Jones, Daniel M.
The contribution of viral and host cell factors to replication of the hepatitis C virus RNA genome.
PhD thesis, University of Glasgow.
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Studies on the hepatitis C virus (HCV) life cycle have been aided by the development of in vitro systems that permit replication of the viral RNA genome and virus particle production. However, the exact functions of the viral proteins, particularly those engaged in RNA synthesis, are poorly understood. It is thought that NS4B, one of the replicase components, induces the formation of replication complexes (RCs) derived from host cell membranes. These RCs appear as punctate foci at the endoplasmic reticulum (ER) membrane and incorporate the viral and cellular proteins necessary for HCV RNA synthesis.
To gain insight into the nature of RCs, green fluorescent protein (GFP) was inserted into the coding region of NS5A, one of the HCV-encoded replicase components. The impact of the GFP insertion was examined in the context of a subgenomic replicon (SGR) based on JFH1, a genotype 2a HCV strain that exhibits efficient RNA replication in cell culture. The resulting construct was capable of robust replication and allowed characterisation of NS5A in live cells that synthesised viral RNA. NS5A displayed a diffuse, ER-like distribution and was also observed in foci. These foci are presumed to represent RCs and NS5A was relatively immobile at these sites. This result was confirmed using SGRs harbouring a photoactivatable derivative of GFP (PAGFP). Utilising plasmid-encoded HCV polyproteins, it was apparent that the targeting of NS5A to these structures was dependent on NS4B. Removal of the NS4B coding region resulted in a diffuse, ER-like distribution of NS5A, with little evidence of the protein within RCs. NS5A was mobile under these conditions, suggesting that the dynamics of NS5A are linked to focus formation by NS4B.
To further investigate these findings, a panel of 15 alanine substitutions was constructed in the C-terminal region of NS4B. Transient replication assays revealed that five mutants were incapable of replication, two displayed an attenuated phenotype, and eight exhibited replication levels comparable to the wild-type (wt) genome. Of the five non-replicating mutants, two were defective in their ability to produce foci, while one failed to generate any foci. Thus, the C-terminus of NS4B is important for RC formation. Loss of NS4B foci correlated with decreased NS5A located in these structures. Furthermore, NS5A hyperphosphorylation was reduced for mutants compromised in foci production. This suggests that the membranous changes induced by NS4B provide a favourable environment for post-translational modifications of NS5A. Interestingly, the remaining two non-replicating mutants displayed no impairment in foci production and the characteristics of NS5A were also unaltered. Therefore, in addition to producing the cellular environment for HCV genome synthesis, NS4B is likely to play a more direct role in RNA replication.
HCV RCs are believed to be relatively enclosed structures that permit limited exchange of materials with the cytoplasm. In support of this hypothesis, previous reports have shown that NS5A is the only replicase component capable of restoring replication to defective genomes when supplied in trans. In those studies, SGRs harbouring replication-lethal NS4B mutations could not be rescued by trans-complementation. Utilising the five novel non-replicating genomes described above, the potential to trans-complement NS4B in transient replication assays was re-examined. Wt protein produced from a functional HCV replicon could trans-complement defective NS4B expressed from two of the five mutants. Moreover, active replication could be reconstituted from two defective viral RNAs harbouring mutations within NS4B and NS5A. These findings have important implications for our understanding of RC formation.
Genome-length JFH1 RNA produces infectious virus particles in Huh-7 cells. Using this system, it has become increasingly apparent that some HCV-encoded replication components are also involved in virus assembly and release. To determine whether NS4B had any influence on these latter stages of the virus life cycle, the NS4B mutations that did not block RNA replication were introduced into the full-length JFH1 genome. While the majority of mutants had no effect on virus production, one mutant consistently enhanced infectious virus titres by up to five-fold compared to wt JFH1. Interestingly, introduction of the same mutation into a chimeric J6-JFH1 genome resulted in repressed virion production. Together, these results suggest that NS4B contributes to virus assembly and release in a genotype-specific manner.
In an attempt to identify novel cellular proteins involved in HCV genome replication, a siRNA library targeting 299 nucleotide-binding proteins was screened. For the screen, a robust system was established using two cell lines (derived from Huh-7 and U2OS cells) that replicated tri-cistronic SGRs. While the U2OS cell line supported HCV RNA replication less efficiently compared to Huh-7 cells, this cell type was efficiently transfected with siRNA. Consequently, increased gene-silencing and greater effects on HCV replication were observed in the U2OS cell line. Thus, U2OS cells may be a suitable alternative to Huh-7 cells for HCV-related siRNA studies. For the library screen, all siRNAs were tested in both cell lines, and cell viability measurements allowed specific effects on viral RNA synthesis to be characterised. The screen identified several cellular proteins that enhanced and suppressed HCV RNA replication. This study provides an important framework for more detailed analyses of these proteins in the future.
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