The putative NTPase/helicase protein from severe acute respiratory syndrome coronavirus (SARS-CoV) is postulated to play a number of crucial roles in the viral life cycle, making it an attractive target for anti-SARS therapy. We have cloned, expressed, and purified this protein as an N-terminal hexahistidine fusion in Escherichia coli and have characterized its helicase and NTPase activities. The enzyme unwinds doublestranded DNA, dependent on the presence of a 5 singlestranded overhang, indicating a 5 to 3 polarity of activity, a distinct characteristic of coronaviridae helicases. We provide the first quantitative analysis of the polynucleic acid binding and NTPase activities of a Nidovirus helicase, using a high throughput phosphate release assay that will be readily adaptable to the future testing of helicase inhibitors. All eight common NTPs and dNTPs were hydrolyzed by the SARS helicase in a magnesium-dependent reaction, stimulated by the presence of either single-stranded DNA or RNA. The enzyme exhibited a preference for ATP, dATP, and dCTP over the other NTP/dNTP substrates. Homopolynucleotides significantly stimulated the ATPase activity (15-25-fold) with the notable exception of poly(G) and poly(dG), which were non-stimulatory. We found a large variation in the apparent strength of binding of different homopolynucleotides, with dT 24 binding over 10 times more strongly than dA 24 as observed by the apparent K m .

The enhancer of zeste homolog 2 (EZH2) is upregulated and has an oncogenic role in several types of human cancer. However, the abnormalities of EZH2 and its underlying mechanisms in the pathogenesis of nasopharyngeal carcinoma (NPC) remain unknown. In this study, we found that high expression of EZH2 in NPC was associated closely with an aggressive and/or poor prognostic phenotype (Po0.05). In NPC cell lines, knockdown of EZH2 by short hairpin RNA was sufficient to inhibit cell invasiveness/metastasis both in vitro and in vivo, whereas ectopic overexpression of EZH2 supported NPC cell invasive capacity with a decreased expression of E-cadherin. In addition, ablation of endogenous Snail in NPC cells virtually totally prevented the repressive activity of EZH2 to E-cadherin, indicating that Snail might be a predominant mediator of EZH2 to suppress E-cadherin. Furthermore, co-immunoprecipitation (IP), chromatin IP and luciferase reporter assays demonstrated that in NPC cells, (1) EZH2 interacted with HDAC1/HDAC2 and Snail to form a repressive complex; (2) these components interact in a linear fashion, not in a triangular fashion, that is, HDAC1 or HDAC2 bridge the interaction between EZH2 and Snail; and (3) the EZH2/HDAC1/2/Snail complex could closely bind to the E-cadherin promoter by Snail, but not YY1, to repress E-cadherin. The data provided in this report suggest a critical role of EZH2 in the control of cell invasion and/or metastasis by forming a co-repressor complex with HDAC1/HDAC2/Snail to repress E-cadherin, an activity that might be responsible, at least in part, for the development and/or progression of human NPCs.

Diallyl sulfide, a major flavor ingredient from garlic, was previously shown to inhibit chemically induced carcinogenesis and cytotoxicity in animal model systems. It modulated cytochrome P-450 compositions by inactivating P-450 2E1 and inducing P-450 2B1. The present studies examined the inhibition of P-450 2E1 mediated p-nitrophenol hydroxylase activity by diallyl sulfide and its putative metabolites diallyl sulfoxide and diallyl sulfone (DASO2). Each compound displayed competitive inhibition of p-nitrophenol hydroxylase activity in incubations using liver microsomes from acetone-pretreated male Sprague-Dawley rats. Preincubation of the microsomes with DASO2 inactivated p-nitrophenol hydroxylase activity in a process that was time- and NADPH-dependent and saturable, exhibited pseudo-first-order kinetics, was protected by alternate substrate, was accompanied by a loss of microsomal P-450-CO binding spectrum, and was unaffected by exogenous nucleophile. The Ki value for DASO2 was 188 microM and the maximal rate of inactivation was 0.32 min-1. DASO2 was ineffective in the inactivation of ethoxyresorufin dealkylase, pentoxyresorufin dealkylase, or benzphetamine demethylase activity. Purified P-450 2E1 in a reconstituted system was inactivated in a time- and NADPH-dependent manner by DASO2. The metabolic conversion of diallyl sulfide to the sulfoxide and sulfone was observed in vivo and in vitro. The results suggest that diallyl sulfide inhibits the metabolism of P-450 2E1 substrates by competitive inhibition mechanisms and by inactivating P-450 2E1 via a suicide-inhibitory action of DASO2.

The recent outbreak of enterovirus 71 (EV71) infected millions of children and caused over 1,000 deaths. To date, neither an effective vaccine nor antiviral treatment is available for EV71 infection. Interferons (IFNs) have been successfully applied to treat patients with hepatitis B and C viral infections for decades but have failed to treat EV71 infections. Here, we provide the evidence that EV71 antagonizes type I IFN signaling by reducing the level of interferon receptor 1 (IFNAR1). We show that the host cells could sense EV71 infection and stimulate IFN-␤ production. However, the induction of downstream IFN-stimulated genes is inhibited by EV71. Also, only a slight interferon response and antiviral effects could be detected in cells treated with recombinant type I IFNs after EV71 infection. Further studies reveal that EV71 blocks the IFN-mediated phosphorylation of STAT1, STAT2, Jak1, and Tyk2 by reducing IFNAR1. Finally, we identified the 2A protease encoded by EV71 as an antagonist of IFNs and show that the protease activity is required for reducing IFNAR1 levels. Taken together, our study for the first time uncovers a mechanism used by EV71 to antagonize type I IFN signaling and provides new targets for future antiviral strategies. E nterovirus 71 (EV71) is a typical positive-strand RNA virus which belongs to the Picornaviridae family (44). The genome of EV71 is approximately 7.5 kb in length and contains a single open reading frame encoding a polyprotein precursor. This polyprotein is cleaved by two viral proteases, 2Apro and 3C pro , to form four structural proteins (VP1, VP2, VP3, and VP4) and seven nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C, and 3D) (39). EV71 infection was a major cause of outbreaks of hand, foot, and mouth disease (HFMD) in infants and young children (19,22). As a typical neurotropic virus, EV71 has a propensity to cause neurological disease during acute infection and may lead to permanent paralysis and even death (1,42,45). The outbreaks and severity of EV71 infection have been frequently reported worldwide (60), and recently EV71 has become a major threat to public health in China (56). The Chinese government reported that there were ϳ1,770,000 cases of HFMD and herpangina with over 900 deaths in 2010 (http://www.moh.gov.cn/publicfiles/business/htmlfiles/zwgkzt /pyq/list.htm). However, to date, no effective vaccine or therapy can be applied to prevent or treat EV71 infection.Type I interferon (IFN), as the first line of host immune response, is critical in mediating antiviral defense. The host recognizes viral invasion and activates the type I IFN response through the recognition of pathogen-associated molecular patterns (PAMPs) by patternrecognition receptors (PRRs) (61). Binding with the corresponding receptors, IFN receptor 1 (IFNAR1) and IFNAR2 (11, 50), the secreted IFNs activate the Janus kinases Jak1 and Tyk2 and then phosphorylate signal transducers and activators of transcription STAT1 and STAT2 (23). The phosphorylated STAT1 and STAT2 form heterodimers and bind with...

Hepatitis B virus (HBV), a serious infectious and widespread human pathogen, represents a major health problem worldwide. Chronic HBV infection has a very high risk of evolving into hepatocellular carcinoma. Although considerable progress was made during the recent past, the pathogenesis of HBV infection is still elusive and a definite diagnosis of HBV infected liver information still relies on biopsy histological test. In this report, we used proteomics technology to globally examine HBV infected serum samples aiming at searching for disease-associated proteins that can be used as serological biomarkers for diagnosis and/or target proteins for pathogenetic study. By comparing with normal and HBV negative serum samples, we found that at least seven proteins were significantly changed in HBV infected sera. These greatly altered proteins were identified to be haptoglobin beta and alpha2 chain, apolipoprotein A-I and A-IV, alpha1-antitrypsin, transthyretin and DNA topoisomerase IIbeta. The alteration of these proteins is displayed not only in quantity but also in patterns (or specificity), which can be correlated with necroinflammatory scores. In particular, apolipoprotein A-I presents heterogeneous change in expression level with different isoforms and alpha1-antitrypsin produces evidently different fragments implying diverse cleavage pathways. These unique phenomena appear specific to HBV infection. A combination simultaneously considering the quantities and isoforms of these proteins could be a useful serum biomarker (or index) for HBV diagnosis and therapy.