Interaction of picornaviral proteinases with eIF4G

Foot-and-Mouth Disease Virus (FMDV) remains an economically important animal pathogen of the picornavirus family. Its replication is entirely dependent on the activity of three virally-encoded proteinases. One of these proteinases is the so-called Leader peptide (now referred to as the Leader (L pro ) proteinase); this papain-like cysteine proteinase cleaves once on the viral polyprotein, the primary product of viral protein synthesis, and also processes at least one cellular protein, the eukaryotic initiation factor (eIF) 4G. As a consequence, protein synthesis from capped mRNA of the host-cell can no longer be initiated; however, synthesis of proteins from the viral mRNA is unaffected, as it does not require a cap-structure but initiates internally. A similar reaction occurs in other members of the picornavirus family such as human rhinoviruses (HRVs, the main causative agents of the common cold) and poliovirus. However, in these picornaviruses, a chymotrypsin-like proteinase, referred to as the 2Apro, carries out the cleavage of eIF4G. It is the aim of this project to employ both biochemistry and structural biology to characterise the interactions of FMDV Lpro and HRV 2Apro with eIF4G. A thorough understanding of this reaction is important as it is a major determinant of virulence in FMDV and swine vesicular disease, an economically important picornavirus of pigs. In addition, proteomics and cell biological experiments will be employed to identify further cellular substrates for Lbpro and to determine the relationship of these cleavages to the events occurring in the host cell during FMDV replication. The experiments proposed are based upon systems developed and results obtained in the previous project, P-13367. Four fundamental breakthroughs were made in this project. First, the three-dimensional structure of Lpro was determined at 1.9 Å. Second, a system was developed in rabbit reticulocyte lysates in which self-processing of Lpro or 2Apro and cleavage of eIF4G takes place at molar ratios similar to those observed during viral replication. Third, this system was used to show that the 18 amino acid long C-terminal extension (CTE) of Lpro , although distant from the active site, is required for eIF4G cleavage. Fourth, a direct interaction of Lpro and 2Apro with a region on eIF4G adjacent to the cleavage site was demonstrated, and which, for Lpro , involved the CTE. However, no information has been obtained on the binding site on the 2Apro for eIF4G. This is just one of many open questions on the interaction between the picornaviral proteinases and eIF4G. This project will address the open questions by introducing defined mutations into regions of Lbpro and 2Apro which are thought to interact with eIF4G. For Lpro , these mutational analyses will be complemented by nuclear magnetic resonance spectroscopy (NMR). This structural work will investigate the possible structures of the Lpro CTE and will also determine the interaction of Lpro with oligopeptides representing the sequences of the cleavage sites on the viral polyprotein and eIF4G. An understanding of the 2Apro of HRVs is complicated by the apparent difference in substrate specificities of these enzymes from HRV serotypes 2 and 14. Mutational and structural approaches (using X-ray crystallography) will be employed to elucidate the reasons for these differences and to identify regions on the 2A pro molecule which are responsible for interacting with the substrate.

Foot-and-Mouth Disease Virus (FMDV) remains an economically important animal pathogen of the picornavirus family. Its replication is entirely dependent on the activity of three virally-encoded proteinases. One of these proteinases is the so-called Leader peptide (now referred to as the Leader (L pro ) proteinase); this papain-like cysteine proteinase cleaves once on the viral polyprotein, the primary product of viral protein synthesis, and also processes at least one cellular protein, the eukaryotic initiation factor (eIF) 4G. As a consequence, protein synthesis from capped mRNA of the host-cell can no longer be initiated; however, synthesis of proteins from the viral mRNA is unaffected, as it does not require a cap-structure but initiates internally. A similar reaction occurs in other members of the picornavirus family such as human rhinoviruses (HRVs, the main causative agents of the common cold) and poliovirus. However, in these picornaviruses, a chymotrypsin-like proteinase, referred to as the 2Apro , carries out the cleavage of eIF4G. In this project, we employed both biochemistry and structural biology to characterise the interactions of FMDV Lpro and HRV 2Apro with eIF4G. A thorough understanding of this reaction is important as it is a major determinant of virulence in FMDV and swine vesicular disease, an economically important picornavirus of pigs. In addition, proteomics and cell biological experiments were employed to attempt to identify further cellular substrates for Lbpro and to determine the relationship of these cleavages to the events occurring in the host cell during FMDV replication. In this project, we used information from the molecular structure of the viral proteinases to understand how they are built up, how they carry out their tasks and how they recognise a cellular protein and modify it. Surprisingly, viral proteinases from both FMDV and HRV use separate mechanisms to recognise viral and cellular proteins. For instance, we could show that FMDV Lpro uses a completely different set of amino acids to recognise eIF4G than it does to interact with its own viral polyprotein. In addition, we also found that the proteins of different HRVs recognise a cellular protein in different ways. Thus, viruses which cause the same disease can differently interact with the host cell. These results help us to understand the mechanisms how these two different proteins have evolved to modulate the physiology of the cells which they infect. Such information forms part of the solution to the larger puzzle how these viruses, encoding at the most 14 mature proteins, can hi-jack a much larger and more complex mammalian cell and turn it within a few hours into a virus producing factory.