Structure determination of N-terminal peptide of nucleoprotein (NP20) of influenza virus H5N1 by nuclear magnetic resonance spectroscopy

Influenza virus has long been a major threat to public health worldwide. The virus can be highly deadly because of antigenic shift. Since the H5N1 outbreak in Hong Kong in 1997, avian flu is regarded as the next pandemic threat. For combating the disease, it is essential to investigate more on the influenza virus, in particular H5N1. Nucleoprotein (NP) is a major component of the ribonucleoprotein complex (RNP) in the influenza virus. NP exhibits both structural and functional roles for influenza virus assembly and propagation and is involved in mediating the transcription-replication process. The NP of the virus binds the RNA genome and acts as a key adapter between the virus and the host cell. It therefore plays important roles and represents an attractive drug target. Recently, the X-ray structure of H5N1 NP was solved to a resolution of 3.3 Å , which provides valuable clues on how NP carries out its functions. However, the N-terminal 1-20 residues were not resolved in the H5N1 NP crystal structure. This N-terminal region is thought to contain a nuclear localization signal (NLS), a cellular splicing factor BAT1/UAP56 binding site, and a nuclear export signal. It has been suggested that the N-terminal NLS binds to importin (a cytosolic protein) for the nuclear import of NP. In the present study, the solution structure of H5N1 NP N-terminal peptide (NP20) in membrane mimetic solvent condition was determined using Circular Dichroism (CD) and Nuclear Magnetic Resonance (NMR) spectroscopies. The CD results show that NP20 adopted an α-helical conformation. The NMR data indicate that NP20 formed a single α-helix spanning from residues Gly5 to Gly16. Surface electrostatic potentials further showed that the NP20 peptide is amphipathic in nature, which may be important for its binding with importin. NMR titration experiments have been carried out between NP20 and importin. Addition of importin into the solution of NP20 peptide caused significant broadening of the NMR signals of NP20 and progressive changes of the chemical shifts of NOE cross-peaks at increasing importin concentration confirm that NP20 could bind with importin. Therefore, the present study supports that NP20 region is the binding site of importin mediating the import of NP into the host cell nucleus. In conclusion, the knowledge gained from this study provides a better understanding on the structure of NP20 and its interaction with the host importin protein, and may serve as a template for the development of novel antiviral drug targeting NP with improved therapeutic index.published_or_final_versionChemistryMasterMaster of Philosoph

Identification of a nonaketide product for the iterative polyketide synthase in biosynthesis of the nine-membered enediyne C-1027

Not octa- but nonaketide: The nine-membered enediyne core polyketide synthase SgcE efficiently synthesizes a nonaketide in the absence of any assisting proteins (see scheme), contrary to the suggestion that an octaketide is the product of the synthase under assistance from a thioesterase. This finding redefines the catalytic functions of the polyketide synthase. CoA=coenzyme A. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

SaeR as a Novel Target for Antivirulence Therapy against Staphylococcus aureus

ABSTRACT:Staphylococcus aureus is a major human pathogen responsible for a wide range of clinical infections. SaeRS is one of the two-component systems in S. aureus that modulate multiple virulence factors. Although SaeR is required for S. aureus to develop an infection, inhibitors have not been reported. Using an in vivo knockdown method, we demonstrated that SaeR is targetable for the discovery of antivirulence agent. HR3744 was discovered through a high-throughput screening utilising a GFP-Lux dual reporter system driven by saeP1 promoter. ,. The antivirulence efficacy of HR3744 was tested using Western blot, Quantitative Polymerase Chain Reaction, leucotoxicity, and hemolysis tests. In electrophoresis mobility shift assay, HR3744 inhibited SaeR-DNA probe binding. WaterLOGSY-NMR test showed HR3744 directly interacted with SaeR's DNA-binding domain When saeR was deleted, HR3744 lost its antivirulence property, validating the target specificity. Virtual docking and mutagenesis were used to confirm the target's specificity. When Glu159 was changed to Asn, the bacteria developed resistance to HR3744. A structure-activity relationship study revealed that a molecule with a slight modification did not inhibit SaeR, indicating the selectivity of HR3744. Interestingly, we found that SAV13, an analogue of HR3744, was four-times more potent than the HR3744 and demonstrated identical antivirulence property and target specificity. In a mouse bacteraemia model, both HR3744 and SAV13 exhibited in vivo effectiveness. Collectively, we identified the first SaeR inhibitor, which exhibited in vitro and in vivo antivirulence property, and proved that SaeR could be a novel target for developing antivirulence drugs against S. aureus infections

Characterization of the Lipidomic Profile of Human Coronavirus-Infected Cells: Implications for Lipid Metabolism Remodeling upon Coronavirus Replication

Lipids play numerous indispensable cellular functions and are involved in multiple steps in the replication cycle of viruses. Infections by human-pathogenic coronaviruses result in diverse clinical outcomes, ranging from self-limiting flu-like symptoms to severe pneumonia with extrapulmonary manifestations. Understanding how cellular lipids may modulate the pathogenicity of human-pathogenic coronaviruses remains poor. To this end, we utilized the human coronavirus 229E (HCoV-229E) as a model coronavirus to comprehensively characterize the host cell lipid response upon coronavirus infection with an ultra-high performance liquid chromatography-mass spectrometry (UPLC–MS)-based lipidomics approach. Our results revealed that glycerophospholipids and fatty acids (FAs) were significantly elevated in the HCoV-229E-infected cells and the linoleic acid (LA) to arachidonic acid (AA) metabolism axis was markedly perturbed upon HCoV-229E infection. Interestingly, exogenous supplement of LA or AA in HCoV-229E-infected cells significantly suppressed HCoV-229E virus replication. Importantly, the inhibitory effect of LA and AA on virus replication was also conserved for the highly pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV). Taken together, our study demonstrated that host lipid metabolic remodeling was significantly associated with human-pathogenic coronavirus propagation. Our data further suggested that lipid metabolism regulation would be a common and druggable target for coronavirus infections

Identification of a Small Molecule Compound Active against Antibiotic-Tolerant <i>Staphylococcus aureus</i> by Boosting ATP Synthesis

Antibiotic tolerance poses a threat to current antimicrobial armamentarium. Bacteria at a tolerant state survive in the presence of antibiotic treatment and account for persistence, relapse and recalcitrance of infections. Antibiotic treatment failure may occur due to antibiotic tolerance. Persistent infections are difficult to treat and are often associated with poor prognosis, imposing an enormous burden on the healthcare system. Effective strategies targeting antibiotic-tolerant bacteria are therefore highly warranted. In this study, small molecule compound SA-558 was identified to be effective against Staphylococcus aureus that are tolerant to being killed by conventional antibiotics. SA-558 mediated electroneutral transport across the membrane and led to increased ATP and ROS generation, resulting in a reduction of the population of antibiotic-tolerant bacteria. In a murine chronic infection model, of which vancomycin treatment failed, we demonstrated that SA-558 alone and in combination with vancomycin caused significant reduction of MRSA abundance. Our results indicate that SA-558 monotherapy or combinatorial therapy with vancomycin is an option for managing persistent S. aureus bacteremia infection and corroborate that bacterial metabolism is an important target for counteracting antibiotic tolerance

Intricate relationship between IL-6, STAT3 activation and LMP1 in EBV-infected NPE cells.

<p>(A) A schematic diagram showing the positive feedback loop of IL-6 activation of STAT3 and LMP1 expression in EBV-infected cell. (B) NP460hTert-EBV, C666-1 and HONE-EBV cells were treated with IL-6 for 4 hours. Total RNA were extracted from treated and untreated cells and examined for mRNA expression levels of LMP1 by quantitative real-time (RT-PCR). Results were normalized to GAPDH. These results were computed from triplicates of individual experiments. * <i>p</i><0.05. (C) EBV-infected NP460hTert cells were stably transduced with STAT3-C or control vector. The mRNA expression levels of LMP1 were analyzed by quantitative RT-PCR. Results were normalized to GAPDH. * <i>p</i><<i>0.05</i>. (D) LMP1 expression was upregulated in NP460hTert-EBV cells after treating with IL-6 for 48 hours. (E) PLNSX-LMP1 vector and PLNSX control vector were introduced into CNE2 cells by retroviral infection. Both LMP1 expression and STAT3 phosphorylation in CNE2 cells were confirmed by Western blotting. (F) HONE1 and EBV-infected HONE1 cells were transiently co-transfected with m67 firefly luciferase reporter construct (luc-m67) in conjunction with a Renilla luciferase vector and STAT3-C plasmid or control plasmid. Cell lysates were assessed for luciferase activity 36 hours after transfection. Relative luciferase activity was calculated by normalizing the firefly luciferase activity to Renilla luciferase activity. These results were computed from triplicate separate experiments. * <i>p</i><0.05 ^#<i>p</i><0.05. (G) HONE1 cells were transiently co-transfected with IL-6 firefly luciferase reporter construct (pGL3-IL-6-Luc) in conjunction with a Renilla luciferase vector and pcDNA3/2117-LMP1 or control vector. These results were computed from triplicate separate experiments. * <i>p</i><0.05.</p

Upregulation of IL-6R in EBV-infected NPE cells is responsible for the enhanced responsiveness to IL-6-induced STAT3 activation.

<p>(A) The expression of IL-6R in EBV-infected and uninfected NP460hTert cells was analyzed by Western blot. Immunoblotting for β-actin was provided as protein loading control. (B) Total RNA was extracted and the expression levels of IL-6R mRNA in NP460hTert and NP460hTert-EBV cells were analyzed by quantitative real time RT-PCR. The mRNA levels of IL-6Rα were normalized to cellular GAPDH mRNA. Data were collected from triplicate separate experiments. * <i>p</i><<i>0.05</i>. (C) NP460hTert cells were infected with retroviral expression vectors, pBabe-IL-6Rα or pBabe empty vectors. Stably transduced cells were treated with IL-6 at 50 ng/ml for 30 minutes. Cell lysates were prepared and examined for expression of IL-6Rα and p-STAT3 (Tyr 705) by Western blot. Immunoblottings for STAT3 and β-actin are shown as controls for protein loading. (D) NP460hTert and NP460hTert-EBV cells were pre-treated with anti-IL-6R antibody at 5 µg/ml for 30 minutes before IL-6 treatment. The expression of p-STAT3 was analyzed by Western blot. Immunoblotting for total levels of STAT3 protein is shown as controls for protein loading. (E) After EBV infection, the EBV-infected NP460hTert cells and its uninfected parental cells were continuously subcultured. Cells lysates were prepared from the cells that had been passaged for 56, 99 and 133 times (designated as early, middle and late passage). The expression of IL-6R was analyzed by Western blotting. Immunoblotting for β-actin was included as control for protein loading. (F) The levels of IL-6R expression in cells were detected by Western blot in several paired uninfected and EBV-infected cell lines, including EBV-infected and non-infected pairs of NP460hTert, NP550-CDK4-hTert, NP361-cyclinD1-hTert, NP550-cyclinD1-hTert. β-actin expressions were shown as protein loading control.</p