Fig 1. Overview of VZV BAC development with a degron insertion and TMP-dependent protein turnover.

(A) A target gene in the VZV BAC is engineered by recombining a degron sequence with an interrupting kanamycin resistance cassette (kan^r) that allows for positive selection in E. coli GS1783 as detailed in the methods. A second induced recombination event in conjunction with expression of the homing endonuclease I-SceI results in markerless excision of the kan^r cassette, so that the degron coding protein is fused to the target gene ORF. BACs are then transfected into human TRPE cell monolayers in the presence of the stabilizing ligand trimethoprim (TMP) to yield infectious VZV. (B) In the presence of TMP (top), TMP (green) is thought to bind the degron and prevent ubiquitin (Ub, red) ligation, thus stabilizing protein and halting turnover. In the absence of TMP (bottom), Ub is ligated to the degron, and the entire protein is targeted for degradation by ubiquitin-proteasome pathway. Created with BioRender.com.