Curriculum Vitae
Publication list
Summary
We all know that viruses are one of the causes why we get sick. There are many different viruses and therefore many different infectious diseases. Once a virus has entered a host-cell, it utilises the processes occurring in the cell to multiply itself, in the end leading to further spread of the virus.
The hepatitis C virus infects cells of the liver, called hepatocytes, causing inflammation and eventually dis-functioning of the liver. This process goes very slow and insidious and often takes 10 to 30 years before clinical symptoms are presented. Worldwide around 130-170 million people are infected with HCV, that is 2-3% of the world population. Unfortunately, there is no vaccine available against HCV and current treatments are for some category patients far from successful. Therefore, it is important to develop new medication. Yet, one first needs to understand the HCV lifecycle to discover possible drug-targets.
The lifecycle of HCV is illustrated in Chapter 1 of this thesis and starts with the entry of the virus into the hepatocyte. To enter, the virus particle binds to proteins on the cell surface. After that, the virus particle releases its genome content, which will be used to produce new viral proteins (translation). Subsequently, these proteins will start making new viral genome copies (replication). This occurs on a site in the cell that is specially created by the virus. The viral non-structural protein NS4b plays an important role in creating this special site by inducing membrane rearrangements of the cell. The other non-structural proteins of the virus are important in copying the genome, including the protein NS3. After completion, the genome is packaged into new virus particles, which leave the cell to infect other hepatocytes. All these steps happen with assistance of host proteins and processes. Within this thesis, I investigated the interactions of the viral proteins with the host cell at several levels. All the viral proteins are associated with membranes of the host cell, which are double-layered sheets of lipids. NS4b is associated with the membrane in a number of ways, through hydrophobic interactions and by domains that span the membrane. We examined the carboxy-terminal domain of the protein and found that this modular domain binds to membranes. When comparing this domain to other proteins, we
Summary
observed similarity with a domain of a bacterial protein that also associates to membranes. Their mode of membrane binding is electrostatic: positively charged amino acids that interact with the negative headgroups of lipids. Experiments in which we mutated positively charged amino acids indicated the same mechanism of association for the carboxy-terminal domain of NS4b. In addition to that, we show that these amino acids, required for membrane interaction, are necessary for replication of the virus.
Several viral proteins have multiple roles in the life cycle, as is the case for NS3. Separate functions need to be regulated and a way to organise this is by protein modifications, these are moieties put onto a protein that modulate a particular function. Not much is known about modifications for NS3. We therefore set up experiments to investigate modifications of NS3 in the context of HCV RNA translation and replication in cells. We made cell lines where we constructed a label to NS3. Using a ‘chemical magnet’ for this label, we pulled NS3 out of these cells. After that, the isolated proteins were examined for protein modifications by several means, including two-dimensional PAGE (poly acrylamide gel electrophoresis) and mass spectrometry techniques. Two-dimensional PAGE showed that multiple forms of NS3 are present and additional experiments showed phosphorylation of the protein. Mass spectrometry furthermore indicated acetylation at the amino terminal end of the protein. These results show that NS3 is post-translationally modified and we postulate that these could be involved in the regulation of its functions.
A third subject of this thesis concerns viral and host protein-protein interactions. The same cell culture system as in the NS3 modification study was used to isolate the tagged version of NS3. To co-purify NS3 interacting proteins with NS3, several methods were employed. We identified two associating partners, GLT25D1 and LH3; both are engaged in the modification (galactosylation) of collagens and collectins. Collagens play a role in strengthening the cells and collectins are involved in the body’s defence mechanism against pathogens. The interactions were further validated, especially as to where the two proteins inside the cell could interact. Since the subcellular localisation was unknown for GLT25D1, we investigated that first. GLT25D1 was shown to contain several signals, such as a signal sequence and a KDEL, that target the protein to the lumen of the endoplasmic reticulum, an organelle in the cell to which several HCV proteins localise. Our data illustrates
that NS3 and GLT25D1 co-localise partially at a specific part of the endoplasmic reticulum. Additional experiments could not reveal the exact protein domain of GLT25D1 required for interaction with NS3, but the signal sequence of GLT25D1 seems to be crucial for the association.
Together these data point out that there is a very complex interplay between HCV and the host-cell. The viral lifecycle is regulated at different levels and this thesis describes several of them, including protein-membrane association, protein modifications, subcellular localisation and protein-protein interactions. To fully understand these interactions and their functions, further investigations of these four new topics in the HCV lifecycle are needed.