Molecular arrows: DNA markers for electron cryotomography

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 conditions caused by these viruses range 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.

One of the best ways to understand viral entry to is to take high-magnification 'snapshots' of viruses and cells. In the last decade, cryogenic electron microscopy and tomography have become important tools 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 data can provide the overall organization of cells and tissues, and can capture pathogens during cell invasion. They contain many different macromolecular complexes in their native environment. Though these techniques have led to many interesting discoveries, extracting all of the potential information can be challenging, as the images are crowded and the contrast is low.

To help identify specific parts of the virus and cell, we will develop a toolkit of 'molecular arrows' that bind specifically to one biomolecule of interest. These molecular arrows will be formed from tightly packed nucleic acids, so they stand out against the background of protein. We will add our arrows to viruses and cells before rapid freezing, and then use electron tomography to determine their 3-dimensional structure. This work will provide a map of the proteins on the virus surface needed for entry into cells, and allow us to try to block their entry. Using these results, we will improve current models of the human herpsevirus entry process, a crucial step towards identifying drug targets. Our novel molecular arrow approach will be applicable to many biological systems and will provide an important tool for cell biology, medicine and beyond.

Thanks to new detectors, electron cryotomography (cryoET) has unprecedented potential to reveal molecular detail inside cells and viruses. Unlike heavy metal stain-based biological electron microscopy, cryoET uses frozen, hydrated samples that retain a near native state. Data interpretation for cryoET remains challenging however, as biological specimens are crowded and composed mainly of light atoms, lowering the contrast. Although strategies have been proposed to overcome these challenges, they have limited applicability and potential toxicity. Here, we propose an alternative labelling toolkit for cryoET, with high contrast and low toxicity, based on DNA nanostructures conjugated to DNA aptamers.

DNA nanostructures are self-assembled DNA structures incorporating stretches of complementary oligonucleotide sequences. They can be designed into many different shapes and sizes, and the phosphate in the DNA backbone provides higher contrast. Nanostructures can be engineered to contain DNA aptamers, short (20-40 nucleotide) single stranded DNA sequences that recognise a biomolecule with high specificity and affinity. We propose to use DNA aptamers to target nanostructures to the entry machinery of herpes simplex virus type 1 (HSV-1).

Herpesviruses are a family of large enveloped viruses, 8 of which cause lifelong infection in humans. These viruses are a major health concern due to their worldwide prevalence, ease of spread, and severity. An important target for therapies is entry of the virus into host cells, which despite much research, is poorly understood. We will use our DNA toolkit to determine the organization of glycoproteins responsible for fusion of the viral and host membranes, and to identify aptamers that block entry. We will then apply our "DNA arrows" intracellularly to follow the packaging of these glycoproteins during HSV-1 infection. This work will develop both a valuable resource for cryoET and provide important information about HSV-1 infection.

Who will benefit from this research?
The proposed research will potentially generate knowledge of value to (i) the wider bioscience and biomedical communities; and (ii) the pharmaceutical/DNA technologies industries.

How will they benefit from this research?
The main contributions from this research will be to (i) develop a toolkit of DNA-based markers to identify features of interest in electron tomograms of frozen, hydrated biological specimens and (ii) to determine the arrangement of glycoproteins on the surface of herpesviruses required for viral-host membrane fusion and to follow the assembly of these proteins into virions in cells. Over 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. At the moment, there is significant knowledge about structures and functions of individual proteins, but much less on how they work together in the cellular context, such tools play an important role. Such information can have valuable implications to the immediate bioscience community.

An immediate goal is also to apply the methods to understand the fusion of 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. 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 work is highly relevant not only to basic researchers but also pharmaceutical industries and the general public and could contribute to the economic competitiveness of the UK in pharmaceutical research and related industries. 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 benefit foreign policy decision-makers regarding overseas aid agencies.

A more native approach to structural biology and new insights resulting from this approach (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, presentation, and public engagement). They will participate in professional seminars and in seminars on communicating sciences to the public provided by the university. 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.