An integrative approach to deciphering the entry process in Herpesviruses

Cellular processes are governed by the intricate coordination and dynamics of biological macromolecules called proteins and nucleic acids. These do not act in isolation but rather interact with each other and assemble to form cellular complexes. Understanding the structures of complexes can be an important step not only in understanding basic cell biology but also disease, as such complexes are also formed between the proteins of invading pathogens like viruses and the host cell proteins. Indeed, to determine how a virus functions, knowledge is needed not only about the molecular arrangement of its own proteins but also about their interactions with the host cell over the course of the viral life cycle. Particularly interesting is the entry process, the earliest stage of infection in the cycle, when the virus comes into first contact with the host cell and introduces viral material into the cell. The goal of our project is to gain a structural view of the entry process in one of the largest and most complex families of viruses that infect humans - the Herpesviruses. The severity of conditions caused by these viruses ranges from cold sores, genital ulcers, and blisters to blindness and life-threatening conditions including fatal encephalitis, meningitis and cancer. This family constitutes a major public health concern due to their worldwide prevalence, ease of spread, and severity of the associated symptoms. To achieve this, we propose a multi-disciplinary approach that integrates computational and experimental methods. The field that aids this project is Structural Biology. It provides 'pictures' of macromolecular complexes and their components through the use of experimental techniques such as X-ray crystallography and nuclear magnetic resonance, each of which has its own limitations to what it can accomplish, depending on the size and purity of sample under investigation, the conditions in which its prepared, and the homogeneity of the complex it contains. In the last decade, cryo electron microscopy and tomography have also become important techniques for observing biological complexes. With these techniques, samples are rapidly frozen using cryogenic liquids and then bombarded with electrons, yielding many images of the 2-dimensional sample that can be combined into a clearer 3-dimensional picture. In tomography, such pictures can provide the overall organization of cells and tissues, and can capture pathogens during cell invasion. They contain many different macromolecular complexes that can be detected in their native environment. Though these techniques have led to many interesting discoveries, here too there are limitations, typically not resulting in near-atomic pictures. In this project, we will study the entry process in human herpesviruses. To this end, we will develop a computational approach that pulls together information from a variety of experimental techniques to construct a clearer and more complete description of structures of complexes imaged initially by cryo electron tomography. The method will have the capability of incorporating information about which protein interacts with which (or how close they are to each other). Such information could come from a variety of techniques, often grouped under the name 'proteomics'. Together, we will fit all the different pieces of information like a jigsaw puzzle, creating a higher resolution picture of the visualised complexes. Obtaining the structures of selected complexes (formed between the proteins placed on the envelope of the virus and between them and their interacting proteins from the host cell) will represent a major advance in our understanding of the molecular and mechanistic details of herpesvirus pathogenesis. This will allow us to improve current models of the entry process, a crucial step towards identifying drug targets. Our novel approach will be applicable to many viral systems and will open the door for similar studies on other pathogens.