Hepatitis C virus (HCV) is a major cause of liver disease. Therapeutic options are limited and preventive strategies are absent. Entry is the first step of infection and requires the cooperative interaction of several host cell factors. Using a functional RNAi kinase screen we identified epidermal growth factor receptor and ephrin receptor A2 as host co-factors for HCV entry. Blocking of kinase function by approved inhibitors broadly inhibited HCV infection of all major HCV genotypes and viral escape variants in cell culture and an animal model in vivo. Receptor tyrosine kinases (RTKs) mediate HCV entry by regulating CD81-claudin-1 co-receptor associations and membrane fusion. These results identify RTKs as novel HCV entry co-factors and uncover that kinase inhibitors have significant antiviral activity. Inhibition of RTK function may constitute a novel approach for prevention and treatment of HCV infection.

Repurposing drugs as treatments for COVID-19 has drawn much attention. Beginning with sigma receptor ligands, and expanding to other drugs from screening in the field, we became concerned that phospholipidosis was a shared mechanism underlying the antiviral activity of many repurposed drugs. For all of the 23 cationic amphiphilic drugs tested, including hydroxychloroquine, azithromycin, amiodarone, and four others already in clinical trials, phospholipidosis was monotonically correlated with antiviral efficacy. Conversely, drugs active against the same targets that did not induce phospholipidosis were not antiviral. Phospholipidosis depends on the physicochemical properties of drugs, and does not reflect specific target-based activities, rather it may be considered a toxic confound in early drug discovery. Early detection of phospholipidosis could eliminate these artifacts, enabling a focus on molecules with therapeutic potential.

SummaryIn eukaryotes, mRNA export involves many evolutionarily conserved factors that carry the nascent transcript to the nuclear pore complex (NPC). The THO/TREX complex couples transcription to mRNA export and recruits the mRNA export receptor NXF1 for the transport of messenger ribonucleoprotein particles (mRNP) to the NPC. The transcription and export complex 2 (TREX-2) was suggested to interact with NXF1 and to shuttle between transcription sites and the NPC. Here, we characterize the dynamics of human TREX-2 and show that it stably associates with the NPC basket. Moreover, the association of TREX-2 with the NPC requires the basket nucleoporins NUP153 and TPR, but is independent of transcription. Differential profiles of mRNA nuclear accumulation reveal that TREX-2 functions similarly to basket nucleoporins, but differently from NXF1. Thus, our results show that TREX-2 is an NPCassociated complex in mammalian cells and suggest that it is involved in putative NPC basket-related functions.

DNA repair is essential in maintaining genome integrity and defects in different steps of the process have been linked to cancer and aging. It is a long lasting question how DNA repair is spatially and temporarily organized in the highly compartmentalized nucleus and whether the diverse nuclear compartments regulate differently the efficiency of repair. Increasing evidence suggest the involvement of nuclear pore complexes in repair of double-strand breaks (DSBs) in yeast. Here, we show that the human nucleoporin 153 (NUP153) has a role in repair of DSBs and in the activation of DNA damage checkpoints. We explore the mechanism of action of NUP153 and we propose its potential as a novel therapeutic target in cancers.Oncogene ( Keywords: DNA repair; nuclear pore; 53BP1 INTRODUCTION DNA double-strand breaks (DSBs) are particularly dangerous as their inefficient or inaccurate repair can result in mutations and chromosomal translocations that may induce cancer. 1 DSBs can be repaired by one of two major pathways: homology-based repair (homologous recombination (HR)) using the intact chromatid as a template present in proximity in S and G2 phases of the cell cycle, or direct joining across the break site (non-homologous end joining (NHEJ)). 2 The coordination between cell cycle progression and DSB repair (DSBR) is regulated by the DNA damage response (DDR) signalling pathway, which activates the cell cycle checkpoints in the presence of DNA breaks. 3 This pathway is initiated by the recruitment of the MRN (MRE11 --RAD50 --NBS1) sensor complex to sites of damage. The recruitment of MRN subsequently activates the ATM kinase, which associates with DSBs and phosphorylates the histone variant H2AX (g-H2AX). 2 MDC1 can then bind to gH2AX and recruit new MRN and ATM proteins, leading to spreading of the repair machinery along the chromosome. MDC1 also recruits ubiquitin ligases, such as RNF8 and RNF168, which facilitate the recruitment of the downstream factors 53BP1 and BRCA1. 2 When the DNA is resected to singlestranded DNA, it is recognized by replication protein A, which results in the recruitment of ATR. 2 Both the ATM and the ATR dependent branches of the pathway lead to the activation of the checkpoint kinases, CHK1 and CHK2, which stall damaged cells in their cell cycle until the lesions are resolved. 3 DNA repair, like all DNA-dependent processes, occur in the highly compartmentalized nucleus. Most nuclear events do not occur ubiquitously, but are limited to defined sites. 2 Several studies in yeast have shown that dedicated DNA repair centres exist as preferential sites of repair. 2,4 Furthermore, persistent DSBs in yeast migrate from their internal nuclear positions to the nuclear periphery, where they associate with nuclear pores. 5,6 This sequestration to the nuclear periphery was shown to require

Mitosis ensures equal segregation of the genome and is controlled by a variety of ubiquitylation signals on substrate proteins. However, it remains unexplored how the versatile ubiquitin code is read out during mitotic progression. Here, we identify the ubiquitin receptor protein UBASH3B as an important regulator of mitosis. UBASH3B interacts with ubiquitylated Aurora B, one of the main kinases regulating chromosome segregation, and controls its subcellular localization but not protein levels. UBASH3B is a limiting factor in this pathway and is sufficient to localize Aurora B to microtubules prior to anaphase. Importantly, targeting Aurora B to microtubules by UBASH3B is necessary for the timing and fidelity of chromosome segregation in human cells. Our findings uncover an important mechanism defining how ubiquitin attachment to a substrate protein is decoded during mitosis.

DNA lesions are sensed by a network of proteins that trigger the DNA damage response (DDR), a signaling cascade that acts to delay cell cycle progression and initiate DNA repair. The Mediator of DNA damage Checkpoint protein 1 (MDC1) is essential for spreading of the DDR signaling on chromatin surrounding Double Strand Breaks (DSBs) by acting as a scaffold for PI3K kinases and for ubiquitin ligases. MDC1 also plays a role both in Non-Homologous End Joining (NHEJ) and Homologous Recombination (HR) repair pathways. Here we identify two novel binding partners of MDC1, the poly (ADP-ribose) Polymerases (PARPs) TNKS1 and 2. We find that TNKSs are recruited to DNA lesions by MDC1 and regulate DNA end resection and BRCA1A complex stabilization at lesions leading to efficient DSB repair by HR and proper checkpoint activation.

SRC-3 is an important coactivator of nuclear receptors including the retinoic acid (RA) receptor α. Most of SRC-3 functions are facilitated by changes in the posttranslational code of the protein that involves mainly phosphorylation and ubiquitination. We recently reported that SRC-3 is degraded by the proteasome in response to RA. Here, by using an RNAi E3-ubiquitin ligase entry screen, we identified CUL-3 and RBX1 as components of the E3 ubiquitin ligase involved in the RA-induced ubiquitination and subsequent degradation of SRC-3. We also show that the RA-induced ubiquitination of SRC-3 depends on its prior phosphorylation at serine 860 that promotes binding of the CUL-3–based E3 ligase in the nucleus. Finally, phosphorylation, ubiquitination, and degradation of SRC-3 cooperate to control the dynamics of transcription. In all, this process participates to the antiproliferative effect of RA.

Papillomavirus (PV) E6 oncoproteins bind and often provoke the degradation of many cellular proteins important for the control of cell proliferation and/or cell death. Structural studies on E6 proteins have long been hindered by the difficulties of obtaining highly concentrated samples of recombinant E6. Here we show that recombinant E6 proteins from eight human and one bovine PV strains exist as oligomeric as well as multimeric species. These species were characterized using a variety of biochemical and biophysical techniques including analytical gel filtration, activity assays, SPR, EM and FTIR. The characterization of E6 oligomers is facilitated by the fusion to the maltose binding protein (MBP), which slows down the formation of higher-order multimeric species. The proportion of each oligomeric form vary depending on the viral strain considered. Oligomers appear to consist of folded units, which, in the case of high-risk mucosal HPV E6, retain binding to the ubiquitin ligase E6AP and the capacity to degrade the pro-apoptotic protein p53. In addition to the small-size oligomers, E6 proteins spontaneously assemble into large organized multimeric structures, a process which is accompanied by a significant increase in the β-sheet secondary structure content. Finally, co-localisation experiments using E6 equipped with different tags further demonstrate the occurrence of E6 self-association in eukaryotic cells. The ensemble of these data suggest that self-association is a general property of E6 proteins which occurs both in vitro and in vivo and might therefore be functionally relevant.