FIG 3.

Three viral noncoding RNAs were identified and confirmed to be directly associated with K8 protein. (A) CLIP-seq reads mapped to the KSHV genome. Enlargement of the K8 cross-linked RNA in the KSHV genomic location showed two peaks (T1.4 RNA and PAN RNA) from the forward strain (left) and one peak (T0.7 RNA) from the reverse strain (right). (B and C) PAN (B) and T1.4 (C) RNAs and their truncation mutants were synthesized in vitro and labeled with biotin. An in vitro RNA pulldown assay was performed to evaluate the abilities of the RNAs and their mutants to be bound by K8. Biotinylated transcripts were mixed with purified GST-K8 protein, and the RNA-protein complexes were precipitated with streptavidin-coupled Dynabeads. The precipitates were analyzed by Western blotting for RNA-bound GST-K8 using anti-GST antibody and by Northern blotting for RNAs using the chemiluminescent nucleic acid detection module.