FIG 10.

Influence of vIRF-2 and vIRF-2-USP7 interaction on innate immune signaling in the context of lytic infection. (A) Precipitation-immunoblot analyses of (lentivirally transduced, tagged) TRAF3 and TRAF6 ubiquitination during lytic reactivation in doxycycline- and sodium butyrate-treated iSLK cells infected with wild-type (WT), vIRF-2.S₂₄₇A-expressing (247), or vIRF-2-null (TTG) BAC16 virus. Replicate cultures were harvested at day 0 (uninduced, latent) or at 1 or 3 days after lytic induction. Experimental procedures for precipitation were as outlined in the legends to Fig. 5 and 7. (B) Relative levels of expression of interferon-β (IFN-β) transcripts, as determined by RT-qPCR, in lytically reactivated iSLK cells infected with wild-type (WT), vIRF-2.S₂₄₇A-expressing (S₂₄₇A), or vIRF-2 initiator ATG-mutated (TTG) BAC16 virus. Data were derived from duplicate cultures; the average level of IFN-β mRNA in BAC16.vIRF-2.S₂₄₇A- and BAC16.vIRF-2.TTG-infected cells harvested 2 days after lytic induction are expressed relative to levels (set at 1) in parallel wild-type virus-infected cells (error bars indicate standard deviations from the mean values; statistical significance was determined by two-tailed Student's t test). (C) Interactions of TRAF3 and TRAF6 with USP7 in iSLK cells infected with BAC16 (WT) or BAC16.vIRF-2.S₂₄₇A (247). The HHV-8⁺ iSLK cultures were transduced with TRAF3-S- or Flag-TRAF6-encoding lentiviral vector, allowing S-protein affinity precipitation (AP) or Flag-immunoprecipitation (IP) from cell lysates 2 days after lytic induction; precipitates were probed for detection of TRAF-interacting USP7 and precipitated TRAFs 3 and 6 (Tr3 and Tr6), and equivalent expression of wild-type and S₂₄₇A vIRF-2 proteins was verified by immunoblotting of cell lysates.