Final Report Summary - HCVPACK (DEVELOPMENT OF TWO COMPLEMENTARY SYSTEMS FOR THE STUDY OF RECOMBINATION AND PACKAGING OF HEPATITIS C VIRUS GENOMIC RNA USING FLUORESCENT PROTEINS AND LIVE IMAGING)
Aim of the project is to develop two complementary and quantitative assays for studying the RNA biology of the hepatitis C virus (HCV). In particular, we intend to develop a viral recombination assay capable of measuring recombination frequencies in different conditions and an RNA labelling assay capable of detecting and tracking genomic RNA in live cells.
After the initiation of the project, novel full-length culture systems for the study of HCV has been developed by our group. The availability of several strains of genotypes 1 and 2 of full-length culture-adapted viruses had prompted us to update our study design to utilize these more advanced systems. Despite the delay in the project timeline, we feel we have achieved important goals during the four years of the fellowship, results that will ensure the successful execution of these studies.
In the area of HCV recombination, we have initially finalized and published a study on HCV recombination, using systems that were current at the onset of the present study. Our qualitative analysis of HCV recombination demonstrated that the virus can recombine efficiently when under heavy selective pressure, even in the absence of replication. Moreover, we have shown that recombinant HCV genomes can undergo additional recombination cycles to obtain higher fitness and remove redundant sequences. Overall, our results showed that recombination is indeed an important mechanism for HCV evolution and life cycle.
Successively, we evaluated the feasibility of original study design that required the insertion of two exogenous genes within the HCV genome. For this purpose, we inserted the mouse heat stable antigen (HSA) gene at three different cleavage sites in the genome of the J6/JFH1-GFP viral construct, which had been previously developed by our group. Viruses encoding HSA at the NS5A/5B junctions displayed the highest viability in the presence or absence of the second exogenous GFP gene, demonstrating the feasibility of our initial approach. During this period, the authors also published a review article analysing the current knowledge on the mechanisms of recombination of HCV and other RNA viruses and retroviruses.
Overall, we have shown that HCV recombination is an important mechanism in the viral life cycle and that recombinant genomes can be promptly detected under proper experimental conditions. In addition, we validated our recombination assay strategy using a well-established HCV viral system expressing GFP.
The second part of the study focuses on RNA genome labelling using fluorescent proteins, to examine the packaging mechanism and efficiency of HCV. In the initial phase of this project we completed a study that investigated the co-localization of Core and NS5A proteins from the 7 major HCV genotypes. Both these proteins are key players in the packaging of HCV genomic RNA and understanding their distribution in different genotypes was a prerequisite for the success of subsequent studies on assembly/packaging. We showed that there are no significant differences in the distribution of Core and NS5A among all major HCV genotypes, suggesting a common assembly pathway for all strains.
The tracking of HCV genomic RNA relies on the capability of inserting specific stem-loop structures within the genomes, which can be specifically recognized by fluorescently labelled RNA-binding proteins. An essential step for the accomplishment of our proposed RNA-labelling assay was the capability of inserting series of specific stem-loop structures within the genome of HCV, as previously shown in other viruses. We succeeded in inserting a series of eighteen Bgl stem-loops, which are specifically recognised by the bacterial BglG protein, within the genome of the widely used J6/JFH1 strain of HCV. Viruses carrying these hairpin series are non-infectious and cannot spread in cell-culture; however, they produce and release large amounts of viral particles upon transfection.
Conventional antibody staining of virions produced by genomes carrying Bgl stem-loops results in particles that can be hardly detected by our microscope system, highlighting sensitivity limitations of the system. Funding for the upgrade of our microscope, including a state-of-the-art EM-CCD camera and an advanced LED illumination-component, has been secured and such upgrades are in the process of being installed. The new components will increase the sensitivity of the system up to single-molecule detection level, ensuring the further advancement of this project.
A second hurdle in the development of fluorescently labelled RNA genomes is the requirement for co-transfection of genomic RNA carrying the proper stem-loops and plasmid DNA encoding fluorescent RNA-binding proteins. Several conventional transfection reagents have been tested with limited success. However, the promising results listed above open the possibility of a bi-cistronic approach that could prove more effective. Namely, that the fluorescently tagged RNA-binding protein could be inserted directly within the genome it is supposed to label, between NS5A and NS5B, removing the co-transfection issue altogether.
Overall, our results indicate that HCV packaging proceeds similarly irrespective of the genotype and that inserting RNA stem-loops in the HCV genome for the purpose of genome tracking is a viable approach. Technical issues related to particle detection and co-transection have been identified and promising solutions are under development.
We are now confident we can proceed in the development of the proposed assays, which will contribute important knowledge to the understanding of HCV life cycle. In particular, viral recombination is an important factor for the development of drug resistance, and a better understanding of this phenomenon could help improving the outcome of future combination therapy of HCV infection.
After the initiation of the project, novel full-length culture systems for the study of HCV has been developed by our group. The availability of several strains of genotypes 1 and 2 of full-length culture-adapted viruses had prompted us to update our study design to utilize these more advanced systems. Despite the delay in the project timeline, we feel we have achieved important goals during the four years of the fellowship, results that will ensure the successful execution of these studies.
In the area of HCV recombination, we have initially finalized and published a study on HCV recombination, using systems that were current at the onset of the present study. Our qualitative analysis of HCV recombination demonstrated that the virus can recombine efficiently when under heavy selective pressure, even in the absence of replication. Moreover, we have shown that recombinant HCV genomes can undergo additional recombination cycles to obtain higher fitness and remove redundant sequences. Overall, our results showed that recombination is indeed an important mechanism for HCV evolution and life cycle.
Successively, we evaluated the feasibility of original study design that required the insertion of two exogenous genes within the HCV genome. For this purpose, we inserted the mouse heat stable antigen (HSA) gene at three different cleavage sites in the genome of the J6/JFH1-GFP viral construct, which had been previously developed by our group. Viruses encoding HSA at the NS5A/5B junctions displayed the highest viability in the presence or absence of the second exogenous GFP gene, demonstrating the feasibility of our initial approach. During this period, the authors also published a review article analysing the current knowledge on the mechanisms of recombination of HCV and other RNA viruses and retroviruses.
Overall, we have shown that HCV recombination is an important mechanism in the viral life cycle and that recombinant genomes can be promptly detected under proper experimental conditions. In addition, we validated our recombination assay strategy using a well-established HCV viral system expressing GFP.
The second part of the study focuses on RNA genome labelling using fluorescent proteins, to examine the packaging mechanism and efficiency of HCV. In the initial phase of this project we completed a study that investigated the co-localization of Core and NS5A proteins from the 7 major HCV genotypes. Both these proteins are key players in the packaging of HCV genomic RNA and understanding their distribution in different genotypes was a prerequisite for the success of subsequent studies on assembly/packaging. We showed that there are no significant differences in the distribution of Core and NS5A among all major HCV genotypes, suggesting a common assembly pathway for all strains.
The tracking of HCV genomic RNA relies on the capability of inserting specific stem-loop structures within the genomes, which can be specifically recognized by fluorescently labelled RNA-binding proteins. An essential step for the accomplishment of our proposed RNA-labelling assay was the capability of inserting series of specific stem-loop structures within the genome of HCV, as previously shown in other viruses. We succeeded in inserting a series of eighteen Bgl stem-loops, which are specifically recognised by the bacterial BglG protein, within the genome of the widely used J6/JFH1 strain of HCV. Viruses carrying these hairpin series are non-infectious and cannot spread in cell-culture; however, they produce and release large amounts of viral particles upon transfection.
Conventional antibody staining of virions produced by genomes carrying Bgl stem-loops results in particles that can be hardly detected by our microscope system, highlighting sensitivity limitations of the system. Funding for the upgrade of our microscope, including a state-of-the-art EM-CCD camera and an advanced LED illumination-component, has been secured and such upgrades are in the process of being installed. The new components will increase the sensitivity of the system up to single-molecule detection level, ensuring the further advancement of this project.
A second hurdle in the development of fluorescently labelled RNA genomes is the requirement for co-transfection of genomic RNA carrying the proper stem-loops and plasmid DNA encoding fluorescent RNA-binding proteins. Several conventional transfection reagents have been tested with limited success. However, the promising results listed above open the possibility of a bi-cistronic approach that could prove more effective. Namely, that the fluorescently tagged RNA-binding protein could be inserted directly within the genome it is supposed to label, between NS5A and NS5B, removing the co-transfection issue altogether.
Overall, our results indicate that HCV packaging proceeds similarly irrespective of the genotype and that inserting RNA stem-loops in the HCV genome for the purpose of genome tracking is a viable approach. Technical issues related to particle detection and co-transection have been identified and promising solutions are under development.
We are now confident we can proceed in the development of the proposed assays, which will contribute important knowledge to the understanding of HCV life cycle. In particular, viral recombination is an important factor for the development of drug resistance, and a better understanding of this phenomenon could help improving the outcome of future combination therapy of HCV infection.