Characterization of RyDEN (C19orf66) as an Interferon-Stimulated Cellular Inhibitor against Dengue Virus Replication

Dengue virus (DENV) is one of the most important arthropod-borne pathogens that cause life-threatening diseases in humans. However, no vaccine or specific antiviral is available for dengue. As seen in other RNA viruses, the innate immune system plays a key role in controlling DENV infection and disease outcome. Although the interferon (IFN) response, which is central to host protective immunity, has been reported to limit DENV replication, the molecular details of how DENV infection is modulated by IFN treatment are elusive. In this study, by employing a gain-of-function screen using a type I IFN-treated cell-derived cDNA library, we identified a previously uncharacterized gene, C19orf66, as an IFN-stimulated gene (ISG) that inhibits DENV replication, which we named Repressor of yield of DENV (RyDEN). Overexpression and gene knockdown experiments revealed that expression of RyDEN confers resistance to all serotypes of DENV in human cells. RyDEN expression also limited the replication of hepatitis C virus, Kunjin virus, Chikungunya virus, herpes simplex virus type 1, and human adenovirus. Importantly, RyDEN was considered to be a crucial effector molecule in the IFN-mediated anti-DENV response. When affinity purification-mass spectrometry analysis was performed, RyDEN was revealed to form a complex with cellular mRNA-binding proteins, poly(A)-binding protein cytoplasmic 1 (PABPC1), and La motif-related protein 1 (LARP1). Interestingly, PABPC1 and LARP1 were found to be positive modulators of DENV replication. Since RyDEN influenced intracellular events on DENV replication and, suppression of protein synthesis from DENV-based reporter construct RNA was also observed in RyDEN-expressing cells, our data suggest that RyDEN is likely to interfere with the translation of DENV via interaction with viral RNA and cellular mRNA-binding proteins, resulting in the inhibition of virus replication in infected cells.     

Intratracheal gene transfer of decorin reduces subpleural fibroproliferation induced by bleomycin

Decorin, a small leucin-rich proteoglycan, is a negative regulator of transforming growth factor-beta, but the antifibrotic effect of decorin gene transfer has not been examined in a mouse model of usual interstitial pneumonia (UIP). We constructed a replication-defective recombinant adenovirus harboring human decorin gene (AdCMV.DC) and administered 1 x l0(9) plaque-forming units of AdCMV.DC intratracheally or intravenously to C57BL/6 mice with intraperitoneal injection of bleomycin, which induces a subpleural fibroproliferation, mimicking UIP, by day 28. Only intratracheal administration of AdCMV.DC increased decorin mRNA expression in the lung and decreased the hydroxyproline content augmented in bleomycin-induced pulmonary fibrosis (1.13 +/- 0.02 to 0.96 +/- 0.02, P = 0.006). In contrast, intravenous administration of AdCMV.DC increased the decorin expression only in the liver, but not in the lung, and without reducing lung fibrosis. These results indicate that adenoviral decorin gene transfer is effective only by direct administration to fibrosing lungs.     

Injection of the insulin receptor alpha subunit increases blood glucose levels in mice

Using the expression vector of the truncated human insulin receptor (hIR), we have constructed a stable Chinese hamster ovary (CHO) cell line which secretes the His-tagged alpha subunit (insulin-binding domain) of hIR into medium. To examine characteristics of the His-tagged hIRalpha, we purified the protein secreted from the CHO cells. The His-tagged hIRalpha was glycosylated and processed a dimer. The molecule bound insulin with an affinity similar to that of the intact hIR. The His-tagged full length of hIR was autophosphorylated by insulin stimulation in CHO cells. Injection of the purified His-tagged hIRalpha into veins of mice increased in the concentration of blood glucose within 30 min. The intraperitoneal glucose tolerance test (ipGTT) done after injection of the purified His-tagged hIRalpha showed evidence of a marked hyperglycemia. These findings provide direct evidence that the presence of hIRalpha in the blood stream inhibits insulin actions by binding with plasma insulin.     

Copper(II) complexes of 1,10-phenanthroline-derived ligands: studies on DNA binding properties and nuclease activity

A series of copper(II) complexes of the type [Cu(L)]2+, where L = N,N'-dialkyl-1,10-phenanthroline-2,9-dimethanamine and R = methyl (L1), n-propyl (L2), isopropyl (L3), sec-butyl (L4), or tert-butyl (L5) group, have been synthesized. The interaction of the complexes with DNA has been studied by DNA fiber electron paramagnetic resonance (EPR) spectroscopy, emission, viscosity and electrochemical measurements and agarose gel electrophoresis. In the X-ray crystal structure of [Cu(HL2)Cl2]NO3, copper(II) is coordinated to two ring nitrogens and one of the two secondary amine nitrogens of the side chains and two chloride ions as well and the coordination geometry is best described as trigonal bipyramidal distorted square based pyramidal (TBDSBP). Electronic and EPR spectral studies reveal that all the complexes in aqueous solution around pH 7 possess CuN3O2 rather than CuN4O chromophore with one of the alkylamino side chain not involved in coordination. The structures of the complexes in aqueous solution around pH 7 change from distorted tetragonal to trigonal bipyramidal as the size of the alkyl group is increased. The observed changes in the physicochemical features of the complexes on binding to DNA suggest that the complexes, except [Cu(L5)]2+, bind to DNA with partial intercalation of the derivatised phen ring in between the DNA base pairs. Electrochemical studies reveal that the complexes prefer to bind to DNA in Cu(II) rather than Cu(I) oxidation state. Interestingly, [Cu(L5)]2+ shows the highest DNA cleavage activity among all the present copper(II) complexes suggesting that the bulky N-tert-butyl group plays an important role in modifying the coordination environment around the copper(II) center, the Cu(II)/Cu(I) redox potential and hence the formation of activated oxidant responsible for the cleavage. These results were compared with those for bis(1,10-phenanthroline)copper(II), [Cu(phen)2]2+.     

Use of layer silicate for protein crystallization: effects of Micromica and chlorite powders in hanging drops

Two kinds of layer silicate powder, Micromica and chlorite, were used to aid protein crystallization by the addition to hanging drops. Using appropriate crystallization buffers, Micromica powder facilitated crystal growth speed for most proteins tested in this study. Furthermore, the addition of Micromica powder to hanging drops allowed the successful crystallization of lysozyme, catalase, concanavalin A, and trypsin even at low protein concentrations and under buffer conditions that otherwise would not generate protein crystals. Except for threonine synthase and apoferritin, the presence of chlorite delayed crystallization but induced the formation of large crystals. X-ray analysis of thaumatin crystals generated by our novel procedure gave better quality data than did that of crystals obtained by a conventional hanging drop method. Our results suggest that the speed of crystal growth and the quality of the corresponding X-ray data may be inversely related, at least for the formation of thaumatin crystals. The effect of Micromica and chlorite powders and the application of layer silicate powder for protein crystallization are discussed.