FIGURE 5.

ORF45/RSK axis is required for efficient assembly of translation initiation complex during KSHV lytic replication. A, KSHV lytic replication induces eIF4B phosphorylation and its association with the translation initiation complex. The iSLK.219 cells maintain strict latency but support robust lytic reactivation upon induction with doxycycline (35). The iSLK.219 and its parental uninfected iSLK cells were treated with Dox. At the indicated time after treatment, cell lysates were prepared and analyzed by Western blot with the specified antibodies (top panel). The lysates were also used in m⁷GTP pulldown assays (lower panel). B, KSHV lytic replication induces phosphorylation of eIF4B through a distinct pathway. The iSLK cells were treated with Dox for 70 h; U0126 (25 μm) and/or rapamycin (Rapa) (50 nm) was added to the medium and incubated for 2 h. The cell lysates were then analyzed by Western blot with antibodies as indicated. C, phosphorylation of eIF4B during KSHV lytic replication requires RSK activity. The iSLK.219 cells were induced with Dox for 70 h and then treated with 10 μm BI-D1870 for 2 h. Cell lysates were analyzed by Western blotting with the antibodies indicated. D, KSHV-induced phosphorylation of eIF4B depends on ORF45. The iSLK cells were transfected with BAC36 or BAC-stop45 and then subjected to hygromycin selection, becoming cells latently infected with wild-type or ORF45-null recombinant KSHV viruses. The cells were treated with doxycycline for 3 days. Cell lysates were then prepared and analyzed by Western blots with antibodies as indicated. LANA, latency-associated nuclear antigen.