Structure and function of picornaviral proteases Lb and 2A

Viruses are obligate intracellular parasites. To use the cell to their own ends, viruses express proteins that interact with the cell`s molecules. Viruses of the picornavirus family are no exception, producing proteins that interfere with protein synthesis of the infected cell. The viral proteins may also affect the transcription of genes encoding stress response proteins and subvert the deubiquitination pathway. This project continues our investigation of two such proteins, namely the leader proteinase (L pro ) of Foot-and-Mouth Disease Virus (FMDV) and the 2A proteinase (2Apro ) of enteroviruses such as poliovirus. Both Lpro and 2Apro prevent the infected cell from translating its own mRNA, whilst leaving viral protein synthesis intact. Inhibition of these enzymes reduces viral yield, implying that compounds specific for these molecules may help to prevent or treat the respective diseases. In this project, we will examine the interaction of the FMDV Lpro with a potent inhibitor developed in the previous project. The inhibitor is an epoxide compound containing amino acids recognised by the substrate binding site of Lpro . We will examine the structure of the Lpro /inhibitor complex using both X-ray and NMR techniques. These results will help us understand how Lpro interacts with its substrate whilst at the same time providing information with which the inhibitor can be improved. In a further set of experiments, we will use NMR to examine the interaction of Lpro with a small fragment derived from eukaryotic initiation factor 4GII (eIF4GII), the only host cell molecule of Lpro that has as yet been convincingly reported. The results should help to explain how this molecule can be efficiently cleaved by Lpro even though the cleavage site on eIF4GII is itself not well recognised on an oligopeptide. Finally, we will also investigate a recent report indicating that Lpro has deubiquitinase activity. Structural information on the interaction of the 2Apro enzyme with its substrate, either in cis or trans, is not at present available. Consequently, our understanding of substrate binding by these enzymes is poor. Such information is vital for the design of potent anti-virals; therefore, we will try to generate this information for the 2A pro of a human rhinovirus, a poliovirus and a coxsackievirus. We will take a three-pronged approach. First, we will express, purify and crystallise selected 2Apro with a short piece of VP1 covalently attached. If successful, this will give us the structure of the cis interaction between enzyme and substrate. Second, we will try to co-crystallise the selected 2Apro with an oligopeptide representing either the polyprotein site or the eIF4GII site to provide us with information on the trans interaction of the enzyme and substrate. Third, we will attempt to generate co-crystals of the 2Apro with small inhibitors such as antipain, elastatinal or chymostatin. These inhibitors were shown previously to inhibit 2Apro in the micromolar range. Using such structural information, it should be possible to develop one of these inhibitors into a more potent and specific anti-viral against 2Apro .

The family of picornaviruses contains important human and animal pathogens such as poliovirus (PV), human rhinovirus (HRV), coxsackievirus (CV) and foot-and-mouth disease virus (FMDV). Vaccines have been used successfully to control infections by PV and FMDV. Vaccines are however not available for HRVs and CVs. In addition, the FMDV vaccines can only be used when certain conditions are met and the PV vaccines may not be sufficiently effective to ensure completion of the PV eradication program without support and backup of anti-viral agents. In this project, we examined two potential anti-viral candidates for the above-mentioned viruses, the proteinase 2Apro from PV, HRV and CV and the leader proteinase (Lpro) from FMDV. The project had two main objectives. The first was to examined the binding of a cellular protein termed eukaryotic initiation factor 4G (eIF4G) with both the 2Apro and Lpro. This binding subsequently leads to cleavage of the host protein; the mechanism of cleavage is however poorly understood. We prepared an appropriate fragment of the eIF4G protein and assigned NMR signals for 90% of the amino acids. We could show that the interaction of the two proteinases with eIF4G alone is not sufficient to form a stable complex. Instead, a stable complex and thus efficient cleavage requires a second cellular protein, eIF4E. Surprisingly, we found that both the 2Apro and the Lpro must interact specifically with both host factors and that these interactions of the 2Apro are different for the HRV2 and CV enzymes. This has implications for the design of inhibitors of the PV enzyme.The second main objective was to understand the mechanism and specificity of the Lpro enzyme. This enzyme possesses 30% fewer amino acids than papain, the prototypic cellular enzyme of the same family. However, the Lpro can perform C-terminal self-processing which papain cannot and Lpro is much more specific. We wished to understand how the protein fold of papain has been modified to fulfil these tasks. To this end, we were able to solve the structure of the Lpro covalently bound to a specific inhibitor that we had developed together with our Brazilian collaborators. This structure revealed that the Lpro recognises a protein to be cleaved across a broader range of amino acids than the prototype papain. Additionally, the acid/base composition of the substrate has to be complementary to that of the Lpro. In this way, only a few, specific proteins gain access to the viral enzymes active site and be efficiently cleaved. One such protein is the cellular protein eIF4G mentioned above.Together, the results explain why the replication of the FMDV can proceed so rapidly and be finished in a cell four hours after infection. After its synthesis on the ribosome, the Lpro cleaves itself off the growing viral protein and binds to the eIF4G and eIF4E that are present on the ribosome. Cleavage of eIF4G ensues, so that a cessation of the translation of host mRNAs occurs, thus preventing the cell from making an innate immune response. The protein synthesis on the viral RNA is not affected because it uses an eIF4G-independent mechanism to recruit ribosomes for translation.