The crystal structure of KSHV ORF57 reveals dimeric active sites important for protein stability and function

Abstract

Kaposi’s sarcoma-associated herpesvirus (KSHV) is a γ-herpesvirus closely associated with Kaposi’s sarcoma, primary effusion lymphoma and multicentric Castleman disease. Open reading frame 57 (ORF57), a viral early protein of KSHV promotes splicing, stability and translation of viral mRNA and is essential for viral lytic replication. Previous studies demonstrated that dimerization of ORF57 stabilizes the protein, which is critical for its function. However, the detailed structural basis of dimerization was not elucidated. In this study, we report the crystal structures of the C-terminal domain (CTD) of ORF57 (ORF57-CTD) in both dimer at 3.5 Å and monomer at 3.0 Å. Both structures reveal that ORF57-CTD binds a single zinc ion through the consensus zinc-binding motif at the bottom of each monomer. In addition, the N-terminal residues 167–222 of ORF57-CTD protrudes a long “arm” and holds the globular domains of the neighboring monomer, while the C-terminal residues 445–454 are locked into the globular domain in cis and the globular domains interact in trans. In vitro crosslinking and nuclear translocation assays showed that either deletion of the “arm” region or substitution of key residues at the globular interface led to severe dimer dissociation. Introduction of point mutation into the zinc-binding motif also led to sharp degradation of KSHV ORF57 and other herpesvirus homologues. These data indicate that the “arm” region, the residues at the globular interface and the zinc-binding motif are all equally important in ORF57 protein dimerization and stability. Consistently, KSHV recombinant virus with the disrupted zinc-binding motif by point mutation exhibited a significant reduction in the RNA level of ORF57 downstream genes ORF59 and K8.1 and infectious virus production. Taken together, this study illustrates the first structure of KSHV ORF57-CTD and provides new insights into the understanding of ORF57 protein dimerization and stability, which would shed light on the potential design of novel therapeutics against KSHV infection and related diseases.

Author summary

KSHV encoded multifunctional protein ORF57 plays a key role in promoting viral mRNA accumulation and translation. Previous biochemical studies showed that ORF57 is stabilized by dimerization, which is critical for its function. To better understand ORF57 dimerization, we resolved the structure of the C-terminal domain (CTD) of ORF57. We found that ORF57-CTD interacts with the neighboring monomer through the “arm” region and the globular interface, and demonstrated that they are responsible for dimerization. In addition, we identified a zinc ion chelated by four residues of a zinc-binding motif within ORF57-CTD and this motif is conserved among herpesvirus homologues, participating in protein folding and stability. By defining the first crystal structure of ORF57-CTD, this study reveals the detailed mechanisms of ORF57 stability and dimerization, and provides novel insights for therapeutic development against KSHV-related diseases.