Fos-like factor capable of inhibiting Tax-mediated activation of the HTLV-1 LTR by interacting with the cellular transcription factor cAMP-response element-binding protein (CREB) and the pleiotropic cellular coactivators p300/CBP. HBZ can also activate cellular transcription through its interaction with p300/CBP. Interestingly, HBZ has also been found to promote T-lymphocyte proliferation. By down-regulating viral expression and by stimulating T-cell proliferation, HBZ could be essential in the establishment of a chronic infection. Antisense transcription also occurs in the closely related HTLV-2 retrovirus as well as in the recently discovered HTLV-3 and HTLV-4. These antisense transcripts are also involved in the production of retroviral proteins that we have termed Antisense Protein of HTLVs (APH). Like HBZ, the APH proteins are localized in the nucleus of transfected cells and repress Tax-mediated viral transcription.

Human T-Lymphotropic Virus Type 1 Tax Protein Complexes with P-TEFb and Competes for Brd4 and 7SK snRNP/HEXIM1 Binding

Positive transcription elongation factor b (P-TEFb) plays an important role in stimulating RNA polymerase II elongation for viral and cellular gene expression. P-TEFb is found in cells in either an active, low-molecular-weight (LMW) form or an inactive, high-molecular-weight (HMW) form. We report here that human T-lymphotropic virus type 1 (HTLV-1) Tax interacts with the cyclin T1 subunit of P-TEFb, forming a distinct Tax/P-TEFb LMW complex. We demonstrate that Tax can play a role in regulating the amount of HMW complex present in the cell by decreasing the binding of 7SK snRNP/HEXIM1 to P-TEFb. This is seen both in vitro using purified Tax protein and in vivo in cells transduced with Tax expression constructs. Further, we find that a peptide of cyclin T1 spanning the Tax binding domain inhibits the ability of Tax to disrupt HMW P-TEFb complexes. These results suggest that the direct interaction of Tax with cyclin T1 can dissociate P-TEFb from the P-TEFb/7SK snRNP/HEXIM1 complex for activation of the viral long terminal repeat (LTR). We also show that Tax competes with Brd4 for P-TEFb binding. Chromatin immunoprecipitation (ChIP) assays demonstrated that Brd4 and P-TEFb are associated with the basal HTLV-1 LTR, while Tax and P-TEFb are associated with the activated template. Furthermore, the knockdown of Brd4 by small interfering RNA (siRNA) activates the HTLV-1 LTR promoter, which results in an increase in viral expression and production. Our studies have identified Tax as a regulator of P-TEFb that is capable of affecting the balance between its association with the large inactive complex and the small active complex.

Analysis of the molecular and regulatory properties of active porcine endogenous retrovirus gamma-1 long terminal repeats in kidney tissues of the NIH-Miniature pig.

The pig genome contains the gamma 1 family of porcine endogenous retroviruses (PERVs), which are a major obstacle to the development of successful xenotransplantation from pig to human. Long terminal repeats (LTRs) found in PERVs are known to be essential elements for the control of the transcriptional activity of single virus by different transcription factors (TFs). To identify transcribed PERV LTR elements, RT-PCR and DNA sequencing analyses were performed. Twenty-nine actively transcribed LTR elements were identified in the kidney tissues of the NIH-Miniature pig. These elements were divided into two major groups (I and II), and four minor groups (I-1, I-2, I-3, and II-1), by the presence of insertion and deletion (INDEL) sequences. Group I elements showed strong transcriptional activity compared to group II elements. Four different LTR elements (PL1, PL2, PL3, and PL4) as representative of the groups were analyzed by using a transient transfection assay. The regulation of their promoter activity was investigated by treatment with M.SssI (CpG DNA methyltransferase) and garcinol (histone acetyltransferase inhibitor). The transcriptional activity of PERV LTR elements was significantly reduced by treatment with M.SssI. These data indicate that transcribed PERV LTR elements harbor sufficient promoter activity to regulate the transcription of a single virus, and the transcriptional activity of PERV LTRs may be controlled by DNA methylation events.

Drastic decrease of transcription activity due to hypermutated long terminal repeat (LTR) region in different HIV-1 subtypes and recombinants

Transcriptional activation of HIV-1 gene expression is partially controlled by the interaction between viral and cellular transcription factors acting at HIV-1 long terminal repeat (LTR) sequences. HIV-1 subtyping at LTR region and nucleotide LTR variability from clinical samples in 48 subjects carrying different HIV-1 subtypes (9A, 5C, 3D, 3F, 21G, 2H, 3J and 2 undefined) at the protease (PR) gene, were performed. LTR sequences from each HIV-1 clade were cloned in luciferase-expression vectors to determine basal and Tat-induced transcriptional activities in the presence and absence of PMA stimulation. A high number (37.8%) of recombinants at LTR/PR regions were identified. All HIV-1 promoters presented low basal transcriptional activity that was nevertheless induced by Tat and PMA. LTR activity was similar across the majority of HIV-1 variants in response to Tat and cell activation. Only subtype C and CRF01_AE LTRs presented higher basal and induced-PMA transcription activities than HXB2 clade B promoter. No basal or Tat/PMA induced activity was found in those promoters presenting G to A hypermutation compared to the wild type promoter activities. G to A hypermutation at some important transcription binding-factor sites within LTR compromised the activity of the viral promoter, decreasing the in vitro viral transcription of the luciferase gene.

Derepression of an endogenous long terminal repeat activates the CSF1R proto-oncogene in human lymphoma

Mammalian genomes contain many repetitive elements, including long terminal repeats (LTRs), which have long been suspected to have a role in tumorigenesis. Here we present evidence that aberrant LTR activation contributes to lineage-inappropriate gene expression in transformed human cells and that such gene expression is central for tumor cell survival. We show that B cell-derived Hodgkin's lymphoma cells depend on the activity of the non-B, myeloid-specific proto-oncogene colony-stimulating factor 1 receptor (CSF1R). In these cells, CSF1R transcription initiates at an aberrantly activated endogenous LTR of the MaLR family (THE1B). Derepression of the THE1 subfamily of MaLR LTRs is widespread in the genome of Hodgkin's lymphoma cells and is associated with impaired epigenetic control due to loss of expression of the corepressor CBFA2T3. Furthermore, we detect LTR-driven CSF1R transcripts in anaplastic large cell lymphoma, in which CSF1R is known to be expressed aberrantly. We conclude that LTR derepression is involved in the pathogenesis of human lymphomas, a finding that might have diagnostic, prognostic and therapeutic implications.

Elevated levels of macrophage migration inhibitory factor (MIF) in the plasma of HIV-1-infected patients and in HIV-1-infected cell cultures: A relevant role on viral replication

The cytokine macrophage migration inhibitory factor (MIF) is involved in the pathogenesis of inflammatory and infectious diseases, however its role in HIV-1 infection is unknown. Here we show that HIV-1-infected patients present elevated plasma levels of MIF, that HIV-1-infected peripheral blood mononuclear cells (PBMCs) release a greater amount of MIF, and that the HIV-1 envelope glycoprotein gp120 induces MIF secretion from uninfected PBMCs. The HIV-1 replication in PBMCs declines when these cells are treated with anti-MIF antibodies, and exposure of HIV-1-infected cells to the ABC-transporter inhibitor probenecid results in inhibition of MIF secretion. The addition of recombinant MIF (rhMIF) to HIV-1-infected PBMCs enhances viral replication of CCR5- or CXCR4-tropic HIV-1 isolates. Using a T CD4(+) cell lineage containing an HIV long terminal repeats (LTR)-Luciferase construct, we detected that rhMIF promotes transcription from HIV-1 LTR. Our results show that HIV-1 induces MIF secretion and suggest that MIF influences the HIV-1 biology through activation of HIV-1 LTR.

Regulation of SIVmac239 Basal Long Terminal Repeat Activity and Viral Replication in Macrophages: FUNCTIONAL ROLES OF TWO CCAAT/ENHANCER-BINDING PROTEIN β SITES IN ACTIVATION AND INTERFERON β-MEDIATED SUPPRESSION

CCAAT/enhancer-binding protein (C/EBP) beta and C/EBP sites in the HIV-1 long terminal repeat (LTR) are crucial for HIV-1 replication in monocyte/macrophages and for the ability of interferon beta (IFN beta) to inhibit ongoing active HIV replication in these cells. This IFN beta-mediated down-regulation involves induction of the truncated, dominant-negative isoform of C/EBP beta referred to as liver-enriched transcriptional inhibitory protein (LIP). Although binding of the C/EBP beta isoform to C/EBP sites in the simian immunodeficiency virus (SIV) LTR has previously been examined, the importance of these sites in core promoter-mediated transcription, virus replication, IFN beta-mediated regulation, and the relative binding of the two isoforms (C/EBP beta and LIP) has not been investigated. Here, we specifically examine two C/EBP sites, JC1 (-100 bp) and DS1 (+134 bp), located within the minimal region of the SIV LTR, required for core promoter-mediated transcription and virus replication in macrophages. Our studies revealed that the JC1 but not DS1 C/EBP site is important for basal level transcription, whereas the DS1 C/EBP site is imperative for productive virus replication in primary macrophages. In contrast, either JC1 or DS1 C/EBP site is sufficient to mediate IFN beta-induced down-regulation of SIV LTR activity and virus replication in these cells. We also characterized the differential binding properties of C/EBP beta and LIP to the JC1 and DS1 sites. In conjunction with previous studies from our laboratory, we demonstrate the importance of these sites in virus gene expression, and we propose a model for their role in establishing latency and persistence in macrophages in the brain.

Xenotropic Murine Leukemia Virus-Related Virus Establishes an Efficient Spreading Infection and Exhibits Enhanced Transcriptional Activity in Prostate Carcinoma Cells

Xenotropic murine leukemia virus-related virus (XMRV) is a novel human gammaretrovirus discovered in association with human prostate tumors. XMRV was first identified in prostate stromal cells surrounding the tumors of patients carrying a mutation in the HPC1 gene locus. To determine the tropism of XMRV in cell culture, we tested the ability of XMRV to spread and replicate in various prostate and nonprostate cell lines. We found that although the expression of XMRV viral proteins and the spread of infectious virus were minimal in a variety of cell lines, XMRV displayed robust expression and infection in LNCaP prostate tumor cells. The transcriptional activity of the XMRV long terminal repeat (LTR) was found to be higher than the Moloney murine leukemia virus LTRs in both LNCaP and WPMY-1 (simian virus 40-transformed prostate stromal cells). The U3 promoter of XMRV and a glucocorticoid response element (GRE) within the U3 were required for the transcriptional activity in LNCaP cells. Coexpression of the androgen receptor and stimulation with dihydrotestosterone stimulated XMRV-LTR-dependent transcription in 293T cells, and the GRE was required for this activity. These data suggest that XMRV may replicate more efficiently in LNCaP cells in part due to the transcriptional environment in LNCaP cells.

The LTR of endogenous retrovirus ev21 retains promoter activity and exhibits tissue specific transcription in chicken

Endogenous viruses integrate into the host genome and influence the expression of neighboring genes through their long terminal repeats (LTRs). In this study, we analyzed the promoter, enhancer and transcriptional activities of the chicken endogenous retrovirus ev21 LTR. The LTR was cloned into pGL3-basic and pGL3-promoter luciferase vectors in forward and reverse orientation separately. The luciferase activities were detected respectively in chicken embryonic fibroblast cell, human embryonic kidney cell line, human lung carcinoma cell line, Chinese hamster ovary cell line, and murine melanoma cell line. Relative luciferase activity analysis indicated that the ev21 LTR retained bi-directional promoter activity but no detectable enhancer activity in these cells. The constructs containing the LTR and F1 region show stronger promoter activity than the constructs containing only LTR. The transcriptional pattern of ev21 LTR varied in tissues of late feathering White Leghorn chicken at post-hatch day one. Skin exhibits the highest expression in the tissues examed. Collectively, our results indicate that the ev21 LTR exhibits tissue-type specific expression in White Leghorn chicken, and it also have regulatory potential.

Comparative functional analysis of Jembrana disease virus Tat protein on lentivirus long terminal repeat promoters: evidence for flexibility at its N-terminus

BackgroundJembrana disease virus (JDV) encodes a potent regulatory protein Tat that strongly stimulates viral expression by transactivating the long terminal repeat (LTR) promoter. JDV Tat (jTat) promotes the transcription from its own LTR as well as non-cognate LTRs, by recruiting host transcription factors and facilitating transcriptional elongation. Here, we compared the sequence requirements of jTat for transactivation of JDV, bovine immunodeficiency virus (BIV) and human immunodeficiency virus (HIV) LTRs.ResultsIn this study, we identified the minimal protein sequence for LTR activation using jTat truncation mutants. We found that jTat N-terminal residues were indispensable for transactivating the HIV LTR. In contrast, transactivation of BIV and JDV LTRs depended largely on an arginine-rich motif and some flanking residues. Competitive inhibition assay and knockdown analysis showed that P-TEFb was required for jTat-mediated LTR transactivation, and a mammalian two-hybrid assay revealed the robust interaction of jTat with cyclin T1. In addition, HIV LTR transactivation was largely affected by fusion protein at the jTat N-terminus despite the fact that the cyclin T1-binding affinity was not altered. Furthermore, the jTat N-terminal sequence enabled HIV Tat to transactivate BIV and JDV LTRs, suggesting the flexibility at the jTat N-terminus.ConclusionThis study showed the distinct sequence requirements of jTat for HIV, BIV and JDV LTR activation. Residues responsible for interaction with cyclin T1 and transactivation response element are the key determinants for transactivation of its cognate LTR. N-terminal residues in jTat may compensate for transactivation of the HIV LTR, based on the flexibility.

The genotoxic potential of retroviral vectors is strongly modulated by vector design and integration site selection in a mouse model of HSC gene therapy

gamma-Retroviral vectors (gammaRVs), which are commonly used in gene therapy, can trigger oncogenesis by insertional mutagenesis. Here, we have dissected the contribution of vector design and viral integration site selection (ISS) to oncogenesis using an in vivo genotoxicity assay based on transplantation of vector-transduced tumor-prone mouse hematopoietic stem/progenitor cells. By swapping genetic elements between gammaRV and lentiviral vectors (LVs), we have demonstrated that transcriptionally active long terminal repeats (LTRs) are major determinants of genotoxicity even when reconstituted in LVs and that self-inactivating (SIN) LTRs enhance the safety of gammaRVs. By comparing the genotoxicity of vectors with matched active LTRs, we were able to determine that substantially greater LV integration loads are required to approach the same oncogenic risk as gammaRVs. This difference in facilitating oncogenesis is likely to be explained by the observed preferential targeting of cancer genes by gammaRVs. This integration-site bias was intrinsic to gammaRVs, as it was also observed for SIN gammaRVs that lacked genotoxicity in our model. Our findings strongly support the use of SIN viral vector platforms and show that ISS can substantially modulate genotoxicity.

O-Linked N -Acetylglucosaminylation of Sp1 Inhibits the Human Immunodeficiency Virus Type 1 Promoter

Human immunodeficiency virus type 1 (HIV-1) gene expression and replication are regulated by the promoter/enhancer located in the U3 region of the proviral 5' long terminal repeat (LTR). The binding of cellular transcription factors to specific regulatory sites in the 5' LTR is a key event in the replication cycle of HIV-1. Since transcriptional activity is regulated by the posttranslational modification of transcription factors with the monosaccharide O-linked N-acetyl-D-glucosamine (O-GlcNAc), we evaluated whether increased O-GlcNAcylation affects HIV-1 transcription. In the present study we demonstrate that treatment of HIV-1-infected lymphocytes with the O-GlcNAcylation-enhancing agent glucosamine (GlcN) repressed viral transcription in a dose-dependent manner. Overexpression of O-GlcNAc transferase (OGT), the sole known enzyme catalyzing the addition of O-GlcNAc to proteins, specifically inhibited the activity of the HIV-1 LTR promoter in different T-cell lines and in primary CD4(+) T lymphocytes. Inhibition of HIV-1 LTR activity in infected T cells was most efficient (>95%) when OGT was recombinantly overexpressed prior to infection. O-GlcNAcylation of the transcription factor Sp1 and the presence of Sp1-binding sites in the LTR were found to be crucial for this inhibitory effect. From this study, we conclude that O-GlcNAcylation of Sp1 inhibits the activity of the HIV-1 LTR promoter. Modulation of Sp1 O-GlcNAcylation may play a role in the regulation of HIV-1 latency and activation and links viral replication to the glucose metabolism of the host cell. Hence, the establishment of a metabolic treatment might supplement the repertoire of antiretroviral therapies against AIDS.

Role of mitogen‐activated protein kinases, nuclear factor‐κB, and interferon regulatory factor 3 in Toll‐like receptor 4‐mediated activation of HIV long terminal repeat

Monocytes/macrophages are known to represent a potential reservoir of human immunodeficiency virus type 1 (HIV-1), which ensures continuous replication of the virus in patients on highly active antiretroviral therapy (HAART). Infected macrophages are a highly productive source of HIV-1 during infections with common opportunistic pathogens. Previous studies report that toll like receptors (TLR)s play a role in HIV-1 replication in macrophages. Here, we investigate the three main pathways activated through TLR4 and the interactions with the HIV-1 long terminal repeat (LTR), using human embryonic kidney (HEK) 293 cells expressing TLR4 and transfected with a luciferase reporter under the control of the HIV-1 LTR. Here, we demonstrate, that TLR4-mediated activation of HIV-LTR is largely governed by the nuclear factor-kappaB pathway. Neither of the mitogen-activated protein kinases ERK1/2, JNK, or p38 nor the transcription factor interferon regulatory factor 3 were involved in the direct transactivation of HIV-LTR through stimulation of TLR4.

Reduced Genotoxicity of Avian Sarcoma Leukosis Virus Vectors in Rhesus Long-term Repopulating Cells Compared to Standard Murine Retrovirus Vectors

Insertional mutagenesis continues to be a major concern in hematopoietic stem-cell gene therapy. Nonconventional gene transfer vectors with more favorable integration features in comparison with conventional retrovirus and lentivirus vectors are being developed and optimized. In this study, we report for the first time a systematic analysis of 198 avian sarcoma leukosis virus (ASLV) insertion sites identified in rhesus long-term repopulating cells, and a comparison of ASLV insertion patterns to Moloney murine leukemia virus (MLV) (n = 396) and simian immunodeficiency virus (SIV) (n = 289) using the newly released rhesus genome databank. Despite a weak preference toward gene-coding regions, ASLV integration is nonclustered, does not favor gene-rich regions, transcription start sites, or CpG islands. There was no propensity for ASLV insertions within or near proto-oncogenes, and most importantly, no insertions close to or within the Mds1-Evi1 locus, which is in contrast to the significant over-representation of this insertion site for MLV vectors in the same transplantation model. Furthermore, ASLV long terminal repeats (LTRs) do not have detectable promoter and enhancer activity in a quantitative luciferase assay to measure neighboring gene activation. The combination of these features is unique for ASLV and suggests that optimized vectors based on this virus could be useful and safe for gene transfer to hematopoietic stem cells and progenitor cells.

Reactive oxygen species activate HIV long terminal repeat via post-translational control of NF-κB

Reduction/oxidation disorder is one of the most common ailments in HIV-infected patients, and such patients are frequently left exposed to chronic oxidative stress after the generation of reactive oxygen species (ROS). Although a variety of clinical trials to inhibit HIV infection have been conducted by focusing on oxidative stress, their precise targets and reaction mechanism have remained unclear. In this study, we demonstrate that H2O2 treatment strongly induced HIV long terminal repeat (LTR)-driven luciferase expression in Jurkat T lymphocytes via NF-κB activation. Treatment with the SN50 peptide or the mutation of NF-κB binding site on LTR resulted in impaired LTR activity in response to ROS. H2O2 induced both IκB degradation and covalent modification of p65. CBP/p300-induced hyperacetylation as well as phosphorylation of p65 was implicated in ROS-mediated LTR activation. The results of our study showed that ROS-induced HIV LTR activation involves immediate early NF-κB activation at the post-translational level.

TLR2-Mediated Cell Stimulation in Bacterial Vaginosis

Bacterial vaginosis (BV) is associated with preterm labor, pelvic inflammatory disease (PID) and increased HIV acquisition, although the pathways that mediate these pathological effects have not been elucidated. To determine the presence of Toll-like receptor (TLR)-ligands and their specificity in BV, genital tract fluids were collected from women with and without BV by cervicovaginal lavage (CVL). The CVL samples were evaluated for their ability to stimulate secretion of proinflammatory cytokines and to activate NFkappaB and the HIV long terminal repeat (LTR), indicators of TLR activation, in human monocytic cells. Stimulation with BV CVLs induced higher levels of IL-8 and TNFalpha secretion, as well as higher levels of HIV LTR and NFkappaB activation, than CVLs from women with normal healthy bacterial flora. To identify which TLRs were important in BV, 293 cells expressing specific TLRs were exposed to CVL samples. BV CVLs induced higher IL-8 secretion by cells expressing TLR2 than CVLs from women without BV. Surprisingly, BV CVLs did not stimulate cells expressing TLR4/MD2, although these cells responded to purified lipopolysaccharide (LPS), a TLR4 ligand. BV CVLs, in cells expressing TLR2, also activated the HIV LTR. Thus, these studies show that soluble factor(s) present in the lower genital tract of women with BV activate cells via TLR2, identifying a pathway through which BV may mediate adverse effects.

Crystal Structure of NFAT Bound to the HIV-1 LTR Tandem κB Enhancer Element

The host factor, nuclear factor of activated T-cells (NFAT), regulates the transcription and replication of HIV-1. Here, we have determined the crystal structure of the DNA binding domain of NFAT bound to the HIV-1 long terminal repeat (LTR) tandem kappaB enhancer element at 3.05 A resolution. NFAT binds as a dimer to the upstream kappaB site (Core II), but as a monomer to the 3' end of the downstream kappaB site (Core I). The DNA shows a significant bend near the 5' end of Core I, where a lysine residue from NFAT bound to the 3' end of Core II inserts into the minor groove and seems to cause DNA bases to flip out. Consistent with this structural feature, the 5' end of Core I become hypersensitive to dimethylsulfate in the in vivo footprinting upon transcriptional activation of the HIV-1 LTR. Our studies provide a basis for further investigating the functional mechanisms of NFAT in HIV-1 transcription and replication.

Molecular basis of the internalization of bovine immunodeficiency virus Tat protein

Bovine Immunodeficiency Virus (BIV) is a nonacute, pathogenic, and horizontally transmitted lentivirus. It shares the parallel properties in morphology and genetics with human immunodeficiency virus type 1 and other lentiviruses. BIV encodes its own transactivator (BTat), which transactivates its cognate long terminal repeat (LTR). However, the mechanism involved in the transactivation is different from that in HIV and other lentiviruses. We determined the mechanisms of BTat internalization by cells and the effect of BTat on neighboring cells. The green fluorescent protein fusion analysis indicated that the internalization of extracellular BTat was a time and dose-dependent, but endocytosis and energy-independent manner. Arginine residues in the arginine-rich motif (ARM) of BTat were definitively responsible for the internalization. Internalized BTat is predominantly present in the nucleus, resulting in LTR activation and NF-kappaB induction. These results propose that the secretion and internalization of BTat facilitates BIV in influencing neighboring cells and makes the cellular environment propitious to viral replication.

CCR5 signaling through phospholipase D involves p44/42 MAP‐kinases and promotes HIV‐1 LTR‐directed gene expression

The chemokine receptor CCR5 plays an important role as an entry gate for the human immunodeficiency virus-1 (HIV-1) and for viral postentry events. Among signal transducers used by chemoattractant receptors, the phosphatidylcholine-specific phospholipase D (PLD) produces large amounts of second messengers in most cell types. However, the relevance of PLD isoforms to CCR5 signaling and HIV-1 infection process remains unexplored. We show here that CCR5 activation by MIP-1beta in HeLa-MAGI cells triggered a rapid and substantial PLD activity, as assessed by mass choline production. This activity required the activation of ERK1/2-MAP kinases and involved both PLD1 and PLD2. MIP-1beta also promoted the activation of an HIV-1 long terminal repeat (LTR) by the transactivator Tat in HeLa P4.2 cells through a process involving ERK1/2. Expression of wild-type and catalytically inactive PLDs dramatically boosted and inhibited the LTR activation, respectively, without altering Tat expression. Wild-type and inactive PLDs also respectively potentiated and inhibited HIV-1(BAL) replication in MAGI cells. Finally, in monocytic THP-1 cells, antisense oligonucleotides to both PLDs dramatically inhibited the HIV-1 replication. Thus, PLD is activated downstream of ERK1/2 upon CCR5 activation and plays a major role in promoting HIV-1 LTR transactivation and virus replication, which may open novel perspectives to anti-HIV-1 strategies.