Phosphorylation of Eukaryotic Translation Initiation Factor 4B (EIF4B) by Open Reading Frame 45/p90 Ribosomal S6 Kinase (ORF45/RSK) Signaling Axis Facilitates Protein Translation during Kaposi Sarcoma-associated Herpesvirus (KSHV) Lytic Replication

Kuang, Ersheng; Fu, Bishi; Liang, Qiming; Myoung, Jinjong; Zhu, Fanxiu

doi:10.1074/jbc.m111.280982

Cited by 70 publications

(87 citation statements)

References 83 publications

(116 reference statements)

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“…Rewiring of the ERK/RSK signaling pathway enables ORF45 to modulate the host cellular environment to a great extent through phosphorylation of a distinct repertoire of protein substrates. We recently identified a prominent cytoplasmic substrate, eIF4B, a translation initiation factor, as a specific substrate of ORF45-activated RSK, revealing a role for ORF45 in translational regulation (22). Here, we show evidence that ORF45 also is involved in transcriptional regulation, because the transcription of a cluster of late genes surrounding ORF25 was depressed in BAC16-45F66A-and BAC16-Stop45-infected cells.…”

Section: Discussionmentioning

confidence: 65%

“…All other antibodies and chemicals used in this study have been described previously (12,(19)(20)(21). Plasmids pKH3-RSK2, pKH3-RSK1, pCR3.1-ORF45, full-length pCMV-ORF45, and derivatives have been described previously (19,20,22). Site-directed mutagenesis of ORF45 was performed by using a QuikChange mutagenesis kit (Stratagene, La Jolla, CA).…”

Section: Methodsmentioning

confidence: 99%

“…Immunoprecipitation and Western blot analysis were performed as previously described (19,20,22). For immunoprecipitation with anti-FLAG or anti-hemagglutinin (HA) antibodies, the cell lysates were incubated with EZview red anti-Flag M2 or anti-HA affinity resin for 4 h or overnight at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…HEK293 and HEK293T cells were cultured under 5% CO 2 at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics. iSLK-puro cells were cultured in DMEM containing 10% FBS, 450 g/ml G418, and 1 g/ml puromycin as previously described (22,23). Transient transfections were performed in 6-well plates with Lipofectamine 2000 (Invitrogen, Carlsbad, CA) or 100-mm dishes with calcium phosphate methods.…”

Section: Methodsmentioning

confidence: 99%

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Activation of p90 Ribosomal S6 Kinases by ORF45 of Kaposi's Sarcoma-Associated Herpesvirus Is Critical for Optimal Production of Infectious Viruses

Kuang

et al. 2015

J Virol

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We have previously shown that ORF45, an immediate-early and tegument protein of Kaposi's sarcoma-associated herpesvirus (KSHV), causes sustained activation of p90 ribosomal S6 kinases (RSKs) and extracellular regulated kinase (ERK) (E. Kuang, Q. Tang, G. G. Maul, and F. Zhu, J Virol 82:1838 -1850, 2008, http://dx.doi.org/10.1128/JVI.02119-07). We now have identified the critical region of ORF45 that is involved in RSK interaction and activation. Alanine scanning mutagenesis of this region revealed that a single F66A point mutation abolished binding of ORF45 to RSK or ERK and, consequently, its ability to activate the kinases. We introduced the F66A mutation into BAC16 (a bacterial artificial chromosome clone containing the entire infectious KSHV genome), producing BAC16-45F66A. In parallel, we also repaired the mutation and obtained a revertant, BAC16-45A66F. The reconstitution of these mutants in iSLK cells demonstrated that the ORF45-F66A mutant failed to cause sustained ERK and RSK activation during lytic reactivation, resulting in dramatic differences in the phosphoproteomic profile between the wildtype virus-infected cells and the mutant virus-infected cells. ORF45 mutation or deletion also was accompanied by a noticeable decreased in viral gene expression during lytic reactivation. Consequently, the ORF45-F66A mutant produced significantly fewer infectious progeny virions than the wild type or the revertant. These results suggest a critical role for ORF45-mediated RSK activation in KSHV lytic replication. IMPORTANCEKSHV is the causative agent of three human malignancies. KSHV pathogenesis is intimately linked to its ability to modulate the host cell microenvironment and to facilitate efficient production of progeny viral particles. We previously described the mechanism by which the KSHV lytic protein ORF45 activates the cellular kinases ERK and RSK. We now have mapped the critical region of ORF45 responsible for binding and activation of ERK/RSK to a single residue, F66. We mutated this amino acid of ORF45 (F66A) and introduced the mutation into a newly developed bacterial artificial chromosome containing the KSHV genome (BAC16). This system has provided us with a useful tool to characterize the functions of ORF45-activated RSK upon KSHV lytic reactivation. We show that viral gene expression and virion production are significantly reduced by F66A mutation, indicating a critical role for ORF45-activated RSK during KSHV lytic replication. K aposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS), the most common malignancy in HIV-infected individuals (1, 2). Besides KS, KSHV is associated with two lymphoproliferative diseases, primary effusion lymphoma and multicentric Castleman's disease (3, 4). KSHV belongs to the Gammaherpesvirinae subfamily in the Herpesviridae family and is closely related to rhesus rhadinovirus (RRV), herpesvirus saimiri (HVS), and murine gammaherpesvirus 68 (MHV-68) in the Rhadinovirus genus (␥2). Its closest relative in humans is Epstein-...

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Section: Discussionmentioning

confidence: 65%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Activation of p90 Ribosomal S6 Kinases by ORF45 of Kaposi's Sarcoma-Associated Herpesvirus Is Critical for Optimal Production of Infectious Viruses

Kuang

et al. 2015

J Virol

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“…These complexes protect the activated phosphorylated forms of ERK and RSK (pERK and pRSK) from dephosphorylation, leading to an increase in persistent activation in KSHV (170). This persistent activation of RSK by ORF45 also causes an increase in phosphorylation and activation of eukaryotic translation factor 4B (eIF4B), which increases viral replication and progeny virion production (168).…”

Section: Kshv Orf45mentioning

confidence: 99%

Role of RRV ORF52 in Virion Maturation

Anderson¹

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Rhesus monkey rhadinovirus (RRV) is a close relative of the human pathogen, Kaposi's Sarcoma-Associated Herpesvirus (KSHV). Primary infection with KSHV in culture is highly inefficient and it predominantly adopts a latent phenotype, expressing only a minimal number of viral genes and producing no progeny virions. This contrasts with RRV in culture, which replicates to high viral titer and produces abundant progeny virions, making it a useful model to study gammaherpesvirus lytic replication, virion structure, and the potential roles of tegument proteins in the biology of gammaherpesviruses. Initial work in our laboratory determined that the RRV virion was composed of 33 virally encoded proteins. Of these, 17 are tegument proteins, five of which are unique to the gammaherpesvirus subfamily. ORF52 is one of these five gammaherpesvirus specific tegument proteins and is the focus of this dissertation. This protein is highly abundant within the virion (approximately 1000 copies per particle) and it is closely associated with the capsid. Our results describe a critical role for ORF52 in the cytoplasmic-based later stages of virion morphogenesis. Without ORF52, capsids fail to undergo tegumentation, which is necessary for final envelopment and production of fully infectious virions. In a later section of this dissertation, we also describe preliminary data proposing that ORF52 may play a direct or indirect role in virion transport through interaction with cellular microtubules.iii ACKNOWLEDGEMENTS I grew up in a small, blue-collar, southern Illinois town where most people stay local and focus on getting by day-to-day, not on things like big cities and advanced degrees. I didn't even know what "graduate school" was until several years into my undergraduate education in Chicago, which, by the way, took me 9 years to complete because I worked full-time and went to school off-and-on and part-time when time and money permitted. The reason I share this is because I didn't get here alone, I didn't even know where "here" was and if I'm honest, I don't think I ever really believed I would earn a PhD, because where I'm from, things like that just don't happen to people like me. There truly are so many people I am honored to thank for helping, and supporting, me at various points in this long, wonderful, horrible, amazing, and trying journey that has been the last 10 years of my life, 7 of those at UVa.

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Kaposi's Sarcoma‐associated Herpesvirus—Antiviral Treatment

Schulz

2021

New Drug Development for Known and Emerging Viruses

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Phosphorylation of Eukaryotic Translation Initiation Factor 4B (EIF4B) by Open Reading Frame 45/p90 Ribosomal S6 Kinase (ORF45/RSK) Signaling Axis Facilitates Protein Translation during Kaposi Sarcoma-associated Herpesvirus (KSHV) Lytic Replication

Cited by 70 publications

References 83 publications

Activation of p90 Ribosomal S6 Kinases by ORF45 of Kaposi's Sarcoma-Associated Herpesvirus Is Critical for Optimal Production of Infectious Viruses

Activation of p90 Ribosomal S6 Kinases by ORF45 of Kaposi's Sarcoma-Associated Herpesvirus Is Critical for Optimal Production of Infectious Viruses

Role of RRV ORF52 in Virion Maturation

Kaposi's Sarcoma‐associated Herpesvirus—Antiviral Treatment

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