Picornaviral proteases and cellular proteins: networks

Viruses are a thousand times smaller than cells and have only between ten to 200 genes, very few compared to the, for instance, 30,000 in a human cell. Nevertheless, viruses are experts in taking over cells and making the machinery of the infected cells work for them. What are the viral strategies involved in hi-jacking and rerouting a cell? One of their tricks is to affect the activity of proteins in the host cell to make them work to the benefit of the virus. Picornaviruses, a family of viruses which includes the common cold virus, poliovirus and foot-and-mouth disease virus, modify the activity of a key protein involved in the control of cellular protein synthesis. This modification prevents the cell from translating its own mRNA, but still allows, or sometimes even enhances, protein synthesis driven from the viral RNA. The virus has therefore created an advantage for itself. How does this reaction take place? Which cellular and viral proteins are involved? How do these proteins interact with each other? What are the structures of these proteins? It is the aim of this project to answer these questions. The proteins known to take part in this reaction are proteinases encoded by foot-and-mouth disease virus, common cold viruses and poliovirus. These enzymes cleave the key cellular protein mentioned above, known as eukaryotic initiation factor (eIF) 4G. Evidence has been accumulating that both the viral proteinases and eIF4G interact with other cellular proteins to facilitate the change in protein synthesis. It will be a major goal of this project to use the techniques of proteomics to identify such cellular proteins. Once the proteins have been identified, experiments can then be performed to see what roles they play in carrying out the modification of protein synthesis in the infected cell and what they do in the uninfected cell. The proteinases encoded by the viruses mentioned above are not identical. The foot-and-mouth disease virus proteinase is related to the plant proteinase papain whereas the common cold virus and poliovirus proteinase is related to the mammalian enzyme chymotrypsin. Recent evidence has indicated that although the proteinase of the common cold virus and poliovirus appear superficially to be closely related, there may be differences in their mechanism of cleavage of eIF4G. Moreover, it seems possible in the light of new data that even amongst the 100 or so common cold virus serotypes, there may be substantial differences in the mechanism of recognition of eIF4GI. It will be a second major aim of this project to discover and understand such differences, using as models the proteinases of the serotypes 2 and 14 which show very divergent amino acid sequences. In a third set of experiments, we will attempt to determine the three-dimensional structure of certain of the viral proteinases as well as the domain of eIF4G which interacts with the viral proteinases. The structure determination will use both X-ray crystallography and nuclear magnetic resonance. Experiments will be performed on the proteinase alone as well as in complexes with each the eIF4G domain. The experiments will deliver significant new biological information as to how the components of the reaction interact with each other and what changes these interactions cause in the structures of the proteins taking part. Thus, the results of this project will help us understand not only a viral strategy but also comprehend more thoroughly cellular proteins. As these cellular proteins are all involved in regulating protein synthesis, this new information will help us to comprehend in closer detail how the control of growth in a healthy cell is orchestrated.

Viruses are a thousand times smaller than cells and have only between ten to 200 genes, very few compared to the, for instance, 30,000 in a human cell. Nevertheless, viruses are experts in taking over cells and making the machinery of the infected cells work for them. What are the viral strategies involved in hi-jacking and rerouting a cell? One of their tricks is to affect the activity of proteins in the host cell to make them work to the benefit of the virus. Picornaviruses, a family of viruses which includes the common cold virus, poliovirus and foot-and-mouth disease virus, modify the activity of a key protein involved in the control of cellular protein synthesis. This modification prevents the cell from translating its own mRNA, but still allows, or sometimes even enhances, protein synthesis driven from the viral RNA. The virus has therefore created an advantage for itself. How does this reaction take place? Which cellular and viral proteins are involved? How do these proteins interact with each other? What are the structures of these proteins? It is the aim of this project to answer these questions. The proteins known to take part in this reaction are proteinases encoded by foot-and-mouth disease virus, common cold viruses and poliovirus. These enzymes cleave the key cellular protein mentioned above, known as eukaryotic initiation factor (eIF) 4G. Evidence has been accumulating that both the viral proteinases and eIF4G interact with other cellular proteins to facilitate the change in protein synthesis. It will be a major goal of this project to use the techniques of proteomics to identify such cellular proteins. Once the proteins have been identified, experiments can then be performed to see what roles they play in carrying out the modification of protein synthesis in the infected cell and what they do in the uninfected cell. The proteinases encoded by the viruses mentioned above are not identical. The foot-and-mouth disease virus proteinase is related to the plant proteinase papain whereas the common cold virus and poliovirus proteinase is related to the mammalian enzyme chymotrypsin. Recent evidence has indicated that although the proteinase of the common cold virus and poliovirus appear superficially to be closely related, there may be differences in their mechanism of cleavage of eIF4G. Moreover, it seems possible in the light of new data that even amongst the 100 or so common cold virus serotypes, there may be substantial differences in the mechanism of recognition of eIF4GI. It will be a second major aim of this project to discover and understand such differences, using as models the proteinases of the serotypes 2 and 14 which show very divergent amino acid sequences. In a third set of experiments, we will attempt to determine the three-dimensional structure of certain of the viral proteinases as well as the domain of eIF4G which interacts with the viral proteinases. The structure determination will use both X-ray crystallography and nuclear magnetic resonance. Experiments will be performed on the proteinase alone as well as in complexes with each the eIF4G domain. The experiments will deliver significant new biological information as to how the components of the reaction interact with each other and what changes these interactions cause in the structures of the proteins taking part. Thus, the results of this project will help us understand not only a viral strategy but also comprehend more thoroughly cellular proteins. As these cellular proteins are all involved in regulating protein synthesis, this new information will help us to comprehend in closer detail how the control of growth in a healthy cell is orchestrated.