Lytic reactivation of KSHV promotes eIF4F assembly.
A. KSHV lytic reactivation increases the incorporation of eIF4E into the eIF4F complex. Non-ionic detergent lysates (INPUT) prepared from TREx BCBL1 or TREx BCBL1-RTA cells either untreated (−) or treated (+) with TPA+DOX for 48 h prior to harvesting were incubated with 7-methyl-GTP (7-M GTP) sepharose. The 7-M GTP cap-bound proteins (CAP) were fractionated by SDS-PAGE and analyzed by immunoblotting using the indicated antisera. B. The abundance of eIF4F-core and associated components remains constant in TREx BCBL1-RTA cells induced to reactivate. Total protein isolated from TREx BCBL1-RTA cells untreated (−) or treated (+) with TPA+DOX for the indicated times was fractionated by SDS-PAGE and analyzed by immunoblotting for the indicated proteins. The asterisk indicates the slower-migrating fully-glycosylated, mature form of K8.1. RhoGDI and actin: loading controls. C. TREx BCBL1-RTA cells contain relatively high levels of eIF4F core and associated proteins. Total protein isolated from equal numbers of live, untreated TREx BCBL1-RTA or primary NHDF cells (1.5×106/ml) was fractionated by SDS-PAGE and analyzed by immunoblotting using the indicated antisera. D. Enrichment of 4E-BP1 hyperphosphorylated isoforms in the 7-M GTP sepharose unbound fraction upon lytic reactivation. As in A except the 7-M GTP sepharose unbound, flow-through fraction (FLOW) was fractionated by SDS-PAGE in 17.5% gels to resolve phosphorylated 4E-BP1 isoforms (hyper- vs. hypophosphorylated indicated by arrowheads) and analyzed by immunoblotting using anti-4E-BP1 antisera. E. PABP redistribution during KSHV lytic reactivation. Left panel: At 48 h post-induction, TREx BCBL1-RTA cells treated as described in A were collected, and immunostained using antisera specific for ORF59 (green signal) or PABP (red signal). Images were viewed with Zeiss LSM510 Meta confocal microscope, and colocalization was evaluated. Right panel: Cells were processed and immunostained for ORF59 and PABP as indicated for the left panel, but additionally counterstained with DAPI and viewed with a Zeiss Axiovert fluorescence microscope. For better contrast, single-stain images are shown in black & white. The Rightmost panel is a merged image showing ORF59 (green), PABP (red), and DAPI (blue).