Characterisation of the structure, function and mode of action of picornaviral leader proteinases and identification of cellular substrates

Foot-and-Mouth Disease Virus (FMDV) remains an economically important animal pathogen. 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 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, the capped mRNA of the host-cell can no longer be translated; however, the viral mRNA is unaffected, as translation does not proceed via a cap-structure but inititates internally. It is the aim of this project to characterise the FMDV Lpro not only structurally and biochemically, but also cell biologically by identifying further cellular substrates for Lbpro and by determining the relationship of these cleavages to the events occurring in the host cell during an FMDV infection. The main achievement in the previous project, P-11222-MED, was the determination of the three-dimensional structure of the Lbpro form of the FMDV leader proteinase (Guarne et al, (1998) Embo J. 7469-7479). The structure shows the overall fold of the protein to be papain-like and indicates how the enzyme recognises its own C-terminus during self-processing from a growing polypeptide chain. Despite these profound insights, three fundamental questions about the Lbpro remain. First, what is the exact enzymatic mechanism? Second, the three- dimensional structure implies that recognition of the Lbpro cleavage site on eIF4G occurs via a basic residue directly C-terminal to the cleaved bond. Thus, the enzyme can make contacts with a substrate through amino acids lying N- or C-terminal to the scissile bond. Which residues are actually recognised by the Lbpro ? Third, this rare ability of the Lbpro to recognise substrates in this way is a strong indication that the enzyme will possess more cellular targets than the presently known eIF4G. What is the nature of these proteins? Does Lpro have additional functions beside self-cleavage from the polyprotein and the inhibition of capped mRNA translation by cleavage of eIF4G? This project will address the above questions. Specifically, the enzymatic mechanism will be investigated by introducing defined mutations into the Lbpro and by the determination of enzymatic parameters using fluorescent oligopeptide substrates based on the sequences of the two known cleavage sites. These studies will also allow amino acids of the Lbpro involved in substrate binding to be pin-pointed. The substrate specificity will be determined by analysing activity on mutated forms of the eIF4G substrate, by cleavage of oligopeptides substrates containing amino acid substitutions and by determining the three-dimensional structure of an oligopeptide substrate complexed to an inactive mutant of Lbpro . Finally, three methods will be employed to identify further targets of the Lbpro in the infected cell. The chosen methods are the detection by two-dimensional electrophoresis of proteins present in mammalian cell extracts which can be cleaved by Lbpro , the establishment of a yeast two-hybrid screen for the Lbpro using an inactive mutant and the expression of the active wild-type Lbpro in yeast under the control of an inducible promoter. Proteins found by these methods to be substrates for the Lbpro will then be examined to determine whether they are indeed cleaved during FMDV replication and to what extent the physiology of the host cell is subsequently reprogrammed.

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 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. 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. 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.