The SWI/SNF Chromatin Regulator BRG1 Modulates the Transcriptional Regulatory Activity of the Epstein-Barr Virus DNA Polymerase Processivity Factor BMRF1

ABSTRACT During the lytic phase of Epstein-Barr virus (EBV), binding of the transactivator Zta to the origin of lytic replication (oriLyt) and the BHLF1 transcript, forming a stable RNA-DNA hybrid, is required to initiate viral DNA replication. EBV-encoded viral DNA replication proteins form complexes to amplify viral DNA. BMRF1, the viral DNA polymerase accessory factor, is essential for lytic DNA replication and also known as a transcriptional regulator of the expression of BHLF1 and BALF2 (single-stranded DNA [ssDNA]-binding protein). In order to determine systematically how BMRF1 regulates viral transcription, a BMRF1 knockout bacmid was generated to analyze viral gene expression using a viral DNA microarray. We found that a subset of Rta-responsive late genes, including BcLF1, BLLF1, BLLF2, and BDLF3, were downregulated in cells harboring a BMRF1 knockout EBV bacmid (p2089ΔBMRF1). In reporter assays, BMRF1 appears to transactivate a subset of viral late promoters through distinct pathways. BMRF1 activates the BDLF3 promoter in an SP1-dependent manner. Notably, BMRF1 associates with the transcriptional regulator BRG1 in EBV-reactivated cells. BMRF1-mediated transactivation activities on the BcLF1 and BLLF1 promoters were attenuated by knockdown of BRG1. In BRG1-depleted EBV-reactivated cells, BcLF1 and BLLF1 transcripts were reduced in number, resulting in reduced virion secretion. BMRF1 and BRG1 bound to the adjacent upstream regions of the BcLF1 and BLLF1 promoters, and depletion of BRG1 attenuated the recruitment of BMRF1 onto both promoters, suggesting that BRG1 is involved in BMRF1-mediated regulation of these two genes. Overall, we reveal a novel pathway by which BMRF1 can regulate viral promoters through interaction with BRG1. IMPORTANCE The cascade of viral gene expression during Epstein-Barr virus (EBV) replication is exquisitely regulated by the coordination of the viral DNA replication machinery and cellular factors. Upon lytic replication, the EBV immediate early proteins Zta and Rta turn on the expression of early proteins that assemble into viral DNA replication complexes. The DNA polymerase accessory factor, BMRF1, also is known to transactivate early gene expression through its interaction with SP1 or Zta on specific promoters. Through a global analysis, we demonstrate that BMRF1 also turns on a subset of Rta-regulated, late structural gene promoters. Searching for BMRF1-interacting cellular partners revealed that the SWI/SNF chromatin modifier BRG1 contributes to BMRF1-mediated transactivation of a subset of late promoters through protein-protein interaction and viral chromatin binding. Our findings indicate that BMRF1 regulates the expression of more viral genes than thought previously through distinct viral DNA replication-independent mechanisms.

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Pin1 Interacts with the Epstein-Barr Virus DNA Polymerase Catalytic Subunit and Regulates Viral DNA Replication

Journal of Virology ◽

10.1128/jvi.02634-12 ◽

2012 ◽

Vol 87 (4) ◽

pp. 2120-2127 ◽

Cited By ~ 25

Author(s):

Y. Narita ◽

T. Murata ◽

A. Ryo ◽

D. Kawashima ◽

A. Sugimoto ◽

...

Keyword(s):

Dna Replication ◽

Dna Polymerase ◽

Epstein Barr Virus ◽

Catalytic Subunit ◽

Viral Dna ◽

Viral Dna Replication ◽

Barr Virus ◽

Epstein Barr

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BGLF4 Kinase Modulates the Structure and Transport Preference of the Nuclear Pore Complex To Facilitate Nuclear Import of Epstein-Barr Virus Lytic Proteins

Journal of Virology ◽

10.1128/jvi.02880-14 ◽

2014 ◽

Vol 89 (3) ◽

pp. 1703-1718 ◽

Cited By ~ 18

Author(s):

Chou-Wei Chang ◽

Chung-Pei Lee ◽

Mei-Tzu Su ◽

Ching-Hwa Tsai ◽

Mei-Ru Chen

Keyword(s):

Dna Replication ◽

Nuclear Import ◽

Epstein Barr Virus ◽

Lytic Replication ◽

Nuclear Pore ◽

Nuclear Targeting ◽

Viral Dna ◽

Viral Dna Replication ◽

Barr Virus ◽

Epstein Barr

ABSTRACTBGLF4 kinase, the only Ser/Thr protein kinase encoded by the Epstein-Barr virus (EBV) genome, phosphorylates multiple viral and cellular substrates to optimize the cellular environment for viral DNA replication and the nuclear egress of nucleocapsids. Previously, we found that nuclear targeting of BGLF4 is through direct interaction with the FG repeat-containing nucleoporins (FG-Nups) Nup62 and Nup153 independently of cytosolic transport factors. Here, we investigated the regulatory effects of BGLF4 on the structure and biological functions of the nuclear pore complex (NPC). In EBV-positive NA cells, the distribution of FG-Nups was modified during EBV reactivation. In transfected cells, BGLF4 changed the staining pattern of Nup62 and Nup153 in a kinase activity-dependent manner. Detection with anti-phospho-Ser/Thr-Pro MPM-2 antibody demonstrated that BGLF4 induced the phosphorylation of Nup62 and Nup153. The nuclear targeting of importin β was attenuated in the presence of BGLF4, leading to inhibition of canonical nuclear localization signal (NLS)-mediated nuclear import. Anin vitronuclear import assay revealed that BGLF4 induced the nuclear import of larger molecules. Notably, we found that BGLF4 promoted the nuclear import of several non-NLS-containing EBV proteins, including the viral DNA-replicating enzymes BSLF1, BBLF2/3, and BBLF4 and the major capsid protein (VCA), in cotransfected cells. The data presented here suggest that BGLF4 interferes with the normal functions of Nup62 and Nup153 and preferentially helps the nuclear import of viral proteins for viral DNA replication and assembly. In addition, the nuclear import-promoting activity was found in cells expressing the BGLF4 homologs of another two gammaherpesviruses but not those from alpha- and betaherpesviruses.IMPORTANCEDuring lytic replication, many EBV genome-encoded proteins need to be transported into the nucleus, not only for viral DNA replication but also for the assembly of nucleocapsids. Because nuclear pore complexes are effective gateways that control nucleocytoplasmic traffic, most EBV proteins without canonical NLSs are retained in the cytoplasm until they form complexes with their NLS-containing partners for nuclear targeting. In this study, we found that EBV BGLF4 protein kinase interacts with the Nup62 and Nup153 and induces the redistribution of FG-Nups. BGLF4 modulates the function of the NPC to inhibit the nuclear import of host NLS-containing proteins. Simultaneously, the nuclear import of non-NLS-containing EBV lytic proteins was enhanced, possibly through phosphorylation of Nup62 and Nup153, nuclear pore dilation, or microtubule reorganization. Overall, our data suggest that BGLF4-induced modification of nuclear pore transport may block nuclear targeting of cellular proteins and increase the import of viral proteins to promote viral lytic replication.

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Uracil DNA Glycosylase BKRF3 Contributes to Epstein-Barr Virus DNA Replication through Physical Interactions with Proteins in Viral DNA Replication Complex

Journal of Virology ◽

10.1128/jvi.00950-14 ◽

2014 ◽

Vol 88 (16) ◽

pp. 8883-8899 ◽

Cited By ~ 14

Author(s):

M.-T. Su ◽

I.-H. Liu ◽

C.-W. Wu ◽

S.-M. Chang ◽

C.-H. Tsai ◽

...

Keyword(s):

Dna Replication ◽

Epstein Barr Virus ◽

Dna Glycosylase ◽

Viral Dna ◽

Uracil Dna Glycosylase ◽

Viral Dna Replication ◽

Replication Complex ◽

Barr Virus ◽

Physical Interactions ◽

Epstein Barr

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Multiple Roles of Epstein-Barr Virus SM Protein in Lytic Replication

Journal of Virology ◽

10.1128/jvi.02665-06 ◽

2007 ◽

Vol 81 (8) ◽

pp. 4058-4069 ◽

Cited By ~ 24

Author(s):

Zhao Han ◽

Elessa Marendy ◽

Yong-Dong Wang ◽

Jing Yuan ◽

Jeffery T. Sample ◽

...

Keyword(s):

Gene Expression ◽

Dna Replication ◽

Epstein Barr Virus ◽

Lytic Replication ◽

Late Gene ◽

Late Gene Expression ◽

Barr Virus ◽

Ebv Dna ◽

Epstein Barr ◽

Sm Protein

ABSTRACT The effect of Epstein-Barr virus (EBV) SM protein on EBV gene expression was examined using a recombinant EBV strain with the SM gene deleted and DNA microarrays representing all known EBV coding regions. Induction of lytic EBV replication in the absence of SM led to expression of approximately 40% of EBV genes, but a block in expression of over 50% of EBV genes. Contrary to previous findings, several early genes were SM dependent, and lytic EBV DNA replication did not occur in the absence of SM. Notably, two genes essential for lytic EBV DNA replication, BSLF1 and BALF5, encoding EBV DNA primase and polymerase, respectively, were SM dependent. Lytic DNA replication was partially rescued by ectopic expression of EBV primase and polymerase, but virion production was not. Rescue of DNA replication only enhanced expression of a subset of late genes, consistent with a direct requirement for SM for late gene expression in addition to its contribution to DNA replication. Therefore, while SM is essential for most late gene expression, the proximate block to virion production by the EBV SM deletion strain is an inability to replicate linear DNA. The block to DNA replication combined with the direct effect of SM on late gene expression leads to a global deficiency of late gene expression. SM also inhibited BHRF1 expression during productive replication in comparison to that of cells induced into lytic replication in the absence of SM. Thus, SM plays a role in multiple steps of lytic cycle EBV gene expression and that it is transcript-specific in both activation and repression functions.

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Homologous Recombinational Repair Factors Are Recruited and Loaded onto the Viral DNA Genome in Epstein-Barr Virus Replication Compartments

Journal of Virology ◽

10.1128/jvi.00049-09 ◽

2009 ◽

Vol 83 (13) ◽

pp. 6641-6651 ◽

Cited By ~ 58

Author(s):

Ayumi Kudoh ◽

Satoko Iwahori ◽

Yoshitaka Sato ◽

Sanae Nakayama ◽

Hiroki Isomura ◽

...

Keyword(s):

Dna Replication ◽

Homologous Recombination ◽

Viral Genome ◽

Epstein Barr Virus ◽

Lytic Replication ◽

Recombinational Repair ◽

Viral Dna ◽

Barr Virus ◽

Homologous Recombinational Repair ◽

Epstein Barr

ABSTRACT Homologous recombination is an important biological process that facilitates genome rearrangement and repair of DNA double-strand breaks (DSBs). The induction of Epstein-Barr virus (EBV) lytic replication induces ataxia telangiectasia-mutated (ATM)-dependent DNA damage checkpoint signaling, leading to the clustering of phosphorylated ATM and Mre11/Rad50/Nbs1 (MRN) complexes to sites of viral genome synthesis in nuclei. Here we report that homologous recombinational repair (HRR) factors such as replication protein A (RPA), Rad51, and Rad52 as well as MRN complexes are recruited and loaded onto the newly synthesized viral genome in replication compartments. The 32-kDa subunit of RPA is extensively phosphorylated at sites in accordance with those with ATM. The hyperphosphorylation of RPA32 causes a change in RPA conformation, resulting in a switch from the catalysis of DNA replication to the participation in DNA repair. The levels of Rad51 and phosphorylated RPA were found to increase with the progression of viral productive replication, while that of Rad52 proved constant. Furthermore, biochemical fractionation revealed increases in levels of DNA-bound forms of these HRRs. Bromodeoxyuridine-labeled chromatin immunoprecipitation and PCR analyses confirmed the loading of RPA, Rad 51, Rad52, and Mre11 onto newly synthesized viral DNA, and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling analysis demonstrated DSBs in the EBV replication compartments. HRR factors might be recruited to repair DSBs on the viral genome in viral replication compartments. RNA interference knockdown of RPA32 and Rad51 prevented viral DNA synthesis remarkably, suggesting that homologous recombination and/or repair of viral DNA genome might occur, coupled with DNA replication to facilitate viral genome synthesis.

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An Epigenetic Journey: Epstein-Barr Virus Transcribes Chromatinized and Subsequently Unchromatinized Templates during Its Lytic Cycle

Journal of Virology ◽

10.1128/jvi.02247-18 ◽

2019 ◽

Vol 93 (8) ◽

Cited By ~ 10

Author(s):

Adityarup Chakravorty ◽

Bill Sugden ◽

Eric C. Johannsen

Keyword(s):

Gene Expression ◽

Dna Replication ◽

Epstein Barr Virus ◽

Dna Amplification ◽

Lytic Cycle ◽

Late Gene ◽

Late Gene Expression ◽

Viral Dna ◽

Barr Virus ◽

Epstein Barr

ABSTRACTThe Epstein-Barr virus (EBV) lytic phase, like those of all herpesviruses, proceeds via an orderly cascade that integrates DNA replication and gene expression. EBV early genes are expressed independently of viral DNA amplification, and several early gene products facilitate DNA amplification. On the other hand, EBV late genes are defined by their dependence on viral DNA replication for expression. Recently, a set of orthologous genes found in beta- and gammaherpesviruses have been determined to encode a viral preinitiation complex (vPIC) that mediates late gene expression. The EBV vPIC requires an origin of lytic replication incis, implying that the vPIC mediates transcription from newly replicated DNA. In agreement with this implication, EBV late gene mRNAs localize to replication factories. Notably, these factories exclude canonical histones. In this review, we compare and contrast the mechanisms and epigenetics of EBV early and late gene expression. We summarize recent findings, propose a model explaining the dependence of EBV late gene expression on lytic DNA amplification, and suggest some directions for future study.

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Architecture of Replication Compartments Formed during Epstein-Barr Virus Lytic Replication

Journal of Virology ◽

10.1128/jvi.79.6.3409-3418.2005 ◽

2005 ◽

Vol 79 (6) ◽

pp. 3409-3418 ◽

Cited By ~ 61

Author(s):

Tohru Daikoku ◽

Ayumi Kudoh ◽

Masatoshi Fujita ◽

Yutaka Sugaya ◽

Hiroki Isomura ◽

...

Keyword(s):

Dna Replication ◽

Viral Replication ◽

Epstein Barr Virus ◽

Protein A ◽

Lytic Replication ◽

Viral Dna ◽

Barr Virus ◽

Epstein Barr ◽

Cellular Replication ◽

Cellular Dna

ABSTRACT Epstein-Barr virus (EBV) productive DNA replication occurs at discrete sites, called replication compartments, in nuclei. In this study we performed comprehensive analyses of the architecture of the replication compartments. The BZLF1 oriLyt binding proteins showed a fine, diffuse pattern of distribution throughout the nuclei at immediate-early stages of induction and then became associated with the replicating EBV genome in the replication compartments during lytic infection. The BMRF1 polymerase (Pol) processivity factor showed a homogenous, not dot-like, distribution in the replication compartments, which completely coincided with the newly synthesized viral DNA. Inhibition of viral DNA replication with phosphonoacetic acid, a viral DNA Pol inhibitor, eliminated the DNA-bound form of the BMRF1 protein, although the protein was sufficiently expressed in the cells. These observations together with the findings that almost all abundantly expressed BMRF1 proteins existed in the DNA-bound form suggest that the BMRF1 proteins not only act at viral replication forks as Pol processive factors but also widely distribute on newly replicated EBV genomic DNA. In contrast, the BALF5 Pol catalytic protein, the BALF2 single-stranded-DNA binding protein, and the BBLF2/3 protein, a component of the helicase-primase complex, were colocalized as distinct dots distributed within replication compartments, representing viral replication factories. Whereas cellular replication factories are constructed based on nonchromatin nuclear structures and nuclear matrix, viral replication factories were easily solubilized by DNase I treatment. Thus, compared with cellular DNA replication, EBV lytic DNA replication factories would be simpler so that construction of the replication domain would be more relaxed.

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