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HITS-CLIP Analysis Uncovers a Link between the Kaposi’s Sarcoma-Associated Herpesvirus ORF57 Protein and Host Pre-mRNA Metabolism

Fig 1

Isolation of cross-linked ORF57-RNA complexes for HITS-CLIP analysis.

Top Nitrocellulose membrane from an ORF57 immunoprecipitation performed under HITS-CLIP conditions. Cross-linked RNA fragments were end-labeled with 32P to be visualized by Phosphoimager. Cells were induced to undergo lytic reactivation, exposed to UV and/or treated with high or low concentrations of MNase as indicated. The samples lacking an ORF57 antibody (lane 1) were precipitated with pre-bleed antibodies from the same rabbit. Lanes 6 and 7 are a dark exposure of lanes 4 and 5. The dashed white box indicates the position of the ORF57 complex cut from the membrane for library preparation. Bottom Western blot of the same HITS-CLIP samples shown in the top panel. Affinity purified rabbit anti-ORF57 was used to detect ORF57. Positions of molecular weight markers are shown on the left. On the right, a single asterisk marks the position of a contaminating ~37 kDa protein; double and triple asterisks mark positions of putative ORF57 homodimers and homotrimers bound to the same RNA. The doublet is likely due to an ORF57 cleavage product [104].

Fig 1