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. Author manuscript; available in PMC: 2021 Feb 1.
Published in final edited form as: Virology. 2019 Dec 30;541:160–173. doi: 10.1016/j.virol.2019.12.011

Figure 3. Construction of recombinant KSHV clones lacking vIRFs.

Figure 3.

(A) WT and vIRF-KO BAC16 DNAs were digested with SbfI and analyzed by PFGE. (B) iSLK cells latently infected by 3xFLAG-vIRF or derivative vIRF-KO recombinant BAC16 clones were treated with 1 μg/ml Dox and 1 mM NaB to induce lytic reactivation for 48 hpi. The loss of vIRF protein expression was confirmed using FLAG-specific immunoblots. RTA and LANA were included as KSHV viral protein controls. (C) To confirm vIRF3 as a lytic protein, 293T cell lines carrying either BAC16-vIRF3–3xFLAG or BAC16-ΔvIRF were treated with 3 mM NaB to induce lytic reactivation. vIRF3 protein expression was determined using FLAG- and vIRF3-specific immunoblots. The asterisk marks a non-specific band in the vIRF3 immunoblot. (D) Immunofluorescence analysis of the expression of vIRF3 using FLAG antibody in latent and reactivated (60 hpi) iSLK-BAC16-vIRF3–3xFLAG cells. (E) Immunofluorescence analysis of vIRF3 expression using vIRF3 antibody in latent and reactivated (60 hpi) iSLK-BAC16 cells.