Kaposi's sarcoma-associated herpesvirus ORF66 is essential for late gene expression and virus production via interaction with ORF34

Kaposi9s sarcoma-associated herpesvirus (KSHV) is closely associated with B-cell and endothelial cell malignancies. After the initial infection, KSHV retains its viral genome in the nucleus of the host cell and establishes a lifelong latency. During lytic infection, KSHV encoded lytic-related proteins are expressed in a sequential manner and are classified as immediate early, early, and late gene transcripts. The transcriptional initiation of KSHV late genes is thought to require the complex formation of the virus specific pre-initiation complex (vPIC), which may consist of at least 6 transcription factors (ORF18, 24, 30, 31, 34, and 66). However, the functional role of ORF66 in vPIC during KSHV replication remains largely unclear. Here, we generated ORF66-deficient KSHV using a BAC system to evaluate its role during viral replication. While ORF66-deficient KSHV demonstrated mainly attenuated late gene expression and decreased viral production, viral DNA replication was unaffected. CHIP analysis showed that ORF66 bound to the promoters of late gene (K8.1), but did not to those of latent gene (ORF72), immediate early gene (ORF16) and early gene (ORF46/47). Furthermore, we found that three highly conserved C-X-X-C sequences and a conserved leucine-repeat in the C-terminal region of ORF66 were essential for interaction with ORF34 and viral production. The interaction between ORF66 and ORF34 occurred in a zinc-dependent manner. Our data support a model, in which ORF66 serves as a critical vPIC component to promote late viral gene expression and viral production.