Hepatitis C virus (HCV) replicates from a ribonucleoprotein (RNP) complex that is associated with the endoplasmic reticulum (ER) membrane. The replication activities of the HCV subgenomic replicon are shown here to induce ER stress. In response to this stress, cells expressing HCV replicons induce the unfolded protein response (UPR), an ER-to-nucleus intracellular signaling pathway. The UPR is initiated by the proteolytic cleavage of a transmembrane protein, ATF6. The resulting cytoplasmic protein fragment of ATF6 functions as a transcription factor in the nucleus and activates selective genes required for an ER stress response. ATF6 activation leads to increased transcriptional levels of GRP78, an ER luminal chaperone protein. However, the overall level of GRP78 protein is decreased. While ER stress is also known to affect translational attenuation, cells expressing HCV replicons have lower levels of phosphorylation of the ␣ subunit of eukaryotic initiation factor 2. Interestingly, cap-independent internal ribosome entry site-mediated translation directed by the 5 noncoding region of HCV and GRP78 is activated in cells expressing HCV replicons. These studies provide insight into the effects of HCV replication on intracellular events and the mechanisms underlying liver pathogenesis.The hepatitis C virus (HCV) is a major worldwide health problem. HCV causes acute and chronic hepatitis, which can lead to chronic active hepatitis, liver cirrhosis, and hepatocellular carcinoma (9). The HCV viral genome consists of a positive-strand RNA, 9.6 kb in length, encoding a 3,000-aminoacid polyprotein (2, 33). The polyprotein is proteolytically cleaved into 10 distinct proteins (2, 33). The structural proteins include the core and highly glycosylated envelope proteins (E1 and E2); the nonstructural proteins (NS2 to NS5) encode enzymatic activities necessary for virus replication (20). The 5Ј noncoding region (5Ј-NCR) and 3Ј-NCR of HCV contain sequences directly involved in RNA replication (10,20). The HCV 5Ј-NCR also functions as an internal ribosome entry site (IRES) permitting cap-independent translation (35,40,41).The absence of a reproducible and efficient cell culture system in HCV studies has hampered efforts to examine alterations in intracellular events by HCV replication. Recently, Lohmann et al. (22) developed selectable HCV subgenomic replicons in a human hepatoma cell line, Huh7, which supported RNA replication. These replicons were bicistronic constructs composed of the HCV IRES (nucleotides 1 to 377 of the 5Ј-NCR); the neomycin phosphotransferase (neo) gene; the encephalomyocarditis IRES, which mediates the translation of HCV nonstructural proteins NS3 through NS5; and the 3Ј-NCR (22) (Fig. 1). HCV replicons do not contain structural proteins and NS2 because they are probably not needed for replication of HCV RNA (22). A high level of subgenomic replicon replication was shown to result from adaptive mutations, which were found in several NS proteins (6, 23). HCV nonstructural proteins, including NS3, NS4, and N...

Hepatitis C virus (HCV) gene expression disrupts normal endoplasmic reticulum (ER) functions and induces ER stress. ER stress results from the accumulation of unfolded or misfolded proteins in the ER; cells can alleviate this stress by degrading or refolding these proteins. The IRE1-XBP1 pathway directs both protein refolding and degradation in response to ER stress. Like IRE1-XBP1, other branches of the ER stress response mediate protein refolding. However, IRE1-XBP1 can also specifically activate protein degradation. We show here that XBP1 expression is elevated in cells carrying HCV subgenomic replicons, but XBP1 trans-activating activity is repressed. This prevents the IRE1-XBP1 transcriptional induction of EDEM (ER degradation-enhancing ␣-mannosidase-like protein). The mRNA expression of EDEM is required for the degradation of misfolded proteins. Consequently, misfolded proteins are stable in cells expressing HCV replicons. HCV may suppress the IRE1-XBP1 pathway to stimulate the synthesis of its viral proteins. IRE1␣-null MEFs, a cell line with a defective IRE1-XBP1 pathway, show elevated levels of HCV IRES-mediated translation. Therefore, HCV may suppress the IRE1-XBP1 pathway to not only promote HCV expression but also to contribute to the persistence of the virus in infected hepatocytes.

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e Current infectious disease molecular tests are largely pathogen specific, requiring test selection based on the patient's symptoms. For many syndromes caused by a large number of viral, bacterial, or fungal pathogens, such as respiratory tract infections, this necessitates large panels of tests and has limited yield. In contrast, next-generation sequencing-based metagenomics can be used for unbiased detection of any expected or unexpected pathogen. However, barriers for its diagnostic implementation include incomplete understanding of analytical performance and complexity of sequence data analysis. We compared detection of known respiratory virus-positive (n ‫؍‬ 42) and unselected (n ‫؍‬ 67) pediatric nasopharyngeal swabs using an RNA sequencing (RNA-seq)-based metagenomics approach and Taxonomer, an ultrarapid, interactive, web-based metagenomics data analysis tool, with an FDA-cleared respiratory virus panel (RVP; GenMark eSensor). Untargeted metagenomics detected 86% of known respiratory virus infections, and additional PCR testing confirmed RVP results for only 2 (33%) of the discordant samples. In unselected samples, untargeted metagenomics had excellent agreement with the RVP (93%). In addition, untargeted metagenomics detected an additional 12 viruses that were either not targeted by the RVP or missed due to highly divergent genome sequences. Normalized viral read counts for untargeted metagenomics correlated with viral burden determined by quantitative PCR and showed high intrarun and interrun reproducibility. Partial or full-length viral genome sequences were generated in 86% of RNA-seq-positive samples, allowing assessment of antiviral resistance, strain-level typing, and phylogenetic relatedness. Overall, untargeted metagenomics had high agreement with a sensitive RVP, detected viruses not targeted by the RVP, and yielded epidemiologically and clinically valuable sequence information. L aboratory diagnosis of infectious diseases has historically taken a syndrome-based approach. Culture of appropriate specimens on a combination of relevant media or cell lines enables detection of certain common bacterial, viral, and fungal pathogens. However, culture requires experienced personnel, requires several days to weeks to yield a definitive answer, depends on viability and appropriate culture conditions, and has limited sensitivity. Molecular tests have superior turnaround times, sensitivity, and taxonomic resolution. However, only targeted pathogens can be detected, and differentiation of clinically or epidemiologically relevant strains or genotypes is limited. Moreover, molecular tests need to be updated when new species or strains are recognized to ensure that newly identified genetic variants can be detected.In contrast, next-generation sequencing-based metagenomic testing combines and extends many advantages of molecular tests and culture-based methods. Host-and pathogen-derived nucleic acids are sequenced without a priori knowledge of expected pathogens, allowing simultaneous detection of a virtually ...

BackgroundHigh-throughput sequencing enables unbiased profiling of microbial communities, universal pathogen detection, and host response to infectious diseases. However, computation times and algorithmic inaccuracies have hindered adoption.ResultsWe present Taxonomer, an ultrafast, web-tool for comprehensive metagenomics data analysis and interactive results visualization. Taxonomer is unique in providing integrated nucleotide and protein-based classification and simultaneous host messenger RNA (mRNA) transcript profiling. Using real-world case-studies, we show that Taxonomer detects previously unrecognized infections and reveals antiviral host mRNA expression profiles. To facilitate data-sharing across geographic distances in outbreak settings, Taxonomer is publicly available through a web-based user interface.ConclusionsTaxonomer enables rapid, accurate, and interactive analyses of metagenomics data on personal computers and mobile devices.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-016-0969-1) contains supplementary material, which is available to authorized users.

Mps1 is a dual specificity protein kinase that is essential for the bipolar attachment of chromosomes to the mitotic spindle and for maintaining the spindle assembly checkpoint until all chromosomes are properly attached. Mps1 is expressed at high levels during mitosis and is abundantly expressed in cancer cells. Disruption of Mps1 function induces aneuploidy and cell death. We report the identification of MPI-0479605, a potent and selective ATP competitive inhibitor of Mps1. Cells treated with MPI-0479605 undergo aberrant mitosis, resulting in aneuploidy and formation of micronuclei. In cells with wild-type p53, this promotes the induction of a postmitotic checkpoint characterized by the ATM-and RAD3-related-dependent activation of the p53-p21 pathway. In both wild-type and p53 mutant cells lines, there is a growth arrest and inhibition of DNA synthesis. Subsequently, cells undergo mitotic catastrophe and/or an apoptotic response. In xenograft models, MPI-0479605 inhibits tumor growth, suggesting that drugs targeting Mps1 may have utility as novel cancer therapeutics. Mol Cancer Ther; 10(12); 2267-75. Ó2011 AACR.

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Background Community-acquired pneumonia (CAP) is a leading cause of pediatric hospitalization. Pathogen identification fails in approximately 20% of children but is critical for optimal treatment and prevention of hospital-acquired infections. We used two broad-spectrum detection strategies to identify pathogens in test-negative children with CAP and asymptomatic controls. Methods Nasopharyngeal/oropharyngeal (NP/OP) swabs from 70 children <5 years with CAP of unknown etiology and 90 asymptomatic controls were tested by next-generation sequencing (RNA-seq) and pan viral group (PVG) PCR for 19 viral families. Association of viruses with CAP was assessed by adjusted odds ratios (aOR) and 95% confidence intervals controlling for season and age group. Results RNA-seq/PVG PCR detected previously missed, putative pathogens in 34% of patients. Putative viral pathogens included human parainfluenza virus 4 (aOR 9.3, P = .12), human bocavirus (aOR 9.1, P < .01), Coxsackieviruses (aOR 5.1, P = .09), rhinovirus A (aOR 3.5, P = .34), and rhinovirus C (aOR 2.9, P = .57). RNA-seq was more sensitive for RNA viruses whereas PVG PCR detected more DNA viruses. Conclusions RNA-seq and PVG PCR identified additional viruses, some known to be pathogenic, in NP/OP specimens from one-third of children hospitalized with CAP without a previously identified etiology. Both broad-range methods could be useful tools in future epidemiologic and diagnostic studies.