Epstein-Barr Virus-Encoded BARF1 Promotes Proliferation of Gastric Carcinoma Cells through Regulation of NF-κB

In Epstein-Barr virus (EBV)-infected gastric carcinoma, EBV-encoded BARF1 has been hypothesized to function as an oncogene. To evaluate cellular changes induced by BARF1, we isolated the full-length BARF1 gene from gastric carcinoma cells that were naturally infected with EBV and transfected BARF1 into EBV-negative gastric carcinoma cells. BARF1 protein was primarily secreted into culture supernatant and only marginally detectable within cells. Compared with gastric carcinoma cells containing empty vector, BARF1-expressing gastric carcinoma cells exhibited increased cell proliferation (P < 0.05). There were no significant differences in apoptosis, invasion, or migration between BARF1-expressing gastric carcinoma cells and empty vector-transfected cells. BARF1-expressing gastric carcinoma cells demonstrated increased nuclear expression of nuclear factor kappa B (NF-κB) RelA protein and increased NF-κB-dependent cyclin D1. The expression of p21^WAF1 was diminished by BARF1 transfection and increased by NF-κB inhibition. Proliferation of naturally EBV-infected gastric carcinoma cells was suppressed by BARF1 small interfering RNA (siRNA) (P < 0.05). Immunohistochemical analysis of 120 human gastric carcinoma tissues demonstrated increased expression of cyclin D1 and reduced expression of p21^WAF1 in EBV-positive samples versus EBV-negative gastric carcinomas (P < 0.05). In conclusion, the secreted BARF1 may stimulate proliferation of EBV-infected gastric carcinoma cells via upregulation of NF-κB/cyclin D1 and reduction of the cell cycle inhibitor p21^WAF1, thereby facilitating EBV-induced cancer progression.     

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

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.     

Structure of the Epstein-Barr virus oncogene BARF1

The Epstein-Barr virus is a human gamma-herpesvirus that persistently infects more than 90% of the human population. It is associated with numerous epithelial cancers, principally undifferentiated nasopharyngeal carcinoma and gastric carcinoma. The BARF1 gene is expressed in a high proportion of these cancers. An oncogenic, mitogenic and immortalizing activity of the BARF1 protein has been shown. We solved the structure of the secreted BARF1 glycoprotein expressed in a human cell line by X-ray crystallography at a resolution of 2.3A. The BARF1 protein consists of two immunoglobulin (Ig)-like domains. The N-terminal domain belongs to the subfamily of variable domains whereas the C-terminal one is related to a constant Ig-domain. BARF1 shows an unusual hexamerisation involving two principal contacts, one between the C-terminal domains and one between the N-terminal domains. The C-terminal contact with an uncommonly large contact surface extends the beta-sandwich of the Ig-domain through the second molecule. The N-terminal contact involves Ig-domains with an unusual relative orientation but with a more classical contact surface with a size in the range of dimer interactions of Ig-domains. The structure of BARF1 is most closely related to CD80 or B7-1, a co-stimulatory molecule present on antigen presenting cells, from which BARF1 must have been derived during evolution. Still, domain orientation and oligomerization differ between BARF1 and CD80. It had been shown that BARF1 binds to hCSF-1, the human colony-stimulating factor 1, but this interaction has to be principally different from the one between CSF-1 and CSF-1 receptor.     

Malignant transformation of Epstein-Barr virus-negative Akata cells by introduction of the BARF1 gene carried by Epstein-Barr virus

Spontaneous loss of the Epstein-Barr virus (EBV) genome in the BL cell line Akata led to loss of tumorigenicity in SCID mice, suggesting an important oncogenic activity of EBV in B cells. We previously showed that introduction of the BARF1 gene into the human B-cell line Louckes induced a malignant transformation in newborn rats (M. X. Wei, J. C. Moulin, G. Decaussin, F. Berger, and T. Ooka, Cancer Res. 54:1843-1848, 1994). Since 1 to 2% of Akata cells expressed lytic antigens and expressed the BARF1 gene, we investigated whether introduction of the BARF1 gene into EBV-negative Akata cells can induce malignant transformation. Here we show that BARF1-transfected, EBV-negative Akata cells activated Bcl2 expression and induced tumor formation when they were injected into SCID mice. In addition, when EBV-positive Akata cells expressing a low level of BARF1 protein were injected into SCID mice, the expression of BARF1, as well as several lytic proteins, such as EA-D, ZEBRA, and a 135-kDa DNA binding protein, increased in tumor cells while no latent LMP1 and late gp220-320 expression was observed in tumor cells. These observations suggest that the BARF1 gene may be involved in the conferral of tumorigenicity by EBV.     

An Epstein-Barr Virus Encoded Inhibitor of Colony Stimulating Factor-1 Signaling Is an Important Determinant for Acute and Persistent EBV Infection

Acute Epstein-Barr virus (EBV) infection is the most common cause of Infectious Mononucleosis. Nearly all adult humans harbor life-long, persistent EBV infection which can lead to development of cancers including Hodgkin Lymphoma, Burkitt Lymphoma, nasopharyngeal carcinoma, gastric carcinoma, and lymphomas in immunosuppressed patients. BARF1 is an EBV replication-associated, secreted protein that blocks Colony Stimulating Factor 1 (CSF-1) signaling, an innate immunity pathway not targeted by any other virus species. To evaluate effects of BARF1 in acute and persistent infection, we mutated the BARF1 homologue in the EBV-related herpesvirus, or lymphocryptovirus (LCV), naturally infecting rhesus macaques to create a recombinant rhLCV incapable of blocking CSF-1 (ΔrhBARF1). Rhesus macaques orally challenged with ΔrhBARF1 had decreased viral load indicating that CSF-1 is important for acute virus infection. Surprisingly, ΔrhBARF1 was also associated with dramatically lower virus setpoints during persistent infection. Normal acute viral load and normal viral setpoints during persistent rhLCV infection could be restored by Simian/Human Immunodeficiency Virus-induced immunosuppression prior to oral inoculation with ΔrhBARF1 or infection of immunocompetent animals with a recombinant rhLCV where the rhBARF1 was repaired. These results indicate that BARF1 blockade of CSF-1 signaling is an important immune evasion strategy for efficient acute EBV infection and a significant determinant for virus setpoint during persistent EBV infection.     

EB病毒感染下调胃癌细胞系NU-GC-3肿瘤坏死因子α的表达

To explore the mechanisms of EBV associated gastric carcinoma, TNF α expression was determined by Northern blot in EBV infected gastric carcinoma cell line NU GC 3 and the significant decrease was found (P<0.01). Furthermore, the determinations were done in the cells transfected by 4 kinds of EBV latency I genes which were expressed in EBV latently infected gastric epithelial cells. It was found that the downregulations were associated with EBV determined nuclear antigen l (EBNA1) gene and EBV encoded small RNAs (EBERs) gene (p<0.01 and p<0.05), but not with BamH I A rightward frame 0 (BARF0) gene and latent membrane protein 2A (LMP2A) gene. EBNA1 and EBERs are known to be correlated with the viral tumorigenicity, while BARF0 and LMP2A are not. TNF α downregulation caused by all the factors related to EBV tumorigenicity suggests that it may be an important mediate step in the signal transduction pathway of gastric epithelial cell canceration.     

The lytic cycle of Epstein-Barr virus in the nonproducer Raji line can be rescued by the expression of a 135-kilodalton protein encoded by the BALF2 open reading frame.

In Epstein-Barr virus (EBV)-carrying nonproducer Raji cells, the induction of the viral replicative cycle by chemical treatment is limited to only the early stage and viral DNA synthesis is totally inhibited. We previously showed the absence of two messenger RNAs that are encoded by the BamHI-A fragment of the EBV genome and that correspond to open reading frames BALF2 and BARF1 in chemically induced Raji cells. Since the BALF2 gene encodes a 135-kDa DNA-binding protein which was immunoprecipitated by antibody against ICP8 protein, a key protein in herpes simplex virus replication, we asked whether the lack of productive cycle in Raji cells is due to the absence of expression of the BALF2 gene. We transfected the Raji cell line with the BALF2 gene. After chemical induction, the BALF2-transfected cells expressed not only early antigens but also late antigens. In these cultures, the viral particles were detected by electron microscopy. The expression of late antigens was completely inhibited by arabinofuranosylthymine, which is a specific inhibitor of viral DNA replication. The BALF2 gene might play an essential role in the induction of the EBV-lytic cycle.     

The Epstein-Barr Virus BARF1 Gene Encodes a Novel, Soluble Colony-Stimulating Factor-1 Receptor

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus associated with infectious mononucleosis and several tumors. The BARF1 gene is transcribed early after EBV infection from the BamHI A fragment of the EBV genome. Evidence shown here indicates that the BARF1 protein is secreted into the medium of transfected cells and from EBV-carrying B cells induced to allow lytic replication of the virus. Expression cloning identified colony-stimulating factor-1 (CSF-1) as a ligand for BARF1. Computer-assisted analyses indicated that subtle amino acid sequence homology exists between BARF1 and c-fms, the cellular proto-oncogene that is the receptor for CSF-1. Recombinant BARF1 protein was found to be biologically active, and it neutralized the proliferative effects of human CSF-1 in a dose-dependent fashion when assayed in vitro. Since CSF-1 is a pleiotropic cytokine best known for its differentiating effects on macrophages, these data suggest that BARF1 may function to modulate the host immune response to EBV infection.     

Epstein-Barr Virus (EBV)-derived BARF1 encodes CD4- and CD8-restricted epitopes as targets for T-cell immunotherapy

Background aims

EBV type II latency tumors, such as Hodgkin lymphoma (HL), Non-Hodgkin lymphoma (NHL) and nasopharyngeal carcinoma, express a limited array of EBV antigens including Epstein-Barr nuclear antigen (EBNA)1, latent membrane protein (LMP)1, LMP2, and BamH1-A right frame 1 (BARF1). Adoptive immunotherapy for these malignancies have focused on EBNA1, LMP1 and LMP2 because little is known about the cellular immune response to BARF1.

Identification and characterization of the product encoded by ORF69 of Kaposi's sarcoma-associated herpesvirus

We report the identification and characterization of p33, the product of Kaposi's sarcoma-associated herpesvirus (KSHV) open reading frame 69 (ORF69), a positional homolog of the conserved herpesvirus protein UL31. p33 is expressed upon induction of viral lytic cycle with early kinetics. Immunofluorescence analysis revealed that in infected cell lines, the protein is localized in the nucleus, both in dotted spots and along the nuclear membrane. Nuclear fractionation experiments showed that p33 partitions with the nuclear matrix, and both immunoblotting of purified virions and immunoelectron microscopy indicated that the novel protein is not a component of the mature virus. Following ectopic expression in KSHV-negative cells, the protein was never associated with the nuclear membrane, suggesting that p33 needs to interact with additional viral proteins to reach the nuclear rim. In fact, after cotransfection with the ORF67 gene, the KSHV positional homolog of UL34, the p33 intranuclear signal changed and the two proteins colocalized on the nuclear membrane. A similar result was obtained when ORF69 was cotransfected with BFRF1, the Epstein-Barr virus (EBV) positional homolog of UL34 and ORF67. Finally, upon cotransfection, ORF69 significantly increased nuclear membrane reduplications induced by BFRF1. The above results indicate that KSHV p33 shares many similarities with its EBV homolog BFLF2 and suggest that functional cross-complementation is possible between members of the gammaherpesvirus subfamily.     

The ORF1 products of tombusviruses play a crucial role in lethal necrosis of virus-infected plants

Hybrids of cymbidium ringspot (CymRSV) and carnation Italian ringspot (CIRV) tombusviruses were used to identify viral symptom determinants responsible for the generalized necrosis in tombusvirus-infected plants. Surprisingly, symptoms of Nicotiana benthamiana infected with CymRSV/CIRV hybrids were distinctly different. It was demonstrated that not all chimeras expressing wild-type (wt) levels of p19 protein caused systemic necrosis as both parents CymRSV and CIRV did. We showed here that hybrids containing chimeric ORF1 were not able to induce lethal necrosis even if the viral replication of these constructs was not altered significantly. However, if a wt p33 (product of ORF1) of CymRSV was provided in trans in transgenic plants expressing p33 and its readthrough product p92, the lethal necrosis characteristic to tombusvirus infection was restored. In addition, the expression of p33 by a potato virus X viral vector in N. benthamiana caused severe chlorosis and occasionally necrosis, indicating the importance of p33 in wt symptoms of tombusviruses. Thus, our results provide evidence that elicitation of the necrotic phenotype requires the presence of the wt p33 in addition to the p19 protein of tombusviruses.     

Isolation and Characterization of a Replication-Competent Molecular Clone of an HIV-1 Circulating Recombinant Form (CRF33_01B)

A growing number of emerging HIV-1 recombinants classified as circulating recombinant forms (CRFs) have been identified in Southeast Asia in recent years, establishing a molecular diversity of increasing complexity in the region. Here, we constructed a replication-competent HIV-1 clone for CRF33_01B (designated p05MYKL045.1), a newly identified recombinant comprised of CRF01_AE and subtype B. p05MYKL045.1 was reconstituted by cloning of the near full-length HIV-1 sequence from a newly-diagnosed individual presumably infected heterosexually in Kuala Lumpur, Malaysia. The chimeric clone, which contains the 5′ LTR (long terminal repeat) region of p93JP-NH1 (a previously isolated CRF01_AE infectious clone), showed robust viral replication in the human peripheral blood mononuclear cells. This clone demonstrated robust viral propagation and profound syncytium formation in CD4⁺, CXCR4-expressing human glioma NP-2 cells, indicating that p05MYKL045.1 is a CXCR4-using virus. Viral propagation, however, was not detected in various human T cell lines including MT-2, M8166, Sup-T1, H9, Jurkat, Molt-4 and PM1. p05MYKL045.1 appears to proliferate only in restricted host range, suggesting that unknown viral and/or cellular host factors may play a role in viral infectivity and replication in human T cell lines. Availability of a CRF33_01B molecular clone will be useful in facilitating the development of vaccine candidates that match the HIV-1 strains circulating in Southeast Asia.     

Neuroprotection by lomerizine, a prophylactic drug for migraine, against hydrogen peroxide-induced hippocampal neurotoxicity

Cervical cancer is the most common cancer in Indian females and is associated with infection with high-risk Human papilloma viruses (HPVs) which encode viral oncoprotein E6 and E7. Estradiol has been established as a risk factor for cervical cancer and has been shown to play a synergistic role with viral oncoproteins. Curcumin (Diferuloyl methane), a chemopreventive agent, is a natural compound extracted from Curcuma longa that allows suppression and retardation of carcinogenesis in many types of cancer and is currently being tested in various human clinical trials as it has been found to be well tolerated at higher doses with a relatively well established safety profile. The objective of this study was to test the effect of curcumin on HPV-positive and negative cervical cancer cell lines HeLa, SiHa, CaSki, and C33A pretreated with estradiol. It was found that HPV-positive cells pretreated with estradiol show reduced apoptosis as compared to curcumin by itself. However, curcumin was able to counteract the proliferative response of estradiol, and induce apoptosis. There was no difference in percentage apoptosis as compared to estradiol pretreatment in HPV-negative cell line C33A. Molecular studies showed elevation of Telomerase, viral oncoproteins E6 and E7, PCNA, p16, Cyclin D1 in HPV-positive cell lines on treatment with estradiol but after treatment with curcumin the level of E7, PCNA, and Cyclin D1 was reduced but the level of E6, Telomerase, and p16 was unaltered. Furthermore, estradiol-pretreated HPV-negative cell line C33A showed reduction in level of Telomerase, PCNA, p16, and activation of both p53 and p73 tumor suppressor proteins, thus, demonstrating the importance of E6 in estradiol-mediated protective effect.     

Polymerization of bacteriophage phi 29 replication protein p1 into protofilament sheets.

Protein p1 (85 amino acids) of the Bacillus subtilis phage phi29 is a membrane-associated protein required for in vivo viral DNA replication. In the present study, we have constructed two fusion proteins, maltose-binding protein (MalE)-p1 and MalE-p1DeltaN33. By using both sedimentation assays and negative-stain electron microscopy analysis, we demonstrated that MalE-p1 molecules self-associated into long filamentous structures, which did not assemble further into larger arrays. These structures were constituted by a core of protein p1 surrounded by MalE subunits. After removal of the MalE component by cleavage with protease factor Xa, the resulting protein p1 filaments tended to associate, forming bundles. The MalE-p1DeltaN33 fusion protein, however, did not self-interact in solution. Nevertheless, after being separated from the MalE domain by factor Xa digestion, protein p1DeltaN33 assembled into long protofilaments that associated in a highly ordered, parallel array forming large two-dimensional sheets. These structures resemble eukaryotic tubulin and bacterial FtsZ polymers. In addition, we show that protein p1 influences the rate of in vivo phi29 DNA synthesis in a temperature-dependent manner. We propose that protein p1 is a component of a viral-encoded structure that associates with the bacterial membrane. This structure would provide an anchoring site for the viral DNA replication machinery.     

Detection of a novel quiescence-dependent protein kinase

We have identified a cell quiescence-specific 33-kDa cytoplasmic protein kinase (p33(QIK), Quiescence-Induced Kinase) based on induction of p33(QIK)-specific kinase activity of cells growth-arrested in the quiescent phase and deactivation upon entry into the cell cycle. Blockage of macromolecular synthesis prevents p33(QIK) from deactivation, indicating a requirement of newly synthesized regulators for deactivation of p33(QIK) during G(0)/G(1) transition. Stress shock induces additional increases of p33(QIK) activity in a quiescence-dependent manner that correlates with induction of apoptosis. Using a specific antibody to Krs1/Mst2 protein, we found that p33(QIK) is related to p63(Krs1) and is distinguishable from a 36-kDa protein kinase, which is induced through proteolytic modification of activated p63(Krs1) in proliferating cells undergoing apoptosis. p33(QIK) is constantly expressed in quiescent, proliferating, and apoptotic quiescent cells. Regulation of p33(QIK) activity involves protein phosphorylation/dephosphorylation in a proteolysis-independent manner. Regulation of p33(QIK) and related p63(Krs1) and p36 appears to involve distinct pathways in quiescent and proliferating cells, respectively. Our results illustrate the relevance of p33(QIK) activity for cell quiescence that may provide a new insight into signaling pathways regulated in cells during quiescence and quiescence-related apoptosis.     

Molecular Anatomy and Number of Antigen Specific CD8 T Cells Required to Cause Type 1 Diabetes

We quantified CD8 T cells needed to cause type 1 diabetes and studied the anatomy of the CD8 T cell/beta (β) cell interaction at the immunologic synapse. We used a transgenic model, in situ tetramer staining to distinguish antigen specific CD8 T cells from total T cells infiltrating islets and a variety of viral mutants selected for functional deletion(s) of various CD8 T cell epitopes. Twenty percent of CD8 T cells in the spleen were specific for all immunodominant and subdominant viral glycoprotein (GP) epitopes. CTLs to the immunodominant LCMV GP33-41 epitope accounted for 63% of the total (12.5% of tetramers). In situ hybridization analysis demonstrated only 1 to 2% of total infiltrating CD8 T cells were specific for GP33 CD8 T cell epitope, yet diabetes occurred in 94% of mice. The immunologic synapse between GP33 CD8 CTL and β cell contained LFA-1 and perforin. Silencing both immunodominant epitopes (GP33, GP276–286) in the infecting virus led to a four-fold reduction in viral specific CD8 CTL responses, negligible lymphocyte infiltration into islets and absence of diabetes.