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.

A detailed description of the structures of macromolecular complexes can be very valuable for understanding cellular processes, including those that are targeted and modified by pathogens. However, due to current limitations of experimental techniques, the only way to achieve a comprehensive characterisation of these structures is to adopt an integrative approach that combines information from multiple sources. At present there are very few methods attempting to do so systematically. Here we aim to integrate multiple experimental and computational approaches to characterise complexes formed between herpesviruses and their host during the entry stage of infection.

The field of 3D EM is increasingly important in molecular and cellular biology, owing to advances in hardware and software that improve image quality and experimental efficiency. One advantage is that the resulting maps often correspond to large and heterogeneous complexes that are difficult to study by other techniques. In tomography in particular, new biological insights arise from structure determination of macromolecular machinery in the native cellular context (here membranes).

Here, we propose to build on Topf group's previous work on data integration and develop a method that efficiently and accurately combines the structures of individual components with low-resolution maps of the complexes they form. These maps will result from cryo electron tomography combined with sub-tomogram averaging and classification performed in the Grünewald lab. The method will allow the inclusion of additional spatial information from chemical cross-linking coupled with mass spectrometry and protein-protein interaction (PPI) networks. The resulting models of sub-complexes mediating herpesvirus entry could potentially lead to the discovery of novel functional details about the infection process. Additionally the Grünewald group will continue to refine image data acquisition and image processing tools and pipelines.

Who will benefit from this research?

The proposed research will potentially generate knowledge of value to (i) the wider bioscience and biomedical communities; (ii) the pharmaceutical industry; and (iii) the general public and government policy makers (e.g., national health and overseas aid).

How will they benefit from this research?

The main contributions from this research will be to (i) elucidate the structure of novel complexes formed during the entry process of herpesviruses and (ii) to provide computational tools and experimental protocols for generating such structures by integration of data from 3D electron microscopy, mass spectrometry and protein interaction networks. In the long term, applying the methods to as many biological complexes as possible will help to generate more fundamental knowledge about basic cell biological processes. This systems level analysis of biological processes is particularly important at the moment, where there is a lot of knowledge about structures and functions of individual proteins, but much less on how they work together in the cellular context. Such information can have valuable implications to the immediate bioscience community.

Additionally, the immediate goal is also to apply the methods to decipher structures of complexes formed between herpesviruses and their host cells. Information generated from such studies is directly related to the understanding of infection and disease and therefore will be of interest to the general public and the pharmaceutical industry. The results on HCMV could reveal new insights into spread of the virus (which presents a particular threat to newborns) and identify new drug targets, which will be beneficial not only to the biomedical community and pharmaceutical industry, but also to the public sector and government policy makers (e.g., national health services and overseas aid). Understanding how herpesviruses work in general is of great importance for improving quality of life and human health, with 5 of the 8 members of the family affecting more than 90% of adult population worldwide. Knowledge about the first step in the infection by these viruses is key to identifying new drug targets and/or to develop a vaccine. This is of high relevance not only for basic researchers but also pharmaceutical industries and the general public. Such contribution could impact the economic competitiveness of the UK in pharmaceutical research and economic development. Ultimately, the results could impact clinicians and patients, through availability of more effective treatments, as well as government policy makers (e.g., in relation to the national health services). Given the importance of diseases caused by the herpesviruses family in developing nations the research could also impact foreign policy decision-makers regarding overseas aid agencies.

The communication about the integrative approach to structural biology and new insights resulting from the research (through the long-term application of the proposed methods to many complexes), whether related to basic cell biology or disease, could all have an impact on the public education of science and arts. This will be done via websites and challenges (e.g., http://autopack.cgsociety.org/autopack/), books and museums.

Finally, highly skilled researchers working on the proposed project will be trained in transferring scientific knowledge to the public and the preparation of materials for communication (e.g., via publications, user-friendly web interfaces, and software documentation). They will participate in professional seminars organised at STRUBI and ISMB, and in seminars on communicating sciences to the public in the centre for learning and professional development (at our universities). They will also be directly involved in the research collaborations. These acquired skills will contribute to the development of their future career not only in bioscience but rather in all employment sectors.