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

Date Published: August 10, 2018

Publisher: Public Library of Science

Author(s): Fei Yuan, Zeng-Qiang Gao, Vladimir Majerciak, Lei Bai, Meng-Lu Hu, Xiao-Xi Lin, Zhi-Ming Zheng, Yu-Hui Dong, Ke Lan, Pinghui Feng.

http://doi.org/10.1371/journal.ppat.1007232

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.

Partial Text

Kaposi’s sarcoma-associated herpesvirus (KSHV, also known as human herpesvirus 8, HHV8) is a human tumor virus belonging to lymphotropic gammaherpesvirus subfamily [1]. KSHV was first discovered from Kaposi’s sarcoma with endothelial origin, and also found as the etiological agent of two lymphoproliferative disorders, primary effusion lymphoma (PEL) and multicentric Castleman’s disease (MCD) [2–4]. Like other herpesviruses, KSHV establishes life-long persistent latent infection. Endogenous and exogenous stress can trigger KSHV reactivation [5] and viral gene expression in a cascade fashion. During reactivation, the viral lytic switch protein RTA (replication and transcription activator) activates the expression of viral early genes including MTA (mRNA transcript accumulation) encoded by open reading frame 57 (ORF57) [6]. ORF57 or MTA translocates into cell nucleus and promotes lytic replication [7]. Thus, ORF57 deletion virus leads to defective expression of viral lytic genes and abortive viral replication, suggesting that ORF57 is indispensable for KSHV lytic replication [8–10].

In the present study and for the first time, we resolved the 3D structure of the KSHV ORF57-CTD. In our experimental system, direct expression of the full-length ORF57 in Escherichia coli led to extremely low yield. After optimization of the conditions, the N-terminal truncation mutant retaining the residues 167 to 455 (ORF57-CTD) became relatively stable and was successfully crystallized. Finally, we obtained a dimeric structure at 3.5 Å and a monomeric structure at 3.0 Å through X-ray crystallography.

 

Source:

http://doi.org/10.1371/journal.ppat.1007232