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. 2008 Jan 9;82(6):2792–2801. doi: 10.1128/JVI.01856-07

FIG. 2.

FIG. 2.

KSHV ORF57 promotes the expression of K-bZIP protein by enhancing K8β RNA splicing. (A) Structures of the full-length KSHV K8β cDNA and K8α RNA, with spliced exons (numbered boxes) and introns (line) illustrated. K8β is an alternative spliced product of K8 pre-mRNA and retains a suboptimal intron (intron 2). The numbers above the cDNA or RNA are the nucleotide positions in the KSHV genome (GenBank accession number U75689 [45]). Splice junctions of intron 1 and intron 3 are indicated in K8β cDNA as 75323/75471 and 75838/76433, respectively. K8β encodes a truncated protein (jagged line) of 190 aa. Splicing of the suboptimal intron from K8β RNA leads to the production of K8α mRNA, which encodes a K-bZIP protein (jagged line) of 238 aa. (B) KSHV ORF57 stimulates K-bZIP expression from K8β cDNA in a dose-dependent manner. HeLa and HEK-293 cells were cotransfected with a KSHV K8β cDNA expression vector, pST3, and a FLAG-tagged KSHV ORF57 expression vector, pVM7, or an empty vector, pcDNA3. The protein samples were prepared 24 or 48 h after transfection and analyzed by Western blotting. (C) KSHV ORF57 promotes RNA splicing of K8β pre-mRNAs. Total RNA from HeLa or HEK-293 cells cotransfected as described above was prepared 48 h after transfection and analyzed for K8 by RT-PCR. (D and E) KSHV ORF57 functionally resembles EBV EB2 protein but differs in activity from HSV ICP27 with respect to its effect on splicing of K8β RNA. HSV ICP27 with or without a FLAG tag was compared with FLAG-tagged KSHV ORF57 and Myc-tagged EBV EB2 by contransfection of HEK-293 cells with K8β cDNA as described above for K8 expression. Protein samples prepared 24 h after cotransfection were analyzed by Western blotting. Lower bands in lane 3 (D) are presumed degradation products of ICP27-FLAG. (F) Diagram of three NLSs in a truncated, FLAG-tagged ORF57 and various mutants in which the indicated residues were replaced with neutral or acidic residues. (G) NLSs of ORF57 play an important role in ORF57-mediated enhancement of K8β RNA splicing. The protein samples prepared 24 h after cotransfection of HEK-293 cells with K8β cDNA plus full-length ORF57 (ORF57FL), ORF57 aa 1 to 251 (ORF57S), or ORF57 aa 1 to 251 with one or two NLS mutations were analyzed by Western blotting for K8 and ORF57 expression. Tubulin from each sample in panels B, D, E, and G served as a sample loading control.