TOX4 and NOVA1 Proteins Are Partners of the LEDGF PWWP Domain and Affect HIV-1 Replication

International audiencePWWP domains are involved in the chromatin attachment of several proteins. They bind to both DNA and proteins and their interaction with specific histone methylation marks define them as a new class of histone code readers. The lens epithelium derived growth factor (LEDGF/p75) contains an N-terminal PWWP domain necessary for its interaction with chromatin but also a C-terminal domain which interacts with several proteins, such as lentiviral integrases. These two domains confer a chromatin-tethering function to LEDGF/p75 and in the case of lentiviral integrases, this tethering participates in the efficiency and site selectivity of integration. Although proteins interacting with LEDGF/p75 C-terminal domain have been extensively studied, no data exist about partners of its PWWP domain regulating its interaction with chromatin. In this study, we report the identification by yeast-two-hybrid of thirteen potential partners of the LEDGF PWWP domain. Five of these interactions were confirmed in mammalian cells, using both a protein complementation assay and co-immunoprecipitation approaches. Three of these partners interact with full length LEDGF/p75, they are specific for PWWP domains of the HDGF family and they require PWWP amino acids essential for the interaction with chromatin. Among them, the transcription activator TOX4 and the splicing cofactor NOVA1 were selected for a more extensive study. These two proteins or their PWWP interacting regions (PIR) colocalize with LEDGF/p75 in Hela cells and interact in vitro in the presence of DNA. Finally, single round VSV-G pseudotyped HIV-1 but not MLV infection is inhibited in cells overexpressing these two PIRs. The observed inhibition of infection can be attributed to a defect in the integration step. Our data suggest that a regulation of LEDGF interaction with chromatin by cellular partners of its PWWP domain could be involved in several processes linked to LEDGF tethering properties, such as lentiviral integration, DNA repair or transcriptional regulation

HEXIM1 and NEAT1 Long Non-coding RNA Form a Multi-subunit Complex that Regulates DNA-Mediated Innate Immune Response

International audienceThe DNA-mediated innate immune response underpins anti-microbial defenses and certain autoimmune diseases. Here we used immunoprecipitation, mass spectrometry, and RNA sequencing to identify a ribonuclear complex built around HEXIM1 and the long non-coding RNA NEAT1 that we dubbed the HEXIM1-DNA-PK-paraspeckle components-ribonucleoprotein complex (HDP-RNP). The HDP-RNP contains DNA-PK subunits (DNAPKc, Ku70, and Ku80) and paraspeckle proteins (SFPQ, NONO, PSPC1, RBM14, and MATRIN3). We show that binding of HEXIM1 to NEAT1 is required for its assembly. We further demonstrate that the HDP-RNP is required for the innate immune response to foreign DNA, through the cGAS-STING-IRF3 pathway. The HDP-RNP interacts with cGAS and its partner PQBP1, and their interaction is remodeled by foreign DNA. Remodeling leads to the release of paraspeckle proteins, recruitment of STING, and activation of DNAPKc and IRF3. Our study establishes the HDP-RNP as a key nuclear regulator of DNA-mediated activation of innate immune response through the cGAS-STING pathway

Interaction Of Tox4 And Nova1 Pirs With Ledgf Pwwp By Gst Pull-Down.

<p>GST pull down were performed using purified GST-PWWP protein and Flag-TOX4 PIR or Flag-NOVA1 PIR expressed and present in 293T cells extracts (A and B) or with His-TOX4 PIR or Flag-NOVA1 PIR expressed in <i>E coli</i> and purified (C). Eluted proteins following pull down were separated through 10% PA SDS-PAGE and analyzed by western blot using M2 anti-Flag antibody (A and B), H1029 anti-His antibody (C) and 4C10 anti-GST antibody (A to C). Purified GST was used as negative control for each experiment. B) Effect of DNA and RNA on interaction with PIRs in extracts was studied by DNAse or RNAse treatment of these extracts. C) Effect of DNA or PN on interaction with purified PIRs was studied by addition of a 2.6 kbp 5SG5E4 DNA fragment or a polynucleosome (PN) asssembled on it.</p

Subcellular Localization Assessed By Cellular Fractionation.

<p>A) Scheme of TOX4 and NOVA1 primary sequence with location of the PIR B) Fractionation protocol (adapted from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081217#pone.0081217-Llano1" target="_blank">[23]</a>). C) Fractionation profile of Hela cells. 30µg of lysates from non-transfected cells (for expression of endogenous LEDGF, TOX4, NOVA1 and α-tubulin) or 5 µg of lysates from cells transfected with HA-LEDGF, FLAG-TOX4 PIR or FLAG-NOVA1 PIR plasmids were used in this fractionation protocol. S1 is triton-soluble fraction, P1 triton-insoluble fraction, S2, DNAse/(NH4)₂SO₄-soluble fraction, P2, (NH4)₂SO₄ fraction. Electrophoretic migrations of endogenous NOVA1 and TOX4 proteins are more precisely described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081217#pone.0081217.s002" target="_blank">Figure S2</a> (lane Hela cells)</p

Localization Of Endogenous And Expressed Tox4 And Nova1 Proteins In Hela Cells.

<p>A) Localization of endogenous TOX4 and NOVA1 in HeLa cells. Fixed cells were co-stained with LEDGF, TOX4, NOVA1 or Coilin antibodies. Merged image is shown on right, with a zoomed panel (far right) corresponding to the red box in the merged image. The right panel corresponds to a cytofluorogram which was used to determine the degree of colocalisation using Pearsons' and Manders' coefficients. Scale bar, 10 um. B) Histogram of Mander's coefficients' for overlap of LEDGF (green) with potential partners TOX4, and NOVA1 or the non-interacting nuclear proteins Coilin and SC35 (red, left panel), or inversely, overlap of partners/Coilin/SC35 (red) with LEDGF (green, right panel). 15 individual cells were measured and error bars represent the standard error of the mean. C) Localization of expressed Flag-TOX4, Flag-NOVA1 and endogenous LEDGF in Hela cells. Fixed cells were co-stained with antibodies against Flag or LEDGF. Merged image is shown on right, with a zoomed panel of the merged image (far right). Scale bar, 10 µm.</p

Interaction Of Tox4 And Nova1, Full Length Or Pir, With Ledgf/P75 By Co-Immunoprecipitation.

<p>Total extracts of cells transiently expressing HA-LEDGF and either 3×Flag-TOX4, 3xFlag-NOVA1, PIR or full length, Flag-HIV Integrase or Flag-Brd4 were immunoprecipitated with anti-Flag M2 coupled agarose beads. Immunoprecipitated proteins were separated on 10% or 7.5% PA-SDS gels and revealed by immunoblot using antibodies indicated on the left side of the panels and more precisely described in Material and Methods section. A) HA-tagged LEDGF co-immunoprecipitates with 3×Flag tagged full-length and PIR constructs of TOX4 and NOVA1 but not with Flag Brd4. B) HA-tagged LEDGF co-immunoprecipitates Flag-HIV1 integrase. C) DNAse (but not RNAse) treatment of cell extracts abolishes HA-tagged LEDGF co-IP with 3×Flag tagged PIR of TOX4 and NOVA1. Cell extracts were digested by nothing (lane 1), DNAse (lane 2) or RNAse (lane 3) before the co-IP protocol (IP (n>3))</p

Effect On Vsv-G Pseudotyped Hiv-1 Infection Of Tox4 And Nova1 Pirs Transiently Overexpressed In Hela Cells.

<p>A) Infectivity in Hela P4 CCR5 over expressing IBD, NOVA 1 PIR and TOX 4 PIR was determined 48 hours post-infection (hpi) by measuring luciferase activity normalized to the amount of protein. B) Infections using the same virus were performed to measure the production of 2LTR circles. For this purpose, 24 hpi total genomic DNA from infected cells was used to measure 2LTR circles by real-time PCR normalized to actin. Infections carried out in the presence of Nevirapine 5 µM led to undetectable levels of both 2-LTR circles. C) Similarly, 24 hpi total genomic DNA was used to determine proviral integration sites by Alu-PCR, as described in Material and Methods. Values presented in this figure are representative of results obtained in three different experiments. Error bars correspond to one experiment performed in triplicate.</p