Cats as a potential source of emerging influenza virus infections

<正>Dear Editor,Historically,the influenza virus has not been regarded as a major pathogen of cats.However,since 2003,natural infections of domestic cats with highly pathogenic H5N1 avian virus causing fatal cases have been reported(Songserm et al.,2006;Yingst et al.,2006;Klopfleisch et al.,2007).Furthermore,infections of this animal with A(H1N1)pdm09 virus,causing respiratory illness with some fatal cases,have also been reported in various parts     

RNA polymerase II acts as an RNA‐dependent RNA polymerase to extend and destabilize a non‐coding RNA

RNA polymerase II (Pol II) is a well‐characterized DNA‐dependent RNA polymerase, which has also been reported to have RNA‐dependent RNA polymerase (RdRP) activity. Natural cellular RNA substrates of mammalian Pol II, however, have not been identified and the cellular function of the Pol II RdRP activity is unknown. We found that Pol II can use a non‐coding RNA, B2 RNA, as both a substrate and a template for its RdRP activity. Pol II extends B2 RNA by 18 nt on its 3′‐end in an internally templated reaction. The RNA product resulting from extension of B2 RNA by the Pol II RdRP can be removed from Pol II by a factor present in nuclear extracts. Treatment of cells with α‐amanitin or actinomycin D revealed that extension of B2 RNA by Pol II destabilizes the RNA. Our studies provide compelling evidence that mammalian Pol II acts as an RdRP to control the stability of a cellular RNA by extending its 3′‐end.     

Modulation of RNA splicing as a potential treatment for cancer

Close to 90% of human genes are transcribed into pre-mRNA that undergoes alternative splicing, producing multiple mRNAs and proteins from single genes. This process is largely responsible for human proteome diversity, and about half of genetic disease-causing mutations affect splicing. Splice-switching oligonucleotides (SSOs) comprise an emerging class of antisense therapeutics that modify gene expression by directing pre-mRNA splice site usage. Bauman et al. investigated an SSO that up-regulated the expression of an anti-cancer splice variant while simultaneously eliminating an over-expressed cancer-causing splice variant. This was accomplished by targeting pre-mRNA of the apoptotic regulator Bcl-x, which is alternatively spliced to express anti- and pro-apoptotic splice variants Bcl-xL and Bcl-xS, respectively. High expression of Bcl-xL is a hallmark of many cancers and is considered a general mechanism used by cancer cells to evade apoptosis. Redirection of Bcl-x pre-mRNA splicing from Bcl-xL to -xS by SSO induced apoptotic and chemosensitizing effects in various cancer cell lines. Importantly, the paper shows that delivery of Bcl-x SSO using a lipid nanoparticle redirected Bcl-x splicing and reduced tumor burden in melanoma lung metastases. This was the first demonstration of SSO efficacy in tumors in vivo. SSOs are not limited to be solely potential anti-cancer drugs.?SSOs were first applied to repair aberrant splicing in thalassemia, a genetic disease, they have been used to create novel proteins (e.g., ?7TNFR1), and they have recently progressed to clinical trials for patients with Duchenne muscular dystrophy.     

Abscisic acid as an inhibitor of RNA synthesis by RNA polymerase in vitro

Abscisic acid interacts with DNA which increases the stabilityof the double strand against heat or alkaline denaturation.Abscisic acid added to RNA polymerase systems isolated fromthe chromatin of coconut nuclei inhibits RNA synthesis by loweringthe template activity of DNA. No interaction with RNA polymeraseor the initiation factor (factor B) is discernible. (Received May 18, 1972; )     

DNA dependent RNA polymerase from Ehrlich ascites tumor cells. V. Characterization of a factor repressing RNA polymerase II as a ribonucleoprotein.

Previously we reported the isolation of a factor, named the R-protein, which strongly repressed RNA polymerase II [EC 2.7.7.6] of Ehrlich ascites tumor cells. In the present work this factor was found to contain much RNA (ratio of RNA to protein, 2.3 to 1). The RNA was G:C rich, with a very high content of guanylic acid (about 38%). On equilibrium density gradient centrifugation in Cs2SO4 solution, the RNA became distributed above free RNA, but after digestion of the R-protein with pronase the RNA cosedimented with free RNA. Thus the R-protein is a complex of RNA and protein.     

RNA polymerase as a molecular machine

Structure and function of DNA-dependent RNA polymerase is considered in terms of a conveying molecular machine. The use of mechanical energy and mechanical devices, such as "power-stroke motor", is supposed unlikely in the conveying function of RNA polymerase, as well as other molecular machines. Brownian motion and thermal mobility of macromolecules and their parts are postulated as the only motive impulse at the molecular level. Binding of substrates and subsequent chemical reaction as the energy input may provide successive selection and fixation of alternative conformational states of the enzyme complex thus providing the directionality of the conveyance ("Brownian ratchet mechanism"). The following sequence of events "substrate binding--fixation of a certain conformational state--chemical reaction--fixation of an alternative conformational state--translocation (dissociation and downstream reassociation) of product-template duplex" is proposed as the principal scheme of the forward movement of RNA polymerase along DNA template.     

A rapid and accurate procedure for assaying DNA polymerase,RNA polymerase,or ribosome dependent protein synthesis

A descending paper chromatographic procedure for separating trichloracetic acid soluble from precipitable material is described. Its use for assaying DNA, RNA, or protein synthesis from a variety of sources with various states of purity attests to its general applicability to reactions of interest to molecular biologists. Large numbers of analyses can be carried out in a short period of time without compromising accuracy or reliability. The use of this procedure for still more enzymes, those involved in modifying DNA, RNA, or proteins, is discussed.     

Discovery of a potent respiratory syncytial virus RNA polymerase inhibitor

Targeting viral polymerases has been a proven and attractive strategy for antiviral drug discovery. Herein we describe our effort in improving the antiviral activity and physical properties of a series of benzothienoazepine compounds as respiratory syncytial virus (RSV) RNA polymerase inhibitors. The antiviral activity and spectrum of this class was significantly improved by exploring the amino substitution of the pyridine ring, resulting in the discovery of the most potent RSV A polymerase inhibitors reported to date.     

Amiloride is a competitive inhibitor of coxsackievirus B3 RNA polymerase

Amiloride and its derivative 5-(N-ethyl-N-isopropyl)amiloride (EIPA) were previously shown to inhibit coxsackievirus B3 (CVB3) RNA replication in cell culture, with two amino acid substitutions in the viral RNA-dependent RNA polymerase 3D(pol) conferring partial resistance of CVB3 to these compounds (D. N. Harrison, E. V. Gazina, D. F. Purcell, D. A. Anderson, and S. Petrou, J. Virol. 82:1465-1473, 2008). Here we demonstrate that amiloride and EIPA inhibit the enzymatic activity of CVB3 3D(pol) in vitro, affecting both VPg uridylylation and RNA elongation. Examination of the mechanism of inhibition of 3D(pol) by amiloride showed that the compound acts as a competitive inhibitor, competing with incoming nucleoside triphosphates (NTPs) and Mg(2+). Docking analysis suggested a binding site for amiloride and EIPA in 3D(pol), located in close proximity to one of the Mg(2+) ions and overlapping the nucleotide binding site, thus explaining the observed competition. This is the first report of a molecular mechanism of action of nonnucleoside inhibitors against a picornaviral RNA-dependent RNA polymerase.     

The HCV non-nucleoside inhibitor Tegobuvir utilizes a novel mechanism of action to inhibit NS5B polymerase function

Tegobuvir (TGV) is a novel non-nucleoside inhibitor (NNI) of HCV RNA replication with demonstrated antiviral activity in patients with genotype 1 chronic HCV infection. The mechanism of action of TGV has not been clearly defined despite the identification of resistance mutations mapping to the NS5B polymerase region. TGV does not inhibit NS5B enzymatic activity in biochemical assays in vitro, suggesting a more complex antiviral mechanism with cellular components. Here, we demonstrate that TGV exerts anti-HCV activity utilizing a unique chemical activation and subsequent direct interaction with the NS5B protein. Treatment of HCV subgenomic replicon cells with TGV results in a modified form of NS5B with a distinctly altered mobility on a SDS-PAGE gel. Further analysis reveals that the aberrantly migrating NS5B species contains the inhibitor molecule. Formation of this complex does not require the presence of any other HCV proteins. The intensity of the aberrantly migrating NS5B species is strongly dependent on cellular glutathione levels as well as CYP 1A activity. Furthermore analysis of NS5B protein purified from a heterologous expression system treated with TGV by mass spectrometry suggests that TGV undergoes a CYP- mediated intracellular activation step and the resulting metabolite, after forming a glutathione conjugate, directly and specifically interacts with NS5B. Taken together, these data demonstrate that upon metabolic activation TGV is a specific, covalent inhibitor of the HCV NS5B polymerase and is mechanistically distinct from other classes of the non-nucleoside inhibitors (NNI) of the viral polymerase.