Structural insight into the herpesvirus core nuclear egress complex - investigating its role as initiator of virus nuclear egress and its potential as an antiviral therapy target.

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Viruses from the herpesviridea family start genomic replication in the host cell nucleus, which is also the place of capsid assembly. Because these nucleocapsids are too large to exit the nucleus via the nuclear pores, herpesviruses rely on an alternative exit mechanism, termed nuclear egress. This is a key step in virus replication and is initiated by the formation of a complex between two viral proteins, namely between an integral nuclear membrane-anchored viral protein, termed pUL50 in human cytomegalovirus (HCMV) and a nucleoplasmic-soluble viral protein, termed pUL53 in HCMV. The complex is thought to collate multiple roles. It (i) forms a platform for the recruitment of additional virus and host cell proteins, (ii) initiates the formation of membrane buds and (iii) recruits the nucleocapsids to the budding sites. We recently published the crystal structure of the HCMV pUL50-pUL53 complex. A specific feature of the complex is a hook-like N-terminal extension in pUL53. The hook (amino acids 59-87) embraces pUL50, contributes 1510 Angs.**2 to the total protein interface area of 1880 Angs.**2 and represents a hallmark of the pUL50-pUL53 interaction. In the crystals the pUL50-pUL53 heterodimers further assemble into hexameric ring-like structures that form a honeycomb-like layer. These assemblies are discussed in the literature as facilitating the bending of the inner nuclear membrane and thereby initiating the budding process. The research field is highly competitive and several core NEC structures, namely from HCMV, HSV-1 and PrV, have been published almost simultaneously.Here, we propose to considerably expand our structural biology knowledge of the core NEC as the virus nuclear egress initiator and at the same time explore the potential of the pUL50-pUL53 complex as an antiviral therapy target. In particular, we aim at determining the crystal structures of the core NEC from Epstein-Barr virus (EBV) and varizella zoster virus (VZV). Both are of high medical relevance, and until now no structure of a gammaherpesvirus core NEC (e.g. EBV) has been solved. In addition, we aim at comparing the NEC complexes from different herpesviruses and their molecular properties using molecular dynamics calculations, solving crystal structures of the core NEC in complex with additional virus or host cell proteins and structurally exploring the interaction between the core NEC and the nuclear capsid proteins as a key step in the initiation of the budding process. Finally, we aim at characterizing the hook-into-groove interaction between HCMV pUL50 and pUL53 as a blueprint for the development of inhibitors of potential therapeutic value. The latter will be achieved by computational design and fragment screening by crystallography. This approach is highly promising due to our recent advances in obtaining crystals of an engineered HCMV core NEC complex diffracting to better than 1.5 Angs. resolution.