Conserved cysteine residues in Kaposi's sarcoma herpesvirus ORF34 are necessary for viral production and viral pre-initiation complex formation.

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.