Title: The mechanism of membrane penetration by rotavirus
Description: The rotavirus outer capsid, comprised of the proteins VP4 and VP7, is an apparatus that has evolved to breach cell membranes and deliver a large replication-competent particle to the cytoplasm. During maturation, VP4 is proteolytically cleaved into two fragments, VP5* and VP8*. VP5* is thought to undergo several conformational rearrangements during virion maturation and cell entry that are reminiscent of the movements of enveloped virus fusion proteins, suggesting a role in membrane penetration. Alternatively, it has been proposed that proteolysis of VP7 after virion uncoating leads to the release of a hydrophobic peptide that mediates membrane penetration. It has been difficult to probe the mechanism of membrane penetration as there is not an efficient technique to specifically mutate rotavirus, largely due to the restrictive mode of rotavirus replication. To circumvent this problem, I have developed a technique for the addition of recombinant VP4 and VP7 to non-infectious, sub-viral particles in vitro to yield highly infectious recoated particles that are similar to authentic virions. Recoating can be used to generate virus particles with mutations in the outer capsid without mutating the viral genome, permitting mutational analysis of functional entry pathways. Ultimately, the role of VP7 in membrane penetration appears questionable, although the sites of cleavage within VP7 that lead to peptide-membrane interaction are defined. I have generated a disulfide-crosslinked VP7 that will likely a viable tool to probe uncoating, as it appears to block entry by stabilizing the outer capsid. Uncoating appears to trigger VP5* membrane interaction and conformational rearrangement.

Membrane interaction by VP5* appears to occur though a short-lived intermediate conformation of the protein and requires the exposure of hydrophobic loops. Membrane interaction by VP5* correlates with many known properties of rotavirus entry, strongly supporting a VP5*-mediated membrane penetration model.