Kaposi’s sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi’s sarcoma, primary effusion lymphoma, and multicentric Castleman’s disease. KSHV establishes a life-long infection in its host and alternates between a latent and lytic infection state. During lytic infection, lytic-related genes are expressed in a temporal manner and categorized as immediate early, early, and late gene transcripts. ORF34 is an early-late gene that interacts with several viral transcription-associated factors, however its physiological importance remains poorly understood. Here, we investigated the role of ORF34 during KSHV infection by generating ORF34-deficient KSHV, using a bacterial artificial chromosome system. Our results reveal that ORF34-deficient KSHV exhibited significantly attenuated late gene expression and viral production but did not affect viral DNA replication. ORF34 interacted with transcription factors ORF18, ORF24, ORF31, and ORF66, and a novel ORF34-interaction partner, ORF23. The C-terminal region of ORF34 was important for interaction with ORF24 and viral production. Our data support a model, in which ORF34 serves as a hub for recruiting a viral transcription complex to ORF24 to promote late viral gene expression.

Kaposi’s 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 (L) gene transcripts. The transcriptional initiation of KSHV late genes is thought to require the complex formation of the viral preinitiation 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 bacterial artificial chromosome (BAC) system to evaluate its role during viral replication. While ORF66-deficient KSHV demonstrated mainly attenuated late gene expression and decreased virus production, viral DNA replication was unaffected. Chromatin immunoprecipitation analysis showed that ORF66 bound to the promoters of a late gene (K8.1) but did not bind to those of a latent gene (ORF72), an immediate early gene (ORF16), or an 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 the interaction with ORF34, the transcription of K8.1, and virus 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 virus production. IMPORTANCE KSHV ORF66 is expressed during the early stages of lytic infection, and ORF66 and vPIC are thought to contribute significantly to late gene expression. However, the physiological importance of ORF66 in terms of vPIC formation remains poorly understood. Therefore, we generated an ORF66-deficient BAC clone and evaluated its viral replication. The results showed that ORF66 plays a critical role in virus production and the transcription of L genes. To our knowledge, this is the first report showing the function of ORF66 in virus replication using ORF66-deficient KSHV. We also clarified that ORF66 interacts with the transcription start site of the K8.1 gene, a late gene. Furthermore, we identified the ORF34-binding motifs in the ORF66 C terminus: three C-X-X-C sequences and a leucine-repeat sequence, which are highly conserved among beta- and gammaherpesviruses. Our study provides insights into the regulatory mechanisms of not only the late gene expression of KSHV but also those of other herpesviruses.

25Kaposi's sarcoma-associated herpesvirus (KSHV) is closely 26 associated with B-cell and endothelial cell malignancies. After the initial 27 infection, KSHV retains its viral genome in the nucleus of the host cell and 28 establishes a lifelong latency. During lytic infection, KSHV encoded lytic-related 29 proteins are expressed in a sequential manner and are classified as immediate 30 early, early, and late gene transcripts. The transcriptional initiation of KSHV late 31 genes is thought to require the complex formation of the virus specific pre-32 initiation complex (vPIC), which may consist of at least 6 transcription factors 33 (ORF18, 24, 30, 31, 34, and 66). However, the functional role of ORF66 in vPIC 34 during KSHV replication remains largely unclear. Here, we generated ORF66-35 deficient KSHV using a BAC system to evaluate its role during viral replication. 36While ORF66-deficient KSHV demonstrated mainly attenuated late gene 37 expression and decreased viral production, viral DNA replication was 38 unaffected. CHIP analysis showed that ORF66 bound to the promoters of late 39 gene (K8.1), but did not to those of latent gene (ORF72), immediate early gene 40 (ORF16) and early gene (ORF46/47). Furthermore, we found that three highly 41 conserved C-X-X-C sequences and a conserved leucine-repeat in the C-42 terminal region of ORF66 were essential for interaction with ORF34 and viral 43 production. The interaction between ORF66 and ORF34 occurred in a zinc-44 dependent manner. Our data support a model, in which ORF66 serves as a 45 critical vPIC component to promote late viral gene expression and viral 46 production. 47 IMPORTANCE 49 KSHV ORF66, a late gene product, and vPIC are thought to contribute 50 significantly to late gene expression during the lytic replication. However, the 51 physiological importance of ORF66 in terms of viral replication and vPIC 52 formation remains poorly understood. Therfore, we generated a ORF66-53 deficient BAC clone and evaluated its viral replication. Results showed that 54 ORF66 played a critical role in virus production and the transcription of L genes. 55To our knowledge, this is the first report showing ORF66 function in virus 56 replication using ORF66-deficient KSHV. We also clarified that ORF66 57 interacted with the transcription start site of K8.1 gene, a late gene. 58 Furthermore, we identified the ORF34-binding motifs in the ORF66 C-terminus: 59 three C-X-X-C sequences and a leucine-repeat sequence, which are highly 60 conserved among -and -herpesviruses. Our study provides insights into the 61 regulatory mechanisms of not only the late gene expression of KSHV but also 62 those of other herpesviruses. 63 64 65 replication. Finally, the transcriptional products of L genes, contribute to viral 90 particle formation by its encoding viral structure proteins (15). 91The viral pre-initiation complex (vPIC) has recently been proposed to 92 regulate L gene expression(16). vPIC is composed of several viral proteins 93 conserved among -and -herpesviruses (17), and has f...

Kaposi’s sarcoma herpesvirus (KSHV) ORF34 is a component of the viral pre-initiation complex (vPIC), a highly conserved piece of machinery essential for late gene expression among beta- and gamma-herpes viruses. KSHV ORF34 is also estimated to be a hub protein, associated with the majority of vPIC components. However, the precise mechanisms underlying how the ORF34 molecule contributes to the vPIC function, including the binding manner to other vPIC components, remain unclear. Therefore, we constructed ORF34 alanine-scanning mutants, in which amino-acid residues that were conserved among other herpesviruses had been replaced by alanine. The mutants were analyzed for their binding functions to other vPIC factors, and then were evaluated for their recovering ability of viral production using the cells harboring ORF34-deficient KSHV-BAC. The results demonstrated that at least four cysteines conserved in ORF34 were crucial for binding to other vPIC components, ORF24 and ORF66, virus production, and late gene transcription and expression. Based on the amino acid sequence of ORF34, these four cysteines were expected to constitute a pair of C-Xn-C consensus motifs. An artificial intelligence-predicted structure model revealed that the four cysteines were present tetrahedrally in an intramolecular fashion. Another prediction algorithm indicated the possible capture of metal cations by ORF34. Furthermore, it was experimentally observed that the elimination of cations by a selective chelator resulted in the loss of ORF34’s binding ability to other vPIC components. In conclusion, our results suggest the functional importance of KSHV ORF34 conserved cysteines for vPIC components assembly and viral replication.