FIG. 4.

KSHV ORF57 preferentially interacts with viral pre-mRNAs. (A) KSHV ORF57 selectively binds to intron-containing viral pre-mRNAs in JSC-1 cells. KSHV ORF57-RNA complexes in JSC-1 cells with or without butyrate (Bu) activation were cross-linked by UV irradiation and immunoprecipitated with anti-ORF57 antibody or nonimmune rabbit serum. RNA extracted from the protein-RNA immunoprecipitates was examined by RT-PCR for the presence of a specific RNA using a pair of gene-specific exon primers (Table 1). The primer pair oST14 and oKY30 was used for LANA detection. (B) KSHV ORF57 selectively binds to K8β pre-mRNAs in the presence of HEK-293 nuclear extract in vitro. FLAG-tagged ORF57 in a full-length (pVM7) or truncated (aa 1 to 251, pVM24) version or the truncated version with point mutations in NLS2 and NLS3 motif (pVM50) purified from HEK-293 cells at 24 h posttransfection was compared to FLAG-tagged ICP27 (pKY15) for RNA binding in vitro. Approximately 200 μg of total protein from each transfection was immobilized on anti-FLAG-conjugated beads before RNA binding. An optimized, ³²P-labeled K8β pre-mRNA (pKY3) (68) transcribed in vitro was spliced for 2 h at 30°C in the presence of HEK-293 nuclear extracts (72). The whole splicing reactions (lanes 3 to 8) or RNAs extracted from the splicing reactions (lanes 11 to 16) were then compared with K8β pre-mRNA (lanes 18 to 23) in RNA binding assays with each protein. Lanes 2, 10, and 24 were the spliced K8β RNA products (pKY3) resolved in an 8% denaturing polyacrylamide gel.