Valérie Martinelli

The AP-1 Binding Sites Located in the pol Gene Intragenic Regulatory Region of HIV-1 Are Important for Viral Replication

Our laboratory has previously identified an important intragenic region in the human immunodeficiency virus type 1 (HIV-1) genome, whose complete functional unit is composed of the 5103 fragment, the DNaseI-hypersensitive site HS7 and the 5105 fragment. These fragments (5103 and 5105) both exhibit a phorbol 12-myristate 13-acetate (PMA)-inducible enhancer activity on the herpes simplex virus thymidine kinase promoter. Here, we characterized the three previously identified AP-1 binding sites of fragment 5103 by showing the PMA-inducible in vitro binding and in vivo recruitment of c-Fos, JunB and JunD to this fragment located at the end of the pol gene. Functional analyses demonstrated that the intragenic AP-1 binding sites are fully responsible for the PMA-dependent enhancer activity of fragment 5103. Moreover, infection of T-lymphoid Jurkat and promonocytic U937 cells with wild-type and mutant viruses demonstrated that mutations of the intragenic AP-1 sites individually or in combination altered HIV-1 replication. Importantly, mutations of the three intragenic AP-1 sites led to a decreased in vivo recruitment of RNA polymerase II to the viral promoter, strongly supporting that the deleterious effect of these mutations on viral replication occurs, at least partly, at the transcriptional level. Single-round infections of monocyte-derived macrophages confirmed the importance of intragenic AP-1 sites for HIV-1 infectivity.

New pyridinone derivatives as potent HIV-1 nonnucleoside reverse transcriptase inhibitors

Several 5-ethyl-6-methyl-4-cycloalkyloxy-pyridin-2(1H)-ones were synthesized and evaluated for their anti HIV-1 activities against wild-type virus and clinically relevant mutant strains. A racemic mixture (10) with methyl substituents at positions 3 and 5 of the cyclohexyloxy moiety had potent antiviral activity against wild-type HIV-1. Subsequent stereoselective synthesis of a stereoisomer displaying both methyl groups in equatorial position was found to have the best EC(50). Further modulations focused on position 3 of the pyridinone ring improved the antiviral activity against mutant viral strains. Compounds bearing a 3-ethyl (22) or 3-isopropyl group (23) had the highest activity against wild-type HIV-1 and displayed low-nanomolar potency against several clinically relevant mutant strains.

Localisation of Nup153 and SENP1 to nuclear pore complexes is required for 53BP1-mediated DNA double-strand break repair

ABSTRACT The nuclear basket of nuclear pore complexes (NPCs) is composed of three nucleoporins: Nup153, Nup50 and Tpr. Nup153 has a role in DNA double-strand break (DSB) repair by promoting nuclear import of 53BP1 (also known as TP53BP1), a mediator of the DNA damage response. Here, we provide evidence that loss of Nup153 compromises 53BP1 sumoylation, a prerequisite for efficient accumulation of 53BP1 at DSBs. Depletion of Nup153 resulted in reduced SUMO1 modification of 53BP1 and the displacement of the SUMO protease SENP1 from NPCs. Artificial tethering of SENP1 to NPCs restored non-homologous end joining (NHEJ) in the absence of Nup153 and re-established 53BP1 sumoylation. Furthermore, Nup50 and Tpr, the two other nuclear basket nucleoporins, also contribute to proper DSB repair, in a manner distinct from Nup153. Similar to the role of Nup153, Tpr is implicated in NHEJ and homologous recombination (HR), whereas loss of Nup50 only affects NHEJ. Despite the requirement of all three nucleoporins for accurate NHEJ, only Nup153 is needed for proper nuclear import of 53BP1 and SENP1-dependent sumoylation of 53BP1. Our data support the role of Nup153 as an important regulator of 53BP1 activity and efficient NHEJ. Highlighted Article: Nup153, previously shown to be required for nuclear import of 53BP1, is also required for sumoylation of 53BP1, acting by displacing the SUMO protease SENP1, contributing to efficient DNA double-strand break repair.

Expression of leukemia-associated Nup98 fusion proteins generates an aberrant nuclear envelope phenotype

Chromosomal translocations involving the nucleoporin NUP98 have been described in several hematopoietic malignancies, in particular acute myeloid leukemia (AML). In the resulting chimeric proteins, Nup98s N-terminal region is fused to the C-terminal region of about 30 different partners, including homeodomain (HD) transcription factors. While transcriptional targets of distinct Nup98 chimeras related to immortalization are relatively well described, little is known about other potential cellular effects of these fusion proteins. By comparing the sub-nuclear localization of a large number of Nup98 fusions with HD and non-HD partners throughout the cell cycle we found that while all Nup98 chimeras were nuclear during interphase, only Nup98-HD fusion proteins exhibited a characteristic speckled appearance. During mitosis, only Nup98-HD fusions were concentrated on chromosomes. Despite the difference in localization, all tested Nup98 chimera provoked morphological alterations in the nuclear envelope (NE), in particular affecting the nuclear lamina and the lamina-associated polypeptide 2α (LAP2α). Importantly, such aberrations were not only observed in transiently transfected HeLa cells but also in mouse bone marrow cells immortalized by Nup98 fusions and in cells derived from leukemia patients harboring Nup98 fusions. Our findings unravel Nup98 fusion-associated NE alterations that may contribute to leukemogenesis.

Structural and theoretical studies of [6-bromo-1-(4-fluorophenylmethyl)-4(1H)-quinolinon-3-yl)]-4-hydroxy-2-oxo-3-butenoïc acid as HIV-1 integrase inhibitor.

Ethyl [6-bromo-1-(4-fluorophenylmethyl)-4(1H)-quinolinon-3-yl]-4-hydroxy-2-oxo-3-butenoate 1 and [6-bromo-1-(4-fluorophenylmethyl)-4(1H)-quinolinon-3-yl)]-4-hydroxy-2-oxo-3-butenoïc acid 2 were synthesized as potential HIV-1 integrase inhibitors and evaluated for their enzymatic and antiviral activity, acidic compound 2 being more potent than ester compound 1. X-ray diffraction analyses and theoretical calculations show that the diketoacid chain of compound 2 is preferentially coplanar with the quinolinone ring (dihedral angle of 0-30 degrees ). Docking studies suggest binding modes in agreement with structure-activity relationships.

Transcriptional regulation of the bovine leukemia virus promoter by the cyclic AMP-response element modulator tau isoform.

Bovine leukemia virus (BLV) expression is controlled at the transcriptional level through three TaxBLV-responsive elements (TxREs) responsive to the viral transactivator TaxBLV. The cAMP-responsive element (CRE)-binding protein (CREB) has been shown to interact with CRE-like sequences present in the middle of each of these TxREs and to play critical transcriptional roles in both basal and TaxBLV-transactivated BLV promoter activity. In this study, we have investigated the potential involvement of the cAMP-response element modulator (CREM) in BLV transcriptional regulation, and we have demonstrated that CREM proteins were expressed in BLV-infected cells and bound to the three BLV TxREs in vitro. Chromatin immunoprecipitation assays using BLV-infected cell lines demonstrated in the context of chromatin that CREM proteins were recruited to the BLV promoter TxRE region in vivo. Functional studies, in the absence of TaxBLV, indicated that ectopic CREMτ protein had a CRE-dependent stimulatory effect on BLV promoter transcriptional activity. Cross-link of the B-cell receptor potentiated CREMτ transactivation of the viral promoter. Further experiments supported the notion that this potentiation involved CREMτ Ser-117 phosphorylation and recruitment of CBP/p300 to the BLV promoter. Although CREB and TaxBLV synergistically transactivated the BLV promoter, CREMτ repressed this TaxBLV/CREB synergism, suggesting that a modulation of the level of TaxBLV transactivation through opposite actions of CREB and CREMτ could facilitate immune escape and allow tumor development.

Mutations in nucleoporin NUP88 cause lethal fetal akinesia deformation sequence

Fetal akinesia deformation sequence (FADS) comprises a spectrum of clinically and genetically heterogeneous disorders with congenital malformations related to impaired fetal movement. FADS often results from mutations in genes affecting the muscle nicotinic acetylcholine receptor (AChR). Here we describe mutations in NUP88 coding for the nucleoporin NUP88 as a novel cause of lethal FADS in two families. A homozygous c.1300G>T (p.D434Y) mutation in two individuals and a compound heterozygous mutation c.1525C>T (p.R509*) and c1899_1901del (p.E634del) in one individual were found. We show that genetic disruption of nup88 in zebrafish results in pleiotropic developmental defects reminiscent of those seen in affected human fetuses, including locomotor defects as well as defects at neuromuscular junctions. Loss of NUP88 coincides with aberrant levels and localization of rapsyn, a key regulator of AChR clustering. These findings expand our understanding of the molecular events causing FADS and suggest that variants in NUP88 should be investigated in cases of FADS.Nucleoporins build the nuclear pore complex (NPC), which, as sole gate for nuclear-cytoplasmic exchange, are of outmost importance for normal cell function. Defects in the process of nucleocytoplasmic transport or in its machinery have been frequently described in human diseases, such as cancer and neurodegenerative disorders, but only in a few cases of developmental disorders. Here we report biallelic mutations in the nucleoporin NUP88 as a novel cause of lethal fetal akinesia deformation sequence (FADS) in two families. FADS comprises a spectrum of clinically and genetically heterogeneous disorders with congenital malformations related to impaired fetal movement. We show that genetic disruption of nup88 in zebrafish results in pleiotropic developmental defects reminiscent of those seen in affected human fetuses, including locomotor defects as well as defects at neuromuscular junctions. Phenotypic alterations become visible at distinct developmental stages, both in affected human fetuses and in zebrafish, whereas early stages of development are apparently normal. The zebrafish phenotypes caused by nup88 deficiency are only rescued by expressing wild-type nup88 and not the disease-linked mutant forms of nup88. Furthermore, using human and mouse cell lines as well as immunohistochemistry on fetal muscle tissue, we demonstrate that NUP88 depletion affects rapsyn, a key regulator of the muscle nicotinic acetylcholine receptor at the neuromuscular junction. Together, our studies provide the first characterization of NUP88 in vertebrate development, expand our understanding of the molecular events causing FADS, and suggest that variants in NUP88 should be investigated in cases of FADS.

The AP-1 binding sites located in the pol gene intragenic regulatory region of HIV-1 are important for virus infectivity

We have previously identified three AP-1 binding sites in the pol gene of human immunodeficiency virus type 1 (HIV-1) and shown that short oligonucleotides containing these sites functioned as phorbol ester-inducible enhancers (Van Lint et al., 1991, J. Virol., 65:7066-7072). These sites are located in a region, called fragment 5103, exhibiting a phorbol ester-inducible enhancing activity on the viral thymidine kinase promoter in HeLa cells. In this study, we have further characterized each of the AP-1 binding sites and have shown that transcription factors cFos, JunB and JunD interacted in vitro with these motifs. For each site, we have identified mutations abolishing AP1 factor binding without altering the underlying amino acid sequence of the HIV-1 reverse transcriptase. By transient transfection assays, we have demonstrated that the intragenic AP-1 binding sites were entirely responsible for the PMA-dependent transcriptional activity of fragment 5103. Moreover, this PMA-stimulated activity of fragment 5103 was inhibited by a dominant-negative A-Fos mutant provided the AP-1 sites were not mutated. Finally, we have investigated the biological significance of the intragenic AP-1 binding sites in HIV-1 replication and have shown that these sites are important for viral infectivity. from Frontiers of Retrovirology: Complex retroviruses, retroelements and their hosts Montpellier, France. 21-23 September 2009