Structures of capsid and capsid-associated tegument complex inside the Epstein-Barr virus.

As the first discovered human cancer virus, Epstein-Barr virus (EBV) causes Burkitt's lymphoma and nasopharyngeal carcinoma. Isolating virions for determining high-resolution structures has been hindered by latency-a hallmark of EBV infection-and atomic structures are thus available only for recombinantly expressed EBV proteins. In the present study, by symmetry relaxation and subparticle reconstruction, we have determined near-atomic-resolution structures of the EBV capsid with an asymmetrically attached DNA-translocating portal and capsid-associated tegument complexes from cryogenic electron microscopy images of just 2,048 EBV virions obtained by chemical induction. The resulting atomic models reveal structural plasticity among the 20 conformers of the major capsid protein, 2 conformers of the small capsid protein (SCP), 4 conformers of the triplex monomer proteins and 2 conformers of the triplex dimer proteins. Plasticity reaches the greatest level at the capsid-tegument interfaces involving SCP and capsid-associated tegument complexes (CATC): SCPs crown pentons/hexons and mediate tegument protein binding, and CATCs bind and rotate all five periportal triplexes, but notably only about one peri-penton triplex. These results offer insights into the EBV capsid assembly and a mechanism for recruiting cell-regulating factors into the tegument compartment as 'cargoes', and should inform future anti-EBV strategies.

CryoEM structure of the tegumented capsid of Epstein-Barr virus.

Epstein-Barr virus (EBV) is the primary cause of infectious mononucleosis and has been shown to be closely associated with various malignancies. Here, we present a complete atomic model of EBV, including the icosahedral capsid, the dodecameric portal and the capsid-associated tegument complex (CATC). Our in situ portal from the tegumented capsid adopts a closed conformation with its channel valve holding the terminal viral DNA and with its crown region firmly engaged by three layers of ring-like dsDNA, which, together with the penton flexibility, effectively alleviates the capsid inner pressure placed on the portal cap. In contrast, the CATCs, through binding to the flexible penton vertices in a stoichiometric manner, accurately increase the inner capsid pressure to facilitate the pressure-driven genome delivery. Together, our results provide important insights into the mechanism by which the EBV capsid, portal, packaged genome and the CATCs coordinately achieve a pressure balance to simultaneously benefit both viral genome retention and ejection.

Atomic structure of the Epstein-Barr virus portal.

Herpesviridae is a vast family of enveloped DNA viruses that includes eight distinct human pathogens, responsible for diseases that range from almost asymptomatic to severe and life-threatening. Epstein-Barr virus infects B-cells and epithelial cells, causing infectious mononucleosis, as well as a number of cancers. Epstein-Barr infection cannot be cured since neither vaccine nor antiviral drug treatments are available. All herpesviruses contain a linear double-stranded DNA genome, enclosed within an icosahedral capsid. Viral portal protein plays a key role in the procapsid assembly and DNA packaging. The portal is the entrance and exit pore for the viral genome, making it an attractive pharmacological target for the development of new antivirals. Here we present the atomic structure of the portal protein of Epstein-Barr virus, solved by cryo-electron microscopy at 3.5 Å resolution. The detailed architecture of this protein suggests that it plays a functional role in DNA retention during packaging.

Why and How Epstein-Barr Virus Was Discovered 50 Years Ago.

An account is given of the experiences and events which led to a search being undertaken for a causative virus in the recently described Burkitt's lymphoma and of the steps which ultimately culminated in the discovery of the new human herpesvirus which came to be known as Epstein-Barr virus (EBV).

Burkitt lymphoma and the discovery of Epstein-Barr virus.

The chance germinal encounter with the first lecture outside Africa on Burkitt lymphoma is described together with the hypothesis of a viral cause. Repeated virological investigations on lymphoma biopsies proved negative, leading to the idea that a latent virus might be activated if lymphoma cells could be cultured, although no human lymphoid cell had at that time ever been maintained in vitro. A chance event reminding of the need for suspension culture with mouse lymphomas led to success. The cultured cells carried a morphologically unequivocal, strangely inert, herpesvirus shown later to be immunologically, biologically and biochemically unique. How this new agent acquired its name, Epstein-Barr virus, is explained.

Latent membrane protein 1 is dispensable for Epstein-Barr virus replication in human embryonic kidney 293 cells.

Epstein Barr Virus (EBV) replicates in oral epithelial cells and gains entry to B-lymphocytes. In B-lymphocytes, EBV expresses a restricted subset of genes, the Latency III program, which converts B-lymphocytes to proliferating lymphoblasts. Latent Membrane Protein 1 (LMP1) and the other Latency III associated proteins are also expressed during virus replication. LMP1 is essential for virus replication and egress from Akata Burkitt Lymphoma cells, but a role in epithelial cell replication has not been established. Therefore, we have investigated whether LMP1 enhances EBV replication and egress from HEK293 cells, a model epithelial cell line used for EBV recombinant molecular genetics. We compared wild type (wt) and LMP1-deleted (LMP1Δ) EBV bacterial artificial chromosome (BAC) based virus replication and egress from HEK293. Following EBV immediate early Zta protein induction of EBV replication in HEK293 cells, similar levels of EBV proteins were expressed in wt- and LMP1Δ-infected HEK293 cells. LMP1 deletion did not impair EBV replication associated DNA replication, DNA encapsidation, or mature virus release. Indeed, virus from LMP1Δ-infected HEK293 cells was as infectious as EBV from wt EBV infected HEK cells. Trans-complementation with LMP1 reduced Rta expression and subsequent virus production. These data indicate that LMP1 is not required for EBV replication and egress from HEK293 cells.

Spatiotemporally different DNA repair systems participate in Epstein-Barr virus genome maturation.

Productive replication of Epstein-Barr virus occurs in discrete sites in nuclei, called replication compartments, where viral DNA replication proteins and host homologous recombinational repair (HRR) and mismatch repair (MMR) factors are recruited. Three-dimensional (3D) surface reconstruction imaging clarified the spatial arrangements of these factors within the replication compartments. Subnuclear domains, designated BMRF1 cores, which were highly enriched in viral polymerase processivity factor BMRF1 could be identified inside the replication compartments. Pulse-chase experiments revealed that newly synthesized viral genomes organized around the BMRF1 cores were transferred inward. HRR factors could be demonstrated mainly outside BMRF1 cores, where de novo synthesis of viral DNA was ongoing, whereas MMR factors were found predominantly inside. These results imply that de novo synthesis of viral DNA is coupled with HRR outside the cores, followed by MMR inside cores for quality control of replicated viral genomes. Thus, our approach unveiled a viral genome manufacturing plant.

Molecular interactions of Epstein-Barr virus capsid proteins.

The capsids of herpesviruses, which comprise major and minor capsid proteins, have a common icosahedral structure with 162 capsomers. An electron microscopic study shows that Epstein-Barr virus (EBV) capsids in the nucleus are immunolabeled by anti-BDLF1 and anti-BORF1 antibodies, indicating that BDLF1 and BORF1 are the minor capsid proteins of EBV. Cross-linking and electrophoresis studies of purified BDLF1 and BORF1 revealed that these two proteins form a triplex that is similar to that formed by the minor capsid proteins, VP19C and VP23, of herpes simplex virus type 1 (HSV-1). Although the interaction between VP23, a homolog of BDLF1, and the major capsid protein VP5 could not be verified biochemically in earlier studies, the interaction between BDLF1 and the EBV major capsid protein, viral capsid antigen (VCA), can be confirmed by glutathione S-transferase (GST) pulldown assay and coimmunoprecipitation. Additionally, in HSV-1, VP5 interacts with only the middle region of VP19C; in EBV, VCA interacts with both the N-terminal and middle regions of BORF1, a homolog of VP19C, revealing that the proteins in the EBV triplex interact with the major capsid protein differently from those in HSV-1. A GST pulldown study also identifies the oligomerization domains in VCA and the dimerization domain in BDLF1. The results presented herein reveal how the EBV capsid proteins interact and thereby improve our understanding of the capsid structure of the virus.

Functional delivery of viral miRNAs via exosomes.

Noncoding regulatory microRNAs (miRNAs) of cellular and viral origin control gene expression by repressing the translation of mRNAs into protein. Interestingly, miRNAs are secreted actively through small vesicles called "exosomes" that protect them from degradation by RNases, suggesting that these miRNAs may function outside the cell in which they were produced. Here we demonstrate that miRNAs secreted by EBV-infected cells are transferred to and act in uninfected recipient cells. Using a quantitative RT-PCR approach, we demonstrate that mature EBV-encoded miRNAs are secreted by EBV-infected B cells through exosomes. These EBV-miRNAs are functional because internalization of exosomes by MoDC results in a dose-dependent, miRNA-mediated repression of confirmed EBV target genes, including CXCL11/ITAC, an immunoregulatory gene down-regulated in primary EBV-associated lymphomas. We demonstrate that throughout coculture of EBV-infected B cells EBV-miRNAs accumulate in noninfected neighboring MoDC and show that this accumulation is mediated by transfer of exosomes. Thus, the exogenous EBV-miRNAs transferred through exosomes are delivered to subcellular sites of gene repression in recipient cells. Finally, we show in peripheral blood mononuclear cells from patients with increased EBV load that, although EBV DNA is restricted to the circulating B-cell population, EBV BART miRNAs are present in both B-cell and non-B-cell fractions, suggestive of miRNA transfer. Taken together our findings are consistent with miRNA-mediated gene silencing as a potential mechanism of intercellular communication between cells of the immune system that may be exploited by the persistent human gamma-herpesvirus EBV.

Primary B-cell infection with a deltaBALF4 Epstein-Barr virus comes to a halt in the endosomal compartment yet still elicits a potent CD4-positive cytotoxic T-cell response.

Epstein-Barr virus (EBV) infection is mediated by several viral envelope glycoproteins. We have assessed gp110's functions during the virus life cycle using a mutant that lacks BALF4 (DeltaBALF4). Exposure of various cell lines and primary cell samples of epithelial or lymphoid lineages to the DeltaBALF4 mutant failed to establish stable infections. The DeltaBALF4 virus, however, did not differ from wild-type EBV in its ability to bind and become internalized into primary B cells, in which it elicited a potent T-cell-specific immune reaction against virion constituents. These findings show that DeltaBALF4 viruses can reach the endosome-lysosome compartment and dovetail nicely with the previously identified contribution of gp110 to virus-cell fusion. Other essential steps of the virus life cycle were unaffected in the viral mutant; DNA lytic replication and viral titers were not altered in the absence of gp110, and DeltaBALF4 viruses complemented in trans transformed infected B cells with an efficiency indistinguishable from that observed with wild-type viruses. All of the steps of virus maturation could be observed in lytically induced 293/DeltaBALF4 cells. Induction of lymphoblastoid cells generated with transiently complemented DeltaBALF4 virus led to the production of rare mature virions. We therefore infer that gp110 is not required for virus maturation and egress in 293 cells or in B cells. The DeltaBALF4 virus's phenotypic traits, an inability to infect human cells coupled with potent antigenicity, potentially qualify this mutant as a live vaccine. It will provide a useful tool for the detailed study of EBV-cell interactions in a physiological context.

Epstein-Barr virus (EBV)-infected monocytes facilitate dissemination of EBV within the oral mucosal epithelium.

Epstein-Barr virus (EBV) causes hairy leukoplakia (HL), a benign lesion of oral epithelium that occurs primarily in the setting of human immunodeficiency virus (HIV)-associated immunodeficiency. However, the mechanisms of EBV infection of oral epithelium are poorly understood. Analysis of HL tissues shows a small number of EBV-positive intraepithelial macrophages and dendritic/Langerhans cells. To investigate a role for these cells in spreading EBV to epithelial cells, we used tongue and buccal explants infected ex vivo with EBV. We showed that EBV first infects submucosal CD14(+) monocytes, which then migrate into the epithelium and spread virus to oral epithelial cells, initiating productive viral infection within the terminally differentiated spinosum and granulosum layers. Incubation of EBV-infected monocytes and oral explants with antibodies to CCR2 receptor and monocyte chemotactic protein 1 prevented entry of monocytes into the epithelium and inhibited EBV infection of keratinocytes. B lymphocytes played little part in the spread of EBV to keratinocytes in our explant model. However, cocultivation of EBV-infected B lymphocytes with uninfected monocytes in vitro showed that EBV may spread from B lymphocytes to monocytes. Circulating EBV-positive monocytes were detected in most HIV-infected individuals, consistent with a model in which EBV may be spread from B lymphocytes to monocytes, which then enter the epithelium and initiate productive viral infection of keratinocytes.

Proteins of purified Epstein-Barr virus.

Mature Epstein-Barr virus (EBV) was purified from the culture medium of infected lymphocytes made functionally conditional for Zta activation of lytic replication by an in-frame fusion with a mutant estrogen receptor. Proteins in purified virus preparations were separated by gradient gel electrophoresis and trypsin-digested; peptides were then analyzed by tandem hydrophobic chromatography, tandem MS sequencing, and MS scans. Potential peptides were matched with EBV and human gene ORFs. Mature EBV was mostly composed of homologues of proteins previously found in a herpes virion. However, EBV homologues to herpes simplex virus capsid-associated or tegument components UL7 (BBRF2), UL14 (BGLF3), and EBV BFRF1 were not significantly detected. Instead, probable tegument components included the EBV and gamma-herpesvirus-encoded BLRF2, BRRF2, BDLF2 and BKRF4 proteins. Actin was also a major tegument protein, and cofilin, tubulin, heat shock protein 90, and heat shock protein 70 were substantial components. EBV envelope glycoprotein gp350 was highly abundant, followed by glycoprotein gH, intact and furin-cleaved gB, gM, gp42, gL, gp78, gp150, and gN. BILF1 (gp64) and proteins associated with latent EBV infection were not detected in virions.

Ulcerative colon T-cell lymphoma: an unusual entity mimicking Crohn's disease and may be associated with fulminant hemophagocytosis.

Primary gastrointestinal T-cell lymphoma is uncommon. Most arise from the small intestine and are usually associated with chronic celiac disease; the so-called enteropathy associated T-cell lymphoma. Primary colon T-cell lymphoma is much more rare. We present two patients with primary colon T-cell lymphoma. Both patients had chronic diarrhea and significant weight loss. Endoscopically, the lymphoma was characterized by the presence of multiple skipped ulcers distributed from the terminal ileum to the descending colon. It was differentiated from Crohn's disease by the absence of fistula or thickening of the intestinal walls. Histologically, the lymphoma was composed of medium to large atypical cells located in the ulcer base with extension to the muscular layer and the adjacent atrophic mucosa. Occasional increased intraepithelial lymphocytes were also seen. Immunohistochemically, the lymphoma cells and intraepithelial lymphocytes were CD3+, CD4-, CD56- and CD8-. It was difficult to diagnosis this unusual lymphoma by biopsy. Because most biopsy specimens showed mixed inflammation within which the lymphoma cell was sometimes hard to identify. Both patients died of fulminant hemophagocytic syndrome and Epstein-Barr virus genome was detected in the lymphoma cells using in situ hybridization on the final surgical specimens. Our study indicates that it is important to recognize this ulcerative colon T-cell lymphoma and to differentiate it from inflammatory bowel disease because of its much more aggressive clinical behavior.

An animal model for Epstein-Barr virus (EBV)-associated lymphomagenesis in the human: malignant lymphoma induction of rabbits by EBV-related herpesvirus from cynomolgus.

It is very important to develop and analyze animal models of Epstein-Barr virus (EBV)-associated tumors in the human. However, only a few reports on the animal models of EBV infection have been reported. Here we review those previous models and describe the details on our newly developed rabbit model of malignant lymphoma induced by EBV-related virus from cynomolgus. In brief, Si-IIA-EBV or Cyno-EBV induced T-cell lymphomas in rabbits inoculated intravenously (77-90%), orally (82-89%), subcutaneously (3/3) and intraperitoneally (2/3) about 2-5 months later. EBV-DNA was detected in peripheral blood by polymerase chain reaction 2 days after oral inoculation of Cyno-EBV while antiviral capsid antigen immunoglobulin G (IgG) was raised 3 weeks after the inoculation. Rabbit lymphomas and their cell lines contained EBV-DNA and expressed EBV-encoded small RNA-1 and EBV-associated nuclear antigen. Rabbit lymphoma cell lines, some of which have specific chromosomal abnormality, showed tumorigenicity in nude mice. The significance and further research subjects of this animal model will be discussed. We believe that the present rabbit model of lymphoma with specific chromosomal abnormalities is very useful for clarifying the role of EBV in human EBV-associated lymphoma and provides a means for studying prophylactic and therapeutic regimens.

Infection of primary human monocytes by Epstein-Barr virus.

Previous studies have reported that infection of monocytes by viruses such as cytomegalovirus and human immunodeficiency virus weakens host natural immunity. In the present study, we demonstrated the capability of Epstein-Barr virus (EBV) to infect and replicate in freshly isolated human monocytes. Using electron microscopy analysis, we observed the presence of EBV virions in the cytoplasm and nuclei of approximately 20% of monocytes. This was confirmed by Southern blot analysis of EBV genomic DNA sequences in isolated nuclei from monocytes. Infection of monocytes by EBV leads to the activation of the replicative cycle. This was supported by the detection of immediate-early lytic mRNA BZLF-1 transcripts, and by the presence of two early lytic transcripts (BALF-2, which appears to function in DNA replication, and BHRF-1, also associated with the replicative cycle). The late lytic BcLF-1 transcripts, which code for the major nucleocapsid protein, were also detected, as well as EBNA-1 transcripts. However, attempts to detect EBNA-2 transcripts have yielded negative results. Viral replication was also confirmed by the release of newly synthesized infectious viral particles in supernatants of EBV-infected monocytes. EBV-infected monocytes were found to have significantly reduced phagocytic activity, as evaluated by the quantification of ingested carboxylated fluoresceinated latex beads. Taken together, our results suggest that EBV infection of monocytes and alteration of their biological functions might represent a new mechanism to disrupt the immune response and promote viral propagation during the early stages of infection.

Productive infection of Epstein-Barr virus (EBV) in EBV-genome-positive epithelial cell lines (GT38 and GT39) derived from gastric tissues.

We characterized the expression of Epstein-Barr virus (EBV) on two epithelial cell lines, GT38 and GT39, derived from human gastric tissues. The EBV nuclear antigen (EBNA) was detected in all cells of both cell lines. The EBV immediate-early BZLF 1 protein (ZEBRA), the early antigen diffuse component (EA-D), and one of the EBV envelope proteins (gp350/220) were expressed spontaneously in small proportions in the cells. EBNA 1, EBNA2, latent membrane protein 1, ZEBRA, and EA-D molecules were then observed by Western blotting in the cells. The lytic cycle was enhanced with treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA) or n-butyrate. The virus particles were observed in the TPA treated GT38 cells by electron microscopy. Infectious EBV was detected with the transformation of cord blood lymphocytes and also with the induction of early antigen to Raji cells by the supernatants of both cells lines. A major single and minor multiple fused terminal fragments and a ladder of smaller fragments of the EBV genome were detected with a Xhol probe in both cell lines. These epithelial cells lines and viruses will be useful in studying their association with EBV in gastric epithelial cells.

Determination of Epstein-Barr virus association with B-cell lymphomas in Japan: study of 72 cases--in situ hybridization, polymerase chain reaction, immunohistochemical studies.

The association of Epstein-Barr virus (EBV) with B-cell lymphoma was examined in 72 human immunodeficiency virus-negative Japanese patients using the polymerase chain reaction (PCR) on DNA obtained from formalin-fixed paraffin-embedded tissues and an in situ hybridization (ISH) technique. EBV-encoded RNA 1 (EBER-1) was detected in 12 of 72 cases (17%); five of 33 cases (15%) of nodal B-cell lymphomas and seven of 39 cases (18%) of extranodal B-cell lymphomas. Three cases of post-bone marrow transplantation and one case of autoimmune disease (Evans syndrome) were included among seven EBER-1 positive extranodal lymphomas. A combined study of immunohistochemistry and EBER-1 revealed that some L26 positive cells were EBER-1 positive. A DNA band was also observed in 13 of 70 examined cases (19%) (four of 33 cases of nodal B-cell lymphomas (12%) and nine of 37 cases of extranodal B-lymphomas (24%)) in the PCR study using primers to detect the Bam HI-W fragment of EBV. In the immunohistochemical study using a monoclonal antibody to the latent membrane protein 1 (LMP-1) of the EBV, one of the EBV-encoded latent gene products, LMP-1, was expressed in six of 34 cases (18%) of extranodal B-lymphomas, but none of the cases with nodal B-cell lymphomas were shown to be LMP-1 positive. Oncoprotein bcl-2 was examined by immunohistochemistry and found to be expressed in seven cases of nodal lymphomas and three cases of extranodal lymphomas, and two of these nodal cases were EBER ISH positive. In EBV serology, only two cases of nodal and one case of extranodal EBER positive B-cell lymphomas revealed a reactivation pattern. In the PCR study using primers to detect the lymphocyte-determined membrane antigen (LYDMA), the same sized monoclonal bands were observed in case 36 in the PCR products from the nose and skin, suggesting the monoclonal proliferation of the tumor. These findings suggested a low incidence of EBV association with B-cell lymphomas unless patients were in an immunologically impaired condition such as post-organ transplantation or autoimmune diseases.

A human B-lymphoblastoid cell line constitutively producing Epstein-Barr herpesvirus and JHK retrovirus.

The human B-lymphoblastoid cell line, designated JHK-3, with pre-B-cell characteristics, chronically produces two viruses, Epstein-Barr virus (EBV) and JHK virus, an apparently novel retrovirus. The JHK-3 cells are much more productive of extracellular EBV than the high-producer marmoset line B95-8. The extracellular virus of the JHK-3 EBV strain is relatively fragile, more broadly dispersed in an ultracentrifuged sucrose gradient than the B95-8 EBV and more susceptible to disruption by combined treatment with urea and dithiothreitol. By restriction fragment length polymorphism analysis, the JHK-3 EBV strain resembles the EBV strain FF-41. The JHK-3 cells also produce an incompletely characterized, relatively fragile, enveloped, icosahedral RNA virus that contains Mn(++)-dependent reverse transcriptase. JHK virions measure 85 nm in ultrathin sections, much smaller than other Retroviridae. The JHK virus exhibits a distinctive morphogenesis, most nearly resembling C-type retroviruses. The JHK-3 cell line provides a human cell model for investigating virus/virus interactions and their pathogenetic affects on host cells which chronically and simultaneously produce DNA and RNA viruses.

Crystal structure of Epstein-Barr virus protein BCRF1, a homolog of cellular interleukin-10.

The crystal structure of Epstein-Barr virus protein BCRF1, an analog of cellular interleukin-10 (IL-10), has been determined at the resolution of 1.9 A and refined to an R-factor 0.191. The structure of this cytokine is similar to that of human IL-10 (hIL-10), forming an intercalated dimer of two 17 kDa polypeptides related by a crystallographic 2-fold symmetry axis. BCRF1 exhibits novel conformations of the N-terminal coil and of the loop between helices A and B compared to hIL-10. These regions are likely to be involved in binding of one or more components of the IL-10 receptor system, and thus the structural differences may account for the lower binding affinity and limited spectrum of biological activities of viral IL-10, compared to hIL-10.