Summary Herpesviruses, which are major human pathogens, establish life-long persistent infections. Although the α-, β-, and γ-herpesviruses infect different tissues and cause distinct diseases, they each encode a conserved serine/threonine kinase critical for virus replication and spread. The extent of substrate conservation and the key common cell signalling pathways targeted by these kinases are unknown. Using a human protein microarray high-throughput approach we identify shared substrates of the conserved kinases from herpes simplex virus, human cytomegalovirus, Epstein-Barr virus (EBV) and Kaposi's sarcoma associated herpesvirus. DNA damage response (DDR) proteins were statistically enriched and the histone acetyltransferase TIP60, an upstream regulator of the DDR pathway, was required for efficient herpesvirus replication. During EBV replication, TIP60 activation by the BGLF4 kinase triggers EBV-induced DDR and also mediates induction of viral lytic gene expression. Identification of key cellular targets of the conserved herpesvirus kinases will facilitate the development of broadly effective anti-viral strategies.
). In this study, we show that BGLF4 interacts with lamin A/C and phosphorylates lamin A protein in vitro. Using a green fluorescent protein (GFP)-lamin A system, we found that Ser-22, Ser-390, and Ser-392 of lamin A are important for the BGLF4-induced disassembly of the nuclear lamina and the EBV reactivation-mediated redistribution of nuclear lamin. Virion production and protein levels of two EBV primary envelope proteins, BFRF1 and BFLF2, were reduced significantly by the expression of GFP-lamin A(5A), which has five Ser residues replaced by Ala at amino acids 22, 390, 392, 652, and 657 of lamin A. Our data indicate that BGLF4 kinase phosphorylates lamin A/C to promote the reorganization of the nuclear lamina, which then may facilitate the interaction of BFRF1 and BFLF2s and subsequent virion maturation. UL kinases of alpha-and betaherpesviruses also induce the disassembly of the nuclear lamina through similar sites on lamin A/C, suggesting a conserved mechanism for the nuclear egress of herpesviruses.Most DNA viruses replicate and assemble their genomes into nucleocapsids in the nuclei of infected cells. To facilitate efficient replication, viruses regulate the nuclear environment by affecting cellular chromatin and nuclear lamina (30,35,40). The nuclear lamina is a thin electron-dense meshwork lining the nucleoplasmic face of the inner nuclear membrane (INM) (14, 20) and provides structural support for the major components of the nuclear envelope (36, 39). The lamina also functions as a transverse scaffold for INM proteins (e.g., emerin and lamin B receptor), chromatin proteins (histone H2A/H2B dimers), and cytoskeleton-interacting proteins (nesprin1/2) (7, 52).The nuclear lamina comprises a series of type V intermediate filaments composed of lamin types A, B1, B2, and C. Types A and C are products of RNA splicing variants of the lmnA transcripts, whereas types B1 and B2 are derived from two other genes, lmnB1 and B2 (15, 22). The INM-associated lamin B layer provides the fundamental structure of the lamina and is essential for the nuclear shape, whereas the lamin A/C layer adjacent to the nucleoplasm has more specialized functions and contributes to nuclear stiffness (7, 23, 52). Similarly to other intermediate filaments, lamins contain globular head and tail domains flanked by a central rod domain (15). The rod domains of two lamin molecules can intertwine to form dimers, whereas regions flanking the head/rod and rod/tail domains potentially interact with other lamin dimers to form longer filaments (45). Physiologically, the nuclear lamina is reorganized dynamically throughout the cell cycle via a mechanism regulated by phosphorylation. Phosphorylation by mitotic Cdc2 kinase at Ser-22, Ser-390, and Ser-392 residues on lamin A/C, or by protein kinase C (PKC) during apoptosis, leads to the depolymerization of lamin (disassembly of the nuclear lamina), which may lead to their release from the INM (11,21,44).The intact meshwork of the nuclear lamina also presents a barrier to most DNA viruses. Upon infection,...
The BGLF4 protein kinase of Epstein-Barr virus (EBV) is a member of the conserved family of herpesvirus protein kinases which, to some extent, have a function similar to that of the cellular cyclin-dependent kinase in regulating multiple cellular and viral substrates. In a yeast two-hybrid screening assay, a splicing variant of interferon (IFN) regulatory factor 3 (IRF3) was found to interact with the BGLF4 protein. This interaction was defined further by coimmunoprecipitation in transfected cells and glutathione S-transferase (GST) pulldown in vitro. Using reporter assays, we show that BGLF4 effectively suppresses the activities of the poly(I: C)-stimulated IFN- promoter and IRF3-responsive element. Moreover, BGLF4 represses the poly(I:C)-stimulated expression of endogenous IFN- mRNA and the phosphorylation of STAT1 at Tyr701. In searching for a possible mechanism, BGLF4 was shown not to affect the dimerization, nuclear translocation, or CBP recruitment of IRF3 upon poly(I:C) treatment. Notably, BGLF4 reduces the amount of active IRF3 recruited to the IRF3-responsive element containing the IFN- promoter region in a chromatin immunoprecipitation assay. BGLF4 phosphorylates GST-IRF3 in vitro, but Ser339-Pro340 phosphorylation-dependent, Pin1-mediated downregulation is not responsible for the repression. Most importantly, we found that three prolinedependent phosphorylation sites at Ser123, Ser173, and Thr180, which cluster in a region between the DNA binding and IRF association domains of IRF3, contribute additively to the BGLF4-mediated repression of IRF3(5D) transactivation activity. IRF3 signaling is activated in reactivated EBV-positive NA cells, and the knockdown of BGLF4 further stimulates IRF3-responsive reporter activity. The data presented here thus suggest a novel mechanism by which herpesviral protein kinases suppress host innate immune responses and facilitate virus replication.The innate immune response is the first-line defense against viral infection. The production of interferons (IFNs) and other cytokines to prevent virus replication and spread is at the center of the antiviral response and requires the activation of multiple transcription activators. The family of IFN regulatory factors (IRFs) is defined by a highly conserved amino-terminal DNA binding domain (DBD) containing five tryptophan repeats and a unique C-terminal domain, the IRF association domain (IAD) (29). IRF3 and IRF7 are two major direct transducers of virus-mediated signaling that induce type I IFNs. IRF3 is a constitutively expressed phosphoprotein of 427 amino acids, which can shuttle into and out of the nucleus in its inactive form. Upon virus infection, cellular TBK-1-and IKKε-mediated phosphorylation of serines 385 and 386 and the serine/threonine cluster between amino acids 396 and 405 of IRF3 lead to its conformational change and activation (19,29,65).The activated IRF3 then undergoes homodimerization or heterodimerization with IRF7, nuclear localization, and association with the coactivator CBP/P300 (29). The phosphor...
The cellular endosomal sorting complex required for transport (ESCRT) machinery participates in membrane scission and cytoplasmic budding of many RNA viruses. Here, we found that expression of dominant negative ESCRT proteins caused a blockade of Epstein-Barr virus (EBV) release and retention of viral BFRF1 at the nuclear envelope. The ESCRT adaptor protein Alix was redistributed and partially colocalized with BFRF1 at the nuclear rim of virus replicating cells. Following transient transfection, BFRF1 associated with ESCRT proteins, reorganized the nuclear membrane and induced perinuclear vesicle formation. Multiple domains within BFRF1 mediated vesicle formation and Alix recruitment, whereas both Bro and PRR domains of Alix interacted with BFRF1. Inhibition of ESCRT machinery abolished BFRF1-induced vesicle formation, leading to the accumulation of viral DNA and capsid proteins in the nucleus of EBV-replicating cells. Overall, data here suggest that BFRF1 recruits the ESCRT components to modulate nuclear envelope for the nuclear egress of EBV.
The Epstein-Barr virus (EBV) open reading frame BGLF4 was identified as a potential Ser/Thr protein kinase gene through the recognition of amino acid sequence motifs characteristic of conserved regions within the catalytic domains of protein kinases. In order to investigate this potential kinase activity, BGLF4 was expressed in Escherichia coli and the purified protein was used to generate a specific antiserum. Recombinant vaccinia virus vTF7-3, which expresses the T7 RNA polymerase, was used to infect 293 and 293T cells after transient transfection with a plasmid containing BGLF4 under the control of the T7 promoter. Autophosphorylation of the BGLF4 protein was demonstrated using the specific antiserum in an immune complex kinase assay. In addition, EBNA-1-tagged BGLF4 and EBNA-1 monoclonal antibody 5C11 were used to demonstrate the specificity of the kinase activity and to locate BGLF4 in the cytoplasm of transfected cells. Manganese ions were found to be essential for autophosphorylation of BGLF4, and magnesium can stimulate the activity. BGLF4 can utilize GTP, in addition to ATP, as a phosphate donor in this assay. BGLF4 can phosphorylate histone and casein in vitro. Among the potential viral protein substrates we examined, the EBV early antigen (EA-D, BMRF1), a DNA polymerase accessory factor and an important transactivator during lytic infection, was found to be phosphorylated by BGLF4 in vitro. Amino acids 1 to 26 of BGLF4, but not the predicted conserved catalytic domain, were found to be essential for autophosphorylation of BGLF4.Protein kinases are known to be involved in the regulation of a wide variety of eukaryotic cellular functions including cell metabolism, cell cycle control, hormone response, and control of transcription and translation. Studying viral protein kinases might therefore lead to an understanding of the mechanisms of virus replication and virus-cell interactions. Most of the protein kinases of the retroviruses are Tyr protein kinases, such as v-src and v-erb, which may contribute to the growth transformation phenotype of the virally infected host cells (for a review, see reference 32). The first protein kinase gene demonstrated in a eukaryotic DNA virus was that contained in the unique short (US) regions of the related human and porcine alphaherpesviruses, herpes simplex virus type 1 (HSV-1), and pseudorabies virus (20). Other protein kinases have been reported in DNA viruses, including protein kinase B1 of the poxviruses (45, 46) and ORF9 of baculovirus (42).Phosphorylation of cellular and viral proteins, which has been observed during lytic infection of cells by herpesviruses, seems to be a common phenomenon which involves a number of different protein kinase activities (21). Two groups of viral protein kinase activities, US3 and UL13, have been identified in alphaherpesviruses. The US3 gene of HSV-1 (37) and the VZV66 gene of varicella-zoster virus (VZV) (19) were predicted to encode protein kinases on the basis of their strong similarity to the family of eukaryotic serine/threon...
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