Severe Acute Respiratory Syndrome Coronavirus Evades Antiviral Signaling: Role of nsp1 and Rational Design of an Attenuated Strain

The severe acute respiratory syndrome (SARS) epidemic was caused by the spread of a previously unrecognized infectious agent, the SARS-associated coronavirus (SARS-CoV). Here we show that SARS-CoV could inhibit both virus- and interferon (IFN)-dependent signaling, two key steps of the antiviral response. We mapped a strong inhibitory activity to SARS-CoV nonstructural protein 1 (nsp1) and show that expression of nsp1 significantly inhibited the activation of all three virus-dependent signaling pathways. We show that expression of nsp1 significantly inhibited IFN-dependent signaling by decreasing the phosphorylation levels of STAT1 while having little effect on those of STAT2, JAK1, and TYK2. We engineered an attenuated mutant of nsp1 in SARS-CoV through reverse genetics, and the resulting mutant virus was viable and replicated as efficiently as wild-type virus in cells with a defective IFN response. However, mutant virus replication was strongly attenuated in cells with an intact IFN response. Thus, nsp1 is likely a virulence factor that contributes to pathogenicity by favoring SARS-CoV replication

Implication of a Chromosome 15q15.2 Locus in Regulating UBR1 and Predisposing Smokers to MGMT Methylation in Lung

O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that protects cells from carcinogenic effects of alkylating agents; however, MGMT is silenced by promoter hypermethylation during carcinogenesis. A single nucleotide polymorphism (SNP) in an enhancer in the MGMT promoter was previously identified to be highly significantly associated with risk for MGMT methylation in lung cancer and sputum from smokers. To further genetic investigations, a genome-wide association and replication study was conducted in two smoker cohorts to identify novel loci for MGMT methylation in sputum that were independent of the MGMT enhancer polymorphism. Two novel trans-acting loci (15q15.2 and 17q24.3) that were identified acted together with the enhancer SNP to empower risk prediction for MGMT methylation. We found that the predisposition to MGMT methylation arising from the 15q15.2 locus involved regulation of the ubiquitin protein ligase E3 component UBR1. UBR1 attenuation reduced turnover of MGMT protein and increased repair of O6-methylguanine in nitrosomethylurea-treated human bronchial epithelial cells (HBEC), while also reducing MGMT promoter activity and abolishing MGMT induction. Overall, our results substantiate reduced gene transcription as a major mechanism for predisposition to MGMT methylation in the lungs of smokers, and support the importance of UBR1 in regulating MGMT homeostasis and DNA repair of alkylated DNA adducts in cells

New World Hantaviruses Activate IFNλ Production in Type I IFN-Deficient Vero E6 Cells

Hantaviruses indigenous to the New World are the etiologic agents of hantavirus cardiopulmonary syndrome (HCPS). These viruses induce a strong interferon-stimulated gene (ISG) response in human endothelial cells. African green monkey-derived Vero E6 cells are used to propagate hantaviruses as well as many other viruses. The utility of the Vero E6 cell line for virus production is thought to owe to their lack of genes encoding type I interferons (IFN), rendering them unable to mount an efficient innate immune response to virus infection. Interferon lambda, a more recently characterized type III IFN, is transcriptionally controlled much like the type I IFNs, and activates the innate immune system in a similar manner.We show that Vero E6 cells respond to hantavirus infection by secreting abundant IFNlambda. Three New World hantaviruses were similarly able to induce IFNlambda expression in this cell line. The IFNlambda contained within virus preparations generated with Vero E6 cells independently activates ISGs when used to infect several non-endothelial cell lines, whereas innate immune responses by endothelial cells are specifically due to viral infection. We show further that Sin Nombre virus replicates to high titer in human hepatoma cells (Huh7) without inducing ISGs.Herein we report that Vero E6 cells respond to viral infection with a highly active antiviral response, including secretion of abundant IFNlambda. This cytokine is biologically active, and when contained within viral preparations and presented to human epithelioid cell lines, results in the robust activation of innate immune responses. We also show that both Huh7 and A549 cell lines do not respond to hantavirus infection, confirming that the cytoplasmic RNA helicase pathways possessed by these cells are not involved in hantavirus recognition. We demonstrate that Vero E6 actively respond to virus infection and inhibiting IFNlambda production in these cells might increase their utility for virus propagation

The IFI-56K and IFI-54K interferon-inducible human genes belong to the same gene family

The IFI-56K and IFI-54K human genes are coordinately regulated by interferon, double-stranded RNA and viruses in a number of cell lines. These genes encode polypeptides of 56 and 54 kDa, respectively, whose function remains to be determined. We analysed the possible structural relatedness between these syntenic and similarly regulated genes. We found that they are very closely related at the protein, mRNA and promoter levels. This suggests that the IFI-56K and IFI-54K genes are members of a gene family, which probably arose from duplication of an ancestor gene.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Identification of residues of SARS-CoV nsp1 that differentially affect inhibition of gene expression and antiviral signaling.

An epidemic of Severe Acute Respiratory Syndrome (SARS) led to the identification of an associated coronavirus, SARS-CoV. This virus evades the host innate immune response in part through the expression of its non-structural protein (nsp) 1, which inhibits both host gene expression and virus- and interferon (IFN)-dependent signaling. Thus, nsp1 is a promising target for drugs, as inhibition of nsp1 would make SARS-CoV more susceptible to the host antiviral defenses. To gain a better understanding of nsp1 mode of action, we generated and analyzed 38 mutants of the SARS-CoV nsp1, targeting 62 solvent exposed residues out of the 180 amino acid protein. From this work, we identified six classes of mutants that abolished, attenuated or increased nsp1 inhibition of host gene expression and/or antiviral signaling. Each class of mutants clustered on SARS-CoV nsp1 surface and suggested nsp1 interacts with distinct host factors to exert its inhibitory activities. Identification of the nsp1 residues critical for its activities and the pathways involved in these activities should help in the design of drugs targeting nsp1. Significantly, several point mutants increased the inhibitory activity of nsp1, suggesting that coronaviruses could evolve a greater ability to evade the host response through mutations of such residues

Geopsy : a user-friendly open-source tool set for ambient vibration processing

Ambient vibrations are nowadays considerably used worldwide for numerous types of engineering applications and scientific research. Geopsy and its companion tools are part of that landscape. Since the first release of the program package in 2005, as outcome of the European Union project Site Effects aSsessment from AMbient noisE, Geopsy has become a mature multiplatform open-source package (released under GNU Public License version 3) that has already been recognized as a reference tool for analyzing ambient vibration data in the context of site characterization studies. The community of users has grown from a core group of researchers up to thousands of seismologists and engineers on every career level and on all continents. The versatility of geopsy allows for the processing of all kinds of data needed in site characterization studies, that is, from single station single trace to three-component array recordings. In all of the aforementioned cases, the steps from field acquisition to the production of publication-ready figures are covered and supported by user-friendly graphical user interfaces or corresponding command-line tools for the automation of the complete processing chain. To avoid black-box usage, a number of lower-level tools guarantee maximum flexibility in accessing and controlling processing results at any stage of the analysis

First round of mutagenesis reveals nsp1-m5 mutant with partial loss of inhibition of host signaling.

<p>SARS-CoV mutants nsp1-m1 through nsp1-m8 were tested for (A) inhibition of host IFN- and virus-dependent signaling using the ISREx3-CAT reporter, followed by inhibition of gene expression using (B) luciferase and (C) β-galactosidase assays. CAT activity values correspond to percent chloramphenicol acetylation using cell extracts diluted 100-fold, luciferase activity is determined in straight extracts and is expressed in RLU and β-galactosidase activity corresponds to released ortho-nitrophenol absorption at 405 nm using extracts diluted 10-fold. Immunoblots of nsp1 mutants are quantitated in (D). Error bars are ± standard error; P-values are result of a t-Test. Mutant nsp1-m5 exhibited attenuated inhibition of host IFN- and virus-dependent signaling (A) while maintaining wildtype inhibition of β-galactosidase (C); P-values for nsp1-m5 are indicated in figure, significance for other mutants is listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062416#pone-0062416-t001" target="_blank">Table 1</a>.</p