LTR Promoter Research Articles

Cellular heat shock factor 1 positively regulates human immunodeficiency virus-1 gene expression and replication by two distinct pathways

Human immunodeficiency virus-1 (HIV-1) infection leads to changes in cellular gene expression, which in turn tend to modulate viral gene expression and replication. Cellular heat shock proteins (HSPs) are induced upon heat shock, UV irradiation and microbial or viral infections. We have reported earlier Nef-dependent induction of HSP40 leading to increased HIV-1 gene expression; however, the mechanism of induction remained to be elucidated. As expression of HSPs is regulated by heat shock factors (HSFs), we have now studied the role of HSF1 not only in Nef-dependent HSP40 induction but also in HIV-1 gene expression. Our results show that HSF1 is also induced during HIV-1 infection and it positively regulates HIV-1 gene expression by two distinct pathways. First, along with Nef it activates HSP40 promoter which in turn leads to increased HIV-1 gene expression. Second, HSF1 directly interacts with newly identified HSF1 binding sequence on HIV-1 LTR promoter and induces viral gene expression and replication. Thus, the present work not only identifies a molecular basis for HSF1-mediated enhancement of viral replication but also provides another example of how HIV-1 uses host cell machinery for its successful replication in the host.

231 The MHC Class II Transactivator, CIITA, is a Viral Restriction Factor for Human Oncogenic Retroviruses

We show that CIITA, the master regulator of MHC-II gene expression, inhibits the replication of HTLV-1 by targeting the viral transactivator Tax-1. CIITA and Tax-1 physically interact in vivo and the first 108 aminoacids of Tax-1 are necessary for this binding. Two adjacent regions (1-252 and 253-410) of CIITA bind independently to Tax-1, but only region 1-252 mediates Tax-1 inhibition, in agreement with the fact that CIITA residues from positions 64-124 are required to block Tax-1 transactivation. The CIITA inhibitory action on Tax-1 function correlates with the nuclear localization of CIITA and is independent of the transcription factor NF-YB, previously involved in CIITA-mediated inhibition of HTLV-2 Tax-2. CIITA severely impairs the physical and functional interaction of Tax-1 with the cellular co-activator PCAF, which is required for the optimal activation of HTLV-1 promoter. Accordingly, CIITA-inhibited transactivation of the viral LTR promoter by Tax-1, is rescued by the over-expression of PCAF. Together with the previously reported inhibition of two other human retroviruses, HIV-1 and HTLV-2, these findings support the idea that CIITA might have evolved as a general defense mechanism of the host against retroviruses, not only because it activates the immune response against the infectious agents, but also because of its intrinsic capacity to act as an endogenous viral restriction factor. This investigation was supported by University of Insubria “FAR” to GT; I.S.S. National Research Project on A.I.D.S. n° 40F.1 and 45G.1 to R.S.A.; Fondazione Cariplo grant 2008-2230 and AIRC, grant IG 8862 to R.S.A.

Down-regulation of HIV-1 infection by inhibition of the MAPK signaling pathway

The human immunodeficiency virus type 1 (HIV-1) can interact with and exploit the host cellular machinery to replicate and propagate itself. Numerous studies have shown that the Mitogen-activated protein kinase (MAPK) signal pathway can positively regulate the replication of HIV-1, but exactly how each MAPK pathway affects HIV-1 infection and replication is not understood. In this study, we used the Extracellular signal-regulated kinase (ERK) pathway inhibitor, PD98059, the Jun N-terminal kinase (JNK) pathway inhibitor, SP600125, and the p38 pathway inhibitor, SB203580, to investigate the roles of these pathways in HIV-1 replication. We found that application of PD98059 results in a strong VSV-G pseudotyped HIV-1(NL4-3) luciferase reporter virus and HIV-1(NL4-3) virus inhibition activity. In addition, SB203580 and SP600125 also elicited marked VSV-G pseudotyped HIV-1(NL4-3) luciferase reporter virus inhibition activity but no HIV-1(NL4-3) virus inhibition activity. We also found that SB203580 and SP600125 can enhance the HIV-1 inhibition activity of PD98059 when cells were treated with all three MAPK pathway inhibitors in combination. Finally, we show that HIV-1 virus inhibition activity of the MAPK pathway inhibitors was the result of the negative regulation of HIV-1 LTR promoter activity.

Vpr-Host Interactions During HIV-1 Viral Life Cycle

Human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) is a multifunctional viral protein that plays important role at multiple stages of the HIV-1 viral life cycle. Although the molecular mechanisms underlying these activities are subject of ongoing investigations, overall, these activities have been linked to promotion of viral replication and impairment of anti-HIV immunity. Importantly, functional defects of Vpr have been correlated with slow disease progression of HIV-infected patients. Vpr is required for efficient viral replication in non-dividing cells such as macrophages, and it promotes, to some extent, viral replication in proliferating CD4+ T cells. The specific activities of Vpr include modulation of fidelity of viral reverse transcription, nuclear import of the HIV-1 pre-integration complex, transactivation of the HIV-1 LTR promoter, induction of cell cycle G2 arrest and cell death via apoptosis. In this review, we focus on description of the cellular proteins that specifically interact with Vpr and discuss their significance with regard to the known Vpr activities at each step of the viral life cycle in proliferating and non-proliferating cells.

A Novel Non-SET Domain Multi-subunit Methyltransferase Required for Sequential Nucleosomal Histone H3 Methylation by the Mixed Lineage Leukemia Protein-1 (MLL1) Core Complex

Gene expression within the context of eukaryotic chromatin is regulated by enzymes that catalyze histone lysine methylation. Histone lysine methyltransferases that have been identified to date possess the evolutionarily conserved SET or Dot1-like domains. We previously reported the identification of a new multi-subunit histone H3 lysine 4 methyltransferase lacking homology to the SET or Dot1 family of histone lysine methyltransferases. This enzymatic activity requires a complex that includes WRAD (WDR5, RbBP5, Ash2L, and DPY-30), a complex that is part of the MLL1 (mixed lineage leukemia protein-1) core complex but that also exists independently of MLL1 in the cell. Here, we report that the minimal complex required for WRAD enzymatic activity includes WDR5, RbBP5, and Ash2L and that DPY-30, although not required for enzymatic activity, increases the histone substrate specificity of the WRAD complex. We also show that WRAD requires zinc for catalytic activity, displays Michaelis-Menten kinetics, and is inhibited by S-adenosyl-homocysteine. In addition, we demonstrate that WRAD preferentially methylates lysine 4 of histone H3 within the context of the H3/H4 tetramer but does not methylate nucleosomal histone H3 on its own. In contrast, we find that MLL1 and WRAD are required for nucleosomal histone H3 methylation, and we provide evidence suggesting that each plays distinct structural and catalytic roles in the recognition and methylation of a nucleosome substrate. Our results indicate that WRAD is a new H3K4 methyltransferase with functions that include regulating the substrate and product specificities of the MLL1 core complex.

Expression of interfering RNAs from an HIV-1 Tat-inducible chimeric promoter

The therapeutic value of antiviral interfering RNAs could be improved by technologies that limit their expression to the infected cell population. The HIV-1 Tat-inducible viral LTR and LTR-containing chimeric promoters have previously been used to drive expression of antiviral RNAs and proteins directed against HIV-1. Here, we characterize an alternative promoter, consisting of a chicken β-actin core promoter fused to the viral TAR element, for the conditional expression of interfering RNAs. This promoter, that we refer to as the CK-TAR promoter, can induce levels of silencing comparable to the viral LTR in response to Tat produced from co-transfected plasmids or during viral replication. While the CK-TAR promoter shows a modest level of basal activity, similar to the viral LTR, it is less responsive to the extracellular stimuli tested including LPS, TNFα, and PMA. The CK-TAR promoter is an alternative Tat-inducible promoter with the potential to minimize the risk of vector mobilization and to drive polycistronic expression of interfering RNAs.

A Ubiquitous Chromatin Opening Element (UCOE) Confers Resistance to DNA Methylation–mediated Silencing of Lentiviral Vectors

DNA methylation may restrict the activity of gene transfer vectors due to inadvertent silencing. In P19 embryonic carcinoma cells in vitro, we found that transgene expression regulated by the SFFV LTR and EF1 alpha promoter declined rapidly within 16 days, but for A2UCOE derived from the human HNRPA2B1-CBX3 housekeeping gene locus, remained completely stable. Silencing correlated with extensive epigenetic methylation of CpG sites, whereas the A2UCOE was almost completely resistant. Linking of the A2UCOE upstream of the SFFV LTR protected this element from both DNA methylation and silencing. Analysis of engrafted hematopoietic cells in vivo transduced with the same vectors revealed a similar pattern. The A2UCOE displayed little or no methylation in either primary or secondary graft recipients, and gene expression profiles were highly conserved between the two groups. These studies provide convincing evidence that DNA methylation plays a direct role in regulating self-inactivating (SIN) lentiviral transgene expression, and that the stability of expression from the A2UCOE is, at least in part, due to methylation resistance. The A2UCOE therefore has considerable utility for gene therapy applications where reliable and sustained gene expression is desirable.

Analysis of infectious virus clones from two HIV-1 superinfection cases suggests that the primary strains have lower fitness

BackgroundTwo HIV-1 positive patients, L and P, participating in the Amsterdam Cohort studies acquired an HIV-1 superinfection within half a year from their primary HIV-1 infection (Jurriaans et al., JAIDS 2008, 47:69-73). The aim of this study was to compare the replicative fitness of the primary and superinfecting HIV-1 strains of both patients. The use of isolate-specific primer sets indicated that the primary and secondary strains co-exist in plasma at all time points after the moment of superinfection.ResultsBiological HIV-1 clones were derived from peripheral blood CD4 + T cells at different time point, and identified as the primary or secondary virus through sequence analysis. Replication competition assays were performed with selected virus pairs in PHA/IL-2 activated peripheral blood mononuclear cells (PBMC's) and analyzed with the Heteroduplex Tracking Assay (HTA) and isolate-specific PCR amplification. In both cases, we found a replicative advantage of the secondary HIV-1 strain over the primary virus. Full-length HIV-1 genomes were sequenced to find possible explanations for the difference in replication capacity. Mutations that could negatively affect viral replication were identified in the primary infecting strains. In patient L, the primary strain has two insertions in the LTR promoter, combined with a mutation in the tat gene that has been associated with decreased replication capacity. The primary HIV-1 strain isolated from patient P has two mutations in the LTR that have been associated with a reduced replication rate. In a luciferase assay, only the LTR from the primary virus of patient P had lower transcriptional activity compared with the superinfecting virus.ConclusionsThese preliminary findings suggest the interesting scenario that superinfection occurs preferentially in patients infected with a relatively attenuated HIV-1 isolate.

ChemInform Abstract: Stresgenin B, an Inhibitor of Heat-Induced Heat Shock Protein Gene Expression, Produced by Streptomyces sp. AS-9.

Stresgenin B was isolated as an inhibitor of heat-induced heat shock protein (HSP) gene expression from a culture broth of Streptomyces sp. AS-9 by silica gel chromatography and HPLC. The molecular formula of the novel compound was determined as C11H13NO5 by high resolution FAB-MS analysis, and the structure was determined by UV, 1H NMR, 13C NMR, HMQC, HMBC, and NOESY spectra. Stresgenin B inhibited heat-induced luciferase reporter-gene expression directed by the human hsp70B promoter in Chinese hamster ovary (CHO) cells at concentrations lower than the concentrations for inhibition of dexamethasone-induced luciferase reporter-gene expression directed by the mouse mammary tumor virus (MMTV)-LTR promoter. The inhibition of heat-induced reporter gene expression was evident even when cells were exposed to stresgenin B only during heat stress treatment. Moreover, the compound inhibited heat-induced syntheses of hsp72/73, hsp90, and hsp110 and thereby suppressed the induction of thermotolerance. Stresgenin B showed moderate cytotoxic activities against several neoplastic cell lines and also showed antibacterial activities against Micrococcus luteus, Bacillus subtilis and Staphylococcus aureus strains.

Heterologous HIV-based lentiviral/adenoviral vectors immunizations result in enhanced HIV-specific immunity

Viral vectors are considered as one of the major means for the induction of strong immune responses against recombinant antigens by genetic immunization. Among these, lentiviral vectors are particularly attractive vehicles, as they can infect a wide variety of cells and can transduce replicating as well as non-replicating cells. We have engineered VRX1023, an HIV-1-based lentiviral vector (LV) vaccine candidate, to deliver HIV-1 Gag, Pol and Rev antigens under control of the native LTR promoter. While VRX1023 has been shown to elicit strong cell-mediated and humoral immunity as a stand-alone vaccine, we report here its combination in a heterologous prime–boost approach. Its combination with an adenovirus serotype 5 (Ad5)-based vector in the mouse model increased the frequency and polyfunctionality of HIV-specific CD4+ and CD8+ T cells. Homologous prime–boost regimens induced high levels of anti-vector neutralizing antibodies in Ad5-immunized mice, whereas the VSV-G-pseudotyped VRX1023 LV elicited low levels of anti-lentiviral vector neutralization. In addition, the heterologous prime–boost strategy resulted in a 5-fold reduction in Ad5-specific vector neutralization as compared to Ad5 homologous immunization. In conclusion, this study demonstrates that LV and Ad5 vector candidates can be combined in a heterologous immunization regimen, yielding dramatically improved immunogenicity while overcoming anti-vector immunity. These findings may have implications for the development of HIV vaccine regimens in populations with elevated Ad5 seroprevalence or when repeated vector administrations are required.

Localization of an hTERT repressor region on human chromosome 3p21.3 using chromosome engineering

Telomerase is a ribonucleoprotein enzyme that synthesizes telomeric DNA. The reactivation of telomerase activity by aberrant upregulation/expression of its catalytic subunit hTERT is a major pathway in human tumorigenesis. However, regulatory mechanisms that control hTERT expression are largely unknown. Previously, we and others have demonstrated that the introduction of human chromosome 3, via microcell-mediated chromosome transfer (MMCT), repressed transcription of the hTERT gene. These results suggested that human chromosome 3 contains a regulatory factor(s) involved in the repression of hTERT. To further localize this putative hTERT repressor(s), we have developed a unique experimental approach by introducing various truncated chromosome 3 regions produced by a novel chromosomal engineering technology into the renal cell carcinoma cell line (RCC23 cells). These cells autonomously express ectopic hTERT (exohTERT) promoted by a retroviral LTR promoter in order to permit cellular division after repression of endogenous hTERT. We found a telomerase repressor region located within a 7-Mb interval on chromosome 3p21.3. These results provide important information regarding hTERT regulation and a unique method to identify hTERT repressor elements.

Novel HIV-1 therapeutics through targeting altered host cell pathways

The emergence of drug-resistant HIV-1 strains presents a challenge for the design of new drugs. Anti-HIV compounds currently in use are the subject of advanced clinical trials using either HIV-1 reverse transcriptase, viral protease or integrase inhibitors. Recent studies show an increase in the number of HIV-1 variants resistant to anti-retroviral agents in newly infected individuals. Targeting host cell factors involved in the regulation of HIV-1 replication might be one way to combat HIV-1 resistance to the currently available anti-viral agents. A specific inhibition of HIV-1 gene expression could be expected from the development of compounds targeting host cell factors that participate in the activation of the HIV-1 LTR promoter. Here we discuss how targeting the host can be accomplished either by using small molecules to alter the function of the host's proteins such as p53 or cdk9, or by utilizing new advances in siRNA therapies to knock down essential host factors such as CCR5 and CXCR4. Finally, we will discuss how the viral protein interactomes should be used to better design therapeutics against HIV-1.

Gene looping between the viral LTRs is a hallmark of transcriptionally active HIV-1 and Mo-MLV proviral DNA

By exploiting the Chromosome Conformation Capture (3C) technique, we have previously shown that the HIV-1 provirus forms a transcription-dependent gene loop structure between the 5' LTR promoter and 3' LTR poly(A) signal. Flavopiridol-mediated inhibition of RNA polymerase II elongation blocked 5' to 3' LTR juxtaposition, indicating that this structure is maintained during transcription [1]. The formation of such high order chromatin structure, if aberrantly generated between a retroviral LTR sequence and the promoter of a neighboring gene, has the potential to activate transcription of the cellular gene. This would provide a molecular explanation to the insertional mutagenesis events observed in several gene therapy clinical trials entailing the use of retroviral vectors, leading to the activation of cellular protoconcogenes by means of long-range chromosomal interactions. Indeed, despite the vast amount of data on the insertion sites of retroviral vectors in preclinical and clinical studies, the molecular mechanisms connecting retrovirus integration to aberrant cellular gene activation are still almost completely obscure. We first wanted to test whether LTR-mediated gene looping is also a characteristics of Mo-MLV-based retroviral vectors. In this respect, compared to HIV-1, Mo-MLV has a much weaker poly(A) signal, having thus higher potential to mobilize cellular sequences due to the frequent 3' poly(A) read-through. We produced and characterized a number of HeLa clones harboring single integrations of a Mo-MLV vector carrying a selectable gene. Using the 3C technique, we observed that, in all the analyzed clones, a gene loop was present juxtaposing the 5' and 3' LTRs of the vector. Thus, gene looping is not a prerogative of HIV-1 but a characteristic also extended to oncoretroviruses. We are currently in the process of analyzing, in our clones, the levels of expression of the cellular genes neighboring the proviral integration sites within regions spanning 50 kb upstream or downstream of the insertions and correlating the eventual changes with the formation of aberrant loops. In addition, 3C experiments are also performed in a series of HaCaT clones in which up-regulation of genes surrounding retroviral integration has already been demonstrated (in collaboration with F. Mavilio, Milan, Italy) and will later be extended to cell clones from patients with SCID-X1 who were enrolled in one of the recent gene therapy clinical trials (with A. Thrasher, London, UK).

Uracil DNA glycosylase 2 negatively regulates HIV-1 LTR transcription

Numerous cellular factors belonging to the DNA repair machineries, including RAD18, RAD52, XPB and XPD, have been described to counteract human immunodeficiency virus type 1 (HIV-1) replication. Recently, Uracil DNA glycosylase 2 (UNG2), a major determinant of the uracil base excision repair pathway, was shown to undergo rapid proteasome-dependent degradation following HIV-1 infection. However, the specific role of intracellular UNG2 depletion during the course of HIV-1 infection is not clearly understood. Our study shows for the first time that overexpression of UNG2 inhibits HIV-1 replication. We demonstrate that this viral inhibition is correlated with a marked decrease in transcription efficiency as shown by monitoring HIV-1 LTR promoter activity and quantification of HIV-1 RNA levels. Interestingly, UNG2 inhibits LTR activity when stimulated by Tat transactivator or TNFα, while barely affected using Phorbol ester activation. Mutational analysis of UNG2 indicates that antiviral activity may require the integrity of the UNG2 catalytic domain and Vpr-interacting domain. Altogether, our data indicate that UNG2 is likely to represent a new host defense factor specifically counteracted by HIV-1 Vpr. The molecular mechanisms involved in the UNG2 antiviral activity still remain elusive but may rely on the sequestration of specific cellular factor(s) critical for viral transcription.

Replication kinetics and persistence of a conditionally live attenuated SIV (SIVrtTA) in vivo confers protection against SIVmac239 wild-type challenge in a rhesus macaque model

Vaccination with live attenuated SIV confers potent protection against wild-type SIV challenge in the SIV/macaque model of HIV. Although safety concerns preclude the direct application of this vaccine approach in humans, a clearer understanding of its mechanism of protection may lead to the development of novel AIDS vaccines. A novel live attenuated SIVmac239 Δnef vaccine (SIVrtTA), dependent on doxycycline administration for replication, has been evaluated in vivo and its ability to protect against pathogenic SIV challenge determined. Two groups of six Indian rhesus macaques (Groups A and B) were vaccinated with SIVrtTA with an oral doxycycline dosing regime for 6 months. In Group A, doxycycline was stopped for 8 weeks before wtSIVmac239 challenge. A third group was vaccinated with a conventional SIVmac239Δnef vaccine. In the presence of doxycycline, all 12 macaques receiving SIVrtTA were infected exhibiting peak SIV RNA levels (log10 3-5 SIV RNA copies/ml) which declined in all macaques but persisted at higher levels in three SIVrtTA vaccinates. Levels of virus replication measured by independent qRT-PCR and qDNA PCR assays were lower in SIVrtTA vaccinates compared with the SIVmac239Δnef vaccine which peaked in the log10 4-6 SIV RNA copies/ml range. No escape from doxycycline dependence was identified. Upon challenge with wild-type SIVmac239 and compared with naive challenge controls (Group D), a significant vaccine effect was observed in all three vaccine groups, assessed by the comparative reduction of circulating viral RNA in plasma. Naive wtSIVmac239 challenge controls exhibited ~7 log10 SIV RNA copies/ml at peak which persisted beyond the acute infection phase. This compared with all three vaccine groups where post wtSIVmac239 viral RNA levels were markedly reduced among SIVrtTA vaccinates with a more pronounced vaccine effect among Group A vaccinates. Viral RNA was un-detectable in one SIVrtTA vaccinate (Group A) and in the 2 log10 range in another (Group B). The highest levels of protection in SIVrtTA vaccinates correlated with an enhanced persistence of SIVrtTA as circulating viral RNA in the immediate post-acute infection vaccine period. Retrospective sequence analysis recovered from SIVrtTA vaccinates indicated a selection of sequence variants in the LTR promoter region and the Tat and rtTA genes that do not affect doxycycline control but which may have influenced replication in vivo. Vaccination with SIVrtTA induced a marked vaccine effect which will enable the role of viral persistence to be evaluated in this vaccination strategy. SIVrtTA represents a new tool to develop and design novel AIDS vaccines.

The viral Tat protein has no essential function in HIV-1 replication other than to activate transcription

It is generally accepted that the Tat protein has a pivotal role in HIV-1 replication because it activates transcription from the viral LTR promoter through binding of the TAR hairpin in the nascent RNA transcript. However, several other functions of Tat have been suggested in e.g. HIV-1 RNA translation, reverse transcription and RNAi suppression. How important these functions are in viral replication is difficult to determine in replication studies with Tat-mutated HIV-1 variants because of the dominant effect of Tat inactivation on gene expression. We therefore used an HIV-1 variant that does not depend on the Tat/TAR components for transcription to reassess whether or not Tat has a second essential function in HIV-1 replication. This HIV-rtTA variant uses the incorporated Tet-On gene expression system for activation of transcription. We previously showed that complete deletion of TAR is compatible with HIV-rtTA replication. Complete inactivation of Tat does however inhibit HIV-rtTA replication and further analysis revealed that Tat stimulates transcription via sequence elements in the U3 promoter region. In agreement with this, evolution of the Tat-minus virus in long-term cultures resulted in U3 changes that increased promoter activity. We generated a novel HIV-rtTA variant that lacks HIV-1 U3 promoter and TAR sequences. This virus replicates efficiently without Tat and spontaneously acquired Tat-inactivating mutations in long-term cultures. Our results show that Tat does not only activate HIV-1 transcription through binding to the TAR RNA hairpin but also through an interaction via U3 DNA elements. Moreover, these results demonstrate that Tat has no other essential function during HIV-1 replication in T cell lines in vitro.

Fourth NF-κB site in HIV-1 subtype-C LTR confers functional advantage to viral gene expression

Subtype-C strains of HIV-1, among the various viral subtypes, are responsible for ~50% of the global and 85-99% of Indian infections. Among others, the most significant molecular feature differentially conserved in the subtype-C promoter is the polymorphism within the enhancer region constituted by NF-κB sites. While the viral promoter of majority of the subtypes contains two NF-κB sites, subtype-C promoter consists of three canonical motifs. Notably, a minority of clade-C primary isolates contain κB or κB-like sites, in addition to the canonical κB sites. Previous studies from our laboratory identified nearly 6% (34/609) of the primary isolates from India to demonstrate κB-site polymorphism in C-LTR. The functional importance of additional κB or κB-like sites in C-LTR has not been evaluated. We confirmed the subtype nature of the viral isolates by sequencing and phylogenetic analysis of LTR, Tat and Env in 20 of 34 samples demonstrating κB-site polymorphism. Sequence analysis of the additional κB- or κB-like sites identified extensive variation among the viral isolates. Among them, a particular sequence variation, constituting the κB-like site GGGACTTTCT, with a C-T variation at position 10, was found to be the most common. The functional importance of the κB-like site in C-LTR has been evaluated by reporter gene expression from isogenic LTR promoters and EMSA. We compared gene expression pattern of LTR constructs in Jurkat cells under different conditions of cell activation including TNF-α, PMA, PHA and combinations that activate NF-κB pathway. The data demonstrate the strongest reporter gene expression from the LTRs containing the additional κB-like sites especially under synergistic activation conditions. Gel shift experiments with TNF-α activated Jurkat cell nuclear extracts showed the recruitment of p50/p65 heterodimer to the variant κB-like site. Our data for the first time provide experimental evidence that the κB-like site in C-LTR is an authentic κB-site and this site confers quantitative and qualitative gain of function. Furthermore, we hypothesize that this gain of function may augment viral fitness and hence contribute to global predominance of subtype-C infections.

Uracil DNA Glycosylase 2 negatively regulates HIV-1 LTR transcription

Numerous cellular factors belonging to the DNA repair machineries, including RAD18, RAD52, XPB and XPD, have been described to counteract human immunodeficiency virus type 1 (HIV-1) replication. Recently, Uracil DNA glycosylase 2 (UNG2), a major determinant of the uracil base excision repair pathway, was shown to undergo rapid proteasome-dependent degradation following HIV-1 infection. However, the specific role of intracellular UNG2 depletion during the course of HIV-1 infection is not clearly understood. Our study shows for the first time that overexpression of UNG2 inhibits HIV-1 replication. We demonstrate that this viral inhibition is correlated with a marked decrease in transcription efficiency as shown by monitoring HIV-1 LTR promoter activity and quantification of HIV-1 RNA levels. Interestingly, UNG2 inhibits LTR activity when stimulated by Tat transactivator or TNFα, while barely affected using Phorbol ester activation. Mutational analysis of UNG2 indicates that antiviral activity may require the integrity of the UNG2 catalytic domain. Altogether, our data indicate that UNG2 is likely to represent a new host defense factor specifically counteracted by HIV-1 Vpr. The molecular mechanisms involved in the UNG2 antiviral activity still remain elusive but may rely on the sequestration of specific cellular factor(s) critical for viral transcription.

Bipartite functions of the CREB co-activators selectively direct alternative splicing or transcriptional activation

The CREB regulated transcription co-activators (CRTCs) regulate many biological processes by integrating and converting environmental inputs into transcriptional responses. Although the mechanisms by which CRTCs sense cellular signals are characterized, little is known regarding how CRTCs contribute to the regulation of cAMP inducible genes. Here we show that these dynamic regulators, unlike other co-activators, independently direct either pre-mRNA splice-site selection or transcriptional activation depending on the cell type or promoter context. Moreover, in other scenarios, the CRTC co-activators coordinately regulate transcription and splicing. Mutational analyses showed that CRTCs possess distinct functional domains responsible for regulating either pre-mRNA splicing or transcriptional activation. Interestingly, the CRTC1-MAML2 oncoprotein lacks the splicing domain and is incapable of altering splice-site selection despite robustly activating transcription. The differential usage of these distinct domains allows CRTCs to selectively mediate multiple facets of gene regulation, indicating that co-activators are not solely restricted to coordinating alternative splicing with increase in transcriptional activity.

PML involvement in the p73-mediated E1A-induced suppression of EGFR and induction of apoptosis in head and neck cancers

Epidermal growth factor receptor (EGFR) tyrosine kinase is commonly overexpressed in human cancers; however, the cellular mechanisms regulating EGFR expression remain unclear. p53, p63 and p73 are transcription factors regulating many cellular targets involved in controlling the cell cycle and apoptosis. p53 activates EGFR expression, whereas TAp63 represses EGFR transcription. The involvement of p73 in the regulation of EGFR has not been reported. Here, a strong correlation between EGFR overexpression and increased levels of the oncogenic DeltaNp73 isoform in head and neck squamous cell carcinoma (HNSCC) cell lines was observed. Ectopic expression of TAp73, particularly TAp73beta, resulted in suppression of the EGFR promoter, significant downregulation of EGFR protein and efficient induction of cell death in all six EGFR-overexpressing HNSCC cell lines. EGFR overexpression from a heterologous LTR promoter protected lung cancer cells from TAp73beta-induced EGFR suppression and apoptosis. Expression of TAp73beta efficiently induced promyelocytic leukaemia (PML) protein expression and PML knockdown by shRNA attenuated the downregulation of EGFR and induction of apoptosis by p73 in HNSCC cells. Furthermore, PML was found to be important for E1A-induced suppression of EGFR and subsequent killing of HNSCC cells. Our data therefore suggest a novel pathway involving PML and p73 in the regulation of EGFR expression.