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  • 1
    Language: English
    In: Antiviral Research, 2011, Vol.89(2), pp.136-148
    Description: Infection with the hepatitis C virus represents a global public health threat given that an estimated 170 million individuals are chronically infected and thus at risk for cirrhosis and hepatocellular carcinoma. A number of direct antiviral molecules are in clinical development. However, side effects, drug resistance and viral genotype-specific differences in efficacy may limit these novel therapeutics. Therefore, a combination of well tolerated drugs with distinct mechanisms of action targeting different steps of the viral replication cycle will likely improve viral response rates and therapy success. To identify small molecules that interfere with different steps of the HCV replication cycle, we developed a novel dual reporter gene assay of the complete HCV life cycle and adapted it to 384-well high-throughput format. The system is based on a highly permissive Huh-7 cell line stably expressing a secreted luciferase. Using these cells and an efficient HCV luciferase reporter virus, perturbations of each step of the viral replication cycle as well as cell viability can be easily and quantitatively determined. The system was validated with a selected set of known HCV entry, replication and assembly inhibitors and then utilized to screen a library of small molecules derived from myxobacteria. Using this approach we identified a number of molecules that specifically inhibit HCV cell entry, or primarily virus assembly and release. Moreover, we also identified molecules that increase viral propagation. These compounds may be useful leads for development of novel HCV inhibitors and could be instrumental for the identification of as yet unknown host-derived viral resistance and dependency factors.
    Keywords: Hepatitis C Virus ; Chronic Liver Disease ; Hcv ; Viral Life Cycle ; Antiviral Therapy ; Tartrolon ; Medicine ; Biology
    ISSN: 0166-3542
    E-ISSN: 1872-9096
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  • 2
    Language: English
    In: 2013, Vol.9(5), p.e1003355
    Description: Hepatitis C virus (HCV) p7 is a membrane-associated ion channel protein crucial for virus production. To analyze how p7 contributes to this process, we dissected HCV morphogenesis into sub-steps including recruitment of HCV core to lipid droplets (LD), virus capsid assembly, unloading of core protein from LDs and subsequent membrane envelopment of capsids. Interestingly, we observed accumulation of slowly sedimenting capsid-like structures lacking the viral envelope in cells transfected with HCV p7 mutant genomes which possess a defect in virion production. Concomitantly, core protein was enriched at the surface of LDs. This indicates a defect in core/capsid unloading from LDs and subsequent membrane envelopment rather than defective trafficking of core to this cellular organelle. Protease and ribonuclease digestion protection assays, rate zonal centrifugation and native, two dimensional gel electrophoresis revealed increased amounts of high-order, non-enveloped core protein complexes unable to protect viral RNA in cells transfected with p7 mutant genomes. These results suggest accumulation of capsid assembly intermediates that had not yet completely incorporated viral RNA in the absence of functional p7. Thus, functional p7 is necessary for the final steps of capsid assembly as well as for capsid envelopment. These results support a model where capsid assembly is linked with membrane envelopment of nascent RNA-containing core protein multimers, a process coordinated by p7. In summary, we provide novel insights into the sequence of HCV assembly events and essential functions of p7. ; Viroporins are small hydrophobic viral membrane proteins which oligomerize and modulate membrane properties to facilitate virus propagation. Within their membrane environment these proteins can form membrane pores or channels which change the permeability of membranes for ions. These properties are known to contribute to release of infectious enveloped virus particles from infected cells and/or to facilitate viral cell entry by catalyzing virus uncoating. In case of HCV, p7 function is essential for production of infectious progeny and its ion channel activity is well documented and in cell-based systems. Recent evidence indicated that p7 channel activity dissipates the low pH of the cellular secretory compartment thus protecting the viral glycoproteins from low pH induced misfolding and inactivation. In this investigation we highlight the involvement of the p7 ion channel in the assembly and envelopment of viral RNA-containing capsids. Our results indicate that p7, likely in concert with the viral envelope proteins, comprises a membrane-bound recipient complex that provides a scaffold to initiate unloading of core protein from lipid droplets for capsid assembly and membrane envelopment. Collectively, these findings highlight novel facets of p7 function in the course of HCV morphogenesis.
    Keywords: Research Article ; Biology
    ISSN: 1553-7366
    E-ISSN: 1553-7374
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  • 3
    Language: English
    In: PLoS Pathogens, 2012, Vol.8(7), p.e1002829
    Description: Hepatitis C virus (HCV) has infected around 160 million individuals. Current therapies have limited efficacy and are fraught with side effects. To identify cellular HCV dependency factors, possible therapeutic targets, we manipulated signaling cascades with pathway-specific inhibitors. Using this approach we identified the MAPK/ERK regulated, cytosolic, calcium-dependent, group IVA phospholipase A2 (PLA2G4A) as a novel HCV dependency factor. Inhibition of PLA2G4A activity reduced core protein abundance at lipid droplets, core envelopment and secretion of particles. Moreover, released particles displayed aberrant protein composition and were 100-fold less infectious. Exogenous addition of arachidonic acid, the cleavage product of PLA2G4A-catalyzed lipolysis, but not other related poly-unsaturated fatty acids restored infectivity. Strikingly, production of infectious Dengue virus, a relative of HCV, was also dependent on PLA2G4A. These results highlight previously unrecognized parallels in the assembly pathways of these human pathogens, and define PLA2G4A-dependent lipolysis as crucial prerequisite for production of highly infectious viral progeny. ; The human genome encodes more than 30 phospholipase A2s. These enzymes cleave fatty acids at the C2 atom of phosphoglycerides and thus modulate membrane properties. Among all PLA2s only PLA2G4A, which is recruited to perinuclear membranes by Ca and activated by extracellular stimuli via the mitogen activated protein kinase pathway, specifically cleaves lipids with arachidonic acid. Metabolism of arachidonic acid yields prostaglandins and leukotriens, important lipid mediators of inflammation. We show that inhibition of PLA2G4A produces aberrant HCV particles and that infectivity is rescued by addition of arachidonic acid. Our results suggest that a specific lipid (arachidonic acid) is essential for production of highly infectious HCV progeny, likely by creating a membrane environment conducive for efficient incorporation of crucial host and viral factors into the lipid envelope of nascent particles. Strikingly, PLA2G4A is also essential for production of highly infectious Dengue Virus (DENV) particles but not for vesicular stomatitis virus (VSV). These observations argue that HCV and DENV which unlike VSV produce particles at intracellular membranes usurp a common host factor (PLA2G4A) for assembly of highly infectious progeny. These findings open new perspectives for antiviral intervention and highlight thus far unrecognized parallels in the assembly pathway of HCV and DENV.
    Keywords: Research Article ; Biology ; Medicine ; Infectious Diseases ; Molecular Biology ; Cell Biology ; Gastroenterology And Hepatology
    ISSN: 1553-7366
    E-ISSN: 1553-7374
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  • 4
    Language: English
    In: PLoS Pathogens, 2016, Vol. 12(4)
    Description: Hepatitis C virus (HCV) particles closely mimic human very-low-density lipoproteins (VLDL) to evade humoral immunity and to facilitate cell entry. However, the principles that govern HCV association with VLDL components are poorly defined. Using an siRNA screen, we identified ABHD5 (α/β hydrolase domain containing protein 5, also known as CGI-58) as a new host factor promoting both virus assembly and release. ABHD5 associated with lipid droplets and triggered their hydrolysis. Importantly, ABHD5 Chanarin-Dorfman syndrome mutants responsible for a rare lipid storage disorder in humans were mislocalised, and unable to consume lipid droplets or support HCV production. Additional ABHD5 mutagenesis revealed a novel tribasic motif that does not influence subcellular localization but determines both ABHD5 lipolytic and proviral properties. These results indicate that HCV taps into the lipid droplet triglyceride reservoir usurping ABHD5 lipase cofactor function. They also suggest that the resulting lipid flux, normally devoted to VLDL synthesis, also participates in the assembly and release of the HCV lipo-viro-particle. Altogether, our study provides the first association between the Chanarin-Dorfman syndrome protein and an infectious disease and sheds light on the hepatic manifestations of this rare genetic disorder as well as on HCV morphogenesis.
    Keywords: Medical And Health Sciences ; Basic Medicine ; Microbiology In The Medical Area ; Medicin Och Hälsovetenskap ; Medicinska Och Farmaceutiska Grundvetenskaper ; Mikrobiologi Inom Det Medicinska Området
    ISSN: 1553-7366
    E-ISSN: 15537374
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