Picornavirus capsid protein VP4: Essential role in cell entry and conserved antiviral target
Lead Research Organisation:
The Pirbright Institute
Department Name: Pirbright Laboratory
Abstract
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.
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.
Technical Summary
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.
Planned Impact
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.
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.
Publications
Panjwani A
(2014)
Capsid protein VP4 of human rhinovirus induces membrane permeability by the formation of a size-selective multimeric pore.
in PLoS pathogens
Guedán A
(2017)
Investigation of the Role of Protein Kinase D in Human Rhinovirus Replication.
in Journal of virology
Kelly JT
(2022)
Membrane Interactions and Uncoating of Aichi Virus, a Picornavirus That Lacks a VP4.
in Journal of virology
Zhu L
(2016)
Structure of human Aichi virus and implications for receptor binding.
in Nature microbiology
Zhu L
(2015)
Structure of Ljungan virus provides insight into genome packaging of this picornavirus.
in Nature communications
Panjwani A
(2016)
The conserved N-terminus of human rhinovirus capsid protein VP4 contains membrane pore-forming activity and is a target for neutralizing antibodies.
in The Journal of general virology
Panjwani A
(2016)
What Is Stopping the Use of Genetically Modified Insects for Disease Control?
in PLoS pathogens
Description | VP4 induces membrane permeability via formation of size selective pores. Structure of Ljungan virus provides insight into genome packaging of this picornavirus. |
Exploitation Route | Use in identifying novel antivirals |
Sectors | Agriculture, Food and Drink,Healthcare,Pharmaceuticals and Medical Biotechnology,Other |
Description | INsect Policy |
Geographic Reach | Europe |
Policy Influence Type | Citation in other policy documents |
Impact | Improved regulatory environment for future use of GM Insects in vector control |
Description | SGM policy committee |
Geographic Reach | National |
Policy Influence Type | Membership of a guideline committee |
Description | HMS |
Organisation | Harvard University |
Department | Harvard Medical School |
Country | United States |
Sector | Academic/University |
PI Contribution | Collaboration |
Collaborator Contribution | Collaboration |
Impact | Collaborative research |
Description | JS - Production of monoclonal antibodies that recognise FMDV VP4 from mouse spleens |
Organisation | University of Edinburgh |
Department | The Roslin Institute |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | VLPs displaying the N-terminal 15 amino acids of FMDV were designed and produced at The Pirbright Institute and then mice were immunised with them. The spleens of the immunised mice were harvested and splenocytes stored. The response to the VP4 sequence was checked by ELISA and the mouse with the best response was selected to send the splenocytes for monoclonal production. |
Collaborator Contribution | The collaborators took the splenocytes obtained from the mouse experiments and carried out the fusions. They then screened the supernatants for positive wells that detected the VP4 sequence. |
Impact | Positive wells have been identified indicating some monoclonals that are specific for VP4 have been generated. These will be further screened for ability to recognise virus and to neutralise infection. |
Start Year | 2017 |
Description | KU Leuven |
Organisation | University of Leuven |
Country | Belgium |
Sector | Academic/University |
PI Contribution | collaboration |
Collaborator Contribution | collaboration |
Impact | collaboration |
Start Year | 2012 |
Description | Leeds FBS |
Organisation | University of Leeds |
Department | Faculty of Biological Sciences |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Research |
Collaborator Contribution | Research and student supervision |
Impact | Research |
Start Year | 2009 |
Description | Oxford - Strubi |
Organisation | University of Oxford |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Collaboration on virus structural biology and joint PhD studentship |
Collaborator Contribution | Collaboration on virus structural biology and joint PhD studentship |
Impact | Collaboration on virus structural biology has produced a number of novel virus structures and new understanding of virus entry and packaging |
Start Year | 2008 |
Description | Diamond (TT) |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Diamond Light Source Open Day - explaining to general public the importance of structural biology and microscopy for understanding viruses and designing improved vaccines. |
Year(s) Of Engagement Activity | 2019 |
Description | Diamond light source open day 7th Nov 2015 |
Form Of Engagement Activity | Participation in an open day or visit at my research institution |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Public/other audiences |
Results and Impact | Representing The Pirbright Institute at the Diamond light source open day 7th Nov 2015 |
Year(s) Of Engagement Activity | 2015 |
Description | JTK - Microbiology Society Conference 2017 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Other audiences |
Results and Impact | Presenting findings at the Microbiology Society annual conference, this stimulated further interest in the work. |
Year(s) Of Engagement Activity | 2017 |
Description | Policy on GM Insects |
Form Of Engagement Activity | Participation in an activity, workshop or similar |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Policymakers/politicians |
Results and Impact | Organisation of a private policy meeting at the House of Lords and an open event at Charles Darwin house on 10th Feb, 2015 on "GM insects and disease control". |
Year(s) Of Engagement Activity | 2015 |
Description | SinoPic TT |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | 'SinoPic': Structural Biology of Picornaviruses meeting in China. Generating interest and collaborations in virus structural biology within China. |
Year(s) Of Engagement Activity | 2018 |
Description | University of Leeds postgraduate symposium titled Capsid protein VP4 of foot-and-mouth disease virus as a novel target for disease control. |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | National |
Primary Audience | Postgraduate students |
Results and Impact | University of Leeds postgraduate symposium presentation titled Capsid protein VP4 of foot-and-mouth disease virus as a novel target for disease control. |
Year(s) Of Engagement Activity | 2015 |