scholarly journals Key motifs in EBV (Epstein–Barr virus)-encoded protein kinase for phosphorylation activity and nuclear localization

2010 ◽  
Vol 431 (2) ◽  
pp. 227-235 ◽  
Author(s):  
Svetlana Gershburg ◽  
Leann Murphy ◽  
Manfred Marschall ◽  
Edward Gershburg
Keyword(s):  
Amino Acids ◽  
Protein Kinase ◽  
Nuclear Localization ◽  
Epstein Barr Virus ◽  
Nuclear Translocation ◽  
Kinase Domain ◽  
Site Directed Mutagenesis ◽  
Barr Virus ◽  
Antiviral Compounds ◽  
Epstein Barr

A sole EBV (Epstein–Barr virus)-encoded protein kinase (EBV-PK) (the BGLF4 gene product) plays important roles in viral infection. Although a number of targets of this protein have been identified, the kinase itself remains largely unstudied with regard to its enzymology and structure. In the present study, site-directed mutagenesis has been employed to generate mutations targeting residues involved in nuclear localization of the EBV-PK, core residues in subdomain III of the protein kinase domain conserved in most protein kinases or residues in subdomain VIa conserved only within the HPK (herpesvirus-encoded protein kinase) group. Deletion of amino acids 389–391 resulted in exclusive cytoplasmic localization of the protein, indicating the involvement of this region in nuclear translocation of the EBV-PK. Mutations at the amino acids Glu113 (core component), Phe175, Leu178, Phe184, Leu185 and Asn186 (conserved in HPKs) resulted in loss of EBV-PK autophosphorylation, protein substrate [EBV EA-D (early antigen diffused)] phosphorylation, and ability to facilitate ganciclovir phosphorylation. These results reiterate the unique features of this group of kinases and present an opportunity for designing more specific antiviral compounds.

scholarly journals Nuclear localization of the Epstein–Barr virus EBNA3B protein

2006 ◽  
Vol 87 (4) ◽  
pp. 789-793 ◽  
Author(s):  
Anita Burgess ◽  
Marion Buck ◽  
Kenia Krauer ◽  
Tom Sculley
Keyword(s):  
Nuclear Localization ◽  
Epstein Barr Virus ◽  
Fluorescent Protein ◽  
Site Directed Mutagenesis ◽  
Nuclear Antigen ◽  
Nuclear Localization Signals ◽  
Barr Virus ◽  
Computer Analyses ◽  
Epstein Barr ◽  
Green Fluorescent

The Epstein–Barr virus nuclear antigen (EBNA) 3B is a hydrophilic, proline-rich, charged protein that is thought to be involved in transcriptional regulation and is targeted exclusively to the cell nucleus, where it localizes to discrete subnuclear granules. Co-localization studies utilizing a fusion protein between enhanced green fluorescent protein (EGFP) and EBNA3B with FLAG-tagged EBNA3A and EBNA3C proteins demonstrated that EBNA3B co-localized with both EBNA3A and EBNA3C in the nuclei of cells when overexpressed. Computer analyses identified four potential nuclear-localization signals (NLSs) in the EBNA3B amino acid sequence. By utilizing fusion proteins with EGFP, deletion constructs of EBNA3B and site-directed mutagenesis, three of the four NLSs (aa 160–166, 430–434 and 867–873) were shown to be functional in truncated forms of EBNA3B, whilst an additional NLS (aa 243–246) was identified within the N-terminal region of EBNA3B. Only two of the NLSs were found to be functional in the context of the full-length EBNA3B protein.

scholarly journals Epstein–Barr virus nuclear antigen 3A contains six nuclear-localization signals

2006 ◽  
Vol 87 (10) ◽  
pp. 2879-2884 ◽  
Author(s):  
Marion Buck ◽  
Anita Burgess ◽  
Roslynn Stirzaker ◽  
Kenia Krauer ◽  
Tom Sculley
Keyword(s):  
Nuclear Localization ◽  
Epstein Barr Virus ◽  
Fluorescent Protein ◽  
Viral Proteins ◽  
Site Directed Mutagenesis ◽  
Nuclear Antigen ◽  
Nuclear Localization Signals ◽  
Barr Virus ◽  
Epstein Barr

The Epstein–Barr nuclear antigen 3A (EBNA3A) is one of only six viral proteins essential for Epstein–Barr virus-induced transformation of primary human B cells in vitro. Viral proteins such as EBNA3A are able to interact with cellular proteins, manipulating various biochemical and signalling pathways to initiate and maintain the transformed state of infected cells. EBNA3A has been reported to have one nuclear-localization signal and is targeted to the nucleus during transformation, where it associates with components of the nuclear matrix. By using enhanced green fluorescent protein-tagged deletion mutants of EBNA3A in combination with site-directed mutagenesis, an additional five functional nuclear-localization signals have been identified in the EBNA3A protein. Two of these (aa 63–66 and 375–381) were computer-predicted, whilst the remaining three (aa 394–398, 573–578 and 598–603) were defined functionally in this study.

scholarly journals Expression and Localization of the Epstein-Barr Virus-Encoded Protein Kinase

2004 ◽  
Vol 78 (22) ◽  
pp. 12140-12146 ◽  
Author(s):  
E. Gershburg ◽  
M. Marschall ◽  
K. Hong ◽  
J. S. Pagano
Keyword(s):  
Protein Kinase ◽  
Expression Pattern ◽  
Nuclear Localization ◽  
Epstein Barr Virus ◽  
Terminal Region ◽  
Lytic Cycle ◽  
Nuclear Fraction ◽  
Barr Virus ◽  
Early Protein ◽  
Epstein Barr

ABSTRACT The protein kinase (PK) encoded by the Epstein-Barr Virus (EBV) BGLF4 gene is the only EBV protein kinase. The expression pattern of EBV PK during the reactivation of the viral lytic cycle and the subcellular localization of the protein were analyzed with a polyclonal antiserum raised against a peptide corresponding to the N terminus of EBV PK. Based on previously published data (E. Gershburg and J. S. Pagano, J. Virol. 76:998-1003, 2002) and the expression pattern described here, we conclude that EBV PK is an early protein that requires viral-DNA replication for maximum expression. By biochemical fractionation, the protein could be detected mainly in the nuclear fraction 4 h after viral reactivation in Akata cells. Nuclear localization could be visualized by indirect immunofluorescence in HeLa cells transiently expressing EBV BGLF4 in the absence of other viral products. Transient expression of 3′-terminal deletion mutants of EBV BGLF4 resulted in cytoplasmic localization, confirming the presence of a nuclear localization site in the C-terminal region of the protein. In contrast to the wild-type EBV PK, all of the mutants were unable to hyperphosphorylate EA-D during coexpression or to phosphorylate ganciclovir, as measured by an in-cell activity assay. Thus, the results demonstrate that the nuclear localization, as well as the kinase activity, of BGFL4 is dependent on an intact C-terminal region.

scholarly journals The Epstein-Barr Virus Lytic Transactivator Zta Interacts with the Helicase-Primase Replication Proteins

1998 ◽  
Vol 72 (11) ◽  
pp. 8559-8567 ◽  
Author(s):  
Zhigang Gao ◽  
Anita Krithivas ◽  
Jon E. Finan ◽  
O. John Semmes ◽  
Sifang Zhou ◽  
...  
Keyword(s):  
Amino Acids ◽  
Epstein Barr Virus ◽  
Nuclear Translocation ◽  
Intracellular Localization ◽  
Activation Domain ◽  
Replication Complex ◽  
Barr Virus ◽  
Amino Terminal ◽  
Epstein Barr ◽  
Primase Complex

ABSTRACT The Epstein-Barr virus transactivator Zta triggers lytic gene expression and is essential for replication of the lytic origin, oriLyt. Previous analysis indicated that the Zta activation domain contributed a replication-specific function. We now show that the Zta activation domain interacts with components of the EBV helicase-primase complex. The three helicase-primase proteins BBLF4 (helicase), BSLF1 (primase), and BBLF2/3 (primase-associated factor) were expressed fused to the Myc epitope. When expression plasmids for BBLF4 or BBLF2/3 plus BSLF1 (primase subcomplex) were separately transfected, the proteins localized to the cytoplasm. Interaction between Zta and the components of the helicase-primase complex was tested by examining the ability of Zta to alter the intracellular localization of these proteins. Cotransfection of Zta with Myc-BBLF4 resulted in nuclear translocation of Myc-BBLF4; similarly, cotransfection of Zta with the primase subcomplex led to nuclear translocation of the Myc-BSLF1 and Myc-BBLF2/3 proteins. This relocalization provides evidence for an interaction between Zta and the helicase and Zta and the primase subcomplex. An affinity assay using glutathioneS-transferase–Zta fusion proteins demonstrated that Myc-BBLF4 and Myc-BBLF2/3 plus BSLF1 bound to the Zta activation domain (amino acids 1 to 133). In the nuclear relocalization assay, the amino-terminal 25 amino acids of Zta were required for efficient interaction with the primase subcomplex but not for interaction with BBLF4. Evidence for interaction between oriLyt bound Zta and the helicase-primase complex was obtained in a superactivation assay using an oriLyt-chloramphenicol acetyltransferase (CAT) reporter. Zta activated expression from a CAT reporter containing the complete oriLyt region and regulated by the oriLyt BHLF1 promoter. Cotransfection of the helicase-primase proteins, one of which was fused to a heterologous activation domain, led to Zta-dependent superactivation of CAT expression. This assay also provided evidence for an interaction between the single-stranded DNA binding protein, BALF2, and the Zta-tethered helicase-primase complex. The helicase-primase interaction is consistent with a role for Zta in stabilizing the formation of an origin-bound replication complex.

scholarly journals Epstein-Barr Virus-Encoded Latent Membrane Protein 1 Activates the JNK Pathway through Its Extreme C Terminus via a Mechanism Involving TRADD and TRAF2

1999 ◽  
Vol 73 (2) ◽  
pp. 1023-1035 ◽  
Author(s):  
Aristides G. Eliopoulos ◽  
Sarah M. S. Blake ◽  
J. Eike Floettmann ◽  
Martin Rowe ◽  
Lawrence S. Young
Keyword(s):  
Amino Acids ◽  
Protein Kinase ◽  
Epstein Barr Virus ◽  
Latent Membrane Protein ◽  
Latent Membrane Protein 1 ◽  
Tnf Receptor ◽  
Barr Virus ◽  
C Terminus ◽  
Epstein Barr ◽  
Jnk Activation

ABSTRACT The transforming Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) activates signalling on the NF-κB axis through two distinct domains in its cytoplasmic C terminus, namely, CTAR1 (amino acids [aa] 187 to 231) and CTAR2 (aa 351 to 386). The ability of CTAR1 to activate NF-κB appears to be attributable to the direct interaction of tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2), while recent work indicates that CTAR2-induced NF-κB is mediated through its association with TNF receptor-associated death domain (TRADD). LMP1 expression also results in activation of the c-Jun N-terminal kinase (JNK) (also known as stress-activated protein kinase) cascade, an effect which is mediated exclusively through CTAR2 and can be dissociated from NF-κB induction. The organization and signalling components involved in LMP1-induced JNK activation are not known. In this study we have dissected the extreme C terminus of LMP1 and have identified the last 8 aa of the protein (aa 378 to 386) as being important for JNK signalling. Using a series of fine mutants in which single amino acids between codons 379 and 386 were changed to glycine, we have found that mutations of Pro379, Glu381, Ser383, or Tyr384 diminish the ability of LMP1 CTAR2 to engage JNK signalling. Interestingly, this region was also found to be essential for CTAR2-mediated NF-κB induction and coincides with the LMP1 amino acid sequences shown to bind TRADD. Furthermore, we have found that LMP1-mediated JNK activation is synergistically augmented by low levels of TRADD expression, suggesting that this adapter protein is critical for LMP1 signalling. TRAF2 is known to associate with TRADD, and expression of a dominant-negative N-terminal deletion TRAF2 mutant was found to partially inhibit LMP1-induced JNK activation in 293 cells. In addition, the TRAF2-interacting protein A20 blocked both LMP1-induced JNK and NF-κB activation, further implicating TRAF2 in these phenomena. While expression of a kinase-inactive mutated NF-κB-inducing kinase (NIK), a mitogen-activated protein kinase kinase kinase which also associates with TRAF2, impaired LMP1 signalling on the NF-κB axis, it did not inhibit LMP1-induced JNK activation, suggesting that these two pathways may bifurcate at the level of TRAF2. These data further define a role for TRADD and TRAF2 in JNK activation and confirm that LMP1 utilizes signalling mechanisms used by the TNF receptor/CD40 family to elicit its pleiotropic activities.

scholarly journals The Novel Nuclear Targeting and BFRF1-Interacting Domains of BFLF2 Are Essential for Efficient Epstein-Barr Virus Virion Release

2019 ◽  
Vol 94 (3) ◽  
Author(s):  
Yu-Ching Dai ◽  
Yen-Tzu Liao ◽  
Yi-Ting Juan ◽  
Yi-Ying Cheng ◽  
Mei-Tzu Su ◽  
...  
Keyword(s):  
Amino Acids ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Epstein Barr Virus ◽  
The Novel ◽  
Nuclear Targeting ◽  
Barr Virus ◽  
Nuclear Egress ◽  
Localization Signal ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) genomic DNA is replicated and packaged into procapsids in the nucleus to form nucleocapsids, which are then transported into the cytoplasm for tegumentation and final maturation. The process is facilitated by the coordination of the viral nuclear egress complex (NEC), which consists of BFLF2 and BFRF1. By expression alone, BFLF2 is distributed mainly in the nucleus. However, it colocalizes with BFRF1 at the nuclear rim and in cytoplasmic nuclear envelope-derived vesicles in coexpressing cells, suggesting temporal control of the interaction between BFLF2 and BFRF1 is critical for their proper function. The N-terminal sequence of BFLF2 is less conserved than that of alpha- and betaherpesvirus homologs. Here, we found that BFLF2 amino acids (aa) 2 to 102 are required for both nuclear targeting and its interaction with BFRF1. Coimmunoprecipitation and confocal analysis indicated that aa 82 to 106 of BFLF2 are important for its interaction with BFRF1. Three crucial amino acids (R47, K50, and R52) and several noncontinuous arginine and histidine residues within aa 59 to 80 function together as a noncanonical nuclear localization signal (NLS), which can be transferred onto yellow fluorescent protein (YFP)-LacZ for nuclear targeting in an importin β-dependent manner. Virion secretion is defective in 293 cells harboring a BFLF2 knockout EBV bacmid upon lytic induction and is restored by trans-complementation of wild-type BFLF2, but not NLS or BFRF1-interacting defective mutants. In addition, multiple domains of BFRF1 were found to bind BFLF2, suggesting multiple contact regions within BFRF1 and BFLF2 are required for proper nuclear egress of EBV nucleocapsids. IMPORTANCE Although Epstein-Barr virus (EBV) BFRF1 and BFLF2 are homologs of conserved viral nuclear egress complex (NEC) in all human herpesviruses, unique amino acid sequences and functions were identified in both proteins. In this study, the nuclear targeting and BFRF1-interacting domains were found within the N terminus of BFLF2. We showed that amino acids (aa) 82 to 106 are the major region required for BFLF2 to interact with BFRF1. However, the coimmunoprecipitation (Co-IP) data and glutathione transferase (GST) pulldown experiments revealed that multiple regions of both proteins contribute to reciprocal interactions. Different from the canonical nuclear localization signal (NLS) in other herpes viral homologs, BFLF2 contains a novel importin-dependent nuclear localization signal, including R47, K50, and R52 and several neighboring discontinuous arginine and histidine residues. Using a bacmid complementation system, we show that both the nuclear targeting and the novel nuclear localization signal within aa 82 to 106 of BFLF2 are required for virion secretion.

scholarly journals Identification and characterization of the conserved nucleoside-binding sites in the Epstein-Barr virus thymidine kinase

2004 ◽  
Vol 379 (3) ◽  
pp. 795-803 ◽  
Author(s):  
Chung-Chun WU ◽  
Min-Che CHEN ◽  
Ya-Ru CHANG ◽  
Tsuey-Ying HSU ◽  
Jen-Yang CHEN
Keyword(s):  
Amino Acids ◽  
Binding Site ◽  
Thymidine Kinase ◽  
Crucial Role ◽  
Metal Ion ◽  
Epstein Barr Virus ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Barr Virus ◽  
Epstein Barr

Thymidine kinase (TK), encoded by EBV (Epstein–Barr virus), is an attractive target for antiviral therapy and provides a novel approach to the treatment of EBV-associated malignancies. Despite the extensive use of nucleoside analogues for the treatment of viral infections and cancer, the structure–function relationship of EBV TK has been addressed rarely. In the absence of any structural information, we sought to identify and elucidate the functional roles of amino acids in the nucleoside-binding site using site-directed mutagenesis. Through alignment with other human herpesviral TK protein sequences, we predicted that certain conserved regions comprise the nucleoside-binding site of EBV TK and, through site-directed mutagenesis, showed significant changes in activity and binding affinity for thymidine of site 3 (-DRH-) and 4 (-VFP-) mutants. For site 3, only mutants D392E (Asp392→Glu) and R393H retain activity, indicating that a negative charge is important for Asp392 and a positive charge is required for Arg393. The increased binding affinities of these two mutants for 3´-deoxy-2´,3´-didehydrothymidine suggest that the two residues are also important for substrate selection. Interestingly, the changed metal-ion usage pattern of D392E reveals that Asp392 plays multiple roles in this region. His394 cannot be compensated by other amino acids, also indicating a crucial role. In site 4, the F402Y mutant retains full activity; however, F402S retains only 60% relative activity. Strikingly, when Phe402 is substituted with serine residue, the original preferred pyrimidine substrates, such as 3´-azido-3´-deoxythymidine, iododeoxyuridine and β-l-5-iododioxolane uracil (l-form substrate), have decreased competitiveness with thymidine, suggesting that Phe402 plays a crucial role in substrate specificity and that the aromatic ring is important for function.

scholarly journals Conserved Regions in the Epstein-Barr Virus Leader Protein Define Distinct Domains Required for Nuclear Localization and Transcriptional Cooperation with EBNA2

2000 ◽  
Vol 74 (21) ◽  
pp. 9953-9963 ◽  
Author(s):  
RongSheng Peng ◽  
Jie Tan ◽  
Paul D. Ling
Keyword(s):  
Amino Acids ◽  
Nuclear Localization ◽  
Nuclear Localization Signal ◽  
Epstein Barr Virus ◽  
Intracellular Localization ◽  
Barr Virus ◽  
Conserved Regions ◽  
Localization Signal ◽  
Epstein Barr ◽  
Stimulation Of

ABSTRACT Epstein-Barr virus (EBV) EBNA-LP is a latent protein whose function is not fully understood. Recent studies have shown that EBNA-LP may be an important EBNA2 cofactor by enhancing EBNA2 stimulation of the latency C and LMP-1 promoters. To further our understanding of EBNA-LP function, we have introduced a series of mutations into evolutionarily conserved regions and tested the mutant proteins for the ability to enhance EBNA2 stimulation of the latency C and LMP-1 promoters. Three conserved regions (CR1 to CR3) are located in the repeat domains that are essential for the EBNA2 cooperativity function. In addition, three serine residues are also well conserved in the repeat domains. Clustered alanine mutations were introduced into CR1 to CR3, and the conserved serines were also changed to alanine residues in an EBNA-LP with two repeats, which is the minimal protein able to cooperate with EBNA2. Mutations introduced into CR1a had no effect on EBNA-LP function, while mutations introduced into CR1b resulted in EBNA-LP with slightly decreased activity. Mutations in CR1c and CR2 resulted in proteins that no longer localized exclusively to the nucleus and also had no EBNA2 cooperation activity. Mutations introduced into conserved serines S5/71 resulted in proteins with slightly higher activity, while mutations introduced into conserved serines S35/101 or in CR3 (which contains S60/126) resulted in EBNA-LP proteins with substantially reduced activity. The potential karyophilic signals within EBNA-LP CR1c and CR2 were also examined by introducing oligonucleotides encoding these positively charged amino acid groupings into a cytoplasmic test protein, herpes simplex virus ΔIE175, and by examining the intracellular localization of the resulting proteins. This assay identified a strong nuclear localization signal between EBNA-LP amino acids 43 and 50 (109 to 117 in the second W repeat) comprising CR2, while EBNA-LP amino acids 29 to 36 (91 to 98 in the second W repeat) were unable to function independently as a nuclear localization signal. However, a combination of amino acids 29 to 50 resulted in more efficient nuclear localization than with amino acids 43 to 50 alone. These results indicate that EBNA-LP has a bipartite nuclear localization signal and that efficient nuclear localization is essential for EBNA2 cooperativity function. Interestingly, EBNA-LP with only a single repeat localized exclusively to the cytoplasm, providing an explanation for why this isoform has no activity. In addition, two conserved serine residues which are distinct from nuclear import functions are important for EBNA2 cooperativity function.

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