FIG 9.

Identification of GGT6 as a pivotal host factor for KSHV lytic reactivation. (A) RT-qPCR measurement of GGT6 expression in shGGT6-treated iSLK cells. (B) RTA expression in shGGT6-treated uninduced and Dox-treated iSLK cells. The quantification of protein expression on the immunoblots is shown as relative fold change. (C) RT-qPCR analysis of latent and reactivated iSLKBAC16 cells treated with different GGT6-specific shRNAs. N.D, not detectable. (D) Cell death analysis of shGGT6-treated reactivated iSLKBAC16 cells at 24 hpi (n.s., not significant). (E) RT-qPCR measurement of the expression of viral genes in the samples described in panel C. (F) Immunoblot analysis of viral protein expression in samples described in panel C. (G and H) iSLKBAC16 cells were treated with shControl or shGGT6 #1 for 48 h, followed by Dox/NaB induction for 72 h, and then viral DNA in the reactivated cells (G) and the virion DNA purified from the supernatant (H) were measured by qPCR. (I) Equal amounts of supernatant from shControl and shGGT6 #1 samples described in panel H were used to infect 293T cells. The KSHV DNA level was measured by qPCR in the infected 293T cells at 24 hpi. Representative immunofluorescence images show 293T cells infected with supernatant derived from shControl- or shGGT6 #1-treated reactivated iSLKBAC16 cells. Green fluorescent protein (GFP) is constitutively expressed from BAC16, which is used for the detection of infected cells. (J) RT-qPCR evaluation of GGT6 expression in shGGT6 #1-treated TRExBCBL1-3×FLAG-RTA cells at 0 hpi (latency) and at 24 hpi (lytic reactivation). (K) TRExBCBL1-3×FLAG-RTA cells were treated with shGGT6 for 2 days followed by Dox induction of 3×FLAG-RTA to trigger lytic viral reactivation for 12 and 24 h. Immunoblot analysis was performed for viral and host proteins indicated on the left. The quantification of protein expression on the immunoblots is shown as relative fold change. N.D, not detectable; *, P < 0.05.