Saturday, February 5, 2011

Advances in Hepatitis C Virus Research: Mutations in JFH-1 isolate and implications on future HCV studies

An established cell culture system is critical to the study of virus replication and ultimately, strategies for clinical intervention. It requires two features: (1)host cells that are able to be infected and (2) a virus that is able to replicate and assemble inside these cells.

In the case of hepatitis C virus (HCV), this has been a major barrier to researching this highly medically important virus. Only after development of chimeric or tissue-adapted variants of the JFH-1 strain has HCV been able to successfully replicate in in Huh-7-derived cell lines (human hepatoma) (2005, Maruyama et al.). Even then, this system is highly limited with inefficient and suboptimal virus production. Incidentally, virus particle production has been linked with viral structure and non-structural proteins, specifically core, NS5A, NS2 NS3 and viral envelope glycoproteins.

In this study, Pokrovskii et al. generate an "adapted" HCV by making mutations to core K78E, NS2 W879R and NS4B V1761L proteins. By serially passing the JFH-1 strain
through naïve Lunet-CD81 cells, they found that the virus becomes better adapted for cell culture while maintaining cytopathic effects.

They go on to identify mutations responsible for enhanced virus production, finding 7 (amino acid) point mutations. To determine whether or not these point mutations are the cause of the increased virus production they ask whether the seven mutations are sufficient to confer adaptive phenotype. Using site-directed mutagenesis to generate a new viral construct they found identical infectivity kinetics with a phenotype that displayed over a 100-fold increase in virus production compared to wildtype (WT).

They continued by making revertants to each of the seven to determine if the mutations were necessary for the added phenotype. Three of the seven revertants reduced the replication and spread of he virus, indicating that these three were primarily responsible. Moreover, they found that these single point mutations were not sufficient to reproduce the effect but a mutant with all-three (Min-3) was. They attempt to determine the mechanism of these mutations by measuring stability and decay. Length of infectivity increased (slower decay) which correlates with increased stability.

This research opens the doors to a number of previously obstructed molecular and biochemical techniques including antibody production, cry-electron microscopy, drug-resistance selection and high throughput screening of HCV inhibitors.

Pokrovskii M , Bush C, Beran R, Robinson M, Cheng G , Tirunagari N, Fenaux M, Greenstein A, Zhong W, Delaney W, Paulson M. Novel mutations in a tissue-culture adapted HCV strain improve infectious virus stability and markedly enhance infection kinetics. J. Virol. published ahead of print on 2 February 2011, doi:10.1128/JVI.01760-10


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