Alquist, Bickham and Johan den Boon Howard Hughes Medical Institute and Institute for Molecular Virology, University of Wisconsin-Madison

Characterization of Newly Identified Kaposi Sarcoma-Associated Herpesvirus (KSHV) Transcripts


The frnk repeat region in the KSHV genome has not previously been predicted to have protein coding potential because there are no start codons for the initiation of translation nor are there stop codons to signal the end of translation in any of its three open reading frames (ORF). Nevertheless, transcriptional activity in this region can be readily detected. Therefore, we hypothesized that proteins might be synthesized from the frnk transcripts using an alternate translation initiation codon. PCR-based single and triple FLAG-epitope tagging and DNA transfection was used to express the frnk RNAs in 293T cells under the direction of the CMV promoter, but no protein production from any of the three open reading frames of the frnk region was detected. The positive control, FLAG-tagged P53, was clearly detected on both the single FLAG western blot and showed increased detection sensitivity on the triple FLAG western blot. Northern blotting used for RNA analysis proved that the frnk transcripts were produced from the CMV promoter and available. Our inability to detect protein synthesis from the frnk RNAs suggests a non-mRNA type function for these novel KSHV transcripts.