CONTRIBUTION OF THE HOST LIPID METABOLISM TO HEPATITIS C VIRUS ASSEMBLY AND RELEASE Background Hepatitis C virus (HCV) infects an estimated 2.2 % of the global population and is responsible for 30% of liver cancers. HCV assembly and release, that is, the production of progeny virions in infected cells, are probably the least understood stages of HCV replication cycle and constitute a burning topic in HCV research. Indeed, these late stages of the viral replication cycle can only be reproduced in the laboratory since 2005, when growth of an HCV isolate in cell culture was finally achieved.
The association of HCV production and the host lipid metabolism is complex and fascinating. First of all, HCV capsid protein covers, in infected cells, the surface of cytosolic lipid droplets, which is considered as an early event for the constitution of assembly complexes, at the junction of lipid droplets and the endoplasmic reticulum (ER). Secondly, HCV uses the lipoprotein secretion pathway for its own release. As a consequence, virions circulating in patient serum or produced in cell culture are associated with lipoproteins. Interestingly, lipoproteins transport lipids that were originally stored in lipid droplets and lipoprotein genesis also takes place at the junction between lipid droplets and the ER. At which stage exactly HCV starts using the lipoprotein metabolism, how virions and lipoproteins are attached, and which properties of lipid droplets are used or hijacked by the HCV production machinery remain opened questions. In this context, we set out to identify ne!
w host factors involved in lipid metabolism that are important for HCV production. Proposed research working plan Using siRNAs, we recently screened a panel of host factors involved in lipid droplet or lipoprotein biology for their importance in HCV production (assembly and release). We identified three new host factors whose knock-down reproductively inhibits HCV production. You will focus on one of these hits. Your aim will be to characterize the step of virus production dependent on this host cell factor, unravel the cellular function of the protein (possibly related to the host lipid metabolism) and analyze how and why this function is essential for HCV production.
To this end, we are currently designing shRNAs against the host factors identified in the screen. Depending on our progress, you will first establish or characterize cell lines stably expressing shRNAs against one host factor. In particular, you will confirm the requirement of the factor for HCV production by correlating the HCV titer released by the cells and the protein expression level. You will confirm the specificity of the shRNA by rescuing protein expression with a shRNA-resistant mutant. The next task will be to check that the host factor is specifically involved in HCV production but not in the other steps of the virus replication cycle (use of retrovirus-based pseudoparticles to evaluate virus entry, and of replicons to assess viral RNA replication). Afterwards, the different steps of virus production will be dissected (e.g. constitution of assembly complexes, encapsidation, envelopment, lipoprotein association, release and properties of the secreted viral particle!
s) using a panel of virological assays previously established in our laboratory. Our hypothesis is that the gene of interest ensures a particular function in the host lipid metabolism that is used by the virus production machinery. Therefore, you will investigate the host lipid metabolism in shRNA-knocked down versus WT cells. As a starting point of this analysis, LD morphology will be observed and lipoprotein secretion assessed. Finally, if relevant, functional or physical interaction between the host factor and viral assembly factors will be assessed.
The project, voluntarily broad, will be adapted depending on its status at the start of the training period and on its developments during the training period.
Environment The host laboratory is located in the Twincore Zentrum, on the MHH campus. It is headed by Pr. Dr. Th. Pietschmann and consists of a team of around 20 people, devoted to the study of various aspects of the HCV replication cycle. For more information, see: http://www.twincore.de/en/working-groups/experimental-virology/ Competences acquired or developed during the training period You will become familiar with a broad spectrum of virological, molecular, cellular and biochemical biology techniques. Virological techniques:
You will mostly work with the infectious HCV system, in S3 containment facilities, and also sporadically utilise the retroviral-based HCV pseudoparticle and replicon systems. You will use the traditional virus titration methods and also the luciferase reporter gene system to assay virus infectivity. Density gradient ultracentrifugations and antibody-neutralization assays may also be relevant to the project. Molecular biology and genetics:
This aspect will include plasmid and RNA preparation, but also cloning of shRNA-resistant gene constructs and possibly of non-functional mutants. You will use or generate and characterize shRNA-expressing cell lines for the host factor of interest. A panel of siRNA against genes involved in lipid metabolism are available in the laboratory and can be used to unravel the pathway involving the host factor of interest. Cellular techniques:
You use traditional cellular biology techniques (cell culture, various transfection transfection methods including electroporations, etc). Protein expression and localization analyses:
You will be using Western Blot or ELISA to monitor protein expression and possibly immunoprecipitation assays to test an eventual interaction between the host factor and viral proteins. Immunofluorescence microscopy, eventually with a confocal microscope, will be useful (i) to determine possible alterations of the lipid droplet phenotype, (ii) to address the subcellular localization of the host factor of interest and of the viral assembly components. In addition to these laboratory techniques, you will further improve your presentation skills with regular lab meetings which are also the opportunity to obtain advice on the project from the whole team. Contact Gabrielle Vieyres
AG Pietschmann
Experimentelle Virologie
Twincore
Feodor-Lynen-Str. 7-9
30625 Hannover
http://www.twincore.de/en/working-groups/experimental-virology/ Email: gabrielle.vieyres@twincore.de
The association of HCV production and the host lipid metabolism is complex and fascinating. First of all, HCV capsid protein covers, in infected cells, the surface of cytosolic lipid droplets, which is considered as an early event for the constitution of assembly complexes, at the junction of lipid droplets and the endoplasmic reticulum (ER). Secondly, HCV uses the lipoprotein secretion pathway for its own release. As a consequence, virions circulating in patient serum or produced in cell culture are associated with lipoproteins. Interestingly, lipoproteins transport lipids that were originally stored in lipid droplets and lipoprotein genesis also takes place at the junction between lipid droplets and the ER. At which stage exactly HCV starts using the lipoprotein metabolism, how virions and lipoproteins are attached, and which properties of lipid droplets are used or hijacked by the HCV production machinery remain opened questions. In this context, we set out to identify ne!
w host factors involved in lipid metabolism that are important for HCV production. Proposed research working plan Using siRNAs, we recently screened a panel of host factors involved in lipid droplet or lipoprotein biology for their importance in HCV production (assembly and release). We identified three new host factors whose knock-down reproductively inhibits HCV production. You will focus on one of these hits. Your aim will be to characterize the step of virus production dependent on this host cell factor, unravel the cellular function of the protein (possibly related to the host lipid metabolism) and analyze how and why this function is essential for HCV production.
To this end, we are currently designing shRNAs against the host factors identified in the screen. Depending on our progress, you will first establish or characterize cell lines stably expressing shRNAs against one host factor. In particular, you will confirm the requirement of the factor for HCV production by correlating the HCV titer released by the cells and the protein expression level. You will confirm the specificity of the shRNA by rescuing protein expression with a shRNA-resistant mutant. The next task will be to check that the host factor is specifically involved in HCV production but not in the other steps of the virus replication cycle (use of retrovirus-based pseudoparticles to evaluate virus entry, and of replicons to assess viral RNA replication). Afterwards, the different steps of virus production will be dissected (e.g. constitution of assembly complexes, encapsidation, envelopment, lipoprotein association, release and properties of the secreted viral particle!
s) using a panel of virological assays previously established in our laboratory. Our hypothesis is that the gene of interest ensures a particular function in the host lipid metabolism that is used by the virus production machinery. Therefore, you will investigate the host lipid metabolism in shRNA-knocked down versus WT cells. As a starting point of this analysis, LD morphology will be observed and lipoprotein secretion assessed. Finally, if relevant, functional or physical interaction between the host factor and viral assembly factors will be assessed.
The project, voluntarily broad, will be adapted depending on its status at the start of the training period and on its developments during the training period.
Environment The host laboratory is located in the Twincore Zentrum, on the MHH campus. It is headed by Pr. Dr. Th. Pietschmann and consists of a team of around 20 people, devoted to the study of various aspects of the HCV replication cycle. For more information, see: http://www.twincore.de/en/working-groups/experimental-virology/ Competences acquired or developed during the training period You will become familiar with a broad spectrum of virological, molecular, cellular and biochemical biology techniques. Virological techniques:
You will mostly work with the infectious HCV system, in S3 containment facilities, and also sporadically utilise the retroviral-based HCV pseudoparticle and replicon systems. You will use the traditional virus titration methods and also the luciferase reporter gene system to assay virus infectivity. Density gradient ultracentrifugations and antibody-neutralization assays may also be relevant to the project. Molecular biology and genetics:
This aspect will include plasmid and RNA preparation, but also cloning of shRNA-resistant gene constructs and possibly of non-functional mutants. You will use or generate and characterize shRNA-expressing cell lines for the host factor of interest. A panel of siRNA against genes involved in lipid metabolism are available in the laboratory and can be used to unravel the pathway involving the host factor of interest. Cellular techniques:
You use traditional cellular biology techniques (cell culture, various transfection transfection methods including electroporations, etc). Protein expression and localization analyses:
You will be using Western Blot or ELISA to monitor protein expression and possibly immunoprecipitation assays to test an eventual interaction between the host factor and viral proteins. Immunofluorescence microscopy, eventually with a confocal microscope, will be useful (i) to determine possible alterations of the lipid droplet phenotype, (ii) to address the subcellular localization of the host factor of interest and of the viral assembly components. In addition to these laboratory techniques, you will further improve your presentation skills with regular lab meetings which are also the opportunity to obtain advice on the project from the whole team. Contact Gabrielle Vieyres
AG Pietschmann
Experimentelle Virologie
Twincore
Feodor-Lynen-Str. 7-9
30625 Hannover
http://www.twincore.de/en/working-groups/experimental-virology/ Email: gabrielle.vieyres@twincore.de