Stability of Structured Kaposi's Sarcoma-Associated Herpesvirus ORF57 Protein Is Regulated by Protein Phosphorylation and Homodimerization

Abstract

Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 plays an essential role in KSHV lytic infection by promoting viral gene expression at the posttranscriptional level. Using bioinformatic and biochemical approaches, we determined that ORF57 contains two structurally and functionally distinct domains: a disordered nonstructural N-terminal domain (amino acids [aa] 1 to 152) and a structured ␣-helix-rich C-terminal domain (aa 153 to 455). The N-terminal domain mediates ORF57 interaction with several RNA-protein complexes essential for ORF57 to function. The N-terminal phosphorylation by cellular casein kinase II (CKII) at S21, T32, and S43, and other cellular kinases at S95 and S97 residues in proximity of the caspase-7 cleavage site, 30-DETD-33, inhibits caspase-7 digestion of ORF57. The structured C-terminal domain mediates homodimerization of ORF57, and the critical region for this function was mapped carefully to ␣-helices 7 to 9. Introduction of point mutations into ␣-helix 7 at ORF57 aa 280 to 299, a region highly conserved among ORF57 homologues from other herpesviruses, inhibited ORF57 homodimerization and led to proteasome-mediated degradation of ORF57 protein. Thus, homodimerization of ORF57 via its C terminus prevents ORF57 from degrading and allows two structure-free N termini of the dimerized ORF57 to work coordinately for host factor interactions, leading to productive KSHV lytic infection and pathogenesis. IMPORTANCEKSHV is a human oncogenic virus linked to the development of several malignancies. KSHV-mediated oncogenesis requires both latent and lytic infection. The KSHV ORF57 protein is essential for KSHV lytic replication, as it regulates the expression of viral lytic genes at the posttranscriptional level. This report provides evidence that the structural conformation of the ORF57 protein plays a critical role in regulation of ORF57 stability. Phosphorylation by CKII on the identified serine/threonine residues at the N-terminal unstructured domain of ORF57 prevents its digestion by caspase-7. The C-terminal domain of ORF57, which is rich in ␣-helices, contributes to homodimerization of ORF57 to prevent proteasome-mediated protein degradation. Elucidation of the ORF57 structure not only enables us to better understand ORF57 stability and functions but also provides an important tool for us to modulate ORF57's activity with the aim to inhibit KSHV lytic replication. K aposi's sarcoma-associated herpesvirus (KSHV) ORF57 (also known as Mta) is expressed early in the KSHV lytic cycle and is required for the efficient expression of a subset of viral genes, including KSHV PAN, ORF59, K8, viral interleukin-6 (vIL-6), ORF47, and others (1-7). A KSHV genome lacking ORF57 expression is associated with a defective lytic cycle incapable of producing infectious virions (8, 9).KSHV ORF57 functions as a posttranscriptional regulator of viral gene expression by affecting RNA stability (PAN, ORF59, and ORF47), splicing (ORF50 and K8), polyadenylation (ORF59), and translation (vIL-6) (1, 2, 4-7, 10) but ...

NP1 Protein of the Bocaparvovirus Minute Virus of Canines Controls Access to the Viral Capsid Genes via Its Role in RNA Processing

“…Because of its specificity, NP1's function and how it regulates RNA processing may be different from these examples. However, a common feature found in HSV-1 ICP27, KSHV ORF57, and HCMV UL69 is an intrinsically disordered region that contains motifs that mediate protein-protein or RNA-protein interaction (53). MVC NP1 has a similar predicted disordered N terminus embedded with SR repeats, which may yet suggest some commonality of function.…”

“…MVC NP1 has a similar predicted disordered N terminus embedded with SR repeats, which may yet suggest some commonality of function. In other systems, posttranslation modification of these regions, including phosphorylation of resident S⁄R residues, have been shown to be important for their function (38,(53)(54)(55)(56). Whether such modifications of NP1 influence its function is currently being investigated.…”

The KSHV RNA regulator ORF57: target specificity and its role in the viral life cycle

“…Indeed, homodimers of ORF57 have been described and recently ICP27 from HSV1 was also shown to exist as a homodimer and the dimerization domain is conserved in ORF57 . In one model an ORF57 homodimer with phosphorylated N‐termini is able to bind a cellular co‐factor and RNA . Here, the cellular protein also binds the RNA and might assist ORF57 target recognition.…”

Alternative RNA splicing of KSHV ORF57 produces two different RNA isoforms

“…After transfer to a nitrocellulose membrane, ORF57 and its fusions were blotted with an anti-ORF57 N- or C-terminal antibody (Majerciak, Kruhlak et al, 2010)(Majerciak, Pripuzova et al, 2015) or an anti-GFP (Clontech) antibody.…”

“…A full-length ORF57 protein, naturally occurring as a homodimer (Majerciak, Pripuzova et al, 2015), is composed of 455 amino acid (aa) residues. In KSHV-infected cells, ORF57 is encoded by an abundant monocistronic transcript initiated from an inducible promoter P 82003 controlled by KSHV transactivator RTA (ORF50) (Lukac, Renne et al, 1998).…”