scholarly journals Tetrameric Ring Formation of Epstein-Barr Virus Polymerase Processivity Factor Is Crucial for Viral Replication

2010 ◽  
Vol 84 (24) ◽  
pp. 12589-12598 ◽  
Author(s):  
Sanae Nakayama ◽  
Takayuki Murata ◽  
Yoshihiro Yasui ◽  
Kazutaka Murayama ◽  
Hiroki Isomura ◽  
...  
Keyword(s):  
Viral Replication ◽  
Epstein Barr Virus ◽  
Ring Formation ◽  
Genome Replication ◽  
Wild Type ◽  
Barr Virus ◽  
Viral Genome Replication ◽  
Processivity Factor ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus BMRF1 DNA polymerase processivity factor, which is essential for viral genome replication, exists mainly as a C-shaped head-to-head homodimer but partly forms a ring-shaped tetramer through tail-to-tail association. Based on its molecular structure, several BMRF1 mutant viruses were constructed to examine their influence on viral replication. The R256E virus, which has a severely impaired capacity for DNA binding and polymerase processivity, failed to form replication compartments, resulting in interference of viral replication, while the C95E mutation, which impairs head-to-head contact in vitro, unexpectedly hardly affected the viral replication. Also, surprisingly, replication of the C206E virus, which is expected to have impairment of tail-to-tail contact, was severely restricted, although the mutant protein possesses the same in vitro biochemical activities as the wild type. Since the tail-to-tail contact surface is smaller than that of the head-to-head contact area, its contribution to ring formation might be essential for viral replication.

scholarly journals Sumoylation of the Epstein-Barr Virus BZLF1 Protein Inhibits Its Transcriptional Activity and Is Regulated by the Virus-Encoded Protein Kinase

2010 ◽  
Vol 84 (9) ◽  
pp. 4383-4394 ◽  
Author(s):  
Stacy R. Hagemeier ◽  
Sarah J. Dickerson ◽  
Qiao Meng ◽  
Xianming Yu ◽  
Janet E. Mertz ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Viral Genome ◽  
Epstein Barr Virus ◽  
Transcriptional Activity ◽  
Genome Replication ◽  
Wild Type ◽  
Barr Virus ◽  
293 Cells ◽  
Viral Genome Replication ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) immediate-early protein BZLF1 (Z) mediates the switch between latent and lytic EBV infection. Z not only activates early lytic viral gene transcription but also plays a direct role in lytic viral genome replication. Although a small fraction of Z is known to be sumoylated, the effects of this posttranslational modification on various different Z functions have not been well defined. In this report, we show that only the lysine at amino acid residue 12 is required for the sumoylation of Z, and that Z can be sumoylated by SUMO isoforms 1, 2, and 3. We also demonstrate that the sumo-defective Z mutants ZK12A and ZK12R have enhanced transcriptional activity. The sumoylated and nonsumoylated forms of Z were found to have a similar cellular location, both being localized primarily within the nuclear matrix. The Z sumo-defective mutants were, however, partially defective for disrupting promyelocytic leukemia (PML) bodies compared to the ability of wild-type Z. In addition, we show that lytic viral genome replication does not require the sumoylation of Z, although a Z mutant altered at both amino acids 12 and 13 is replication defective. Furthermore, we show that the sumoylation of Z is greatly increased (from less than 1 to about 11%) in lytically induced 293 cells infected with an EBV mutant virus deleted for the EBV-encoded protein kinase (EBV-PK) compared to that of 293 cells infected with wild-type EBV, and that the overexpression of EBV-PK leads to the reduced sumoylation of Z in EBV-negative cells. Our results suggest that the sumoylation of Z helps to promote viral latency, and that EBV-PK inhibits Z sumoylation during viral reactivation.

scholarly journals The Epstein-Barr Virus BMRF1 Gene Is Essential for Lytic Virus Replication

2006 ◽  
Vol 80 (10) ◽  
pp. 5078-5081 ◽  
Author(s):  
Bernhard Neuhierl ◽  
Henri-Jacques Delecluse
Keyword(s):  
Epstein Barr Virus ◽  
Lytic Replication ◽  
Wild Type ◽  
Reading Frame ◽  
Barr Virus ◽  
Wild Type Phenotype ◽  
Late Protein ◽  
Processivity Factor ◽  
Epstein Barr ◽  
Lytic Dna Replication

ABSTRACT The Epstein-Barr virus (EBV) BMRF1 protein is a DNA polymerase processivity factor. We have deleted the BMRF1 open reading frame from the EBV genome and assessed the ΔBMRF1 EBV phenotype. ΔBMRF1 viruses were replication deficient, but the wild-type phenotype could be restored by BMRF1 trans-complementation. The replication-deficient phenotype included impaired lytic DNA replication and late protein expression. ΔBMRF1 and wild-type viruses were undistinguishable in terms of their ability to transform primary B cells. Our results provide genetic evidence that BMRF1 is essential for lytic replication of the EBV genome.

scholarly journals Epstein-Barr Virus BNRF1 Protein Allows Efficient Transfer from the Endosomal Compartment to the Nucleus of Primary B Lymphocytes

2006 ◽  
Vol 80 (19) ◽  
pp. 9435-9443 ◽  
Author(s):  
R. Feederle ◽  
B. Neuhierl ◽  
G. Baldwin ◽  
H. Bannert ◽  
B. Hub ◽  
...  
Keyword(s):  
B Cells ◽  
Epstein Barr Virus ◽  
Wild Type ◽  
Barr Virus ◽  
Fold Reduction ◽  
Viral Particles ◽  
Viral Mutant ◽  
Tumor Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) is a tumor virus with marked B lymphotropism. After crossing the B-cell membrane, the virus enters cytoplasmic vesicles, where decapsidation takes place to allow transfer of the viral DNA to the cell nucleus. BNRF1 has been characterized as the EBV major tegument protein, but its precise function is unknown. We have constructed a viral mutant that lacks the BNRF1 gene and report here its in vitro phenotype. A recombinant virus devoid of BNRF1 (ΔBNRF1) showed efficient DNA replication and production of mature viral particles. B cells infected with the ΔBNRF1 mutant presented viral lytic antigens as efficiently as B cells infected with wild-type or BNRF1 trans-complemented ΔBNRF1 viruses. Antigen presentation in B cells infected with either wild-type (EBV-wt) or ΔBNRF1 virus was blocked by leupeptin addition, showing that both viruses reach the endosome/lysosome compartment. These data were confirmed by direct observation of the mutant virus in endosomes of infected B cells by electron microscopy. However, we observed a 20-fold reduction in the number of B cells expressing the nuclear protein EBNA2 after infection with a ΔBNRF1 virus compared to wild-type infection. Likewise, ΔBNRF1 viruses transformed primary B cells much less efficiently than EBV-wt or BNRF1 trans-complemented viruses. We conclude from these findings that BNRF1 plays an important role in viral transport from the endosomes to the nucleus.

scholarly journals Terminal Differentiation into Plasma Cells Initiates the Replicative Cycle of Epstein-Barr Virus In Vivo

2005 ◽  
Vol 79 (2) ◽  
pp. 1296-1307 ◽  
Author(s):  
Lauri L. Laichalk ◽  
David A. Thorley-Lawson
Keyword(s):  
Viral Replication ◽  
Epstein Barr Virus ◽  
Plasma Cells ◽  
Acute Stress ◽  
Cytotoxic T Lymphocyte ◽  
Lytic Cycle ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT In this paper we demonstrate that the cells which initiate replication of Epstein-Barr virus (EBV) in the tonsils of healthy carriers are plasma cells (CD38hi, CD10−, CD19+, CD20lo, surface immunoglobulin negative, and cytoplasmic immunoglobulin positive). We further conclude that differentiation into plasma cells, and not the signals that induce differentiation, initiates viral replication. This was confirmed by in vitro studies showing that the promoter for BZLF1, the gene that begins viral replication, becomes active only after memory cells differentiate into plasma cells and is also active in plasma cell lines. This differs from the reactivation of BZLF1 in vitro, which occurs acutely and is associated with apoptosis and not with differentiation. We suggest that differentiation and acute stress represent two distinct pathways of EBV reactivation in vivo. The fraction of cells replicating the virus decreases as the cells progress through the lytic cycle such that only a tiny fraction actually release infectious virus. This may reflect abortive replication or elimination of cells by the cellular immune response. Consistent with the later conclusion, the cells did not down regulate major histocompatibility complex class I molecules, suggesting that this is not an immune evasion tactic used by EBV and that the cells remain vulnerable to cytotoxic-T-lymphocyte attack.

scholarly journals Epstein-Barr Virus BGLF4 Kinase Induces Disassembly of the Nuclear Lamina To Facilitate Virion Production

2008 ◽  
Vol 82 (23) ◽  
pp. 11913-11926 ◽  
Author(s):  
Chung-Pei Lee ◽  
Yu-Hao Huang ◽  
Su-Fang Lin ◽  
Yao Chang ◽  
Yu-Hsin Chang ◽  
...  
Keyword(s):  
Epstein Barr Virus ◽  
Fluorescent Protein ◽  
Nuclear Lamina ◽  
Lamin A ◽  
Genome Replication ◽  
Barr Virus ◽  
Virion Production ◽  
Cellular Environment ◽  
Epstein Barr

ABSTRACT DNA viruses adopt various strategies to modulate the cellular environment for efficient genome replication and virion production. Previously, we demonstrated that the BGLF4 kinase of Epstein-Barr virus (EBV) induces premature chromosome condensation through the activation of condensin and topoisomerase IIα (C. P. Lee, J. Y. Chen, J. T. Wang, K. Kimura, A. Takemoto, C. C. Lu, and M. R. Chen, J. Virol. 81:5166-5180, 2007). In this study, we show that BGLF4 interacts with lamin A/C and phosphorylates lamin A protein in vitro. Using a green fluorescent protein (GFP)-lamin A system, we found that Ser-22, Ser-390, and Ser-392 of lamin A are important for the BGLF4-induced disassembly of the nuclear lamina and the EBV reactivation-mediated redistribution of nuclear lamin. Virion production and protein levels of two EBV primary envelope proteins, BFRF1 and BFLF2, were reduced significantly by the expression of GFP-lamin A(5A), which has five Ser residues replaced by Ala at amino acids 22, 390, 392, 652, and 657 of lamin A. Our data indicate that BGLF4 kinase phosphorylates lamin A/C to promote the reorganization of the nuclear lamina, which then may facilitate the interaction of BFRF1 and BFLF2s and subsequent virion maturation. UL kinases of alpha- and betaherpesviruses also induce the disassembly of the nuclear lamina through similar sites on lamin A/C, suggesting a conserved mechanism for the nuclear egress of herpesviruses.

scholarly journals Quantitative evaluation of the role of Epstein–Barr virus immediate-early protein BZLF1 in B-cell transformation

2009 ◽  
Vol 90 (10) ◽  
pp. 2331-2341 ◽  
Author(s):  
Koichi Ricardo Katsumura ◽  
Seiji Maruo ◽  
Yi Wu ◽  
Teru Kanda ◽  
Kenzo Takada
Keyword(s):  
B Cells ◽  
Epstein Barr Virus ◽  
Lytic Replication ◽  
Immediate Early ◽  
Wild Type ◽  
Barr Virus ◽  
Ebv Infection ◽  
Early Protein ◽  
Epstein Barr

The Epstein–Barr virus (EBV) immediate-early transactivator BZLF1 plays a key role in switching EBV infection from the latent to the lytic form by stimulating the expression cascade of lytic genes; it also regulates the expression of several cellular genes. Recently, we reported that BZLF1 is expressed in primary human B cells early after EBV infection. To investigate whether this BZLF1 expression early after infection plays a role in the EBV-induced growth transformation of primary B cells, we generated BZLF1-knockout EBV and quantitatively evaluated its transforming ability compared with that of wild-type EBV. We found that the 50 % transforming dose of BZLF1-knockout EBV was quite similar to that of wild-type EBV. Established lymphoblastoid cell lines (LCLs) harbouring BZLF1-knockout EBV were indistinguishable from LCLs harbouring wild-type EBV in their pattern of latent gene expression and in their growth in vitro. Furthermore, the copy numbers of EBV episomes were very similar in the LCLs harbouring BZLF1-knockout EBV and in those harbouring wild-type EBV. These data indicate that disrupting BZLF1 expression in the context of the EBV genome, and the resultant inability to enter lytic replication, have little impact on the growth of LCLs and the steady-state copy number of EBV episomes in established LCLs.

scholarly journals The Epstein-Barr Virus (EBV)-Encoded Protein Kinase, EBV-PK, but Not the Thymidine Kinase (EBV-TK), Is Required for Ganciclovir and Acyclovir Inhibition of Lytic Viral Production

2010 ◽  
Vol 84 (9) ◽  
pp. 4534-4542 ◽  
Author(s):  
Qiao Meng ◽  
Stacy R. Hagemeier ◽  
Joyce D. Fingeroth ◽  
Edward Gershburg ◽  
Joseph S. Pagano ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Viral Replication ◽  
Thymidine Kinase ◽  
Epstein Barr Virus ◽  
Raji Cell ◽  
Virus Production ◽  
Mutant Virus ◽  
Wild Type ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Ganciclovir (GCV) and acyclovir (ACV) are guanine nucleoside analogues that inhibit lytic herpesvirus replication. GCV and ACV must be monophosphorylated by virally encoded enzymes to be converted into nucleotides and incorporated into viral DNA. However, whether GCV and/or ACV phosphorylation in Epstein-Barr virus (EBV)-infected cells is mediated primarily by the EBV-encoded protein kinase (EBV-PK), the EBV-encoded thymidine kinase (EBV-TK), or both is controversial. To examine this question, we constructed EBV mutants containing stop codons in either the EBV-PK or EBV-TK open reading frame and selected for stable 293T clones latently infected with wild-type EBV or each of the mutant viruses. Cells were induced to the lytic form of viral replication with a BZLF1 expression vector in the presence and absence of various doses of GCV and ACV, and infectious viral titers were determined by a green Raji cell assay. As expected, virus production in wild-type EBV-infected 293T cells was inhibited by both GCV (50% inhibitory concentration [IC50] = 1.5 μM) and ACV (IC50 = 4.1 μM). However, the EBV-PK mutant (which replicates as well as the wild-type (WT) virus in 293T cells) was resistant to both GCV (IC50 = 19.6 μM) and ACV (IC50 = 36.4 μM). Expression of the EBV-PK protein in trans restored GCV and ACV sensitivity in cells infected with the PK mutant virus. In contrast, in 293T cells infected with the TK mutant virus, viral replication remained sensitive to both GCV (IC50 = 1.2 μM) and ACV (IC50 = 2.8 μM), although susceptibility to the thymine nucleoside analogue, bromodeoxyuridine, was reduced. Thus, EBV-PK but not EBV-TK mediates ACV and GCV susceptibilities.

scholarly journals Epstein-Barr Virus BARF1 Protein Is Dispensable for B-Cell Transformation and Inhibits Alpha Interferon Secretion from Mononuclear Cells

1999 ◽  
Vol 73 (9) ◽  
pp. 7627-7632 ◽  
Author(s):  
Jeffrey I. Cohen ◽  
Kristen Lekstrom
Keyword(s):  
B Cells ◽  
Recombinant Virus ◽  
Epstein Barr Virus ◽  
Mononuclear Cells ◽  
Cell Transformation ◽  
Alpha Interferon ◽  
Wild Type ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) BARF1 gene encodes a soluble colony-stimulating factor 1 (CSF-1) receptor that neutralizes the effects of CSF-1 in vitro. To study the effect of BARF1 on EBV-induced transformation, we added recombinant BARF1 to B cells in the presence of EBV. BARF1 did not enhance transformation of B cells by EBV in vitro. To study the role of BARF1 in the context of EBV infection, we constructed a recombinant EBV mutant with a large deletion followed by stop codons in the BARF1 gene as well as a recombinant virus with a wild-type BARF1 gene. While BARF1 has previously been shown to act as an oncogene in several cell lines, the EBV BARF1 deletion mutant transformed B cells and initiated latent infection, and the B cells transformed with the BARF1 mutant virus induced tumors in SCID mice with an efficiency similar to that of the wild-type recombinant virus. Since human CSF-1 stimulates secretion of alpha interferon from mononuclear cells and BARF1 encodes a soluble CSF-1 receptor, we examined whether recombinant BARF1 or BARF1 derived from EBV-infected B cells could inhibit alpha interferon secretion. Recombinant BARF1 inhibited alpha interferon secretion by mononuclear cells in a dose-dependent fashion. The B cells transformed with mutant BARF1 EBV showed reduced inhibition of alpha interferon secretion by human mononuclear cells when compared with the B cells transformed with wild-type recombinant virus. These experiments indicate that BARF1 expressed from the EBV genome directly inhibits alpha interferon secretion, which may modulate the innate host response to the virus.

scholarly journals Helper cell-independent cytotoxic clones in man.

1982 ◽  
Vol 156 (6) ◽  
pp. 1854-1859 ◽  
Author(s):  
S L Wee ◽  
L K Chen ◽  
G Strassmann ◽  
F H Bach
Keyword(s):  
T Cell ◽  
Epstein Barr Virus ◽  
Helper Cell ◽  
Cytotoxic T Cell ◽  
Lymphoblastoid Cell Lines ◽  
Barr Virus ◽  
Mediate Cytotoxicity ◽  
Epstein Barr ◽  
And Function

We report here a class of helper cell-independent cytotoxic T cell (HITc) clones in man that can proliferate in response to antigenic stimulation as well as mediate cytotoxicity. HITc appear to be rare among clones derived from primary in vitro allosensitized culture, but constitute the majority of clones derived from cells sensitized to autologous Epstein-Barr virus-transformed lymphoblastoid cell lines. The implications of the derivation and function of HITc clones are discussed.

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