Experimental Evolution Reveals a Genetic Basis for Membrane-Associated Virus Release

Many animal viruses replicate and are released from cells in close association to membranes. However, whether this is a passive process or is controlled by the virus remains poorly understood. Importantly, the genetic basis and evolvability of membrane-associated viral shedding have not been investigated. To address this, we performed a directed evolution experiment using coxsackievirus B3, a model enterovirus, in which we repeatedly selected the free-virion or the fast-sedimenting membrane-associated viral subpopulations. The virus responded to this selection regime by reproducibly fixing a series of mutations that altered the extent of membrane-associated viral shedding, as revealed by full-genome ultra-deep sequencing. Specifically, using site-directed mutagenesis, we showed that substitution N63H in the viral capsid protein VP3 reduced the ratio of membrane-associated to free viral particles by 2 orders of magnitude. These findings open new avenues for understanding the mechanisms and implications of membrane-associated viral transmission.