Phosphoproteomic Profiling Reveals Epstein-Barr Virus Protein Kinase Integration of DNA Damage Response and Mitotic Signaling

Li, Renfeng; Liao, Gangling; Nirujogi, Raja Sekhar; Pinto, Sneha M.; Shaw, Patrick G.; Huang, Tai‐Chung; Wan, Jun; Qian, Jiang; Gowda, Harsha; Wu, Xinyan; Lv, Dongwen; Zhang, Kun; Manda, Srikanth S.; Pandey, Akhilesh; Hayward, S. Diane

doi:10.1371/journal.ppat.1005346

Cited by 57 publications

(62 citation statements)

References 121 publications

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“…However, these studies relied on in vitro assays, and consequently, the substrates identified might not be representative of the true substrates during viral replication. Recently, the same group published data obtained from phosphoproteomic profiling of BGLF4-expressing cells (55). Such experiments would be invaluable in delineating the substrates of KSHV ORF36, as well as other herpesviral kinases.…”

Section: Discussionmentioning

confidence: 99%

Discovery of a Coregulatory Interaction between Kaposi's Sarcoma-Associated Herpesvirus ORF45 and the Viral Protein Kinase ORF36

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Pifer

et al. 2016

J Virol

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Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of three human malignancies. KSHV ORF36 encodes a serine/threonine viral protein kinase, which is conserved throughout all herpesviruses. Although several studies have identified the viral and cellular substrates of conserved herpesvirus protein kinases (CHPKs), the precise functions of KSHV ORF36 during lytic replication remain elusive. Here, we report that ORF36 interacts with another lytic protein, ORF45, in a manner dependent on ORF36 kinase activity. We mapped the regions of ORF36 and ORF45 involved in the binding. Their association appears to be mediated by electrostatic interactions, since deletion of either the highly basic N terminus of ORF36 or an acidic patch of ORF45 abolished the binding. In addition, the dephosphorylation of ORF45 protein dramatically reduced its association with ORF36. Importantly, ORF45 enhances both the stability and kinase activity of ORF36. Consistent with previous studies of CHPK homologs, we detected ORF36 protein in extracellular virions. To investigate the roles of ORF36 in the context of KSHV lytic replication, we used bacterial artificial chromosome mutagenesis to engineer both ORF36-null and kinase-dead mutants. We found that ORF36-null/mutant virions are moderately defective in viral particle production and are further deficient in primary infection. In summary, our results uncover a functionally important interaction between ORF36 and ORF45 and indicate a significant role of ORF36 in the production of infectious progeny virions. IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus with a significant public health burden. KSHV ORF36 encodes a serine/threonine viral protein kinase, whose functions throughout the viral life cycle have not been elucidated. Here, we report that ORF36 interacts with another KSHV protein, ORF45. We mapped the regions of ORF36 and ORF45 involved in their association and further characterized the consequences of this interaction. We engineered ORF36 mutant viruses in order to investigate the functional roles of ORF36 in the context of KSHV lytic replication, and we confirmed that ORF36 is a component of KSHV virions. Moreover, we found that ORF36 mutants are defective in virion production and primary infection. In summary, we discovered and characterized a functionally important interaction between KSHV ORF36 and ORF45, and our results suggest a significant role of ORF36 in the production of infectious progeny virions, a process critical for KSHV pathogenesis. K aposi's sarcoma-associated herpesvirus (KSHV) is a human tumor virus and the causative agent of Kaposi's sarcoma (KS), as well as two lymphoproliferative disorders (1-3). All herpesviruses encode at least one serine/threonine protein kinase that is conserved throughout the three subfamilies (alpha-, beta-, and gammaherpesviruses), collectively referred to as conserved herpesvirus protein kinases (CHPKs) (reviewed in references 4 and 5). Orthologs of CHPKs include UL13 of herpes simp...

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Section: Discussionmentioning

confidence: 99%

Discovery of a Coregulatory Interaction between Kaposi's Sarcoma-Associated Herpesvirus ORF45 and the Viral Protein Kinase ORF36

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Tepper

Pifer

et al. 2016

J Virol

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“…The Akata-BX1 (EBV + ) and Akata-4E3 (EBV -) cells were grown in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS) (Gibco) in 5% CO 2 at 37°C (Li et al, 2011(Li et al, , 2012b (Li et al, 2015). Cells were cultured for at least six doubling times for complete incorporation.…”

Section: Cell Culture and Treatmentmentioning

confidence: 99%

“…23227; Pierce). Peptides were prepared by an in-solution tryptic digestion protocol (Li et al, 2015). Trypsin digestion efficiency was assessed by SDS-PAGE and Coomassie Brilliant Blue staining before further processing.…”

Section: Sample Preparationmentioning

confidence: 99%

“…The enriched peptides were analyzed on an LTQ-Orbitrap Elite mass spectrometer interfaced with Easy-nLC II nanoflow LC system (Thermo Scientific) using the same parameters as preciously described (Li et al, 2015).…”

Section: Et Almentioning

confidence: 99%

“…In this study, we employed stable isotope labeling by amino acid in cell culture (SILAC)-based quantitative proteomics (Li et al, 2015;Pinto et al, 2015;Zhong et al, 2012) to monitor the dynamic changes in protein ubiquitination in EBV + and EBV -B cells upon IgG cross-linking-mediated BCR activation. For the first time, this report describes the long-term effects of BCR activation on protein ubiquitination, which offers a basis for dissecting the cellular environment during the course of EBV lytic replication.…”

Section: Introductionmentioning

confidence: 99%

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Understanding Epstein-Barr Virus Life Cycle with Proteomics: A Temporal Analysis of Ubiquitination During Virus Reactivation

Zhong

Zhang

et al. 2017

OMICS: A Journal of Integrative Biology

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Epstein-Barr virus (EBV) is a human c-herpesvirus associated with cancer, including Burkitt lymphoma, nasopharyngeal, and gastric carcinoma. EBV reactivation in latently infected B cells is essential for persistent infection whereby B cell receptor (BCR) activation is a physiologically relevant stimulus. Yet, a global view of BCR activation-regulated protein ubiquitination is lacking when EBV is actively replicating. We report here, for the first time, the long-term effects of IgG cross-linking-regulated protein ubiquitination and offer a basis for dissecting the cellular environment during the course of EBV lytic replication. Using the Akata-BX1 (EBV + ) and Akata-4E3 (EBV -) Burkitt lymphoma cells, we monitored the dynamic changes in protein ubiquitination using quantitative proteomics. We observed temporal alterations in the level of ubiquitination at *150 sites in both EBV + and EBV -B cells post-IgG cross-linking, compared with controls with no cross-linking. The majority of protein ubiquitination was downregulated. The upregulated ubiquitination events were associated with proteins involved in RNA processing. Among the downregulated ubiquitination events were proteins involved in apoptosis, ubiquitination, and DNA repair. These comparative and quantitative proteomic observations represent the first analysis on the effects of IgG cross-linking at later time points when the majority of EBV genes are expressed and the viral genome is actively being replicated. In all, these data enhance our understanding of mechanistic linkages connecting protein ubiquitination, RNA processing, apoptosis, and the EBV life cycle.

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The anaphase‐promoting complex: A key mitotic regulator associated with somatic mutations occurring in cancer

Melloy

2019

Genes Chromosomes & Cancer

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The anaphase‐promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase that helps control chromosome separation and exit from mitosis in many different kinds of organisms, including yeast, flies, worms, and humans. This review represents a new perspective on the connection between APC/C subunit mutations and cancer. The complex nature of APC/C and limited mutation analysis of its subunits has made it difficult to determine the relationship of each subunit to cancer. In this work, cancer genomic data were examined to identify APC/C subunits with a greater than 5% alteration frequency in 11 representative cancers using the cBioPortal database. Using the Genetic Determinants of Cancer Patient Survival database, APC/C subunits were also studied and found to be significantly associated with poor patient prognosis in several cases. In comparing these two kinds of cancer genomics data to published large‐scale genomic analyses looking for cancer driver genes, ANAPC1 and ANAPC3/CDC27 stood out as being represented in all three types of analyses. Seven other subunits were found to be associated both with >5% alteration frequency in certain cancers and being associated with an effect on cancer patient prognosis. The aim of this review is to provide new approaches for investigators conducting in vivo studies of APC/C subunits and cancer progression. In turn, a better understanding of these APC/C subunits and their role in different cancers will help scientists design drugs that are more precisely targeted to certain cancers, using APC/C mutation status as a biomarker.

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Phosphoproteomic Profiling Reveals Epstein-Barr Virus Protein Kinase Integration of DNA Damage Response and Mitotic Signaling

Cited by 57 publications

References 121 publications

Discovery of a Coregulatory Interaction between Kaposi's Sarcoma-Associated Herpesvirus ORF45 and the Viral Protein Kinase ORF36

Discovery of a Coregulatory Interaction between Kaposi's Sarcoma-Associated Herpesvirus ORF45 and the Viral Protein Kinase ORF36

Understanding Epstein-Barr Virus Life Cycle with Proteomics: A Temporal Analysis of Ubiquitination During Virus Reactivation

The anaphase‐promoting complex: A key mitotic regulator associated with somatic mutations occurring in cancer

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