High resolution microscopy studies of the architecture of sites of hepatitis C virus RNA replication

Hepatitis C Virus (HCV) is a major cause of chronic liver disease, with an estimated 170 million people worldwide currently infected with the virus. Infection leads to chronicity in the majority of cases, resulting in fibrosis, cirrhosis and hepatocellular carcinoma. It is now the leading cause of liver transplantation in developed countries. HCV, a member of the Flaviviridae virus family, has a single stranded, positive sense, RNA genome encoding a unique polyprotein of 3000 amino acids in length. Genome replication of all positive-strand RNA viruses occur in association with the cytoplasmic surface of host cell membranes. Expression of the viral non-structural proteins induces complex structural changes within the membrane forming a framework for replication of the RNA genome. Expression of the isolated HCV non-structural protein 4B has been shown to induce specific alterations to the endoplasmic reticulum (ER) membrane, recruiting a number of host factors including lipid droplets, double and single membrane vesicles. Until recently HCV genome replication was thought to occur in structurally complex ER membrane invaginations into the lumen of the organelle, in a manner similar to the other members of the Flaviviridae family such as dengue virus. However a growing body of evidence suggests that such membrane alterations protrude into the cytoplasm of the cell connected by a thin neck like structure. Using a number of novel optical and electron microscopy imaging techniques, this study sought to investigate the interaction and architecture of the viral replication complex with the host membrane through both qualitative and quantitative methods. Using the increased resolution of the OMX structured illumination microscopy (SIM) system, this study examined the interaction of multiple components of the viral replication complex (RC). Utilising the Huh-7/SGR-JFH1 and Huh-7/SGR-JFH1NS5AGFP cell line, a previously sub-resolution hollow structure formed by the NS5A protein harbouring the dsRNA replication intermediate was identified. The structure, interaction and intracellular location of these viral components are indicative of the viral RCs common to all positive-strand RNA viruses. During this investigation it was noted that two distinct NS5A populations were apparent within the cell; a small population of 300nm barrel structures identified as putative replication foci and a larger population of smaller diameter indistinct objects. Utilising the quantitative modeling capabilities of the Imaris software package, these objects were identified, filtered for diameter and the individual populations quantified. However limitations in the object identification algorithms constrained accurate quantification of the two populations. Efforts to develop the comparative colocalisation as a quantitative methodology identified issues with OMX channel alignment affecting correlation values. Correction of this alignment issue increased coefficient values but contrary to expectation these values were found to be inversely correlated to microscope resolution. Following an in-depth investigation and comparison of the CLSM and OMX image capture technologies and colocalisation methodologies a number of factors affecting correlation values were identified. Through this process it became apparent that the differences in correlation coefficient values between the two imaging systems were not necessarily a decrease in colocalisation of the target proteins but an inherent variation of the imaging technology combined with the correlation algorithms. Following accurate characterisation of the OMX microscopy datasets, the study went on to examine the interaction of the hollow barrel-shaped RC structures with the surface of the ER membrane. Utilising the GFP tagged peripheral ER protein Sec61β representative of the membrane surface, the viral RC was identified in a location distal to the membrane surface, residing as a spherule within the cytoplasm connected by a small membrane “neck”. This result supports the recently published evidence suggesting that the HCV replication foci mirror the membrane protrusions found in poliovirus rather than the other members of the flavivirus family. The data processing pipeline developed throughout the study was used to provide quantitative analysis of the correlation between two forms of NS5A from different cistrons in subgenomic replicon. Through this analytical process it was shown that the NS3-4B proteins were required for targeting and integration of the NS5A protein into the viral RC. The application and validation of such statistical methods of colocalisation analysis provided a platform for a more robust and non-subjective test of correlation. Alongside the development of super resolution microscopy in this study, a novel method for bridging the resolution gap between optical and electron microscopy was investigated. A number of correlative light and electron microscopy (CLEM) techniques were assessed to provide the optimal balance of optical immunofluorescence specificity with the ultrastructural detail provided by transmission electron microscopy (TEM). The high quantum yield and electron dense core make quantum dots an ideal novel dual modality probe for CLEM. Despite a number of failed attempts to develop an accurate probe conjugate and staining methodology, recent advances in probe conjugation methods alongside access to a highly purified primary antibody allowed production of a novel probe specific for the viral NS5A protein. Using the QD-NS5A antibody probe it was possible to correlate replication sites imaged through CLSM with the ultrastructural micrograph generated through TEM. These images revealed that regions of high intensity fluorescence mapped to a complex region of ER membrane interwoven throughout a number of lipid droplets and vesicles. Parallel to the above imaging studies, proteomic analysis was conducted on Huh-7 and Huh-7.5 cells to try to determine whether the greater permissiveness of Huh-7.5 cells for viral replication was a result of differential protein expression. Utilising a SILAC labeling methodology, cell line proteomes were isotopically labeled following development of a specific protocol for stable growth and replication. Although preliminary screening of fractionated proteome samples failed to identify any significant differences between cell lines, the development of a successful maintenance protocol provides the opportunity for further analysis of cell lines and organelle proteomes.