Next-generation sequencing reveals a controlled immune response to Zaire Ebola virus challenge in cynomolgus macaques immunized with vesicular stomatitis virus expressing Zaire Ebola virus glycoprotein (VSVΔG/EBOVgp).

Vesicular stomatitis virus expressing Zaire Ebola virus (EBOV) glycoprotein (VSVΔG/EBOVgp) could be used as a vaccine to meet the 2014 Ebola virus outbreak. To characterize the host response to this vaccine, we used mRNA sequencing to analyze peripheral blood mononuclear cells (PBMCs) from cynomolgus macaques after VSVΔG/EBOVgp immunization and subsequent EBOV challenge. We found a controlled transcriptional response that transitioned to immune regulation as the EBOV was cleared. This observation supports the safety of the vaccine.

The draft genome sequence of the ferret (Mustela putorius furo) facilitates study of human respiratory disease.

The domestic ferret (Mustela putorius furo) is an important animal model for multiple human respiratory diseases. It is considered the 'gold standard' for modeling human influenza virus infection and transmission. Here we describe the 2.41 Gb draft genome assembly of the domestic ferret, constituting 2.28 Gb of sequence plus gaps. We annotated 19,910 protein-coding genes on this assembly using RNA-seq data from 21 ferret tissues. We characterized the ferret host response to two influenza virus infections by RNA-seq analysis of 42 ferret samples from influenza time-course data and showed distinct signatures in ferret trachea and lung tissues specific to 1918 or 2009 human pandemic influenza virus infections. Using microarray data from 16 ferret samples reflecting cystic fibrosis disease progression, we showed that transcriptional changes in the CFTR-knockout ferret lung reflect pathways of early disease that cannot be readily studied in human infants with cystic fibrosis disease.

Drug repurposing: a better approach for infectious disease drug discovery?

The advent of publicly available databases containing system-wide phenotypic data of the host response to both drugs and pathogens, in conjunction with bioinformatics and computational methods now allows for in silico predictions of FDA-approved drugs as treatments against infection diseases. This systems biology approach captures the complexity of both the pathogen and drug host response in the form of expression patterns or molecular interaction networks without having to understand the underlying mechanisms of action. These drug repurposing techniques have been successful in identifying new drug candidates for several types of cancers and were recently used to identify potential therapeutics against influenza, the newly discovered Middle Eastern Respiratory Syndrome coronavirus and several parasitic diseases. These new approaches have the potential to significantly reduce both the time and cost for infectious diseases drug discovery.

Host regulatory network response to infection with highly pathogenic H5N1 avian influenza virus.

During the last decade, more than half of humans infected with highly pathogenic avian influenza (HPAI) H5N1 viruses have died, yet virus-induced host signaling has yet to be clearly elucidated. Airway epithelia are known to produce inflammatory mediators that contribute to HPAI H5N1-mediated pathogenicity, but a comprehensive analysis of the host response in this cell type is lacking. Here, we leveraged a system approach to identify and statistically validate signaling subnetworks that define the dynamic transcriptional response of human bronchial epithelial cells after infection with influenza A/Vietnam/1203/2004 (H5N1, VN1203). Importantly, we validated a subset of transcripts from one subnetwork in both Calu-3 cells and mice. A more detailed examination of two subnetworks involved in the immune response and keratinization processes revealed potential novel mediators of HPAI H5N1 pathogenesis and host response signaling. Finally, we show how these results compare to those for a less virulent strain of influenza virus. Using emergent network properties, we provide fresh insight into the host response to HPAI H5N1 virus infection and identify novel avenues for perturbation studies and potential therapeutic interventions for fatal HPAI H5N1 disease.

PSGL-1 and mTOR regulate translation of ROCK-1 and physiological functions of macrophages.

Rho-associated kinases (ROCKs) are critical molecules involved in the physiological functions of macrophages, such as chemotaxis and phagocytosis. We demonstrate that macrophage adherence promotes rapid changes in physiological functions that depend on translational upregulation of preformed ROCK-1 mRNA, but not ROCK-2 mRNA. Before adherence, both ROCK mRNAs were present in the cytoplasm of macrophages, whereas ROCK proteins were undetectable. Macrophage adherence promoted signaling through P-selectin glycoprotein ligand-1 (PSGL-1)/Akt/mTOR that resulted in synthesis of ROCK-1, but not ROCK-2. Following synthesis, ROCK-1 was catalytically active. In addition, there was a rapamycin/sirolimus-sensitive enhanced loading of ribosomes on preformed ROCK-1 mRNAs. Inhibition of mTOR by rapamycin abolished ROCK-1 synthesis in macrophages resulting in an inhibition of chemotaxis and phagocytosis. Macrophages from PSGL-1-deficient mice recapitulated pharmacological inhibitor studies. These results indicate that receptor-mediated regulation at the level of translation is a component of a rapid set of mechanisms required to direct the macrophage phenotype upon adherence and suggest a mechanism for the immunosuppressive and anti-inflammatory effects of rapamycin/sirolimus.

The transcriptome and its translation during recovery from cell cycle arrest in Saccharomyces cerevisiae.

Complete genome sequences together with high throughput technologies have made comprehensive characterizations of gene expression patterns possible. While genome-wide measurement of mRNA levels was one of the first applications of these advances, other important aspects of gene expression are also amenable to a genomic approach, for example, the translation of message into protein. Earlier we reported a high throughput technology for simultaneously studying mRNA level and translation, which we termed translation state array analysis, or TSAA. The current studies test the proposition that TSAA can identify novel instances of translation regulation at the genome-wide level. As a biological model, cultures of Saccharomyces cerevisiae were cell cycle-arrested using either alpha-factor or the temperature-sensitive cdc15-2 allele. Forty-eight mRNAs were found to change significantly in translation state following release from alpha-factor arrest, including genes involved in pheromone response and cell cycle arrest such as BAR1, SST2, and FAR1. After the shift of the cdc15-2 strain from 37 degrees C to 25 degrees C, 54 mRNAs were altered in translation state, including the products of the stress genes HSP82, HSC82, and SSA2. Thus, regulation at the translational level seems to play a significant role in the response of yeast cells to external physical or biological cues. In contrast, surprisingly few genes were found to be translationally controlled as cells progressed through the cell cycle. Additional refinements of TSAA should allow characterization of both transcriptional and translational regulatory networks on a genomic scale, providing an additional layer of information that can be integrated into models of system biology and function.

Regulated translation termination at the upstream open reading frame in s-adenosylmethionine decarboxylase mRNA.

The upstream open reading frame (uORF) in the mRNA encoding S-adenosylmethionine decarboxylase is a cis-acting element that confers feedback control by cellular polyamines on translation of this message. Recent studies demonstrated that elevated polyamines inhibit synthesis of the peptide encoded by the uORF by stabilizing a ribosome paused in the vicinity of the termination codon. These studies suggested that polyamines act at the termination step of uORF translation. In this paper, we demonstrate that elevated polyamines stabilize an intermediate in the termination process, the complete nascent peptide linked to the tRNA that decodes the final codon. The peptidyl-tRNA molecule is found associated with the ribosome fraction, and decay of this molecule correlated with release of the paused ribosome from the message. Furthermore, the stability of this complex is influenced by the same parameters that influence regulation by the uORF in vivo, namely the concentration of polyamines and the sequence of the uORF-encoded peptide. These results suggest that the regulated step in uORF translation is after formation of the peptidyl-tRNA molecule but before hydrolysis of the peptidyl-tRNA bond. This regulation may involve an interaction between the peptide, polyamines, and a target in the translational apparatus.