Mutation of a C-Terminal Motif Affects Kaposi's Sarcoma-Associated Herpesvirus ORF57 RNA Binding, Nuclear Trafficking, and Multimerization

Taylor, Adam; Jackson, Brian R.; Noerenberg, Marko; Hughes, David J.; Boyne, James R.; Verow, Mark; Harris, Mark; Whitehouse, Adrian

doi:10.1128/jvi.00138-11

Cited by 17 publications

(29 citation statements)

References 51 publications

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“…Introduction of point mutations into the conserved residues within the region blocked ORF57 homodimerization and triggered rapid degradation of ORF57. These data are in contrast to the report that the RGG2 motif (aa 372 to 374) in ␣-helix 10 is critical for ORF57 multimerization (52). Experimentally, we were unable to confirm the role of the RGG2 motif in ␣-helix 10 in ORF57 homodimerization because using a peptide vm3 corresponding to ␣-helix 10 did not affect dimerization of ORF57 (Fig.…”

Section: Discussioncontrasting

confidence: 99%

“…ORF57 does not form a heterodimer with the EBV EB2 protein, a closely related homologue that also forms a homodimer in mammalian cells. By comparing the N-terminal half to the C-terminal half of ORF57 in nuclear translocation of a cytoplasmic reporter protein, the C-terminal half of ORF57 was also identified for the homodimerization of ORF57 as reported previously (47,52). By using small peptides derived from the ORF57 C terminus, we further identified the peptide vm5-corresponding ␣-helix 7, which is highly conserved among ORF57 homologues, as an interaction interface for the ORF57 homodimer.…”

Section: Discussionsupporting

confidence: 55%

“…7B). It might be that the RGG2 mutation changes the overall ORF57 structure, as those author suggested (52).…”

Section: Discussionmentioning

confidence: 96%

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

Majerčiak

Pripuzova

Chan

et al. 2015

J Virol

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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 ...

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Section: Discussioncontrasting

confidence: 99%

Section: Discussionsupporting

confidence: 55%

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

Majerčiak

Pripuzova

Chan

et al. 2015

J Virol

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“…KH domains contain essential ␤-sheets and overall have different folds than seen in our structure (49). A fold for the KSHV ORF57 was also generated using computational methods, which is very different from the fold value observed in our structure (50). Many herpes proteins lack significant homology to known structures, but this warrants caution when using weak fold prediction methods for generating working models for their structural information.…”

Section: Figmentioning

confidence: 76%

Structure of the C-Terminal Domain of the Multifunctional ICP27 Protein from Herpes Simplex Virus 1

Patel

Dahlroth

Rajakannan

et al. 2015

J Virol

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Herpesviruses are nuclear-replicating viruses that have successfully evolved to evade the immune system of humans, establishing lifelong infections. ICP27 from herpes simplex virus is a multifunctional regulatory protein that is functionally conserved in all known human herpesviruses. It has the potential to interact with an array of cellular proteins, as well as intronless viral RNAs. ICP27 plays an essential role in viral transcription, nuclear export of intronless RNAs, translation of viral transcripts, and virion host shutoff function. It has also been implicated in several signaling pathways and the prevention of apoptosis. Although much is known about its central role in viral replication and infection, very little is known about the structure and mechanistic properties of ICP27 and its homologs. We present the first crystal structure of ICP27 C-terminal domain at a resolution of 2.0 Å. The structure reveals the C-terminal half of ICP27 to have a novel fold consisting of ␣-helices and long loops, along with a unique CHCC-type of zinc-binding motif. The two termini of this domain extend from the central core and hint to possibilities of making interactions. ICP27 essential domain is capable of forming self-dimers as seen in the structure, which is confirmed by analytical ultracentrifugation study. Preliminary in vitro phosphorylation assays reveal that this domain may be regulated by cellular kinases. IMPORTANCE ICP27 is a key regulatory protein of the herpes simplex virus and has functional homologs in all known human herpesviruses.Understanding the structure of this protein is a step ahead in deciphering the mechanism by which the virus thrives. In this study, we present the first structure of the C-terminal domain of ICP27 and describe its novel features. We critically analyze the structure and compare our results to the information available form earlier studies. This structure can act as a guide in future experimental designs and can add to a better understanding of mechanism of ICP27, as well as that of its homologs. Herpes simplex virus (HSV) and other human herpesviruses (HHVs; e.g., Epstein-Barr virus and varicella-zoster virus) have evolved a unique strategy of virion host shutoff, in which they efficiently suppress the host protein synthesis. At the onset of the lytic phase, viral immediate-early (IE) genes are expressed first. These IE proteins are responsible for inducing the expression of viral early and late genes. The key IE protein of HSV, infectious cell protein 27 (ICP27), helps in the host shutoff function (1, 2). This 63-kDa protein consists of 512 amino acids and is involved in multiple functions, such as stalling host pre-RNA processing, exporting intronless viral mRNAs out of the nucleus, shuttling between host nucleus and cytoplasm facilitated by its nuclear export signal (NES) and nuclear localization signal (NLS), and interacting with RNA via its RGG box (3, 4). Most of these functions are conserved across the Herpesviridae family, and ICP27 homologs are present in all kno...

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“…To maximise the data obtained from the study, cellular fractionation was performed to separate the major cellular compartments where ORF57 is known to be present: the cytoplasm, nucleoplasm and nucleoli (102). Predictably, as ORF57 is a known viral RNA processing protein, its expression led to an enrichment of proteins involved in RNA processing, RNA post-transcriptional modification and gene expression, particularly in the nucleolar fraction, where ORF57 is highly abundant (103,104). Importantly, the analysis also identified an enrichment in DNA repair proteins that led to the discovery of a novel role for ORF57 in genome instability (105).…”

Section: Elucidating the Effects Of Fully Competent γ-Herpesvirus Infmentioning

confidence: 99%

Utilising proteomic approaches to understand oncogenic human herpesviruses (Review)

Owen

Hughes

Baquero-Pérez

et al. 2014

Molecular and Clinical Oncology

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Abstract. The γ-herpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus are successful pathogens, each infecting a large proportion of the human population. These viruses persist for the life of the host and may each contribute to a number of malignancies, for which there are currently no cures. Large-scale proteomic-based approaches provide an excellent means of increasing the collective understanding of the proteomes of these complex viruses and elucidating their numerous interactions within the infected host cell. These large-scale studies are important for the identification of the intricacies of viral infection and the development of novel therapeutics against these two important pathogens.

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Mutation of a C-Terminal Motif Affects Kaposi's Sarcoma-Associated Herpesvirus ORF57 RNA Binding, Nuclear Trafficking, and Multimerization

Cited by 17 publications

References 51 publications

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

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

Structure of the C-Terminal Domain of the Multifunctional ICP27 Protein from Herpes Simplex Virus 1

Utilising proteomic approaches to understand oncogenic human herpesviruses (Review)

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