Essential ionic triggers for enveloped virus entry

In order to cause disease, all viruses must gain entry to their target cells. A common way viruses do this is by hijacking a cellular system called the endocytic network, which is normally used by cells to take up nutrients from the external environment. This network consists of tiny compartments called endosomes and many viruses have evolved the ability to trick cells that they are useful cargos in order to enter the network, and thus gain entry to the cell. However, once a virus enters an endosome, it is faced with a problem; it is effectively trapped, and so the virus must escape from the endosome in order to start an infection. The aim of this proposal is to understand in detail how viruses are able to escape the endosomes, and this work is based on exciting new research findings from our laboratory. Viruses escape endosomes using specialised spike proteins that cover the virion exterior and cause a process called fusion. To cause fusion, these spikes interact with specific chemicals within endosomes and dramatically change shape. After changing shape, the fusion spikes interact with the endosomal membrane from the inside, and force this membrane to mix with the viral envelope. When these two membranes merge together, fusion has occurred and the viral genome is released into the cytoplasm to continue the viral replication cycle. In simple terms, the ability of these viral spikes to change shape is critical in order for a virus to cause fusion, to escape the endosome and thus to continue the infection process. Despite this critical role, the mechanism by which spikes change shape and then cause fusion is poorly understood. Highlighting this poor understanding, we recently showed for the first time that some viruses within an important class of viruses known as bunyaviruses (which include many haemorrhagic fever viruses and viruses that are transmitted by mosquitoes - an increasing risk in the UK due to global warming) require potassium ions (K+) to cause the spikes to change shape and produce fusion. This finding represents a critical and previously overlooked requirement of the fusion mechanism.
This proposal describes a set of experiments that will first reveal whether the requirement for K+ is a general characteristic of other viruses within the broader bunyavirus group. Next, we will investigate whether any other ions within endosomes have the same effect as K+ during virus entry, and then use state-of-the-art cryo-electron microscopy and X-ray crystallography techniques to reveal the high-resolution structure of the spikes in the inactive (pre-fusion) and activated shapes. Finally, we will use a variety of genetic techniques to identify parts of the spikes that are critical for responding to the biochemical signals, and also for mediating the shape changes themselves. Taken together, these experiments will provide a major advance in the understanding of the fusion mechanism, revealing in high detail how spikes are able to respond to chemical signals within endosomes, to change shape and cause fusion. This information is required to provide an essential foundation on which to design strategies to block spike fusogenesis; drugs that can do this would prevent infection and disease.

To cause disease, viruses must first enter cells and many viruses do this by hijacking the endocytic network by mimicking cellular cargos. Once inside an endosome, a virus is effectively trapped, and must escape to continue its lifecycle. Here, we propose experiments that will provide new understanding of the endosome escape mechanism, based on our exciting new research findings. Viruses escape from endosomes using protein spikes that cover their exterior and mediate the merging of the viral and endosomal membranes (fusion). The spikes respond to chemical signals within endosomes causing structural changes, switching spikes from a pre-fusion to a fusion-active conformation. This allows spikes to interact with the endosomal membrane from the inside, and soon after, fusion occurs allowing the viral genome to enter the cell. How spikes respond to chemical signals is poorly understood, and highlighting this, we recently showed that an important class of viruses known as bunyaviruses require potassium ions (K+) to escape from endosomes. We showed K+ is required to switch spikes from pre-fusion to fusion-active states, which represents a paradigm shift in the understanding of virus entry. In this project, we will examine whether the K+ requirement is a characteristic of the wider bunyavirus group and whether other ions within endosomes mediate similar spike changes. Next, we will use cryo-EM and X-ray crystallography to reveal high-resolution structures of spikes in their pre-fusion and fusion-active conformations. Finally, we will use genetic techniques to identify spike residues that mediate these structural changes. Together these experiments will characterise endosomal fusion triggers and reveal the molecular details of how spikes become fusogenic, which is critical for virus entry. Improved understanding of endosome escape is required to aid the rational design of strategies to block spike fusogenesis; drugs that can do this would prevent infection and disease.

Experiments described within the current proposal will provide impact in three major areas:

1. Research impact. The focus of this proposal is to elucidate the requirement of specific biochemical signals for entry of enveloped viruses into cells and the associated structural rearrangement in the viral glycoprotein spikes. We have recently shown that selected bunyaviruses require the influx of K+ to allow endosomal escape, and release of the viral genome into cells. The proposed research will first examine whether this K+ requirement is a general characteristic of enveloped bunyaviruses, and second determine the precise molecular mechanism by which K+ influences the virus entry. This basic research will represent high impact world-leading science, using cutting edge technologies. We believe this work will have profound impact on the understanding of virus entry, which is a critical phase of the life cycle of all enveloped viruses, which include major human and animal pathogens (e.g. HIV, Ebola, Influenza, etc). In addition, our demonstration that this process can be blocked using small molecules, will have potentially massive impact in disease prevention.
This work will impact the research of academic and government institutions with scientists studying enveloped viruses, as well as scientists working in the broad fields of cellular and structural biology, as well as host-pathogen interactions. These experiments will reveal new and critical details of the mechanism of virus entry, and this information can be exploited towards the development of new anti-viral therapies.

2. Commercial impact. The identification of a new requirement for virus entry that can be impeded by anti-viral treatments will open up opportunities for commercial exploitation. Ultimately this will enhance the economic competiveness of the UK, improve the skill sets of UK based scientists, and improve or safeguard the health of the at-risk human population. To facilitate the transition from basic research to translational research, the University of Leeds (UoL) has several mechanisms already in place, including seed funding, dedicated personnel with industrial and knowledge transfer expertise, and previously established commercial/academic partnerships. The ways in which we will exploit these resources is detailed in the 'pathways to impact' document that accompanies this proposal.

We will define a process critical for viral life cycle that depends on host ion channels, and that the activity of these channels can be blocked by clinically approved drugs, therefore there is the potential to use such existing drugs through re-purposing to treat outbreaks and possible future epidemics of new emerging viruses belonging to this broad group of viruses.

3. Impact to public awareness of science. The work in this proposal will also impact the public by enhancing our ability to communicate the ability of science to improve lives. Public awareness of viruses in general and viral epidemics is high. At the UoL and Astbury Centre for Structural Molecular Biology, we strive to reach out to the public through several types of media including printed copy, radio, the internet and TV. These interactions are facilitated by dedicated personnel expert in the UoL press office, who provide assistance in generating press releases to gather initial interest from diverse media outlets. We also have strong links with local museums, particularly the Thackray Museum (The Museum of Modern Medicine, Leeds). We regularly work with the museum on biologically and medically relevant science undertaken at the UoL. The most recent being an exhibit on structural molecular biology and its role in discovery and the life of William Astbury, a former Professor at UoL who was one of the pioneers of applying X-ray analysis to biological material. In this way, the public will be made aware of the efforts of academic and government institutions to safeguard the nation's heath.