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In bioRxiv : the preprint server for biology

UNLABELLED : 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ORF34 alanine-scanning mutants, in which amino-acid residues that were conserved among other herpesviruses had been replaced by alanine.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.

IMPORTANCE : The gamma- and beta-herpesvirus family conserve the viral-factor based mechanism for initiating viral late gene transcription. This viral pre-initiation complex (vPIC) is a functional analog to cellular PIC consisting of general transcriptional factors. We focused on KSHV ORF34, an essential factor for viral replication as a vPIC component. The precise mechanism underlying vPIC formation and critical domain structure of ORF34 for its function are presently unclear. Therefore, we investigated the contribution of conserved amino-acid residues among ORF34 homologs to virus production, late gene expression, and interaction with other vPIC components. We demonstrated for the first time that four conserved cysteines (C170, C175, C256, and C259) in ORF34 are essential for vPIC formation, late gene transcription, and viral production. Importantly, the predicted structure model and biochemical experiment provide evidence showing that these four conserved cysteines are present in a tetrahedral formation which helped to maintain metal cation.

Watanabe Tadashi, Narahari Akshara, Bhardwaj Esha, Kuriyama Kazushi, Nishimura Mayu, Izumi Taisuke, Fujimuro Masahiro, Ohno Shinji