The chromatin landscape at the HIV-1 provirus integration site determines viral expression

HIV-1 persists lifelong in memory cells of the immune system as latent provirus that rebounds upon treatment interruption. Therefore, the latent reservoir is the main target for an HIV cure. Here, we studied the direct link between integration site and transcription using LEDGINs and Barcoded HIV-ensembles (B-HIVE). LEDGINs are antivirals that inhibit the interaction between HIV-1 integrase and the chromatin-tethering factor LEDGF/p75. They were used as a tool to retarget integration, while the effect on HIV expression was measured with B-HIVE. B-HIVE tracks insert-specific HIV expression by tagging a unique barcode in the HIV genome. We confirmed that LEDGINs retarget integration out of gene-dense and actively transcribed regions. The distance to H3K36me3, the marker recognized by LEDGF/p75, clearly increased. LEDGIN treatment reduced viral RNA expression and increased the proportion of silent provirus. Finally, silent proviruses obtained after LEDGIN treatment were located further away from epigenetic marks associated with active transcription. Interestingly, proximity to enhancers stimulated transcription irrespective of LEDGIN treatment, while the distance to H3K36me3 only changed after treatment with LEDGINs. The fact that proximity to these markers are associated with RNA expression support the direct link between provirus integration site and viral expression.

PIM1 (Moloney Murine Leukemia Provirus Integration Site) Inhibition Decreases the Nonhomologous End-Joining DNA Damage Repair Signaling Pathway in Pulmonary Hypertension

Objective: Pulmonary arterial hypertension (PAH) is a fatal disease characterized by the narrowing of pulmonary arteries (PAs). It is now established that this phenotype is associated with enhanced PA smooth muscle cells (PASMCs) proliferation and suppressed apoptosis. This phenotype is sustained in part by the activation of several DNA repair pathways allowing PASMCs to survive despite the unfavorable environmental conditions. PIM1 (Moloney murine leukemia provirus integration site) is an oncoprotein upregulated in PAH and involved in many prosurvival pathways, including DNA repair. The objective of this study was to demonstrate the implication of PIM1 in the DNA damage response and the beneficial effect of its inhibition by pharmacological inhibitors in human PAH-PASMCs and in rat PAH models. Approach and Results: We found in vitro that PIM1 inhibition by either SGI-1776, TP-3654, siRNA (silencer RNA) decreased the phosphorylation of its newly identified direct target KU70 (lupus Ku autoantigen protein p70) resulting in the inhibition of double-strand break repair (Comet Assay) by the nonhomologous end-joining as well as reduction of PAH-PASMCs proliferation (Ki67-positive cells) and resistance to apoptosis (Annexin V positive cells) of PAH-PASMCs. In vivo, SGI-1776 and TP-3654 given 3× a week, improved significantly pulmonary hemodynamics (right heart catheterization) and vascular remodeling (Elastica van Gieson) in monocrotaline and Fawn-Hooded rat models of PAH. Conclusions: We demonstrated that PIM1 phosphorylates KU70 and initiates DNA repair signaling in PAH-PASMCs and that PIM1 inhibitors represent a therapeutic option for patients with PAH.

Antiretroviral APOBEC3 Cytidine Deaminases Alter HIV-1 Provirus Integration Site Profiles

ABSTRACTAPOBEC3 (A3) proteins are host-encoded deoxycytidine deaminases that provide an innate immune barrier to retroviral infection, notably against HIV-1. While the catalytic activity of these proteins can induce catastrophic hypermutation in proviral DNA leading to near-total restriction of infection, sublethal levels of deamination contribute to the genetic evolution of HIV-1. So far, little is known about how A3 might impact HIV-1 integrations into human chromosomal DNA. Using a deep sequencing approach, we analyzed the influence A3F and A3G on HIV-1 integration site selections. DNA editing was detected at the extremities of the long terminal repeat regions of the virus. Both catalytic active and non-catalytic A3 enzymes decreased insertions into gene coding sequences and increased integration sites into SINE elements, oncogenes and transcription-silencing non-B DNA features. Our data implicate A3 as host factors that influence HIV-1 integration site selection and promote insertions into genomic sites that are transcriptionally less active.GRAPHICAL ABSTRACTSchematic depicting the influence of APOBEC3 (A3) proteins on HIV integration site targeting.Left, in the absence of A3, HIV has a strong preference for integrating into genes. Right, both catalytic active and non-catalytic A3 mutants decrease integration into genes and increase integration into SINE elements and in transcription-silencing non-B DNA features.

The selectivity of inhibitors of protein kinase CK2: an update

CK2 (casein kinase 2) is a very pleiotropic serine/threonine protein kinase whose abnormally high constitutive activity has often been correlated to pathological conditions with special reference to neoplasia. The two most widely used cell permeable CK2 inhibitors, TBB (4,5,6,7-tetrabromo-1H-benzotriazole) and DMAT (2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole), are marketed as quite specific CK2 blockers. In the present study we show, by using a panel of approx. 80 protein kinases, that DMAT and its parent compound TBI (or TBBz; 4,5,6,7-tetrabromo-1H-benzimidazole) are potent inhibitors of several other kinases, with special reference to PIM (provirus integration site for Moloney murine leukaemia virus)1, PIM2, PIM3, PKD1 (protein kinase D1), HIPK2 (homeodomain-interacting protein kinase 2) and DYRK1a (dual-specificity tyrosine-phosphorylated and -regulated kinase 1a). In contrast, TBB is significantly more selective toward CK2, although it also inhibits PIM1 and PIM3. In an attempt to improve selectivity towards CK2 a library of 68 TBB/TBI-related compounds have been tested for their ability to discriminate between CK2, PIM1, HIPK2 and DYRK1a, ending up with seven compounds whose efficacy toward CK2 is markedly higher than that toward the second most inhibited kinase. Two of these, K64 (3,4,5,6,7-pentabromo-1H-indazole) and K66 (1-carboxymethyl-2-dimethylamino-4,5,6,7-tetrabromo-benzimidazole), display an overall selectivity much higher than TBB and DMAT when tested on a panel of 80 kinases and display similar efficacy as inducers of apoptosis.

Ahi-1, a Novel Gene Encoding a Modular Protein with WD40-Repeat and SH3 Domains, Is Targeted by the Ahi-1 and Mis-2 Provirus Integrations

ABSTRACT The Ahi-1 locus was initially identified as a common helper provirus integration site in Abelson pre-B-cell lymphomas and shown to be closely linked to the c-myb proto-oncogene. Since no significant alteration of c-myb expression was found in Abelson murine leukemia virus-induced pre-B-lymphomas harboring a provirus inserted within the Ahi-1 locus, this suggested that it harbors another gene whose dysregulation is involved in tumor formation. Here we report the identification of a novel gene (Ahi-1) targeted by these provirus insertional mutations and the cloning of its cDNA. The Ahi-1 proviral insertions were found at the 3′ end of the gene, in an inverse transcriptional orientation, with most of them located around and downstream of the last exon, whereas another insertion was within intron 22. In addition, another previously identified provirus insertion site, Mis-2, was found to map within the 16th intron of the Ahi-1 gene. The Ahi-1 cDNA encodes a 1,047-amino-acid protein. The predicted Ahi-1 protein is a modular protein that contains one SH3 motif and seven WD40 repeats. The Ahi-1 gene is conserved in mammals and encodes two major RNA species of 5 and 4.2 kb and several other shorter splicing variants. The Ahi-1 gene is expressed in mouse embryos and in several organs of the mouse and rat, notably at high levels in the brain and testes. In tumor cells harboring insertional mutations in Ahi-1, truncated Ahi-1/viral fused transcripts were identified, including some splicing variants with deletion of the SH3 domain. Therefore, Ahi-1 is a novel gene targeted by provirus insertion and encoding a protein that exhibits several features of a signaling molecule. Thus, Ahi-1 may play an important role in signal transduction in normal cells and may be involved in tumor development, possibly in cooperation with other oncogenes (such as v-abl and c-myc) or with a tumor suppressor gene (Nf1), since Ahi-1 insertion sites were identified in tumors harboring v-abl defective retroviruses or a c-myc transgene or in tumors exhibiting deletion of Nf1.

A majority of mice show long-term expression of a human beta-globin gene after retrovirus transfer into hematopoietic stem cells

Murine bone marrow was infected with a high-titer retrovirus vector containing the human beta-globin and neomycin phosphotransferase genes. Anemic W/Wv mice were transplanted with infected marrow which in some cases had been exposed to the selective agent G418. Human beta-globin expression was monitored in transplanted animals by using a monoclonal antibody specific for human beta-globin polypeptide, and hematopoietic reconstitution was monitored by using donor and recipient mice which differed in hemoglobin type. In some experiments all transplanted mice expressed the human beta-globin polypeptide for over 4 months, and up to 50% of peripheral erythrocytes contained detectable levels of polypeptide. DNA analysis of transplanted animals revealed that virtually every myeloid cell contained a provirus. Integration site analysis and reconstitution of secondary marrow recipients suggested that every mouse was reconstituted with at least one infected stem cell which had extensive repopulation capability. The ability to consistently transfer an active beta-globin gene into mouse hematopoietic cells improves the feasibility of using these techniques for somatic cell gene therapy in humans.

A majority of mice show long-term expression of a human beta-globin gene after retrovirus transfer into hematopoietic stem cells.

Murine bone marrow was infected with a high-titer retrovirus vector containing the human beta-globin and neomycin phosphotransferase genes. Anemic W/Wv mice were transplanted with infected marrow which in some cases had been exposed to the selective agent G418. Human beta-globin expression was monitored in transplanted animals by using a monoclonal antibody specific for human beta-globin polypeptide, and hematopoietic reconstitution was monitored by using donor and recipient mice which differed in hemoglobin type. In some experiments all transplanted mice expressed the human beta-globin polypeptide for over 4 months, and up to 50% of peripheral erythrocytes contained detectable levels of polypeptide. DNA analysis of transplanted animals revealed that virtually every myeloid cell contained a provirus. Integration site analysis and reconstitution of secondary marrow recipients suggested that every mouse was reconstituted with at least one infected stem cell which had extensive repopulation capability. The ability to consistently transfer an active beta-globin gene into mouse hematopoietic cells improves the feasibility of using these techniques for somatic cell gene therapy in humans.