Structure, Cell Entry and Egress of Lassa Fever Virus

Lassa virus is the causative agent of a serious disease called Lassa fever, an acute and hemorrhagic fever affecting up to 500,000 people and killing thousands of people in West-Africa each year. It is carried by rats scavenging around homes in epidemic areas, and spreads to humans often through contaminated food or inhaled dried rat urine or feces. Due to its high mortality, and the fact that it can spread through air as aerosols, it is also a serious risk that Lassa virus could be used as a biological weapon. Despite its high medical relevance and potential biological threats, there are no approved vaccines or specific anti-viral therapies against Lassa virus.

Several basic questions about Lassa virus biology remain unanswered. This is largely due to the high pathogenicity of this virus, necessitating all handling to be carried out in laboratories with ultra-high biosafety, which are not widely available. In this project, we are collaborating with a group of scientist studying Lassa virus at University of Marburg, Germany, where such as laboratory is available. There, infectious viruses can be produced prior to chemical inactivation to allow us to carry out further analysis at University of Oxford.

We will address several important but unanswered questions in LASV basic research: How does the virus identify and enter its host cell? How do different viral components come together in an infected cell to form a new virus particle? Firstly, we will use state-of-the-art electron microscopy at Oxford Particle Imaging Centre to study purified viruses to decipher the interactions between different viral building blocks in the assembled virus particle. Secondly, we will use the same technique to study cells infected with the virus to study the formation of new virus particles at different stages of the process. Thirdly, we will use the synchrotron radiation at the Diamond Light Source to solve the atomic structures of protein components of the virus which are responsible for finding new cells for infection and for the entry of the viral genetic material in to the cell to understand these key processes.

Answering these questions will increase our understanding on Lassa virus infection. Furthermore, it will allow us to identify rational targets for drug development. Virus entry into the host cell, and thus infection, could be hindered by small molecule drugs preventing the virus either from binding to or from entering in to the host cell. Formation of new virus particles inside the infected host cell is another key step that could be targeted by drugs. Thus, in the long term our results will contribute towards curing Lassa fever.

Lassa virus (LASV) is a deadly zoonotic pathogen which kills thousands of people each year. Despite the threat that it poses, little is known about the molecular mechanisms it employs for entry into and egression from host cells. Such information is invaluable for the rational development of therapeutics that can prevent and treat LASV infection. Here, we will address this paucity of information using a combined structural approach that incorporates electron cryo-microscopy (cryo-EM) and X-ray crystallography.

Cryo-EM will be utilized for medium resolution (10-30 A) imaging of unstained LASV (fully deactivated) and non-pathogenic virus-like-particles (VLP). This technique is highly suited for this work as samples will be kept in a vitreous, near physiological state. As LASV and its VLPs are pleomorphic in shape, we will employ tomographic data acquisition and sub-volume averaging, methods which will allow the structure of the entire virus and subcomponent complexes of structural proteins (e.g. attachment-fusion glycoprotein and nucleocapsid-RNA complexes), to be solved in three-dimensions. Additionally, using cellular cryo-EM we will examine how progeny virus particles assemble and bud from host cells.

X-ray crystallography will serve as a complementary method to cryo-EM and will allow high resolution (2-3 A) structural studies of the LASV envelope glycoproteins, GP1 and GP2. These glycoproteins are involved in viral attachment and fusion, respectively, and will be produced using the in-house mammalian expression system designed to control highly heterogeneous N-linked sugars for structural work. Following crystallization of these individual glycoproteins alone and in complex with their cellular receptor, a-dystroglycan, protein crystals will be subjected to synchrotron radiation for structural elucidation at Diamond Light Source.

Ultimately, this multidisciplinary work will reveal structural targets for the rational antiviral and vaccine design.

Background

Lassa virus (LASV), a member of Arenaviridae, is a causative agent of Lassa fever, an acute hemorrhagic fever endemic in West-Africa and infecting up to 500,000 people per year. Due to its high morbidity and mortality, and the fact that it can spread as aerosols, LASV is also a serious potential biological weapon agent. Currently there are no approved vaccines or specific antiviral treatments against LASV. The research proposed here aims to answer two questions: What are the molecular interactions between different structural components in the virus? How do new viruses form and how are they released from the infected cells? These are very basic questions in Arenavirus biology and crucial to understanding the viral infection mechanism at cellular level, and yet they have remain unanswered. Vaccine and drug development stems from basic research, and these developments would have two major impacts in the long term: 1. improved human health in endemic areas and 2. nation's improved readiness to face bio-weapon threats. Details who will benefit from the research, and how, are summarized below.

Impact on human health in endemic areas

Although the proposed research is strongly rooted in basic science, the findings are expected make an impact on human health in the long term. Lassa fever is severely affecting large human populations in some of the World's poorest regions. Basic research, as outlined in this proposal, will contribute key knowledge required for drug design and vaccine development. For example, the solved structure LASV will provide valuable information for projects aiming at developing specific anti-viral therapies by rational drug design. Thus mid-term beneficiaries will be in research and development teams in pharmaceutical industry and governmental organizations. If their drug and vaccine developments will be successful, in the long term the beneficiaries will be people affected by Lassa fever in the endemic areas. Furthermore, vaccination against Lassa fever would enhance the quality of life, health and economy in these areas and provide protection to hospital personnel.

Impact on readiness to face bio-weapon threats

Another important aspect is the serious threat that Lassa virus could be used as a biological weapon against any nation, including the UK. Successful development of antivirals would improve nation's readiness to face such threats and to treat citizens in the case of an actual attack. Furthermore, as symptoms of Lassa fever are extremely dreadful, the psychological effect of even an implied risk would lead to great economic losses and impair nation's economy. On the other hand, existence of an effective anti-viral drug would have a greatly calming effect and positive impact on economy and human well-being in such a crisis.