Cis and Trans acting factors for HHV8 lytic replication

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DESCRIPTION (provided by applicant): Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8) lytic DNA replication is directed by one of two cis acting lytic origins within the genome. OriLyt-L is located between open reading frames (ORFs) K4.2 and K5 and OriLyt-R is located between ORF69 and vFLIP. Transient assays determined that two A+T rich DNA sequences, three AP1 transcription factor binding sites, an ORF50 response element (RE) and a downstream TATA consensus sequence are all required for efficient amplification of oriLyt. Using one of the lytic origins as a reporter we established a cotransfection-replication assay and elucidated 8 required proteins necessary and sufficient to amplify cloned oriLyt. The ORFs are: ORF6 (single-stranded DNA binding protein), ORF9 (DNA polymerase), ORF40/41 (primase-associated factor), ORF44 (helicase), ORF56 (primase), ORF59 (pol processivity factor), ORF50/K-Rta (transactivator) and K8 (unknown function). Previous studies have demonstrated that K-Rta and RAP interact in infected cells. We now show that the ORF50 RE within oriLyt interacts with K-Rta and this region acts as a powerful promoter in transient assays. In addition, RNA transcripts can be detected from a region just downstream of this promoter region. We have also constructed a recombinant HHV8 BAC that does not express the ORF50 gene product, K-Rta. This virus fails to produce infectious virus and does not accumulate viral DNA upon treatment with TPA, an inducer of the viral lytic cycle. This data suggests that K-Rta has a dual role in the viral lytic cycle; induction of viral genes required for lytic replication and participation in viral lytic DNA replication by direct interaction with oriLyt. The hypothesis for this proposal is that RAP performs an essential replication function by interacting with the lytic origin of replication and K-Rta facilitates lytic replication by contributing a transactivator function within oriLyt. To address this hypothesis, in this proposal we will: i) define any additional essential cis acting sequences within oriLyt; ii) determine the role of RAP and K-Rta in the context of the viral genome; and iii) determine RAP and K-Rta binding sites within oriLyt and elucidate protein domains responsible for transactivation and/or DNA replication.