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

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.

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

10.21203/rs.3.rs-45964/v1 ◽

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.

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Faculty Opinions recommendation of Spontaneous lytic replication and epitheliotropism define an Epstein-Barr virus strain found in carcinomas.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718140900.793529960 ◽

2017 ◽

Author(s):

Micah Luftig

Keyword(s):

Virus Strain ◽

Epstein Barr Virus ◽

Lytic Replication ◽

Barr Virus ◽

Epstein Barr

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Nuclear localization of the Epstein–Barr virus EBNA3B protein

Journal of General Virology ◽

10.1099/vir.0.81640-0 ◽

2006 ◽

Vol 87 (4) ◽

pp. 789-793 ◽

Cited By ~ 7

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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Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen 1 Mimics Epstein-Barr Virus EBNA1 Immune Evasion through Central Repeat Domain Effects on Protein Processing

Journal of Virology ◽

10.1128/jvi.00411-07 ◽

2007 ◽

Vol 81 (15) ◽

pp. 8225-8235 ◽

Cited By ~ 70

Author(s):

Hyun Jin Kwun ◽

Suzane Ramos da Silva ◽

Ishita M. Shah ◽

Neil Blake ◽

Patrick S. Moore ◽

...

Keyword(s):

Antigen Processing ◽

Kaposi's Sarcoma ◽

Epstein Barr Virus ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Nuclear Antigen ◽

Barr Virus ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Epstein Barr ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8 [HHV8]) and Epstein-Barr virus (EBV/HHV4) are distantly related gammaherpesviruses causing tumors in humans. KSHV latency-associated nuclear antigen 1 (LANA1) is functionally similar to the EBV nuclear antigen-1 (EBNA1) protein expressed during viral latency, although they have no amino acid similarities. EBNA1 escapes cytotoxic lymphocyte (CTL) antigen processing by inhibiting its own proteosomal degradation and retarding its own synthesis to reduce defective ribosomal product processing. We show here that the LANA1 QED-rich central repeat (CR) region, particularly the CR2CR3 subdomain, also retards LANA1 synthesis and markedly enhances LANA1 stability in vitro and in vivo. LANA1 isoforms have half-lives greater than 24 h, and fusion of the LANA1 CR2CR3 domain to a destabilized heterologous protein markedly decreases protein turnover. Unlike EBNA1, the LANA1 CR2CR3 subdomain retards translation regardless of whether it is fused to the 5′ or 3′ end of a heterologous gene construct. Manipulation of sequence order, orientation, and composition of the CR2 and CR3 subdomains suggests that specific peptide sequences rather than RNA structures are responsible for synthesis retardation. Although mechanistic differences exist between LANA1 and EBNA1, the primary structures of both proteins have evolved to minimize provoking CTL immune responses. Simple strategies to eliminate these viral inhibitory regions may markedly improve vaccine effectiveness by maximizing CTL responses.

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Elevated blood levels of soluble tumor necrosis factor receptors in nasopharyngeal carcinoma: correlation with humoral immune response to lytic replication of Epstein-Barr virus.

International Journal of Oncology ◽

10.3892/ijo.15.1.167 ◽

1999 ◽

Cited By ~ 1

Author(s):

P Feng ◽

S H Chan ◽

E E Ooi ◽

M Y Soo ◽

K S Loh ◽

...

Keyword(s):

Tumor Necrosis ◽

Epstein Barr Virus ◽

Lytic Replication ◽

Blood Levels ◽

Elevated Blood ◽

Tumor Necrosis Factor Receptors ◽

Humoral Immune ◽

Barr Virus ◽

Epstein Barr ◽

Necrosis Factor

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Curcumin Inhibits Proliferation of Epstein–Barr Virus-Associated Human Nasopharyngeal Carcinoma Cells by Inhibiting EBV Nuclear Antigen 1 Expression

BioMed Research International ◽

10.1155/2019/8592921 ◽

2019 ◽

Vol 2019 ◽

pp. 1-10

Author(s):

Limei Liu ◽

Jiaomin Yang ◽

Wuguang Ji ◽

Chao Wang

Keyword(s):

Nasopharyngeal Carcinoma ◽

Epstein Barr Virus ◽

Lytic Replication ◽

Nuclear Antigen ◽

Mrna Levels ◽

Curcumin Treatment ◽

Antitumor Effects ◽

Barr Virus ◽

Cycle Arrest ◽

Epstein Barr

This investigation aims to study the effect of curcumin on the proliferation, cycle arrest, and apoptosis of Epstein–Barr virus- (EBV-) positive nasopharyngeal carcinoma (NPC) cells. EBV+ NPC cells were subjected to curcumin treatment. The cell viability was evaluated with the CCK-8. Cell cycle and apoptosis were analyzed by flow cytometry analysis. Expression (protein and mRNA) levels were detected with western blotting and quantitative real-time PCR, respectively. Curcumin efficiently reduced the viability of EBV+ NPC cells. Curcumin induced the cycle arrest of the HONE1 and HK1-EBV cells positive for EBV. Moreover, curcumin treatment promoted the NPC cell apoptosis, via the mitochondria- and death receptor-mediated pathways. Furthermore, curcumin decreased the expression of EBNA1 in the HONE1 and HK1-EBV cells and inhibited the transcriptional level of EBNA1 in the HeLa cells. Curcumin induced EBNA1 degradation via the proteasome-ubiquitin pathway. In addition, curcumin inhibited the proliferation of HONE1 and HK1-EBV cells positive for EBV, probably by decreasing the expression level of EBNA1. In both the HONE1 and HK1-EBV cells, curcumin inhibited the EBV latent and lytic replication. Curcumin could reduce the EBNA1 expression and exert antitumor effects against NPC in vitro.

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Establishment and characterization of an Epstein-Barr virus transformed cell line with strong phagocytic activity

Blood ◽

10.1182/blood.v74.1.430.430 ◽

1989 ◽

Vol 74 (1) ◽

pp. 430-436 ◽

Cited By ~ 4

Author(s):

P von den Driesch ◽

R Bhardwaj ◽

HD Flad ◽

DC Neugebauer ◽

HJ Pielken ◽

...

Keyword(s):

Cell Line ◽

Phagocytic Activity ◽

Antigen Processing ◽

Epstein Barr Virus ◽

Interleukin 1 ◽

Immunoglobulin M ◽

Nuclear Antigen ◽

Barr Virus ◽

Transformed Cell Line ◽

Epstein Barr

Abstract An immunoglobulin M (IgM)-positive cell line, Ms 28, apparently spontaneously transformed by Epstein-Barr virus (EBV) was established from peripheral blood cells of a patient with immature myeloblastic leukemia. It has been characterized according to phenotype, cytochemistry, and membrane antigen pattern. The cell line expresses lymphoid markers like CD 19, CD 22, and CD 30 and synthesizes and secretes IgM. Monocyte markers CD 11c, CD 14, and CD 15 are absent. Neither interleukin-1 (IL-1), nor tumor necrosis factor (TNF-alpha) are produced. But Ms 28 cells show strong phagocytic activity and engulf Latex particles and sheep RBCs (SRBCs) that need not to be opsonized. The phagocytic activity can be inhibited by chloroquine. Both phagocytosis and EBV nuclear-antigen (EBNA) expression can be observed in one and the same cell. Ms 28 cells might be useful to study immunologic activities like antigen processing and presentation.

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CD8+ immunodominance among Epstein-Barr virus lytic cycle antigens directly reflects the efficiency of antigen presentation in lytically infected cells

Journal of Experimental Medicine ◽

10.1084/jem.20041542 ◽

2005 ◽

Vol 201 (3) ◽

pp. 349-360 ◽

Cited By ~ 90

Author(s):

Victoria A. Pudney ◽

Alison M. Leese ◽

Alan B. Rickinson ◽

Andrew D. Hislop

Keyword(s):

T Cell ◽

Epstein Barr Virus ◽

Cd8 T Cell ◽

Lytic Replication ◽

Lytic Cycle ◽

E Proteins ◽

Barr Virus ◽

Infected Cells ◽

Epstein Barr ◽

L Proteins

Antigen immunodominance is an unexplained feature of CD8+ T cell responses to herpesviruses, which are agents whose lytic replication involves the sequential expression of immediate early (IE), early (E), and late (L) proteins. Here, we analyze the primary CD8 response to Epstein-Barr virus (EBV) infection for reactivity to 2 IE proteins, 11 representative E proteins, and 10 representative L proteins, across a range of HLA backgrounds. Responses were consistently skewed toward epitopes in IE and a subset of E proteins, with only occasional responses to novel epitopes in L proteins. CD8+ T cell clones to representative IE, E, and L epitopes were assayed against EBV-transformed lymphoblastoid cell lines (LCLs) containing lytically infected cells. This showed direct recognition of lytically infected cells by all three sets of effectors but at markedly different levels, in the order IE > E ≫ L, indicating that the efficiency of epitope presentation falls dramatically with progress of the lytic cycle. Thus, EBV lytic cycle antigens display a hierarchy of immunodominance that directly reflects the efficiency of their presentation in lytically infected cells; the CD8+ T cell response thereby focuses on targets whose recognition leads to maximal biologic effect.

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