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.

Isolating the Epstein–Barr virus (EBV) gp350/220 binding site on complement receptor type 2 (CR2/CD21) using site-directed mutagenesis

2007 ◽  
Vol 44 (1-3) ◽  
pp. 180-181
Author(s):  
Jonathan P. Hannan ◽  
Kendra A. Young ◽  
Gerda Szakonyi ◽  
Michael Klein ◽  
Xiaojiang S. Chen ◽  
...  
Keyword(s):  
Binding Site ◽  
Epstein Barr Virus ◽  
Site Directed Mutagenesis ◽  
Receptor Type ◽  
Directed Mutagenesis ◽  
Complement Receptor ◽  
Complement Receptor Type ◽  
Barr Virus ◽  
Epstein Barr

scholarly journals Site-directed mutagenesis in a conserved motif of Epstein–Barr virus DNase that is homologous to the catalytic centre of type II restriction endonucleases

2003 ◽  
Vol 84 (3) ◽  
pp. 677-686 ◽  
Author(s):  
Ming-Tsan Liu ◽  
Hsien-Ping Hu ◽  
Tsuey-Ying Hsu ◽  
Jen-Yang Chen
Keyword(s):  
Dna Binding ◽  
Epstein Barr Virus ◽  
Nuclease Activity ◽  
Restriction Endonucleases ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Type Ii ◽  
Conserved Motif ◽  
Barr Virus ◽  
Epstein Barr

Sequence alignment of human herpesvirus DNases revealed that they share several conserved regions. One of these, the conserved motif D203…E225XK227 (D…EXK) in the sequence of Epstein–Barr virus (EBV) DNase, has a striking similarity to the catalytic sites of some other nucleases, including type II restriction endonucleases, λ exonuclease and MutH. The predicted secondary structures of these three residues were shown to resemble the three catalytic residues of type II restriction endonucleases. Site-directed mutagenesis was carried out to replace each of the acidic residues near the motif by residues with different properties. All substitutions of D203, E225 and K227 were shown to cause significant reductions in nuclease activity. Six other acidic residues, within the conserved regions, were also replaced by Asn or Gln. Five of these six variants retained nuclease activity and mutant D195N alone lost nuclease activity. The four charged residues, D195, D203, E225 and K227, of EBV DNase were found to be important for nuclease activity. Biochemical analysis indicated that the preference for divalent cations was altered from Mg2+ to Mn2+ for mutant E225D. The DNA-binding abilities of D203E, E225D and E225Q were shown to be similar to that of wild-type. However, K227 mutants were found to have variable DNA-binding abilities: K227G and K227N mutants retained, K227E and K227D had reduced and K227R lost DNA-binding ability. Comparison of the biochemical properties of the corresponding substitutions among EBV DNase and type II restriction enzymes indicated that the D…EXK motif is most likely the putative catalytic centre of EBV DNase.

Characterizing the complement receptor type 2 (CR2)–Epstein-Barr virus interaction: A site-directed mutagenesis and molecular docking approach

2007 ◽  
Vol 44 (16) ◽  
pp. 3929 ◽  
Author(s):  
Jonathan P. Hannan ◽  
Kendra A. Young ◽  
Andrew P. Herbert ◽  
Xiaojiang S. Chen ◽  
Paul N. Barlow ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Complement Receptor ◽  
Barr Virus ◽  
Docking Approach ◽  
Virus Interaction ◽  
Epstein Barr ◽  
A Site

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.

Characterization of an Epstein-Barr virus-induced thymidine kinase.

1986 ◽  
Vol 57 (3) ◽  
pp. 1105-1112 ◽  
Author(s):  
M de Turenne-Tessier ◽  
T Ooka ◽  
G de The ◽  
J Daillie
Keyword(s):  
Thymidine Kinase ◽  
Epstein Barr Virus ◽  
Barr Virus ◽  
Epstein Barr

scholarly journals EBV-LMP1 promotes radioresistance by inducing protective autophagy through BNIP3 in nasopharyngeal carcinoma

2021 ◽  
Vol 12 (4) ◽  
Author(s):  
San Xu ◽  
Zhuan Zhou ◽  
Xingzhi Peng ◽  
Xuxiu Tao ◽  
Peijun Zhou ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Crucial Role ◽  
Epstein Barr Virus ◽  
Interacting Protein ◽  
Multiple Tumors ◽  
Barr Virus ◽  
Signaling Axis ◽  
Epstein Barr ◽  
Adenovirus E1b

AbstractStudies have indicated that dysfunction of autophagy is involved in the initiation and progression of multiple tumors and their chemoradiotherapy. Epstein–Barr virus (EBV) is a lymphotropic human gamma herpes virus that has been implicated in the pathogenesis of nasopharyngeal carcinoma (NPC). EBV encoded latent membrane protein1 (LMP1) exhibits the properties of a classical oncoprotein. In previous studies, we experimentally demonstrated that LMP1 could increase the radioresistance of NPC. However, how LMP1 contributes to the radioresistance in NPC is still not clear. In the present study, we found that LMP1 could enhance autophagy by upregulating the expression of BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3). Knockdown of BNIP3 could increase the apoptosis and decrease the radioresistance mediated by protective autophagy in LMP1-positive NPC cells. The data showed that increased BNIP3 expression is mediated by LMP1 through the ERK/HIF1α signaling axis, and LMP1 promotes the binding of BNIP3 to Beclin1 and competitively reduces the binding of Bcl-2 to Beclin1, thus upregulating autophagy. Furthermore, knockdown of BNIP3 can reduce the radioresistance promoted by protective autophagy in vivo. These data clearly indicated that, through BNIP3, LMP1 induced autophagy, which has a crucial role in the protection of LMP1-positive NPC cells against irradiation. It provides a new basis and potential target for elucidating LMP1-mediated radioresistance.

scholarly journals Comparative Mutagenesis of Pseudorabies Virus and Epstein-Barr Virus gH Identifies a Structural Determinant within Domain III of gH Required for Surface Expression and Entry Function

2015 ◽  
Vol 90 (5) ◽  
pp. 2285-2293 ◽  
Author(s):  
Britta S. Möhl ◽  
Christina Schröter ◽  
Barbara G. Klupp ◽  
Walter Fuchs ◽  
Thomas C. Mettenleiter ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Surface ◽  
B Cell ◽  
Epstein Barr Virus ◽  
Pseudorabies Virus ◽  
Surface Expression ◽  
Structural Motif ◽  
Cell Surface Expression ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACTHerpesviruses infect cells using the conserved core fusion machinery composed of glycoprotein B (gB) and gH/gL. The gH/gL complex plays an essential but still poorly characterized role in membrane fusion and cell tropism. Our previous studies demonstrated that the conserved disulfide bond (DB) C278/C335 in domain II (D-II) of Epstein-Barr virus (EBV) gH has an epithelial cell-specific function, whereas the interface of D-II/D-III is involved in formation of the B cell entry complex by binding to gp42. To extend these studies, we compared gH of the alphaherpesvirus pseudorabies virus (PrV) with gH of the gammaherpesvirus EBV to identify functionally equivalent regions critical for gH function during entry. We identified several conserved amino acids surrounding the conserved DB that connects three central helices of D-III of PrV and EBV gH. The present study verified that the conserved DB and several contacting amino acids in D-III modulate cell surface expression and thereby contribute to gH function. In line with this finding, we found that DB C404/C439 and T401 are important for cell-to-cell spread and efficient entry of PrV. This parallel comparison between PrV and EBV gH function brings new insights into how gH structure impacts fusion function during herpesvirus entry.IMPORTANCEThe alphaherpesvirus PrV is known for its neuroinvasion, whereas the gammaherpesvirus EBV is associated with cancer of epithelial and B cell origin. Despite low amino acid conservation, PrV gH and EBV gH show strikingly similar structures. Interestingly, both PrV gH and EBV gH contain a structural motif composed of a DB and supporting amino acids which is highly conserved within theHerpesviridae. Our study verified that PrV gH uses a minimal motif with the DB as the core, whereas the DB of EBV gH forms extensive connections through hydrogen bonds to surrounding amino acids, ensuring the cell surface expression of gH/gL. Our study verifies that the comparative analysis of distantly related herpesviruses, such as PrV and EBV, allows the identification of common gH functions. In addition, we provide an understanding of how functional domains can evolve over time, resulting in subtle differences in domain structure and function.

Diagnosis of Nasopharyngeal Carcinoma Using the Epstein-Barr Virus Coded Alkaline Dnase, Membrane Antigen and Thymidine Kinase

1991 ◽  
pp. 353-356
Author(s):  
Edward Littler ◽  
Sally A. Baylis ◽  
Yvonne Connolly ◽  
Margaret J. Conway ◽  
Michael Mackett ◽  
...  
Keyword(s):  
Nasopharyngeal Carcinoma ◽  
Thymidine Kinase ◽  
Epstein Barr Virus ◽  
Barr Virus ◽  
Epstein Barr

scholarly journals Finding the Binding Site of Peloruside A and its Secondary Effects in Saccharomyces Cerevisiae using a  Chemical Genetics Approach

2021 ◽  
Author(s):  
◽  
Reem Hanna
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Amino Acids ◽  
Flow Cytometry ◽  
Binding Site ◽  
Mammalian Cells ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Peloruside A ◽  
Microtubule Function

<p>Peloruside A, a natural product isolated from the marine sponge Mycale hentscheli, is a microtubule-stabilising agent that has a similar mechanism of action to the anticancer drug paclitaxel and is cytotoxic to cultured mammalian cells. Peloruside appears to bind to a distinct site on mammalian tubulin that is different from that of the taxoid-site drugs. Because of the high sequence homology between yeast and mammalian tubulin, Saccharomyces cerevisiae (S. cerevisiae) was used as a model organism to characterise the peloruside-binding site with the aim of advancing our understanding about this site on mammalian tubulin. Wild type S. cerevisiae (BY4741) was sensitive to peloruside at uM concentrations; however, a strain that lacks the mad2 (Mitotic Arrest Deficient 2) gene showed increased sensitivity to the drug at much lower uM concentrations. This gene is a component of the spindle-assembly checkpoint complex that delays the onset of anaphase in cells with defects in mitotic spindle assembly. The main aims of this project were to define the binding site of peloruside A using yeast tubulin to see if microtubule function and/or morphology is altered in yeast by peloruside, and to identify any secondary drug targets "friends of the target" through chemical genetic interactions profiling (Homozygous deletion profiling microarray). Site-directed mutagenesis was used to mutate two conserved amino acids (A296T; R306H) known to confer resistance to peloruside in mammalian cells. Based on a published computer model of the peloruside binding site on mammalian tubulin, we also mutated three other amino acids, two that were predicted to affect peloruside binding (Q291M and N337L), and one that was predicted to affect laulimalide binding but have little affect on peloruside binding (V333W). We also included a negative control that was predicted to have no effect on peloruside binding (R282Q) and would affect epothilone binding. We found that of the six point mutations, only Q291M failed to confer resistance in yeast and instead it increased the inhibition to the drug. Using a bud index assay, confocal microscopy, and flow cytometry, 40-50 uM peloruside was shown to block cells in G2/M of the cell cycle, confirming a direct action of the drug on microtubule function. Homozygous profiling (HOP) microarray analysis of a deletion mutant set of yeast genes was also carried out to identify gene products that interact with peloruside in order to link the drug to specific networks or biochemical pathways in the cells. From site-directed mutagenesis, we concluded that peloruside binds to yeast B-tubulin in the region predicted by the published model of the binding site, and therefore mapping the site on yeast tubulin could provide useful information about the mammalian binding site for peloruside. The bud index, flow cytometry, and confocal microscopy experiments provided further evidence that peloruside interacts with yeast tubulin. From HOP we found that peloruside has roles in the cell cycle, as expected, and has effects on protein transport, secretion, cell wall synthesis, and steroid biosynthesis pathways.</p>

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.

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