Protein Array Identification of Substrates of the Epstein-Barr Virus Protein Kinase BGLF4

The Epstein-Barr virus BGLF4 and BGLF5 genes encode a protein kinase and an alkaline exonuclease, respectively. Both proteins were previously found to regulate multiple steps of virus replication, including lytic DNA replication and primary egress. However, while inactivation of BGLF4 led to the downregulation of several viral proteins, the absence of BGLF5 had the opposite effect. Using recombinant viruses that lack both viral enzymes, we confirm and extend these initial observations, e.g., by showing that both BGLF4 and BGLF5 are required for proper phosphorylation of the DNA polymerase processivity factor BMRF1. We further found that neither BGLF4 nor BGLF5 is required for baseline viral protein production. Complementation with BGLF5 downregulated mRNA levels and translation of numerous viral genes, though to various degrees, whereas BGLF4 had the opposite effect. BGLF4 and BGLF5 influences on viral expression were most pronounced for BFRF1 and BFLF2, two proteins essential for nuclear egress. For most viral genes studied, cotransfection of BGLF4 and BGLF5 had only a marginal influence on their expression patterns, showing that BGLF4 antagonizes BGLF5-mediated viral gene shutoff. To be able to exert its functions on viral gene expression, BGLF4 must be able to escape BGLF5's shutoff activities. Indeed, we found that BGLF5 stimulated the BGLF4 gene's transcription through an as yet uncharacterized molecular mechanism. The BGLF4/BGLF5 enzyme pair builds a regulatory loop that allows fine-tuning of virus protein production, which is required for efficient viral replication.The Epstein-Barr virus (EBV) is a large double-stranded DNA virus that has more than 100 genes, the large majority of which are required for virus replication (29). Lytic genes are sequentially expressed and therefore classified as immediateearly, early, or late genes. Expression of EBV lytic genes is positively regulated by transactivators such as the immediateearly BZLF1 and BRLF1 (5, 9, 20), but the alkaline exonuclease BGLF5 (AE) has also been identified as a negative regulator (8). Through its ability to reduce expression of major histocompatibility complex class I and class II genes, BGLF5 was initially identified as a mediator of immune evasion (41, 54). BGLF5 host shutoff properties were subsequently found to extend to viral genes; expression of the BFLF2 gene, an early gene required for primary egress, was found to be enhanced in a ⌬BGLF5 recombinant EBV (8, 17). Conversely, reintroduction of BGLF5 reverted this effect. The study of this mutant revealed multiple functions for AE during virus replication; the viral exonuclease was required for optimal total DNA synthesis and efficient processing of linear viral genomes. In the absence of BGLF5, viral encapsidation, nuclear egress, and virus production were substantially reduced (8).These phenotypic traits are strongly reminiscent of those previously reported for an EBV producer cell line in which the BGLF4 gene, the only hitherto-identified EBV protein kinase (PK) gene, had been knocke...

Section: Vol 83 2009 Regulation Of Ebv Protein Synthesis 10889mentioning

confidence: 99%

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confidence: 99%

The Epstein-Barr Virus Protein Kinase BGLF4 and the Exonuclease BGLF5 Have Opposite Effects on the Regulation of Viral Protein Production

Feederle

Mehl-Lautscham

Bannert

et al. 2009

“…Indeed, half of the EBV-PK targets identified by a protein array in a recent study were also found to be in vitro substrates for the cellular kinase CDK1/cyclin B (63). Overexpression of EBV-PK in cells has been shown to result in phosphorylation of a number of different cellular proteins, including the cell cycle regulatory proteins p27 (19) and pRB (C. V. Kuny and R. F. Kalejta, unpublished data); nuclear lamin A/C (32); interferon regulatory factor 3 (IRF3) (54); cellular translational elongation factor 1␦ (24,26); MCM4 (part of the MCM4-MCM6-MCM7 cellular replication origin binding complex) (29); and histone protein H2AX (52).…”

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confidence: 99%

Simian Virus 40 T/t Antigens and Lamin A/C Small Interfering RNA Rescue the Phenotype of an Epstein-Barr Virus Protein Kinase (BGLF4) Mutant

Qiao

Hagemeier²,

Kuny

et al. 2010

The Epstein-Barr virus (EBV)-encoded viral protein kinase, EBV-PK (the BGLF4 gene product), is required for efficient nuclear viral egress in 293 cells. However, since EBV-PK phosphorylates a number of different viral and cellular proteins (including lamin A/C), the relative importance of each target during lytic viral replication remains unclear. We show here that an EBV PK mutant (PKmut; containing stop codons at residues 1 and 5 in EBV-PK) is highly defective for release of infectious virus from 293 cells but not 293T cells. Furthermore, the phenotype of the PKmut in 293 cells is substantially reversed by expression of the simian virus 40 (SV40) large (T) and small (t) T antigens. Efficient rescue requires the presence of both SV40 T/t proteins. We show that 293T cells have a much higher level of constitutive lamin A/C phosphorylation than do 293 cells over residues (S22 and S392) that promote phosphorylation-dependent nuclear disassembly and that both large T and small t contribute to enhanced lamin A/C phosphorylation. Finally, we demonstrate that knockdown of lamin A/C expression using small interfering RNA also rescues the PKmut phenotype in 293 cells. These results suggest that essential roles of EBV-PK during lytic viral replication include the phosphorylation and dispersion of lamin A/C.The Epstein-Barr virus (EBV) BGLF4 gene product, EBV-PK, is homologous to the cytomegalovirus (CMV) UL97 kinase and belongs to a conserved family of herpesviral protein kinases. EBV-PK is the only protein kinase encoded by the EBV genome (6) and is expressed as an early lytic viral protein during viral reactivation (11). EBV-PK is also packaged into the virion tegument and thus is delivered in an active form to cells immediately after virus penetration (3). In the context of the intact virus, several recent studies indicate that a major and essential role of EBV-PK is to promote viral egress from the nucleus during lytic infection (10,13,41). Although EBV-PK phosphorylates a number of different viral and cellular proteins in vitro and when overexpressed in cells, the relative importance of the potential EBV-PK cellular and viral targets in the context of the intact viral genome has not been well studied.The nuclear EBV-PK protein is a serine/threonine kinase that shares many targets with cyclin-dependent cellular kinases. Indeed, half of the EBV-PK targets identified by a protein array in a recent study were also found to be in vitro substrates for the cellular kinase CDK1/cyclin B (63). Overexpression of EBV-PK in cells has been shown to result in phosphorylation of a number of different cellular proteins, including the cell cycle regulatory proteins p27 (19) and pRB (C. V. Kuny and R. F. Kalejta, unpublished data); nuclear lamin A/C (32); interferon regulatory factor 3 (IRF3) (54); cellular translational elongation factor 1␦ (24, 26); MCM4 (part of the MCM4-MCM6-MCM7 cellular replication origin binding complex) (29); and histone protein H2AX (52). The ability of EBV-PK to phosphorylate and inactivate two key cell cycl...

“…We previously used protein microarrays to identify substrates of human kinases, monitor changes in the human phospho-proteome, identify kinases that phosphorylate viral proteins, and identify cellular and viral substrates of viral kinases (61)(62)(63)(64)(65). We have now adapted this ap-proach to identify proteins that are potentially ERK-primed GSK-3 substrates.…”

Section: Establishment Of the Screening Assaymentioning

confidence: 99%

A Screen for Extracellular Signal-Regulated Kinase-Primed Glycogen Synthase Kinase 3 Substrates Identifies the p53 Inhibitor iASPP

Woodard

Liao

Goodwin

et al. 2015

Self Cite

The Kaposi's sarcoma-associated herpesvirus (KSHV) LANA protein is essential for the replication and maintenance of virus genomes in latently KSHV-infected cells. LANA also drives dysregulated cell growth through a multiplicity of mechanisms that include altering the activity of the cellular kinases extracellular signal-regulated kinase (ERK) and glycogen synthase kinase 3 (GSK-3). To investigate the potential impact of these changes in enzyme activity, we used protein microarrays to identify cell proteins that were phosphorylated by the combination of ERK and GSK-3. The assays identified 58 potential ERK-primed GSK-3 substrates, of which 23 had evidence for in vivo phosphorylation in mass spectrometry databases. Two of these, SMAD4 and iASPP, were selected for further analysis and were confirmed as ERK-primed GSK-3 substrates. Cotransfection experiments revealed that iASPP, but not SMAD4, was targeted for degradation in the presence of GSK-3. iASPP interferes with apoptosis induced by p53 family members. To determine the importance of iASPP to KSHV-infected-cell growth, primary effusion lymphoma (PEL) cells were treated with an iASPP inhibitor in the presence or absence of the MDM2 inhibitor Nutlin-3. Drug inhibition of iASPP activity induced apoptosis in BC3 and BCBL1 PEL cells but did not induce poly(ADP-ribose) polymerase (PARP) cleavage in virus-negative BJAB cells. The effect of iASPP inhibition was additive with that of Nutlin-3. Interfering with iASPP function is therefore another mechanism that can sensitize KSHV-positive PEL cells to cell death. IMPORTANCEKSHV is associated with several malignancies, including primary effusion lymphoma (PEL). The KSHV-encoded LANA protein is multifunctional and promotes both cell growth and resistance to cell death. LANA is known to activate ERK and limit the activity of another kinase, GSK-3. To discover ways in which LANA manipulation of these two kinases might impact PEL cell survival, we screened a human protein microarray for ERK-primed GSK-3 substrates. One of the proteins identified, iASPP, showed reduced levels in the presence of GSK-3. Further, blocking iASPP activity increased cell death, particularly in p53 wild-type BC3 PEL cells.T he Kaposi's sarcoma-associated herpesvirus (KSHV) latencyassociated nuclear antigen (LANA) is expressed in all KSHVinfected cells, including the tumor cells of the KSHV-associated malignancies Kaposi's sarcoma, primary effusion lymphoma (PEL), and muticentric Castleman disease. LANA functions in the replication and maintenance of latent, episomal KSHV genomes (1, 2) by binding to the KSHV origin of replication in the terminal repeats (3, 4), recruiting cellular replication proteins (5-11), and tethering the KSHV episomal genomes to host chromosomes (12-18). Sequences in the KSHV terminal repeats are bound by the C terminus of LANA, and the X-ray crystal structures of the human and murine gammaherpesvirus 68 (MHV68) LANA DNA-binding domains have been solved (19)(20)(21).KSHV infection leads to a global reprogramming of cel...