Presentation description

Sindbis Virus is an alphavirus used for directed evolution. Directed evolution allows us to evolve or modify proteins for engineering or biochemical purposes. These evolved proteins are subsequently used in a variety of biological systems to reprogram cellular biology. Evolving these proteins within the intended target cell context is critical for achieving high efficacy tools for cellular engineering. However, the infectivity of Sindbis virus is limited across cell types, impairing its use as a directed evolution vector in those cells. Mutations to the Sindbis virus glycoprotein coat, which mediates cell entry, have been observed to improve viral infectivity in various cell types. Many other mutations have been observed to fix and stabilize in continuous passaged Sindbis virus populations. Few of these mutations have been tested to evaluate if they improve viral infectivity in cultured cells.
To address this question, I synthesized three mutated versions of the glycoprotein to see if the altered versions allowed for better viral infectivity. Firstly, I synthesized the glycoprotein-coding plasmid DNA containing our desired mutants, categorized as mutant 1, mutant 2, and mutant 3 (or M1, M2, and M3, respectively). Secondly, I made RNA from the DNA mutant plasmids that were created using in vitro transcription. To test infectivity, I will electroporate BHK21 cells with a three part viral system consisting of (1) RNA expressing the replication machinery and a gene of interest (tTA), (2) RNA expressing the capsid protein, and (3) four different versions of RNA expressing the glycoprotein: wild-type (WT), M1, M2, and M3. Then, I will harvest the virus and quantify the amount using RT-qPCR. Lastly, I will infect fresh cells with equal concentrations of the virus from each harvested condition and measure how much infection occurs with an infection sensor. With this work, I hope to increase infectivity of the sindbis virus to increase the overall efficacy of the directed evolution system used in the English Lab.