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Scripps Florida scientists develop a process to disrupt hepatitis C virion production
Date:3/21/2008

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Before more effective therapies can be developed, scientists need to understand, at the molecular level, the detailed mechanisms HCV uses to infect cells, replicate itself, assemble progeny virus, and exit the cell. Each of these processes could potentially be a target for a new drug to eliminate HCV infection. HCV, like all viruses, requires the normal cellular machinery for its replication and has developed strategies to utilize normal cell physiology for its own benefit (often to the detriment of the host).

The Tellinghuisen team, which includes Research Assistants Katie L. Foss and Jason Treadaway, has focused recent efforts on NS5A to understand the regulation of events used by the virus to assemble infectious copies of itself and exit the cell. NS5A is a three-domain protein, which means it is comprised of three compactly folded regions roughly 50 to 300 amino acids in length. The requirement of domains I and II for RNA replication is well documented. NS5A domain III, however, is not required for RNA replication, and the function of this region in the HCV life cycle is unknown.

Using standard molecular biology, the researchers removed from domain III of NS5A a coding sequence corresponding to roughly 15 amino acids. Then they generated a clone of the virus, transcribed the RNA from that clone, and purified the RNA. This RNA, which is directly infectious, was then transfected into a liver cell line where it produced all the HCV proteins that are encoded by that RNA genome.

Those proteins assemble in the cell to make a structure called a replicase that then copies the viral RNA, Tellinghuisen explains. We measured that RNA accumulation and observed no defect in RNA replication, but found, surprisingly, that no infectious viral particles were released from the cells. The team also found that no viral RNA nor nucleocapsid protein are released from cells, indicating that an early event in virus assembly had been affected.
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Contact: Keith McKeown
kmckeown@scrpps.edu
858-784-8134
Scripps Research Institute
Source:Eurekalert

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