Nuclear Import of Epstein-Barr virus BLLF2 is Mediated by an Importin β1-Dependent Mechanism

2020 ◽  
Author(s):  
Meili Li ◽  
Yingjie Guo ◽  
Yangxi Deng ◽  
Yiwen Li ◽  
Xiaowen Ou ◽  
...  
Keyword(s):  
Nuclear Membrane ◽  
Nuclear Import ◽  
Antigen Processing ◽  
Epstein Barr Virus ◽  
Lytic Replication ◽  
Viral Dna ◽  
Nuclear Localization Signals ◽  
Barr Virus ◽  
Epstein Barr ◽  
Dependent Pathway

Abstract Background: Epstein-Barr virus (EBV), the pathogen of several human malignancies, encodes many proteins that require to be transported into the nucleus for viral DNA reproduction and nucleocapsids assembly in the lytic replication cycle. A nuclear membrane phosphoprotein encoded by EBV BLLF2, is believed to associate with viral DNA packaging and primary egress across the nuclear membrane. Results: Here, fluorescence microscope, mutation analysis, interspecies heterokaryon assays, co-immunoprecipitation assays and western blot were performed to explore the nuclear import mechanism of BLLF2. As results, BLLF2 was shown to be a nucleocytoplasmic shuttling protein, which was mediated neither by chromosomal region maintenance 1 (CRM1)- nor transporter associated with antigen processing (TAP)-dependent pathway. Yet, two functional nuclear localization signals (NLSs) of BLLF2, NLS1 (16KRQALETVPHPQNRGR31) and NLS2 (48PPVAKRRR58), were identified, whereas the predicted NES was nonfunctional. Finally, BLLF2 was proved to transport into the nucleus via Ran-dependent and importin β1-dependent pathway. Conclusions: This mechanism may contribute to a more extensive insight of the assembly and synthesis of EB virions in the nucleus, thus affording a new direction for the treatment of viruses.

scholarly journals Subcellular Localization of Epstein–Barr Virus BLLF2 and Its Underlying Mechanisms

2021 ◽  
Vol 12 ◽  
Author(s):  
Jingjing Li ◽  
Yingjie Guo ◽  
Yangxi Deng ◽  
Li Hu ◽  
Bolin Li ◽  
...  
Keyword(s):  
Antigen Processing ◽  
Epstein Barr Virus ◽  
Lytic Replication ◽  
Nucleocytoplasmic Shuttling ◽  
Nuclear Localization Signals ◽  
Barr Virus ◽  
Epstein Barr ◽  
Underlying Mechanisms ◽  
Dependent Pathway ◽  
Insight Into

Epstein–Barr virus (EBV), the pathogen of several human malignancies, encodes many proteins required to be transported into the nucleus for viral DNA reproduction and nucleocapsids assembly in the lytic replication cycle. Here, fluorescence microscope, mutation analysis, interspecies heterokaryon assays, co-immunoprecipitation assay, RNA interference, and Western blot were performed to explore the nuclear import mechanism of EBV encoded BLLF2 protein. BLLF2 was shown to be a nucleocytoplasmic shuttling protein neither by a chromosomal region maintenance 1 (CRM1)- nor by a transporter associated with antigen processing (TAP)-dependent pathway. Yet, BLLF2’s two functional nuclear localization signals (NLSs), NLS1 (16KRQALETVPHPQNRGR31) and NLS2 (44RRPRPPVAKRRRFPR58), were identified, whereas the predicted NES was nonfunctional. Finally, BLLF2 was proven to transport into the nucleus via a Ran-dependent and importin β1-dependent pathway. This mechanism may contribute to a more extensive insight into the assembly and synthesis of EBV virions in the nucleus, thus affording a new direction for the treatment of viruses.

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

2014 ◽  
Vol 89 (3) ◽  
pp. 1703-1718 ◽  
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.

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

2009 ◽  
Vol 83 (13) ◽  
pp. 6641-6651 ◽  
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.

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

2005 ◽  
Vol 79 (6) ◽  
pp. 3409-3418 ◽  
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.

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

2017 ◽  
Vol 91 (9) ◽  
Author(s):  
Mei-Tzu Su ◽  
Ya-Ting Wang ◽  
Yen-Ju Chen ◽  
Su-Fang Lin ◽  
Ching-Hwa Tsai ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Epstein Barr Virus ◽  
Lytic Replication ◽  
Viral Gene ◽  
Viral Dna ◽  
Viral Dna Replication ◽  
Barr Virus ◽  
Epstein Barr

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.

scholarly journals Inhibition of Epstein-Barr Virus Replication by a Benzimidazole l-Riboside: Novel Antiviral Mechanism of 5,6-Dichloro-2-(Isopropylamino)-1-β-l-Ribofuranosyl-1H-Benzimidazole

1999 ◽  
Vol 73 (9) ◽  
pp. 7271-7277 ◽  
Author(s):  
Valerie L. Zacny ◽  
Eduard Gershburg ◽  
Michelle G. Davis ◽  
Karen K. Biron ◽  
Joseph S. Pagano
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Epstein Barr Virus ◽  
Blot Hybridization ◽  
Lytic Replication ◽  
Viral Dna ◽  
Barr Virus ◽  
Latently Infected ◽  
Ebv Dna ◽  
Epstein Barr

ABSTRACT Although a number of antiviral drugs inhibit replication of Epstein-Barr virus (EBV) in cell culture, and acyclovir (ACV) suppresses replication in vivo, currently available drugs have not proven effective for treatment of EBV-associated diseases other than oral hairy leukoplakia. Benzimidazole riboside compounds represent a new class of antiviral compounds that are potent inhibitors of human cytomegalovirus (HCMV) replication but not of other herpesviruses. Here we characterize the effects of two compounds in this class against lytic replication of EBV induced in a Burkitt lymphoma cell line latently infected with EBV. We analyzed linear forms of EBV genomes, indicative of lytic replication, and episomal forms present in latently infected cells by terminal probe analysis followed by Southern blot hybridization as well as the high-molecular-weight unprocessed viral DNA by pulsed-field gel electrophoresis. d-Ribofuranosyl benzimidazole compounds that act as inhibitors of HCMV DNA maturation, including BDCRB (5,6-dichloro-2-bromo-1-β-d-ribofuranosyl-1H-benzimidazole), did not affect the accumulation of high-molecular-weight or monomeric forms of EBV DNA in the induced cells. In contrast, the generation of linear EBV DNA as well as precursor viral DNA was sensitive to thel-riboside 1263W94 [5,6-dichloro-2-(isopropylamino)-1-β-l-ribofuranosyl-1H-benzimidazole]. The 50% inhibitory concentration range for 1263W94 was 0.15 to 1.1 μM, compared with 10 μM for ACV. Thus, 1263W94 is a potent inhibitor of EBV. In addition, 1263W94 inhibited the phosphorylation and the accumulation of the essential EBV replicative cofactor, early antigen D.

scholarly journals Characterization of the Nucleocytoplasmic Transport Mechanisms of Epstein-Barr Virus BFLF2

2018 ◽  
Vol 51 (4) ◽  
pp. 1500-1517 ◽  
Author(s):  
Meili Li ◽  
Tao Chen ◽  
Xingmei Zou ◽  
Zuo Xu ◽  
Yuanfang Wang ◽  
...  
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Antigen Processing ◽  
Epstein Barr Virus ◽  
Antiviral Drug ◽  
Nuclear Localization Sequence ◽  
Transport Mechanisms ◽  
Barr Virus ◽  
Epstein Barr

Background/Aims: Epstein-Barr virus (EBV) BFLF2, the homologue of herpes simplex virus 1 (HSV-1) UL31, is crucial for the efficient viral DNA packaging and primary egress across the nuclear membrane. However, we still do not know its subcellular transport mechanisms. Methods: Interspecies heterokaryon assays were utilized to detect the nucleocytoplasmic shuttling of BFLF2, and mutation analysis, plasmid transfection and fluorescence microscopy assays were performed to identify the functional nuclear localization sequence (NLS) and nuclear export sequence (NES) of BFLF2 in live cells. Furthermore, the nuclear import and export of BFLF2 were assessed by confocal microscopy, co-immunoprecipitation and immunoblot assays. Results: BFLF2 was confirmed to shuttle between the nucleus and cytoplasm. Two predicted NESs were shown to be nonfunctional, yet we proved that the nuclear export of BFLF2 was mediated through transporter associated with antigen processing (TAP), but not chromosomal region maintenance 1 (CRM1) dependent pathway. Furthermore, one functional NLS, 22RRLMHPHHRNYTASKASAH40, was identified, and the aa22-23, aa22-25, aa28-30 and aa37-40 had an important role in the nuclear localization of BFLF2. Besides, the nuclear import of BFLF2 was demonstrated through Ran-, importin α7-, importin β1- and transportin-1-dependent mechanism that does not require importin α1, α3 and α5. Conclusion: These works are of significance for the further study of the functions of BFLF2 during EBV infection, as well as for further insights into the design of new antiviral drug target and vaccine development against EBV.

scholarly journals BFRF1 of Epstein-Barr Virus Is Essential for Efficient Primary Viral Envelopment and Egress

2005 ◽  
Vol 79 (6) ◽  
pp. 3703-3712 ◽  
Author(s):  
Antonella Farina ◽  
Regina Feederle ◽  
Salvatore Raffa ◽  
Roberta Gonnella ◽  
Roberta Santarelli ◽  
...  
Keyword(s):  
Nuclear Membrane ◽  
Epstein Barr Virus ◽  
Molecular Mechanisms ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Wild Type ◽  
Barr Virus ◽  
Viral Mutant ◽  
Epstein Barr ◽  
Viral Titers

ABSTRACT The molecular mechanisms that underlie maturation and egress of Epstein-Barr virus (EBV) virions are only partially characterized. We have recently shown that the BFRF1 gene, the EBV positional homolog of herpes simplex virus type 1 and pseudorabies virus UL34, is expressed early during EBV lytic replication and that it is found predominantly on the nuclear membrane (A. Farina, R. Santarelli, R. Gonnella, R. Bei, R. Muraro, G. Cardinali, S. Uccini, G. Ragona, L. Frati, A. Faggioni, and A. Angeloni, J. Virol. 74:3235-3244, 2000). These data suggest that the BFRF1 protein might be involved in viral primary envelopment. To precisely determine the function of this protein, we have constructed an EBV mutant devoid of the BFRF1 gene (BFRF1-KO). 293 cells carrying BFRF1-KO showed no differences in comparison with wild-type EBV in terms of DNA lytic replication or expression of late viral proteins upon induction of the lytic cycle. However, binding assays and infection experiments using cell lines or human cord blood lymphocytes showed a clear reduction in the viral mutant titers. Complementation experiments with BFRF1-KO and a BFRF1 expression vector restored viral titers to levels similar to those for the wild-type control, showing that the modifications that we introduced were limited to the BFRF1 gene. Electron microscopic observations showed that the reduction in viral titers was due to sequestration of EBV nucleocapsids in the nuclei of lytically induced cells. This suggests that BFRF1 is involved in transport of the maturing virion across the nuclear membrane. This hypothesis was further supported by the observation that BFRF1 is present in maturing intracellular virions but not in their extracellular counterparts. This implies that BFRF1 is a key protein for EBV maturation.

scholarly journals Low Level of Lytic Replication in a Recombinant Epstein-Barr Virus Carrying an Origin of Replication Devoid of BZLF1-Binding Sites

2004 ◽  
Vol 78 (21) ◽  
pp. 12082-12084 ◽  
Author(s):  
R. Feederle ◽  
H. J. Delecluse
Keyword(s):  
Binding Sites ◽  
Epstein Barr Virus ◽  
Lytic Replication ◽  
Origin Of Replication ◽  
Viral Dna ◽  
Lower Efficiency ◽  
Viral Dna Replication ◽  
Barr Virus ◽  
Viral Transactivator ◽  
Epstein Barr

ABSTRACT Binding of the BZLF1 viral transactivator to Epstein-Barr virus (EBV) oriLyt has been reported to be essential for viral DNA replication. We have constructed a recombinant virus (E2-oriLyt-R) in which the oriLyt BZLF1-binding sites (ZRE) were exchanged against papilloma E2-binding sites. A fusion protein between the BZLF1 protein-transactivating domain and the E2 protein-binding domain was able to reactivate lytic replication in E2-oriLyt-R. However, BZLF1 alone could also induce E2-oriLyt-R, albeit with much lower efficiency. ZRE are therefore important but not absolutely essential cis elements for lytic replication. This shows the importance of recombinants to evaluate viral functions.

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