Madison Edwards Barnett, Wake Forest University

Madison Edwards Barnett and Sarah M. Esstman, Department of Biology, Wake Forest University

Virus Replication: Entry, Exit and Everything in Between

The rotavirus virion consists of three capsid layers: an inner VP2 core shell layer, an intermediate VP6 layer, and an outer VP7-VP4 layer. Rotavirus assembly first yields a double-layered particle (DLP), which becomes a virion following outer layer addition. However, the DLP assembly mechanism is poorly understood, and pre-DLP assembly intermediates are incompletely characterized. Here, we sought to optimize biochemical techniques for isolating pre-DLP intermediates from infected cells to study their compositions and ultrastructures. Wildtype (WT) strain SA11-infected monkey kidney cells were lysed using a Dounce homogenizer, and subviral particles (SVPs) were pelleted by ultracentrifugation. Following incubation of this SVP pellet for 10 minutes at 50-65℃ and native agarose gel electrophoresis, two putative pre-DLP intermediates were resolved. One intermediate migrated in the gel similar to the control DLP, but it showed a difference in its thermostability. Specifically, the “DLP-like” intermediate was stable at 65℃, while the DLP was unstable, suggesting that these are compositionally different particles. A second intermediate migrated similar to the single-layered core. Both the control core and the “core-like” intermediate were stable at 65℃, suggesting similarity between these particles. To further investigate these intermediates, we performed experiments using tsVP6, an SA11 mutant with a temperature-sensitive block in DLP assembly. Following incubation of tsVP6 SVPs at 65℃, only the “core-like” intermediate was resolved. This result suggests that the “DLP-like” intermediate of tsVP6, but not that of WT, is thermosensitive. Future work will employ this isolation procedure with WT and tsVP6 to reveal new insights into rotavirus DLP assembly.