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Molecular characterization of the HCV core protein mobility to/from lipid droplets and its role in their formation

Final Activity Report Summary - HCVCORE2005 (Molecular characterization of the HCV core protein mobility to/from lipid droplets and its role in their formation)

Hepatitis C virus (HCV) infection affects about 170 million people. The virus attacks the liver where it usually establishes a lifelong, chronic infection. Chronic infection often leads to liver disease and can result in either cirrhosis or liver cancer. Therefore, HCV is very important as a cause of human disease. Although there are treatments to combat infection, these are often unsuccessful. Consequently, new approaches are needed to tackle HCV, which rely on understanding how the virus survives in the liver.

A key process for any virus is its ability to make new virus particles. This involves enclosing the virus genome (the genetic information carried by the virus) within a protein shell called the capsid. In HCV, the capsid is composed of a virus protein called core. Understanding core's properties and how it associates with the HCV genome may lead to novel ways of interfering with production of new virus, thereby either eradicating the virus from infected people or preventing serious liver disease.

HCV core is unusual in that it attaches to storage compartments within liver cells, called lipid droplets. These organelles play an important part in the properties of liver cells. Core probably binds to lipid droplets to make use of these properties for producing infectious virus particles.

This study addressed 3 major questions:

How does core attach to lipid droplets?
By changing the amino acids that make up HCV core, we identified a region at the end of the protein (called D2), which was responsible for its binding to lipid droplets and we determined the structure of D2. We were able also to apply a new method that allows HCV production in cells in the laboratory. Using this approach, core that could not attach to lipid droplets failed to make any virus. Therefore, for the first time, lipid droplets were shown to be important in the life cycle of a virus.

Is binding to lipid droplets by core reversible or irreversible?
This question is important since core protein must be released from lipid droplets to allow virus particles to leave the cell. By adding a foreign protein (called GFP) to core protein and D2, we showed that GFP could also be targeted to lipid droplets. Using special properties of GFP along with biophysical methods, we demonstrated that core was able to move between lipid droplets located at different positions in the cell. The results indicated also that the ability of D2 to fold correctly was very important for mobility. Core proteins that could not fold properly, and were unable to release from droplets, failed to make any infectious virus. This approach again underlined the importance of the association of core with lipid droplets to production of new virus.

Is the behaviour of lipid droplets influenced by the attachment of core?
To address how lipid droplets may be used to make new virus particles, changes in their location were studied in virus-infected cells. Lipid droplets, which were coated with core, formed clumps and moved towards the region of the cell containing the nucleus. This movement towards the nucleus came from altered interactions between lipid droplets and transport networks within the cell. The change in lipid droplet distribution also brought about close positioning of core and the HCV genome within the cell. This close connection between core and the virus genome may represent sites where the first steps in assembly of new virus particles takes place. The ability of core to influence lipid droplets could be relevant also to disease states, which occur in infected livers, in particular steatosis. This condition is an accumulation of lipid droplets in liver cells, and often predicts the development of more serious liver disease.

These results have an important bearing on our understanding of HCV assembly. Armed with this information, it may be possible to develop compounds that interfere with the binding of core to lipid droplets and disrupt the ability of the virus to grow in liver cells.