Fig. 6.

5′-triphosphate containing vtRNAs block KSHV lytic reactivation. a iSLK.219 cells were mock transfected, or transfected with 100 ng in vitro transcribed vtRNAs with or without CIP treatment, or a RIG-I ligand RNA (3pRNA) and reactivated by adding Dox 4 h posttransfection. GFP and RFP images were captured 48 h postreactivation. Bar indicates 300 μm. b Quantification of RFP positive cells in (a). c Expression of the indicated viral genes was determined in latent and 48 h post-Dox treatment cells by RT-qPCR. d Expression of the indicated genes was quantified in latent and 24 h post-Dox treatment cells by RT-qPCR. c, d The gene expression was normalized to the level of 18S rRNA and Mock in latent cells was set as 1. Error bars in all panels represent mean ± SD from three independent experiments. p Values were determined by the Student’s t test, ^*p < 0.05, ^**p < 0.01. e The model depicting how RLRs-MAVS pathway is activated and restricts KSHV lytic reactivation. In latency, DUSP11 removes 5′ triphosphates of vtRNAs, thus preventing their recognition by RIG-I. During lytic reactivation, 5′-end processing of vtRNAs is attenuated due a reduction in DUSP11 expression. 5′-ppp-vtRNAs and long dsRNAs are sensed by RIG-I and MDA5, respectively. RLRs elicit an antiviral gene expression program through MAVS and downstream phosphorylation of IRF3