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We describe regio- and enantioselective bromocyclization of difluoroalkenes catalyzed by chiral bisphosphine oxides. Owing to the simultaneous activation of both the brominating reagent and amide substrate, the desired cyclization reaction proceeds smoothly even at low temperature to provide bromodifluoromethyl-containing oxazolines with a chiral quaternary center in a highly enantioselective fashion (up to 99% ee). This protocol features the use of commercially available brominating reagents and readily accessible chiral catalysts. The regioselectivity and enantioselectivity are influenced by the catalyst structure, the brominating reagent, and the reaction temperature. Under the optimal conditions, 5-exo cyclization proceeds preferentially compared with 6-endo cyclization, depending on the electronic properties of the alkene substrates. A gram-scale synthesis of chiral oxazoline was achieved with as little as 1 mol % of the catalyst.

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HM1.24 (also known as BST-2, CD317, and Tetherin) is a type II single-pass transmembrane glycoprotein, which traverses membranes using an N-terminal transmembrane helix and is anchored in membrane lipid rafts via a C-terminal glycosylphosphatidylinositol (GPI). HM1.24 plays a role in diverse cellular functions, including cell signaling, immune modulation, and malignancy. In addition, it also functions as an interferon-induced cellular antiviral restriction factor that inhibits the replication and release of diverse enveloped viruses, and which is counteracted by Vpu, an HIV-1 accessory protein. Vpu induces down-regulation and ubiquitin conjugation to the cytoplasmic domain of HM1.24. However, evidence for ubiquitination site(s) of HM1.24 remains controversial. We demonstrated that HM1.24 is constitutively poly-ubiquitinated at the N-terminal cytoplasmic domain, and that the mutation of all potential ubiquitination sites, including serine, threonine, cysteine, and lysine in the cytoplasmic domain of HM1.24, does not affect the ubiquitination of HM1.24. We further demonstrated that although a GPI anchor is necessary and sufficient for HM1.24 antiviral activities and virion-trapping, the deleted mutant of GPI does not influence the ubiquitination of HM1.24. These results suggest that the lipid raft localization of HM1.24 is not a prerequisite for the ubiquitination. Collectively, our findings demonstrate that the ubiquitination of HM1.24 occurs at the N-terminal amino acid in the cytoplasmic domain and indicate that the constitutive ubiquitination machinery of HM1.24 may differ from the Vpu-induced machinery.

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LGP85/LIMP-2 is a type III transmembrane glycoprotein of lysosomes, which traverses the membrane twice with an N-terminal uncleaved signal sequence and C-terminal hydrophobic domain. In addition to functioning as a receptor for a lysosomal enzyme ß-glucocerebrosidase and for several enteroviruses, LGP85 plays a key role in the biogenesis and maintenance of endosomal/lysosomal compartments (ELCs). Our previous studies have demonstrated that overexpression of rat LGP85 into COS cells results in the enlarged ELCs, from where membrane trafficking is impaired. We show here that rat LGP85 is polyubiquitinated at the N-terminal short cytoplasmic domain that comprises of only three amino acid residues, alanine, arginine, and cysteine. Replacement of either arginine or cysteine with alanine within the N-terminal cytoplasmic domain did not influence the ubiquitination of LGP85, thereby indicating that ubiquitin (Ub) is conjugated to the α-NH2 group of the N-terminal alanine residue. Furthermore, we were able to define a domain necessary for ubiquitination in a region ranging from the amino acids 156 to 255 within the lumenal domain of LGP85. This is the first report showing that the integral lysosomal membrane protein LGP85 is ubiquitinated.

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Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes lethal encephalitis in humans. We previously reported that the V protein, one of the three accessory proteins encoded by the P gene, is one of the key determinants of the pathogenesis of NiV in a hamster infection model. Satterfield B.A. et al. have also revealed that V protein is required for the pathogenicity of henipavirus in a ferret infection model. However, the complete functions of NiV V have not been clarified. In this study, we identified UBX domain-containing protein 1 (UBXN1), a negative regulator of RIG-I-like receptor signaling, as a host protein that interacts with NiV V. NiV V interacted with the UBX domain of UBXN1 via its proximal zinc-finger motif in the C-terminal domain. NiV V increased the level of UBXN1 protein by suppressing its proteolysis. Furthermore, NiV V suppressed RIG-I and MDA5-dependent interferon signaling by stabilizing UBXN1 and increasing the interaction between MAVS and UBXN1 in addition to directly interrupting the activation of MDA5. Our results suggest a novel molecular mechanism by which the induction of interferon is potentially suppressed by NiV V protein via UBXN1.

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In rare cases, measles virus (MV) in children leads to fatal neurological complications such as primary measles encephalitis, post-acute measles encephalitis, subacute sclerosing panencephalitis and measles inclusion-body encephalitis. To investigate the pathogenesis of MV-induced encephalitis, rodent brain-adapted MV strains CAM/RB and CAMR40 were generated. These strains acquired mutations to adapt to the rodent brain during 40 passages in rat brain. However, it is still unknown which genes confer the neurovirulence of MV. We previously established a rescue system for recombinant MVs possessing the backbone of wild-type strain HL, an avirulent strain in mice. In the present study, to identify the genes in CAMR40 that elicit neurovirulence, we generated chimeric recombinant MVs based on strain HL. As a result, recombinant wild-type MV in which the haemagglutinin (H) gene was substituted with that of CAMR40 caused a non-lethal mild disease in mice, while additional substitution of the HL phosphoprotein (P) gene with that of strain CAMR40 caused lethal severe neurological signs comparable to those of CAMR40. These results clearly indicated that, in addition to the H gene, the P gene is required for the neurovirulence of MV CAMR40.

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Nipah virus (NiV) causes severe encephalitis in humans, with high mortality. NiV nonstructural C protein (NiV-C) is essential for its pathogenicity, but its functions are unclear. In this study, we focused on NiV-C trafficking in cells and found that it localizes predominantly in the cytoplasm but partly in the nucleus. An analysis of NiV-C mutants showed that amino acids 2, 21-24 and 110-139 of NiV-C are important for its localization in the cytoplasm. Inhibitor treatment indicates that the nuclear export determinant is not a classical CRM1-dependent nuclear export signal. We also determined that amino acids 60-75 and 72-75 were important for nuclear localization of NiV-C. Furthermore, NiV-C mutants that had lost their capacity for nuclear localization inhibited the interferon (IFN) response more strongly than complete NiV-C. These results indicate that the IFN-antagonist activity of NiV-C occurs in the cytoplasm.

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Nipah virus belongs to the genus Henipavirus in the family Paramyxoviridae, and its RNA genome is larger than those of other paramyxoviruses because it has long untranslated regions (UTRs) in each gene. However, the functions of these UTRs are not fully understood. In this study, we investigated the functions of the 5' UTRs and found that the 5' UTR of the M gene upregulated the translation of a reporter gene. Using an RNA pull-down assay, we showed that eukaryotic elongation factor 1-beta (EEF1B2) interacts with nucleotides 81-100 of the M 5' UTR and specifically enhances its translation efficiency. Our results suggest that the M 5' UTR promotes the production of M protein and viral budding by recruiting EEF1B2.

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