Structural and mechanistic characterisation of DNA packaging motors from human Cytomegalovirus and related viruses

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During the assembly of many infectious viruses, such as Cytomegalovirus, Epstein-Barr virus and other herpes viruses, a DNA packaging motor inserts the viral genome into a protective protein capsid. The large terminase (LT) protein is a key component of the motor, representing a viable target for drug development. This PhD project will use a combination of protein engineering, structural biology (both X-ray and Cryo-EM), biophysics and biochemistry to define the assembly state, structure and function of these large molecular machines. By engineering a multipurpose in vitro system in which to study the DNA packaging processes of large dsDNA viruses, we will address the BBSRC remit for promoting World Class Underpinning Bioscience as we dissect the mechanism of ATP-powered DNA translocation and the physical principles that govern the tight packaging of DNA within the viral capsid. In addition, this system will allow efficient screening of compound libraries for potential inhibitors of DNA translocation and packaging as a method of anti-viral drug discovery. This PhD project is a collaboration with an industrial partner and will include a 3-month placement for the student at Inspiralis Ltd where several functional biochemical assays will be carried out with wild type and mutant proteins, using expertise in ATPase and helicase activity assays available at Inspiralis. The student will benefit from training from an academic / industry partnership providing a platform to develop as a "highly skilled researcher" while the research itself will contribute to UK "strength in core underpinning disciplines such as cellular, molecular and structural biology" whilst building "new tools and technologies that enable researchers to push the boundaries".Objectives(i) define mutant LT proteins and chemical inhibitors that will enhance our mechanistic understanding and produce stable complexes of nuclease and ATPase reaction intermediates; (ii) characterize these complexes using biophysical, biochemical and high-resolution structural approaches; (iii) use this information to model reaction mechanism and design new mutants and/or inhibitors and to assay the consequences of perturbation in vivo.NoveltyIt is unknown how LT protein interacts with DNA, how its ATPase and nuclease activities are switched on and off, and how these could be inhibited by small molecules. The research is expected to produce a library of high-resolution structural 'snapshots' of the reaction mechanism.TimelinessRecent advances have contributed to further understanding of the reaction mechanisms and novel inhibitor development in related enzymes with RNAse H-like and ATPase folds. Application of this information to viral LT proteins is invaluable for the development of novel approaches for fighting infections caused by herpesviruses.Experimental ApproachesThis project will use LT protein from cytomegalovirus, Epstein Barr, and other herpes viruses. If herpes LT proteins prove too challenging for structural and mechanistic stadues (i.e. solubility or stability issues) the project will refocus on tractable homologous LT proteins from mesophilic bacterial viruses SPP1 and HK97, for which purification procedure has already been established in Antson lab. Objectives will be addressed via a combination of biochemical, biophysical, and structural approaches. The ability of mutant LT proteins to translocate and cut DNA will be monitored using established in vitro and in vivo assays.