Abstract
The specific packaging of the hepatitis C virus (HCV) genome is hypothesised to be driven by Core-RNA interactions. To identify the regions of the viral genome involved in this process, we used SELEX (systematic evolution of ligands by exponential enrichment) to identify RNA aptamers which bind specifically to Core in vitro. Comparison of these aptamers to multiple HCV genomes revealed the presence of a conserved terminal loop motif within short RNA stem-loop structures. We postulated that interactions of these motifs, as well as sub-motifs which were present in HCV genomes at statistically significant levels, with the Core protein may drive virion assembly. We mutated 8 of these predicted motifs within the HCV infectious molecular clone JFH-1, thereby producing a range of mutant viruses predicted to possess altered RNA secondary structures. RNA replication and viral titre were unaltered in viruses possessing only one mutated structure. However, infectivity titres were decreased in viruses possessing a higher number of mutated regions. This work thus identified multiple novel RNA motifs which appear to contribute to genome packaging. We suggest that these structures act as cooperative packaging signals to drive specific RNA encapsidation during HCV assembly.
Highlights
Hepatitis C virus (HCV) is the leading cause of chronic liver disease, hepatocellular carcinoma and liver transplants in the developed world
It is apparent that the RNA replication and virion assembly processes are spatially distinct within the cellular microenvironment and interactions between the replication complex and the Core protein are required for efficient packaging[8]
We provide evidence that the abrogation of short motifs located across the hepatitis C virus (HCV) genome has a significant effect on RNA encapsidation, manifested by a decrease in infectious titre
Summary
Hepatitis C virus (HCV) is the leading cause of chronic liver disease, hepatocellular carcinoma and liver transplants in the developed world. Direct-acting antivirals (DAAs) targeting 3 viral non-structural proteins are available for treatment of HCV infection. It is apparent that the RNA replication and virion assembly processes are spatially distinct within the cellular microenvironment and interactions between the replication complex and the Core protein are required for efficient packaging[8]. The packaging of replication-defective genomes (which are not presented by a replication complex) is notoriously inefficient in HCV It is this inability to study assembly without concurrent replication which has hindered the identification of RNA structures contributing solely to assembly. Unlike prototypic packaging signals such as those found in the retroviruses, alphaviruses and coronaviruses, a single RNA structure which is both essential and sufficient to target non-viral RNAs to a nascent HCV nucleocapsid particle has not been identified
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