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.

CRISPR/Cas9-Mediated Knockout and In Situ Inversion of the ORF57 Gene from All Copies of the Kaposi's Sarcoma-Associated Herpesvirus Genome in BCBL-1 Cells

“…Detrimental effect of ORF57 KO on virus production in iSLK/Bac16 cells. After demonstrating that ORF57 KO affects virus genome instability and the expression of various viral lytic genes in BCBL-1 cells, we examined the effect of ORF57 KO on KSHV virion production in iSLK/Bac16 cells carrying a KSHV-GFP genome and an inducible ORF50 transgene under the control of a Tet-On promoter; thus, an infectious KSHVgreen fluorescent protein (GFP) virus could be induced from the KSHV Bac16 genome in the presence of doxycycline (7,36,47). Because puromycin was used to stably maintain the KSHV Bac16 genome in iSLK cells, we added a blasticidin resistance gene to our dual gRNA-expressing vector (gRNAs 2 and 4).…”

Structural identification of conserved RNA binding sites in herpesvirus ORF57 homologs: implications for PAN RNA recognition

“…A dimeric state of KSHV ORF57 was previously indicated by cross-linking experiments (42), and later both monomer and dimer states were observed in crystal structures (49). Therefore we characterized the solution oligomeric state of purified hvsORF57Δ146 and ksORF57Δ153 proteins by SEC-MALS.…”

“…A low-resolution (3.5 Å) structure of ksORF57 (Protein Data Bank code 5ZB3) was recently published which revealed a dimeric fold with a domain swap mediated by an N-terminal arm (49). The ksORF57 structure closely resembled that of hvsORF57 with near-identical secondary structure composition (Figure 4) and superposition of the dimers indicating a Cα RMSD of 2.6 Å, calculated for both chains of the homo-dimer residues using residues 147–417 hvsORF57 (Figure 5C).…”

“…The ICP27 IHD structure revealed a stable homo-dimer, a feature also detected in ksORF57 and other homologs in α- and γ-herpesviruses (41,42,45,46), while the β-herpesvirus homolog UL69 from HCMV forms a tetramer (47,48). Recently, both monomeric and dimeric crystal structures of the C-terminal domain of ksORF57 were solved at 3.1 and 3.5 Å resolution, respectively (49). Proteins from within the same α-, β- or γ-subfamily share closer sequence homology to each other than between subfamilies.…”

Minute Virus of Canines NP1 Protein Interacts with the Cellular Factor CPSF6 To Regulate Viral Alternative RNA Processing

“…3G, compare lanes 2 and 4). The N terminus of NP1 is intrinsically disordered (16), a feature that favors oligomerization and interactions common in many RNA and DNA binding proteins that modulate RNA processing and viral genome replication (41)(42)(43)(44).…”