Viral Capsid-Membrane Interactions Propel Non-Brownian Movements of Non-enveloped Reoviruses during Entry

Understanding how non-enveloped viruses breach host cell membranes is critical for developing strategies to block viral entry, a key step in infection. Despite extensive study, how viral capsids and host lipid membranes dynamically cooperate during membrane penetration remains poorly defined. Here, using reovirus as a model non-enveloped virus and planar model membranes, we identify previously unrecognized non-Brownian membrane motions of infectious subvirion particles (ISVPs) by single-virus tracking. We then integrate experiments with computational modeling to dissect the stepwise, processive capsid-membrane interactions encoded in these distinct dynamics. We show that ISVP motion transitions from an initial phase of directed translocation to progressively confined diffusion. This behavior reflects a multistep entry mechanism in which initial capsid-membrane contact triggers release of the membrane-active 1N peptide. As 1N accumulates within the bilayer, it generates membrane-associated viral retention sites that promote further virus adsorption and increasingly constrain particle mobility. By directly visualizing these motion signatures, we resolve transient and cooperative capsid-membrane interactions that are difficult to capture using conventional biochemical approaches. Together, these findings provide new insight into early membrane penetration events of non-enveloped viruses. SIGNIFICANCE STATEMENTNon-enveloped viruses must penetrate host cell membranes to initiate infection, yet the dynamic mechanisms by which viral capsids engage and remodel lipid membranes remain poorly understood. By directly visualizing the motion of individual reovirus particles on model membranes, we uncover previously unrecognized non-Brownian dynamics that encode stepwise capsid-membrane interactions during viral entry. Integrating single-virus tracking with computational modeling reveals how peptide-mediated membrane remodeling feeds back to regulate viral engagement. This work provides the first direct visualization of processive, stepwise capsid-membrane interactions during non-enveloped virus entry and establishes viral particle dynamics as a quantitative readout for dissecting membrane penetration mechanisms.