Real-time single particle analysis of reovirus-membrane interactions that drive infection

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Real-time single particle analysis of reovirus-membrane interactions that drive infection To deliver their genomic material into the host cells for replication, viruses must cross a host membrane. For non-enveloped viruses that lack a lipid membrane, successful entry requires perforation of the host membrane. However, due the absence of methods to analyze membrane-associated events in entry that occur transiently, this step is poorly understood. In this project, we will develop a new approach to quantify and analyze interactions between non-enveloped viruses and host membranes in real time and at a single particle resolution. We will use reovirus as our experimental model. Entry of reovirus involves initial disassembly to expose the membrane penetration protein µ1 and generate an infectious subvirion particle (ISVP). ISVPs undergo a conformational transition to generate ISVP*s and release a myristylated, µ1 N-terminal fragment, myr-µ1N. The function of myr-µ1N is essential for infection. The myr-µ1N peptide associates with the membrane, recruits ISVPs to the membrane, and promotes the release of additional myr-µ1N peptides via a positive feedback loop. Myr-µ1N is also involved in pore formation. How myr-µ1N mediates all of these functions is unknown. Our hypothesis is that the processive three-way interactions between ISVPs, membrane, and membrane associated myr-µ1N, is required for efficient infection. We will test this hypothesis with the aid of two Specific Aims. In Aim 1, we will identify determinants within myr-µ1N that are important for membrane binding, pore formation, and ISVP recruitment. We will also determine the consequences of disrupting each of these myr-µ1N functions on virus infection. Our preliminary evidence indicates that ISVPs incubated with supported lipid bilayers display progressive interactions - including random motion, non-random motion, and ultimately stable attachment. In Aim 2, using mutant viruses that release different amounts of myr-µ1N or those that release myr-µ1N at different rates, we will define the relationship between myr-µ1N release and particle motion. We will also determine how changing progressive interactions affects virus infectivity. Completion of this work will illuminate the elusive aspects of non-enveloped virus entry. Further, the development of new tools to study these membrane-proximal steps in entry will be transformational for uncovering membrane penetration events of other non-enveloped viruses.