Picornavirus capsid protein VP4: Essential role in cell entry and conserved antiviral target

The picornavirus family includes viruses such as human rhinovirus, poliovirus and enterovirus 71. Human rhinovirus (HRV) infects humans more frequently than any other virus and is responsible for approximately 70% of all subclinical respiratory infections (the common cold) which costs the UK £billions every year. Despite decades of research there remains no licensed drug to prevent or reduce infection. Poliovirus (PV) is the subject of an ongoing world-wide eradication campaign but as we reach closer to the final stages of eradication, some experts believe there is an urgent need for additional novel control strategies for the post-eradication era. Enterovirus 71 was until recently only thought to cause a generally mild disease of young children (hand-foot-and-mouth disease). However, in recent years huge outbreaks of EV71 have swept across China and Southeast Asia with cases in the 100,000s including more severe disease and hundreds of child deaths. No vaccine or antiviral is available.

Picornaviruses infect cells by hijacking cellular machinery in order to be taken into the cell within an internalised membrane vesicle. For infection to begin, the virus genome (the blueprint for making new virus) must be delivered through the membrane of this vesicle, to reach the cytoplasm, the compartment of the cell where virus replication occurs. The mechanism used by the virus for genome delivery remains unclear. Understanding this process in more detail will provide valuable insights for the development of antiviral agents that interfere with cell entry.

The viral genome is contained within a protein coat or capsid. The capsid protects the genome from environmental damage and is also a dynamic structure which plays a crucial role in the cell entry process. Experiments have shown that during cell entry, one of the capsid proteins, VP4, comes out of the virus and interacts with the membrane. Experiments with mutated viruses have confirmed that VP4 is involved in the entry process. We have used model membranes and recombinant VP4 which provide a convenient system for investigating protein-membrane interactions and membrane permeability. We have demonstrated that VP4 is able to interact with liposomes and induce membrane permeability by forming a multimeric pore. We therefore propose that VP4 functions as a membrane pore during cell entry: the 'hole' in the membrane through which the virus genome is delivered into the cytoplasm. We believe that we should study the role of VP4 in more detail in order to develop molecules or drugs that interact with VP4, block its function and prevent infection.

The picornavirus capsid comprises sixty copies of each of VP1, VP2, VP3 (which form the icosahedral particle) and VP4 which is a small, myristoylated internal protein which stabilises the capsid structure. The capsid is dynamic and undergoes a process of 'breathing' whereby internal components the N-terminus of VP1 (VP1N) and the N-terminus of VP4 are transiently exposed at the surface. During cell entry, these components become irreversibly externalised and interact with membranes. The VP1N-membrane interaction tethers the particle to the membrane while VP4 is completely externalised from the particle and also associates with the membrane. There is biochemical evidence for the involvement of VP4 in the cell entry process and genetic evidence for its involvement in delivery of the viral RNA genome across the membrane into the cytoplasm. The applicants (and collaborators) have developed the use of model membranes and recombinant proteins as convenient systems for studying virus-membrane and VP4-membrane interactions. The results of our recent studies demonstrate for the first time that VP4 interacts with liposomes to make them permeable by the formation of multimeric membrane pores. The pores are size-selective, with a cut-off consistent with a pore that could transport single stranded RNA. We propose that during virus entry, VP4 forms a channel through which the viral RNA is delivered into the cytoplasm for infection to begin. Although VP4 is internal in the virion, capsid breathing exposes it at the surface, where it can interact with antibodies that neutralise infectivity. Importantly, VP4 is highly conserved and inhibitors therefore have good potential to be cross-protective. We therefore believe there is good justification for investigation of VP4 structure & function and proof-of-principle studies to understand and validate VP4 as an antiviral target.

Beyond the academic scientific community, the proposed research may also realise tangible benefits of a social and economic nature. These will be of benefit to the Institute for Animal Health (IAH), the MRC and its stakeholders such as the UK Department of Health and equivalent organizations worldwide. In addition, the outcomes of the research will be of interest to other groups such as the World Health Organization and Global Poliovirus Eradication Initiative, general practitioners, healthcare workers, students and the general public. Engagement with these diverse groups will be achieved via meetings, articles in the trade press, tailored web pages, press releases to the media and travelling shows.

Our proposed studies may lead to new antiviral drugs or new approaches for developing drugs. If such measures are identified, additional funding will be sought from MRC and other sources for further development. There is extensive experience within IAH of patent applications and commercialisation; new opportunities will feed into an established system for technology development and knowledge transfer by the IAH Business Development group, and where appropriate in combination with MRC Technology, the MRC affiliated technology transfer company.