Flavivirus RNA replication occurs within a replication complex (RC) that assembles on ER membranes and comprises both non-structural (NS) viral proteins and host cofactors. As the largest protein component within the flavivirus RC, NS5 plays key enzymatic roles through its N-terminal methyltransferase (MTase) and C-terminal RNA-dependent-RNA polymerase (RdRp) domains, and constitutes a major target for antivirals. We determined a crystal structure of the full-length NS5 protein from Dengue virus serotype 3 (DENV3) at a resolution of 2.3 Å in the presence of bound SAH and GTP. Although the overall molecular shape of NS5 from DENV3 resembles that of NS5 from Japanese Encephalitis Virus (JEV), the relative orientation between the MTase and RdRp domains differs between the two structures, providing direct evidence for the existence of a set of discrete stable molecular conformations that may be required for its function. While the inter-domain region is mostly disordered in NS5 from JEV, the NS5 structure from DENV3 reveals a well-ordered linker region comprising a short 310 helix that may act as a swivel. Solution Hydrogen/Deuterium Exchange Mass Spectrometry (HDX-MS) analysis reveals an increased mobility of the thumb subdomain of RdRp in the context of the full length NS5 protein which correlates well with the analysis of the crystallographic temperature factors. Site-directed mutagenesis targeting the mostly polar interface between the MTase and RdRp domains identified several evolutionarily conserved residues that are important for viral replication, suggesting that inter-domain cross-talk in NS5 regulates virus replication. Collectively, a picture for the molecular origin of NS5 flexibility is emerging with profound implications for flavivirus replication and for the development of therapeutics targeting NS5.

The six-subunit origin recognition complex (ORC) binds to DNA to mark the site for the initiation of replication in eukaryotes. Here we report a 3 Å cryo-electron microscopy structure of the Saccharomyces cerevisiae ORC bound to a 72-base-pair origin DNA sequence that contains the ARS consensus sequence (ACS) and the B1 element. The ORC encircles DNA through extensive interactions with both phosphate backbone and bases, and bends DNA at the ACS and B1 sites. Specific recognition of thymine residues in the ACS is carried out by a conserved basic amino acid motif of Orc1 in the minor groove, and by a species-specific helical insertion motif of Orc4 in the major groove. Moreover, similar insertions into major and minor grooves are also embedded in the B1 site by basic patch motifs from Orc2 and Orc5, respectively, to contact bases and to bend DNA. This work pinpoints a conserved role of ORC in modulating DNA structure to facilitate origin selection and helicase loading in eukaryotes.

Background: NS3-NS5 interaction is important for the dengue virus life cycle. Results: NS3 residue Asn-570 is essential for its interaction with NS5; mutation in an infectious cDNA abolished virus production and reduced positive-strand RNA synthesis. Conclusion: NS3-NS5 interaction may be required for coordinated positive-and negative-strand RNA synthesis. Significance: NS3-NS5 interaction may be a target for rational design of antiviral drugs.

Dengue virus NS5 is the most highly conserved amongst the viral non-structural proteins and is responsible for capping, methylation and replication of the flavivirus RNA genome. Interactions of NS5 with host proteins also modulate host immune responses. Although replication occurs in the cytoplasm, an unusual characteristic of DENV2 NS5 is that it localizes to the nucleus during infection with no clear role in replication or pathogenesis. We examined NS5 of DENV1 and 2, which exhibit the most prominent difference in nuclear localization, employing a combination of functional and structural analyses. Extensive gene swapping between DENV1 and 2 NS5 identified that the C-terminal 18 residues (Cter18) alone was sufficient to direct the protein to the cytoplasm or nucleus, respectively. The low micromolar binding affinity between NS5 Cter18 and the nuclear import receptor importin-alpha (Impα), allowed their molecular complex to be purified, crystallised and visualized at 2.2 Å resolution using x-ray crystallography. Structure-guided mutational analysis of this region in GFP-NS5 clones of DENV1 or 2 and in a DENV2 infectious clone reveal residues important for NS5 subcellular localization. Notably, the trans conformation adopted by Pro-884 allows proper presentation for binding Impα and mutating this proline to Thr, as present in DENV1 NS5, results in mislocalizaion of NS5 to the cytoplasm without compromising virus fitness. In contrast, a single mutation to alanine at NS5 position R888, a residue conserved in all flaviviruses, resulted in a completely non-viable virus, and the R888K mutation led to a severely attenuated phentoype, even though NS5 was located in the nucleus. R888 forms a hydrogen bond with Y838 that is also conserved in all flaviviruses. Our data suggests an evolutionarily conserved function for NS5 Cter18, possibly in RNA interactions that are critical for replication, that is independent of its role in subcellular localization.

An effective, template-free synthesis methodology has been developed for preparing mesoporous nitrogen-doped SrTiO 3 (meso-STON) using glycine as both a nitrogen source and a mesopore creator. The N-doping, large surface area and developed porosity endow meso-STON with excellent activity in visible-light-responsive photodegradation of organic dyes.The exceptional electro-optical properties and physicochemical stability of the perovskite SrTiO 3 (STO) give rise to its attractive performance in photocatalytic applications of solar power, including photocatalytic degradation of organic pollutants, water splitting and photoreduction of CO 2 .1-3 However, the intrinsic large bandgap energy (E g = 3.2 eV) of SrTiO 3 allows only the utilization of UV light, encompassing approximately 5.0% energy of the sunlight. 4 A variety of transition metals (TM) have been doped into a STO's crystal matrix in efforts to tune its electronic bandgap for harvesting visible light. 5,6Unfortunately, TM-doping can also bring about either phase impurity or fast recombination of photogenerated charge carriers. Nonmetal-doping represents another effective strategy to realize visible-light response.7-9 Indeed, it was found that N-doped SrTiO 3 (SrTiO 3Àx N x , STON) exhibited excellent photoreactivity and stability under visible-light irradiation. 10Mesoporous-structured photocatalysts are highly desirable in photocatalysis since their large specific surface area (SSA) and mesoporous channels greatly facilitate adsorption, diffusion and surface reaction of the reactants.11 STO perovskite belongs to the cubic crystal system, and typically has low SSA and poor porosity. Moreover, the porosity of STO could be further destroyed by the known processes of nitriding STO to STON. 5,12Although mesoporous STO has been prepared via templatedirected synthesis using various soft (e.g. surfactant or polymer 13 ) and hard (e.g. inorganic salts 14 ) templates, the synthesis of mesoporous STON has rarely been achieved.Here we report a novel, template-free synthesis methodology to prepare mesoporous STON using glycine as both a nitrogen source and a mesopore creator. Aqueous solution of glycine and Sr(NO 3 ) 2 was dropped into ethanol solution of titanium butoxide under stirring, followed by solvent evaporation and subsequent calcinations at 550 1C for 2 hours. The obtained STON was characterized by XRD, TEM, FTIR, UV-vis, and XPS techniques and used for the photodegradation of three refractory organic dyes under visible-light irradiation.Only a strong single peak appears in the small angle XRD pattern (Fig. 1) of the STON sample, suggesting that it possesses disordered wormlike mesopores. 15 The TEM image in Fig. 2A nicely confirms such mesoporosity. The HRTEM image inserted in Fig. 2A reveals that the walls of the mesopores are comprised of single crystal perovskite STON. The labelled lattice distances are consistent with those of (100) and (110) diffractions obtained from XRD tests (Fig. S1, ESIw). In contrast, the STO sample presents poor mesoporous featu...

Transition metal nitrogen carbon based single‐atom catalysts (SACs) have exhibited superior activity and selectivity for CO2 electroreduction to CO. A favorable local nitrogen coordination environment is key to construct efficient metal‐N moieties. Here, a facile plasma‐assisted and nitrogen vacancy (NV) induced coordinative reconstruction strategy is reported for this purpose. Under continuous plasma striking, the preformed pentagon pyrrolic N‐defects around Ni sites can be transformed to a stable pyridinic N dominant Ni‐N2 coordination structure with promoted kinetics toward the CO2‐to‐CO conversion. Both the CO selectivity and productivity increase markedly after the reconstruction, reaching a high CO Faradaic efficiency of 96% at mild overpotential of 590 mV and a large CO current density of 33 mA cm‐2 at 890 mV. X‐ray adsorption spectroscopy and density functional theory (DFT) calculations reveal this defective local N environment decreases the restraint on central Ni atoms and provides enough space to facilitate the adsorption and activation of CO2 molecule, leading to a reduced energy barrier for CO2 reduction.

Dengue virus (DENV) causes several hundred million human infections and more than 20,000 deaths annually. Neither an efficacious vaccine conferring immunity against all four circulating serotypes nor specific drugs are currently available to treat this emerging global disease. Capping of the DENV RNA genome is an essential structural modification that protects the RNA from degradation by 5′ exoribonucleases, ensures efficient expression of viral proteins, and allows escape from the host innate immune response. The large flavivirus nonstructural protein 5 (NS5) (105 kDa) has RNA methyltransferase activities at its N-terminal region, which is responsible for capping the virus RNA genome. The methyl transfer reactions are thought to occur sequentially using the strictly conserved flavivirus 5′ RNA sequence as substrate (G ppp AG-RNA), leading to the formation of the 5′ RNA cap:To elucidate how viral RNA is specifically recognized and methylated, we determined the crystal structure of a ternary complex between the full-length NS5 protein from dengue virus, an octameric cap-0 viral RNA substrate bearing the authentic DENV genomic sequence (5′-m7 G 0ppp A 1 G 2 U 3 U 4 G 5 U 6 U 7 -3′), and S-adenosyl-L-homocysteine (SAH), the by-product of the methylation reaction. The structure provides for the first time, to our knowledge, a molecular basis for specific adenosine 2′-O-methylation, rationalizes mutagenesis studies targeting the K61-D146-K180-E216 enzymatic tetrad as well as residues lining the RNA binding groove, and offers previously unidentified mechanistic and evolutionary insights into cap-1 formation by NS5, which underlies innate immunity evasion by flaviviruses.dengue virus | nonstructural protein 5 methyltransferase-polymerase | 2′-O-ribose methyltransferase | cap-0 RNA | innate immunity evasion S everal members of the Flavivirus genus from the Flaviviridae family are major human pathogens, such as the four serotypes of dengue virus (DENV1-4), West Nile virus (WNV), Japanese encephalitis virus (JEV), and yellow fever virus (YFV). Recent largescale DENV vaccine trials using a tetravalent formulation and three dose injections have shown only limited protection against the four DENV serotypes, and no specific antiviral drug has reached the market so far (1-3). The flavivirus genome consists of a (+)-sense singlestranded RNA of ∼11 kb with a type 1 cap structure, followed by the strictly conserved dinucleotide sequence "AG": 5′-m7 G ppp A m2′-O-G-3′ (4, 5). Addition of the cap moiety to the 5′ end of the viral genome is crucial for viral replication, because this structure ensures efficient production of viral polyproteins by the host translation machinery and protection against degradation by 5′-3′ exoribonucleases, and also because it conceals the triphosphorylated (or diphosphorylated) end from recognition by host cell innate immune sensors (6-9). Following (+)-strand RNA synthesis by the C-terminal RNA-dependent RNA polymerase (RdRp) domain of nonstructural protein 5 (NS5), cap formation in flaviviruses resu...

The administration of TG4010 was generally well tolerated in patients with metastatic tumors, and transient disease stabilization was observed in several patients, warranting further clinical studies with the product.