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. 2014 Sep 3;8:86. doi: 10.3389/fnana.2014.00086

Figure 1.

Figure 1

Design and construction of the IE180-null PRV mutant. (A) Recombineering strategy for deletion of both copies of IE180 from the PRV pBecker3 BAC carrying the PRV genome. (B) Inducible IE180 trans-complementation system for packaging IE180-null PRV. To make the PK15-IE180 cell line we first stably expressed the reverse Tet-transactivator rtTA in PK15 cells. We then inserted IE180 under the control of the Tet regulated pTreTIGt promoter. The addition of doxycycline to the cell growth media results in IE180 expression and thus viral production. (C) PK15-rtTA cells and PK15-IE180 cells were infected with IE180-null PRV and imaged 48 h later. Cytopathic effects associated with PRV replication were observed only in PK15-IE180 cells and were enhanced by the addition of doxycycline. (D) Titers of viral supernatants produced by PK15 and PK15-IE180 cells infected with IE180-null PRV (harvested 3 days after infection) at MOI of 1, 0.1 or 0.01 quantified on PK15-IE180 cells. No infectious material can be detected in supernatants of PK15 cultures lacking IE180.