FIG. 3.
Triangular interaction among LANA, TR DNA, and SUV39H1. (A) Biotinylated-DNA pull-down assay. 293 cells were transfected with expression vectors for LANA (wild type [wt]) or its mutant (LNΔN), which lacked the SUV39H1-interacting site, and pCMV5-flagSUV39H1. Biotinylated DNA of either the LBS or the MCS of pBS-SKII (Stratagene) as a control DNA fragment was incubated with NE from the transfectants and bound to streptavidin-Sepharose (Amersham Bioscience). Precipitates were analyzed on Western blots (WB) probed with a mouse anti-V5 monoclonal antibody and a mouse anti-FLAG monoclonal antibody. The DNA sequence of the LBS is shown. (B) IFA of 293LANA cells with or without the BAC-TR construct. The focal concentration of LANA was detected in 293LANA cells carrying BAC-TR. (C) Colocalization of LANA with SUV39H1 in 293 cells bearing the artificial KSHV DNA (BAC-TR). 293LANA cells were transiently transfected with pCMV5-FLAG-SUV39H1 with (panels 1 to 3) or without (panels 4 to 6) BAC-TR. (D) Association between LANA and heterochromatin in Vero cells stably expressing GFP-LANA with BAC-TR or the control BAC construct (BAC-neor). Parental Vero cell (panels 1 to 3), Vero/gfpLANA cells with the empty BAC (panels 4 to 11), and Vero/gfpLANA cells with BAC-TR (panels 12 to 15) were stained with an anti-HP-1α antibody (panels 1, 4, 8, and 12). The IFA was performed as described in Materials and Methods.