Investigation of the biophysical mechanisms and membrane determinants of respirovirus binding and fusion using artificial lipid membranes and isolated physiological membranes as targets

PROJECT SUMMARY/ABSTRACT The broad objectives of this project are A) to investigate the biophysical mechanisms of the initial steps of

respirovirus infection (binding to the host cell and subsequent membrane fusion), and B) to develop/validate biophysical methods used to study viral binding/fusion more broadly. Respiroviruses are members of the paramyxovirus family, and

are a leading cause of respiratory infection, especially in children. Although respiroviruses present a significant health

burden, there are no licensed respirovirus-specific antivirals or vaccines to date. Deeper fundamental understanding of the

respirovirus infection mechanisms will be important to develop therapeutic approaches. To study respirovirus binding and

fusion, this project will use murine respirovirus (a.k.a. Sendai virus), which has been used as a model respirovirus for

some time. Aside from its utility as a model respirovirus, Sendai virus itself is also of considerable interest – its wide

tissue tropism in human cells and lack of pathogenicity in humans has made it an attractive clinical and laboratory vector for gene therapy and vaccine development. Therefore, this study aims not only to expand mechanistic understanding of respirovirus binding and fusion, but also of this useful laboratory tool.

To investigate the biophysical mechanisms of respirovirus binding and fusion, single virus measurements will be employed, observing interactions between single virus particles and host cell membrane mimics called model lipid membranes. Model lipid membranes are lipid bilayers formed from a few lipid components, allowing the experimenter to

investigate key molecular interactions. In Aim 1, this approach will be utilized to investigate the molecular mechanism of viral binding to its receptor in the host cell membrane, how that binding then triggers the virus to initiate membrane fusion, and the role that cholesterol plays in these processes.

Aim 2 will develop and use analytical chemistry methods to study the chemical composition of the model membranes themselves. Model lipid membranes are not only used as host cell membrane mimics in this project, but are

also used as cell membrane mimics in many fields, including in drug delivery, drug formulation, and basic science studies.

They can afford the experimenter precise control over the membrane composition, but it is rare for researchers to examine the composition produced by their preparation method, especially in single virus studies. Therefore, this project will

identify best practices to minimize variability and produce robust results both in single virus studies, and in other fields. Finally, this project also aims to develop new model membrane platforms, using membranes derived from human

red blood cells (Aim 3). These platforms will be used to investigate additional biophysical questions about the influence of membrane environment on respirovirus binding and fusion, and will enable precise control and measurement of single

viruses in the context of a native membrane environment. They will also be useful more broadly to study viral binding and fusion for other viruses as well.