Substrate recognition by picornaviral proteases L and 2A

All viruses must in some way interact with the macromolecules and organelles of the host cell to ensure successful replication of the viral genome and production of new infectious particles. For all RNA viruses, modulation of the protein synthesis is of paramount importance to ensure that the RNA genome can be translated efficiently in the hostile environment of the cell. In addition, the inhibition of protein synthesis from cellular mRNAs prevents production of important proteins for the innate immune system of the host, allowing viruses to overcome host defences. This effect can be enhanced by inhibition of the activation of NF-B to prevent transcription of genes encoding inflammation response proteins and by impairing the deubiquitination pathway. This project extends our investigation of two proteins of picornaviruses, the leader proteinase (Lpro) of Foot-and-Mouth Disease Virus (FMDV) and the 2A proteinase (2Apro) of enteroviruses such as coxsackievirus and human rhinovirus, that modulate the protein synthesis of the host cell as well as interfering with the innate immune system. In addition, the project will also focus on the structure and function of the host translation initiation factor eIF4G that is specifically cleaved by the two proteinases. Using nuclear magnetic resonance (NMR), we showed that a short fragment derived from eIF4G interacts with both Lpro and 2Apro as well as with the cellular protein eIF4E that recruits the eukaryotic mRNA via eIF4G to the ribosome. In addition, we showed that a stable trimeric complex between eIF4GII and the picornaviral proteases is only formed in the presence of eIF4E. Unexpectedly, Lbpro directly contacts both eIF4GII and eIF4E and the 2Apro even forms a stable complex with eIF4E in the absence of eIF4GII. Therefore, we conclude that a triangular trimeric complex is formed that targets the active translation initiation factors. These contrasting protein-protein interactions by Lbpro and 2Apro indicate that their mechanisms to interact with the eIF4GII/eIF4E have evolved independently. Based on this information, we will investigate the detailed mechanisms by which eIF4GII, eIF4E and the eIF4GII/eIF4E complex are targeted by the proteases and how these mechanisms differ. The interaction between eIF4GII and eIF4E also represents an important control point for cellular growth and is often disturbed in cancer cells. Thus, the results of the investigation will also be relevant to processes leading to uncontrolled growth in mammalian cells. For the Lpro, we will also search for further substrates in the infected cell using a novel technique that labels newly generated N-termini as well as examining reports that Lpro can deubiquitinate host proteins in an attempt to subvert the immune response. For the enteroviral 2Apro, we will use NMR to examine self-processing and to examine peptide substrate binding with a view to developing specific inhibitors of these viruses.

The family of picornaviruses contains important human and animal pathogens such as poliovirus (PV), human rhinovirus (HRV), 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. In addition, the FMDV vaccines can only be used under certain conditions and the PV vaccines may not be sufficiently effective to ensure completion of the PV eradication program without support of anti-viral agents. Here, we examined two potential candidates for anti-viral drugs for the above-mentioned viruses, the proteinase 2Apro from HRV and the leader proteinase (Lpro) from FMDV. The project had two objectives. The first examined the 2Apro from two genetic groups of human rhinoviruses. The 2Apro from these groups show only 50% identity or less at the amino acid level and possess different specificities. Investigations into the differences had been hampered by the inability to purify a 2Apro from the genetic group B. In this project, we overcame this problem and were able to examine the activity of 2Apro from both groups on different substrates. Although both 2Apro are from human rhinoviruses, the 2Apro do indeed have different activities towards cellular substrates, implying that viruses of the two groups interact differently with the infected cell. Nevertheless we previously designed a molecule that inhibited both enzymes. Now, we succeeded in determining the molecular structure of the enzyme from genetic group A with the inhibitor. This will enable more active compounds against these viral enzymes to be developed. Comparison with models of the poliovirus enzyme suggest that the knowledge learnt with rhinoviruses will be applicable to poliovirus. The second objective was to use our understanding of the specificity of the Lpro enzyme to investigate its role in suppressing the innate immunity and as a tool to investigate the architecture of ubiquitin modifications. Together with colleagues in Utrecht and Cambridge, we showed that Lpro of FMDV actively disrupts the innate immune response by removing the innate immune marker protein ISG15 from proteins. Furthermore, the removal of ISG15 leaves a special amino acid signal behind which can be used to detect the presence of an FMDV infection. We also demonstrated that the Lpro degrades specifically certain key proteins of the innate immune system that signal the presence of an infection. With our understanding of the Lpro, we were also able to modify its specificity to allow it to process the related marker protein ubiquitin. With the modified enzyme, we could examine how ubiquitin is bound to an important protein in Parkinson's disease called parkin and further our understanding of this important molecule. This result reveals how the investigation of a vital viral enzyme can enhance the understanding of human disease.