KSHV 2.0: A Comprehensive Annotation of the Kaposi's Sarcoma-Associated Herpesvirus Genome Using Next-Generation Sequencing Reveals Novel Genomic and Functional Features
Figure 5
KSHV employs multiple strategies to expand and regulate its coding repertoire.
(A–C) Novel splice variant of ORF57 detected by mRNA-Seq. Tracks for mRNA-Seq and Ribo-Seq in CHX treated cells (48 hpi) show the new, shorter-splice variant of ORF57, ORF57A (red box). Splicing of the second intron in ORF57 removes the UAA stop codon, resulting in the generation of a 33 aa C-terminal exon. (B) Zoom in of the region highlighted in (A). Note that ribosomes accumulate downstream of the ORF57 stop codon, indicating that the new exon may be translated. (C) Verification of ORF57A splicing by end-point PCR in lytic iSLK-219 (72 hpi) and LEC-219 cells. The primers used for amplification flank the intron boundaries and are shown in panel (A) as solid black lines. The high molecular weight product (∼800 bp) corresponds to the unspliced form of ORF57. The low molecular weight product (165 bp), corresponds to ORF57A, from which the second intron has been removed. (D–E) mRNA editing in the RTA and Kaposin transcripts. Sanger sequencing of RTA (D) and Kaposin (E) genomic DNA (top panel) and cDNAs (48 hpi) (bottom panel) from iSLK-219 cells. The edited nucleotide is bold, italic font. (F–G) ORF54 translation starts from two alternative initiation codons. (F) Ribo-Seq tracks for cycloheximide (CHX blue) and harringtonine (Harr teal) treated cells at 72 hpi. The dotted lines indicate the two translation initiation sites used for ORF54 translation. (G) Immunoblot for ORF54 in latent and lytic (48 hpi) iSLK cells, infected with Wt KSHV or an ORF54 knock-out virus (ΔORF54). *Non-specific product. See also Figures S6, S7 and S8.