• Animals
• Mice
• Hepatitis A virus/genetics
• Hepatitis A virus/metabolism
• Protein Biosynthesis
• Loxapine
• Virus Replication/genetics
• RNA/metabolism
• Viral Proteins/metabolism
• RNA, Viral/genetics
• RNA, Viral/metabolism

• Ministry of Health, Labour and Welfare
• Japan Society for the Promotion of Science
• Japan Agency for Medical Research and Development

• Hepatitis C virus
• RNA dependent RNA polymerase
• molecular docking
• molecular dynamics simulations
• protein-ligand interaction profiler.
• virtual screening

• Humans
• Hepacivirus
• Molecular Docking Simulation
• RNA-Dependent RNA Polymerase/chemistry
• RNA-Dependent RNA Polymerase/therapeutic use
• Hepatitis C, Chronic
• Ligands
• Hepatitis C/drug therapy
• Molecular Dynamics Simulation
• RNA, Viral
• DNA-Directed RNA Polymerases/therapeutic use
• Antiviral Agents/pharmacology
• Antiviral Agents/chemistry

• 8-256-2014/STEDIR/2013 Department of Science, Technology and Environment, Goa State, India

• FoSpc2
• Fusarium odoratissimum
• extracellular enzymes
• light response
• signal peptidase

• Humans
• Virulence/genetics
• Photophobia
• Fungal Proteins/genetics
• Fungal Proteins/metabolism
• Serine Endopeptidases/metabolism
• Membrane Proteins/metabolism
• Protein Sorting Signals

• Program of Fujian Key Laboratory for Monitoring and Integrated Management of Crop Pests
• Technology Innovation of Fujian Agriculture and Forestry University
• MOST | National Natural Science Foundation of China (NSFC)
• Natural Science Foundation of Fujian Province (Fujian Provincial Natural Science Foundation)

• Cell Line
• HEK293 Cells
• Hepacivirus/metabolism
• Hepatitis C/virology
• Host Microbial Interactions
• Humans
• Membrane Proteins/chemistry
• Membrane Proteins/metabolism
• Molecular Dynamics Simulation
• Protein Conformation
• Viral Envelope Proteins/chemistry
• Viral Envelope Proteins/metabolism
• Viroporin Proteins/chemistry
• Viroporin Proteins/metabolism
• Virus Assembly
• Virus Replication

• R01 AI110358 NIAID NIH HHS
• R01 AI146227 NIAID NIH HHS
• National Institute of Allergy and Infectious Diseases
• King Saud bin Abdulaziz University
• Friends of the HZI

• ER translocon
• crosslinking mass spectrometry
• cryo-EM
• membrane thinning
• molecular dynamics simulations
• protein maturation
• protein secretion
• secretory pathway
• signal peptidase complex
• signal peptide

The hepatitis C virus (HCV), an obligatory intracellular pathogen, highly depends on its host cells to propagate successfully. The HCV life cycle can be simply divided into several stages including viral entry, protein translation, RNA replication, viral assembly and release. Hundreds of cellular factors involved in the HCV life cycle have been identified over more than thirty years of research. Characterization of these cellular factors has provided extensive insight into HCV replication strategies. Some of these cellular factors are targets for anti-HCV therapies. In this review, we summarize the well-characterized and recently identified cellular factors functioning at each stage of the HCV life cycle.

• Hepatitis C virus
• Cellular factor
• Viral entry
• Translation
• Replication
• Assembly
• Release

• Animals
• Hepacivirus
• Hepatitis C/drug therapy
• Humans
• Life Cycle Stages
• Virus Assembly
• Virus Replication

• SPCS1
• Signal peptidase
• Signal sequence
• Spc1
• Transmembrane

• Biomarker discovery
• HIV
• Latency
• Proteomics
• SWATH-MS

• Biomarkers/analysis
• Cell Line
• HIV-1/chemistry
• HIV-1/genetics
• Humans
• Jurkat Cells
• Mass Spectrometry/methods
• Proteomics/methods
• T-Lymphocytes/virology
• U937 Cells
• Virus Latency

• R01 DA043258 NIDA NIH HHS

Candidatus Liberibacter solanacerum (CLso), transmitted by Bactericera trigonica in a persistent and propagative mode causes carrot yellows disease, inflicting hefty economic losses. Understanding the process of transmission of CLso by psyllids is fundamental to devise sustainable management strategies. Persistent transmission involves critical steps of adhesion, cell invasion, and replication before passage through the midgut barrier. This study uses a transcriptomic approach for the identification of differentially expressed genes with CLso infection in the midguts, adults, and nymphs of B. trigonica and their putative involvement in CLso transmission. Several genes related to focal adhesion and cellular invasion were upregulated after CLso infection. Interestingly, genes involved with proper functionality of the endoplasmic reticulum (ER) were upregulated in CLso infected samples. Notably, genes from the endoplasmic reticulum associated degradation (ERAD) and the unfolded protein response (UPR) pathway were overexpressed after CLso infection. Marker genes of the ERAD and UPR pathways were also upregulated in Diaphorina citri when infected with Candidatus Liberibacter asiaticus (CLas). Upregulation of the ERAD and UPR pathways indicate induction of ER stress by CLso/CLas in their psyllid vector. The role of ER in bacteria–host interactions is well-documented; however, the ER role following pathogenesis of CLso/CLas is unknown and requires further functional validation.

• ER stress
• ERAD
• Liberibacter
• UPR
• bacterial pathogen
• insect vector
• psyllid

• HCV
• NS5B
• PKR
• RdRp
• eIF2α

• Carcinoma, Hepatocellular/enzymology
• Carcinoma, Hepatocellular/virology
• Cell Line, Tumor
• Enzyme Activation
• Gene Expression Regulation, Neoplastic
• Gene Expression Regulation, Viral
• Hepacivirus/enzymology
• Humans
• Liver Neoplasms
• Plasmids
• RNA, Viral/genetics
• RNA-Dependent RNA Polymerase/genetics
• RNA-Dependent RNA Polymerase/metabolism
• eIF-2 Kinase/genetics
• eIF-2 Kinase/metabolism

Osteoarthritis (OA) is a complex degenerative joint disorder, which is caused by both environmental and genetic factors. Previous studies have indicated that the GNL3 gene is associated with knee osteoarthritis (KOA) susceptibility in Europeans; however, the exact molecular mechanism is still unclear. In the present study, we investigated the potential genetic association of GNL3 with KOA in a two-stage sample of 6,704 individuals from the Han Chinese population. Subjects containing 1,052 KOA patients and 2,117 controls were considered the discovery dataset, while subjects consisting of 1,173 KOA patients and 2,362 controls were utilized as the replication dataset. Single-SNP association, imputation, and haplotypic association analyses were performed. The SNP of rs11177 in GNL3 was identified to be significantly associated with KOA after accounting for age, gender and BMI in both stages. The imputed SNP of rs6617 in SPCS1 was found to be strongly associated with KOA risk, and the significant association signal was confirmed in the replication stage. Moreover, a haplotype-based analysis also indicated a positive genetic effect of GNL3 on KOA susceptibility. In summary, our results proved that GNL3 plays an important role in the etiology of KOA, suggesting that GNL3 is a potential genetic modifier for KOA development.

• assembly
• flavivirus
• host factor
• protein-protein interactions
• viral replication

• Tribolium castaneum
• apoptosis
• cell proliferation
• midgut
• signal peptidase complex

• Animals
• Female
• Gastrointestinal Tract/enzymology
• Hemocytes/metabolism
• Insect Proteins/metabolism
• Melanins/metabolism
• Membrane Proteins/metabolism
• RNA Interference
• Serine Endopeptidases/metabolism
• Tribolium/enzymology
• Tribolium/growth & development

• Amino Acid Sequence
• Cell Line
• HEK293 Cells
• Hepacivirus/genetics
• Hepacivirus/pathogenicity
• Hepacivirus/physiology
• Hepatitis C/etiology
• Hepatitis C/virology
• Host-Pathogen Interactions/genetics
• Host-Pathogen Interactions/physiology
• Humans
• Models, Biological
• Mutation
• Protein Processing, Post-Translational
• Viral Envelope Proteins/chemistry
• Viral Envelope Proteins/genetics
• Viral Envelope Proteins/physiology
• Viral Nonstructural Proteins/physiology
• Viral Proteins/chemistry
• Viral Proteins/genetics
• Viral Proteins/physiology
• Virulence/genetics
• Virulence/physiology
• Virus Assembly/genetics
• Virus Assembly/physiology

• R01 AI087925 NIAID NIH HHS
• European Research Council (BE)
• Agence Nationale de la Recherche sur le SIDA et les hépatites virales
• Agence Nationale de la Recherche (FR)

The classical swine fever virus (CSFV) nonstructural protein p7 is crucial for virus production, yet precisely how the p7 modulates this process is unclear. In this study, we first identified the interactions of p7 with E2 and NS2. The key binding regions of both p7 and NS2 mapped to the first transmembrane (TM1) domain of two proteins. Three amino acid substitutions in the TM1 region of p7 (p7^{TDI18/19/20AAA}, p7^{EVV21/22/23AAA} and p7^{YFY25/26/30AAA}) impaired infectious virus production and reduced the interaction of p7 with the NS2 protein. The E2p7 processing and mature p7, but not the E2p7 precursor, are essential for infectious virus production. Bicistronic mutants (pSM/E2/IRES) with single substitutions at residues 1 to 9 of p7 exhibited a significantly increased infectious CSFV titer compared to their counterparts in the context of pSM. Viral genomic RNA copies of the mutants exhibited similar levels compared with the wt CSFV. Our results demonstrated that CSFV p7 and its precursor E2p7 modulate viral protein interactions and infectious virus production without influencing viral RNA replication.

Here we identified PUF60, a splicing factor and a U2 small nuclear ribonucleoprotein auxiliary factor, as a versatile regulator of transcriptional and post-transcriptional steps in expression of hepatitis B virus (HBV) 3.5 kb, precore plus pregenomic RNA. We demonstrate that PUF60 is involved in: 1) up-regulation of core promoter activity through its interaction with transcription factor TCF7L2, 2) promotion of 3.5 kb RNA degradation and 3) suppression of 3.5 kb RNA splicing. When the 1.24-fold HBV genome was introduced into cells with the PUF60-expression plasmid, the 3.5 kb RNA level was higher at days 1–2 post-transfection but declined thereafter in PUF60-expressing cells compared to viral replication control cells. Deletion analyses showed that the second and first RNA recognition motifs (RRMs) within PUF60 are responsible for core promoter activation and RNA degradation, respectively. Expression of PUF60 mutant deleting the first RRM led to higher HBV production. To our knowledge, this is the first to identify a host factor involved in not only positively regulating viral gene expression but also negative regulation of the same viral life cycle. Functional linkage between transcriptional and post-transcriptional controls during viral replication might be involved in mechanisms for intracellular antiviral defense and viral persistence.

Directed differentiation of human stem cells including induced pluripotent stem cells into hepatic cells potentially leads to acquired susceptibility to hepatitis C virus (HCV) infection. However, cellular determinants that change their expression during cell reprogramming or hepatic differentiation and are pivotal for supporting the HCV life cycle remain unclear. In this study, by introducing a set of reprogramming factors, we established HuH-7-derived oval-like cell lines, Hdo-17 and -23, which possess features of bipotential liver precursors. Upon induction of hepatocyte differentiation, expression of mature hepatocyte markers and hepatoblast markers in cells increased and decreased, respectively. In contrast, in response to cholangiocytic differentiation induction, gene expression of epithelium markers increased and cells formed round cysts with a central luminal space. Hdo cells lost their susceptibility to HCV infection and viral RNA replication. Hepatic differentiation of Hdo cells potentially led to recovery of permissiveness to HCV RNA replication. Gene expression profiling showed that most host-cell factors known to be involved in the HCV life cycle, except CD81, are expressed in Hdo cells comparable to HuH-7 cells. HCV pseudoparticle infectivity was significantly but partially recovered by ectopic expression of CD81, suggesting possible involvement of additional unidentified factors in HCV entry. In addition, we identified miR200a-3p, which is highly expressed in Hdo cells and stem cells but poorly expressed in differentiated cells and mature hepatocytes, as a novel negative regulator of HCV replication. In conclusion, our results showed that epigenetic reprogramming of human hepatoma cells potentially changes their permissivity to HCV.

• Immune response
• Immunity
• Immunology and Microbiology Section
• epigenetic reprogramming
• hepatitis C virus
• hepatoma cell
• miR200a
• oval cell

Hepatitis C virus (HCV) is a leading cause of chronic liver disease affecting over 170 million people worldwide. Chronic infection with HCV progresses to liver fibrosis, cirrhosis, and hepatocellular carcinoma. HCV exploits host cellular factors for viral propagation. To investigate the cellular factors required for HCV propagation, we screened a siRNA library targeting human cell cycle genes using cell culture grown HCV-infected cells. In the present study, we selected and characterized a gene encoding Rad51. Rad51, a member of a conserved recombinase family, is an essential factor for homologous recombination and repair of double-strand DNA breaks. We demonstrated that siRNA-mediated knockdown of Rad51 significantly inhibited HCV propagation without affecting HCV RNA replication. Silencing of Rad51 impaired secretion of infectious HCV particles and thus intracellular viruses were accumulated. We showed that HCV NS3 specifically interacted with Rad51 and accumulated Rad51 in the cytosol. Furthermore, Rad51 was coprecipitated with NS3 and HCV RNA. By employing membrane flotation and protease protection assays, we also demonstrated that Rad51 was co-fractionated with HCV NS3 on the lipid raft. These data indicate that Rad51 may be a component of the HCV RNA replication complex. Collectively, these data suggest that HCV may exploit cellular Rad51 to promote viral propagation and thus Rad51 may be a potential therapeutic target for HCV.

• NS3
• Rad51
• hepatitis C virus
• siRNA screening
• therapeutic target
• viral production

• HCV
• RNA replication
• adaptive mutation
• cell culture
• infectious virus production

• ATP
• Endoplasmic reticulum
• Hepatitis C virus
• Membrane vesicle
• Mitochondria
• Replication factory
• Virus-host interaction

• EGFRvIII
• External secretion
• F25P preproinsulin
• Glioblastoma

Hepatitis C virus (HCV) infection frequently leads to chronic liver disease, liver cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms by which HCV infection leads to chronic liver disease and HCC are not well understood. The infection cycle of HCV is initiated by the attachment and entry of virus particles into a hepatocyte. Replication of the HCV genome inside hepatocytes leads to accumulation of large amounts of viral proteins and RNA replication intermediates in the endoplasmic reticulum (ER), resulting in production of thousands of new virus particles. HCV-infected hepatocytes mount a substantial stress response. How the infected hepatocyte integrates the viral-induced stress response with chronic infection is unknown. The unfolded protein response (UPR), an ER-associated cellular transcriptional response, is activated in HCV infected hepatocytes. Over the past several years, research performed by a number of laboratories, including ours, has shown that HCV induced UPR robustly activates autophagy to sustain viral replication in the infected hepatocyte. Induction of the cellular autophagy response is required to improve survival of infected cells by inhibition of cellular apoptosis. The autophagy response also inhibits the cellular innate antiviral program that usually inhibits HCV replication. In this review, we discuss the physiological implications of the HCV-induced chronic ER-stress response in the liver disease progression.

• Autophagy
• Chaperon-mediated autophagy (CMA)
• Endoplasmic reticulum stress (ER-stress)
• Hepatitis C virus (HCV)
• Hepatocellular carcinoma (HCC)
• Interferon (IFN)
• Interferon-alpha receptor-1 (IFNAR1)
• Unfolded protein response (UPR)
• chronic liver disease (CLD)

Although information regarding morphogenesis of the hepatitis C virus (HCV) is accumulating, the mechanism(s) by which the HCV genome encapsidated remains unknown. In the present study, in cell cultures producing HCV, the molecular ratios of 3’ end- to 5’ end-regions of the viral RNA population in the culture medium were markedly higher than those in the cells, and the ratio was highest in the virion-rich fraction. The interaction of the 3’ untranslated region (UTR) with Core in vitro was stronger than that of the interaction of other stable RNA structure elements across the HCV genome. A foreign gene flanked by the 3’ UTR was encapsidated by supplying both viral NS3-NS5B proteins and Core-NS2 in trans. Mutations within the conserved stem-loops of the 3’ UTR were observed to dramatically diminish packaging efficiency, suggesting that the conserved apical motifs of the 3´ X region are important for HCV genome packaging. This study provides evidence of selective packaging of the HCV genome into viral particles and identified that the 3’ UTR acts as a cis-acting element for encapsidation.

Although cell culture systems provide a powerful tool for deciphering the life cycle of the hepatitis C virus (HCV), the mechanisms of encapsidation of the viral genome into infectious particles remain to be uncovered. The HCV genome is a positive RNA with one single reading frame flanked by 5’- and 3’ untranslated regions (UTRs). Thus far, there is no direct evidence that HCV employs a packaging-signal dependent- or replication-coupled mechanism of encapsidation of its genome. The possible overlap of RNA sequences that function in RNA replication with those that function in encapsidation may present an obstacle to investigation of the cis-elements for RNA packaging. In this study, we characterized the properties of HCV RNAs in a cell culture system by determining their integrity in virus-replicating cells and in culture supernatants, and we found that over-distributed 5’-subgenomes were negatively selected during virus assembly in the cells. Using trans-packaging systems with replication defective subgenomes, we identified the 3’UTR as a cis-acting element that was sufficient for packaging of not only a HCV subgenome but also a foreign gene into infectious particles. Mutagenesis analyses, together with an in vitro binding assay with Core demonstrated that, whereas the best encapsidation occurs with the entire 3’ UTR, the loop sequences of the 3’ X region appear to be essential for encapsidation. Our work opens new perspectives for understanding the molecular mechanisms that regulate the HCV life cycle and potentially paves a way to a new anti-viral therapy.

Recognition and elimination of tumor cells by the immune system is crucial for limiting tumor growth. Natural killer (NK) cells become activated when the receptor NKG2D is engaged by ligands that are frequently upregulated in primary tumors and on cancer cell lines. However, the molecular mechanisms driving NKG2D ligand expression on tumor cells are not well defined. Using a forward genetic screen in a tumor-derived human cell line, we identified several novel factors supporting expression of the NKG2D ligand ULBP1. Our results show stepwise contributions of independent pathways working at multiple stages of ULBP1 biogenesis. Deeper investigation of selected hits from the screen showed that the transcription factor ATF4 drives ULBP1 gene expression in cancer cell lines, while the RNA-binding protein RBM4 supports ULBP1 expression by suppressing a novel alternatively spliced isoform of ULBP1 mRNA. These findings offer insight into the stress pathways that alert the immune system to danger.

DOI:http://dx.doi.org/10.7554/eLife.08474.001

Cancer is caused by a series of mutations that result in uncontrolled cell growth and division. Yet, the body's immune system can often detect and destroy abnormal cells before they cause tumors and disease. Natural killer cells are part of the immune system and have receptors on their surface that allow them to tell the difference between healthy host cells and host cells that are stressed or abnormal. Some of these receptors activate the natural killer cells when they bind to their target molecules. Other receptors have the opposite effect and inhibit the natural killer cells. Activation occurs when the signaling from the activating receptors is stronger than the signals from the inhibitory receptors.

One of the well-studied activating receptors recognizes a number of proteins and molecules that are produced by abnormal or tumor cells, including a protein called ULBP1. This protein is absent from the surface of healthy cells but is found in abundance on tumor cells. However, it is still not clear what drives tumor cells to produce ULBP1 (or other molecules) that are recognized by natural killer cell receptors.

Now, Gowen et al. report on a genetic screen that has revealed numerous genes that regulate the levels of ULBP1 in human cells. Many of these genes had independent effects that when added together accounted for most of the ULBP1 present on the cell surface.

Gowen et al. then explored some of the ‘regulators’ encoded by these genes in more detail. One called ATF4, which had previously been linked to stress responses, was shown to increase the expression of the gene for ULBP1 in cancer cells. Another regulator called RBM4 instead acted in a different way and at a later stage in ULBP1 production.

All together, these findings offer insight into the stress pathways that alert the immune system to abnormal cells. The next challenge will be investigating how these pathways might be exploited for cancer immunotherapy.

DOI:http://dx.doi.org/10.7554/eLife.08474.002

• CRISPR/Cas9
• NKG2D
• alternative splicing
• cellular stress
• human
• immunology
• natural killer cells
• tumor immunology

• enzyme inhibitor
• mutant
• protein processing
• protein synthesis
• protein trafficking
• secretory protein
• signal peptidase
• viral polypeptide

Hepatitis C virus (HCV) is a hepatotrophic virus and a major cause of chronic liver disease, including hepatocellular carcinoma, worldwide. The life cycle of HCV is closely associated with the metabolism of lipids and lipoproteins. The main function of lipoproteins is transporting lipids throughout the body. Triglycerides, free cholesterol, cholesteryl esters, and phospholipids are the major components of the transported lipids. The pathway of HCV assembly and secretion is closely linked to lipoprotein production and secretion, and the infectivity of HCV particles largely depends on the interaction of lipoproteins. Moreover, HCV entry into hepatocytes is strongly influenced by lipoproteins. The key lipoprotein molecules mediating these interactions are apolipoproteins. Apolipoproteins are amphipathic proteins on the surface of a lipoprotein particle, which help stabilize lipoprotein structure. They perform a key role in lipoprotein metabolism by serving as receptor ligands, enzyme co-factors, and lipid transport carriers. Understanding the association between the life cycle of HCV and lipoprotein metabolism is important because each step of the life cycle of HCV that is associated with lipoprotein metabolism is a potential target for anti-HCV therapy. In this article, we first concisely review the nature of lipoprotein and its metabolism to better understand the complicated interaction of HCV with lipoprotein. Then, we review the outline of the processes of HCV assembly, secretion, and entry into hepatocytes, focusing on the association with lipoproteins. Finally, we discuss the clinical aspects of disturbed lipid/lipoprotein metabolism and the significance of dyslipoproteinemia in chronic HCV infection with regard to abnormal apolipoproteins.

• Hepatitis C virus
• Apolipoprotein
• Lipo-viral particle
• Dyslipoproteinemia
• Lipoprotein

• Animals
• Hepacivirus/growth & development
• Hepacivirus/metabolism
• Hepacivirus/pathogenicity
• Hepatitis C, Chronic/diagnosis
• Hepatitis C, Chronic/metabolism
• Hepatitis C, Chronic/virology
• Humans
• Lipoproteins/metabolism
• Liver/metabolism
• Liver/virology
• Virus Internalization

• Envelope glycoproteins
• Hepatitis C virus
• Membrane fusion
• Receptor
• Virus entry

• Hepatitis C virus
• Viral life cycle
• Virus entry
• Virus-host interactions

• Cell Biology/trends
• Hepacivirus/genetics
• Hepacivirus/growth & development
• Hepacivirus/pathogenicity
• Hepatitis C/pathology
• Hepatitis C/physiopathology
• Hepatocytes/pathology
• Hepatocytes/virology
• Humans
• Life Cycle Stages/physiology
• Lipid Metabolism/physiology
• RNA, Viral/physiology
• Virology/trends
• Virus Replication/physiology