Epstein-Barr Virus Productive Cycle Research Articles

Oral Microbiota Alteration and Roles in Epstein-Barr Virus Reactivation in Nasopharyngeal Carcinoma.

Microbiota has recently emerged as a critical factor associated with multiple malignancies. Nasopharyngeal carcinoma (NPC) is highly associated with Epstein-Barr virus (EBV); the oncovirus resides and is transmitted in the oral cavity. However, the alternation of oral microbiota in NPC patients and its potential link to EBV reactivation and host cell response under the simultaneous existence of EBV and specific bacteria is largely unknown. Here, oral microbiota profiles of 303 NPC patients and controls with detailed clinical information, including serum EBV anti-virus capsid antigen (VCA) IgA level, were conducted. A distinct microbial community with lower diversity and imbalanced composition in NPC patients was observed. Notably, among enriched bacteria in patients, Streptococcus sanguinis was associated with anti-VCA IgA, an indicator of NPC risk and EBV reactivation. By measuring the concentration of its metabolite, hydrogen peroxide (H2O2), in the saliva of clinical patients, we found the detection rate of H2O2 was 2-fold increased compared to healthy controls. Further coculture assay of EBV-positive Akata cells with bacteria in vitro showed that S. sanguinis induced EBV lytic activation by its metabolite, H2O2. Host and EBV whole genome-wide transcriptome sequencing and EBV methylation assays showed that H2O2 triggered the host cell signaling pathways, notably tumor necrosis factor alpha (TNF-α) via NF-κB, and induced the demethylation of the global EBV genome and the expression of EBV lytic-associated genes, which could result in an increase of virus particle release and potential favorable events toward tumorigenesis. In brief, our study identified a characterized oral microbial profile in NPC patients and established a robust link between specific oral microbial alteration and switch of latency to lytic EBV infection status in the oral cavity, which provides novel insights into EBV's productive cycle and might help to further clarify the etiology of NPC. IMPORTANCE EBV is classified as the group I human carcinogen and is associated with multiple cancers, including NPC. The interplays between the microbiota and oncovirus in cancer development remain largely unknown. In this study, we investigate the interactions between resident microbes and EBV coexistence in the oral cavity of NPC patients. We identify a distinct oral microbial feature for NPC patients. Among NPC-enriched bacteria, we illustrated that a specific species, S. sanguinis, associated with elevated anti-IgA VCA in patients, induced EBV lytic activation by its by-product, H2O2, and activated the TNF-α/NF-κB pathway of EBV-positive B cells in vitro, together with increased detection rate of H2O2 in patients' oral cavities, which strengthened the evidence of bacteria-virus-host interaction in physiological circumstances. The effects of imbalanced microbiota on the EBV latent-to-lytic switch in the oral cavity might create the likelihood of EBV infection in epithelial cells at the nasopharynx and help malignant transition and cancer development.

The Nuclear and Adherent Junction Complex Component Protein Ubinuclein Negatively Regulates the Productive Cycle of Epstein-Barr Virus in Epithelial Cells

The Epstein-Barr Virus (EBV) productive cycle is initiated by the expression of the viral trans-activator EB1 (also called Zebra, Zta, or BZLF1), which belongs to the basic leucine zipper transcription factor family. We have previously identified the cellular NACos (nuclear and adherent junction complex components) protein ubinuclein (Ubn-1) as a partner for EB1, but the function of this complex has never been studied. Here, we have evaluated the consequences of this interaction on the EBV productive cycle and find that Ubn-1 overexpression represses the EBV productive cycle whereas Ubn-1 downregulation by short hairpin RNA (shRNA) increases virus production. By a chromatin immunoprecipitation (ChIP) assay, we show that Ubn-1 blocks EB1-DNA interaction. We also show that in epithelial cells, relocalization and sequestration of Ubn-1 to the tight junctions of nondividing cells allow increased activation of the productive cycle. We propose a model in which Ubn-1 is a modulator of the EBV productive cycle: in proliferating epithelial cells, Ubn-1 is nuclear and inhibits activation of the productive cycle, whereas in differentiated cells, Ubn-1 is sequestrated to tight junctions, thereby allowing EB1 to fully function in the nucleus.

Epstein-Barr Virus mRNA Export Factor EB2 Is Essential for Intranuclear Capsid Assembly and Production of gp350

Most human herpesviruses, including Epstein-Barr virus (EBV), express a protein which functions primarily as an mRNA export factor. Previously, we deleted the gene for the Epstein-Barr virus mRNA export factor EB2 from the EBV genome and then introduced the mutated genome into 293 cells. Using a transcomplementation assay in which ectopic expression of the transcription factor EB1/ZEBRA was sufficient to induce the EBV productive cycle, we showed that Ori-Lyt-dependent replication of the EBV DNA occurs in the absence of EB2, indicating that EB2 is not essential for the expression and export of early mRNAs. However, in the absence of EB2, no infectious viral particles are produced (H. Gruffat, J. Batisse, D. Pich, B. Neuhierl, E. Manet, W. Hammerschmidt, and A. Sergeant, J. Virol. 76:9635-9644, 2002). In this report, we now show that EB2 is essential for the nuclear export of most, but not all, late mRNAs produced from intronless genes that translate into proteins involved in intranuclear capsid assembly and maturation. As a consequence, we show that EB2 is essential for the proper assembly of intranuclear capsids. Interestingly, the late BLLF1 gene contains an intron, and both unspliced and spliced mRNAs must be exported to the cytoplasm to be translated into gp350 and gp220, respectively (M. Hummel, D. A. Thorley-Lawson, and E. Kieff, J. Virol. 49:413-417, 1984). Our results also demonstrate that although BLLF1 spliced mRNAs are exported to the cytoplasm independently of EB2, EB2 is essential for the nuclear export of unspliced BLLF1 mRNA. In the same assay, herpes simplex virus 1 ICP27 completely inhibited the nuclear export of BLLF1 spliced mRNAs whereas unspliced BLLF1 mRNAs were exported, confirming that in a physiological assay, ICP27 inhibits splicing.

A Region of the Epstein-Barr Virus (EBV) mRNA Export Factor EB2 Containing an Arginine-rich Motif Mediates Direct Binding to RNA

The Epstein-Barr virus (EBV) protein EB2 (also called Mta, SM, or BMLF1) has properties in common with mRNA export factors and is essential for the production of EBV infectious virions. However, to date no RNA-binding motif essential for EB2-mediated mRNA export has been located in the protein. We show here by Northwestern blot analysis that the EB2 protein purified from mammalian cells binds directly to RNA. Furthermore, using overlapping glutathione S-transferase (GST)-EB2 peptides, we have, by RNA electrophoretic mobility shift assays (REMSAs) and Northwestern blotting, located an RNA-binding motif in a 33-amino acid segment of EB2 that has structural features of the arginine-rich RNA-binding motifs (ARMs) also found in many RNA-binding proteins. A synthetic peptide (called Da), which contains this EB2 ARM, bound RNA in REMSA. A GST-Da fusion protein also bound RNA in REMSA without apparent RNA sequence specificity, because approximately 10 GST-Da molecules bound at multiple sites on a 180-nucleotide RNA fragment. Importantly, a short deletion in the ARM region impaired both EB2 binding to RNA in vivo and in vitro and EB2-mediated mRNA export without affecting the shuttling of EB2 between the nucleus and the cytoplasm. Moreover, ectopic expression of ARM-deleted EB2 did not rescue the production of infectious virions by 293 cells carrying an EBVDeltaEB2 genome, which suggests that the binding of EB2 to RNA plays an essential role in the EBV productive cycle.

A novel function for the Epstein-Barr virus transcription factor EB1/Zta: induction of transcription of the hIL-10 gene.

Interleukin-10 (IL-10) plays a critical role in Epstein-Barr virus (EBV) biology. Indeed, the EBV genome contains a gene (BCRF1) with homology to the human IL-10 (hIL-10) gene. In addition to viral IL-10, which is secreted late in the productive cycle, hIL-10 production is also induced in B cells infected by EBV. The EBV protein LMP-1 and the viral small non-polyadenylated RNAs (EBERs) expressed during latency are involved in hIL-10 induction. In this study, we show that in B cells the viral transcription factor EB1, which is the main inducer of the EBV productive cycle, also activates transcription of the hIL-10 gene and secretion of the hIL-10 protein. Accordingly, EB1 bound directly to specific DNA sequences in the hIL-10 minimal promoter. Moreover, specific disruption of EB1 binding to some of these sites impaired EB1-mediated activation of transcription at the hIL-10 promoter in a transient expression assay. Therefore, an increase in IL-10 production occurs during latency and early and late during the productive cycle. This production of IL-10 might favour the survival of EBV-infected cells in vivo and/or create a microenvironment required for efficient de novo infection of B lymphocytes by EBV virions.

A Novel Early Antigen Associated with Epstein-Barr Virus Productive Cycle

A murine monoclonal antibody (mAb) 92A recognized a 48-kilodalton Epstein-Barr virus (EBV) early antigen (EA). The mAb stained nuclei of EBV-activated P3HR-1, B95-8 and Akata cells in a distinctive, microgranular immunofluorescence pattern. The 92A antigen was sensitive to methanol-fixation. Expression of the 92A antigen in those cells paralleled diffuse (EA-D) and restricted (EA-R) components of EA, and viral DNA (vDNA) replication. Phosphonoacetic acid did not inhibit expression of the 92A antigen. The colocalization of 92A antigen, EA-D, and vDNA was observed in viral replication compartments of B95-8 cells. On the other hand, in P3HR-1 virus-superinfected Raji cells the percentages of 92A antigen-positive cells were at much lower levels than were EA-D and -R positive cells. I mmunofluorescence staining with 92A mAb was blocked by pretreatment with EBV-positive human sera, but not with EBV-negative sera. We conclude that 92A mAb recognizes a novel EA which may function in vDNA replication.

Epstein-Barr virus gene expression in P3HR1-superinfected Raji cells.

The pattern of Epstein-Barr virus (EBV) RNAs expressed in Raji cells superinfected with P3HR1 EBV was examined. RNAs whose expression was of an immediate-early type (resistant to treatment of the cells with anisomycin) were identified. These RNAs, encoding the EBV reading frames BZLF1 and BRLF1, were probably expressed from defective virus within the P3HR1 preparation, and some of them were responsible for the induction of the EBV productive cycle in the Raji cells. The structures of the B95-8 RNAs equivalent to the anisomycin-resistant RNAs were determined. The RNA encoding the BZLF1 reading frame contained two splices which extended and modified the reading frame from that previously described.

Epstein-Barr virus activation of human epithelial—Nasopharyngeal carcinoma hybrid cells

The authors used indirect immunofluorescence and electron microscopic techniques to study the effects of 5-iododeoxyuridine (IUDR) and the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) on the epithelial hybrid cell line (NPC-KT) derived from nasopharyngeal carcinoma. The hybrid cells contained Epstein-Barr virus (EBV)-induced early antigens at a very low level but not Epstein-Barr viral capsid antigens in spontaneous production; the hybrid cells responded significantly to both IUDR and TPA induction. The chemicals induced both early antigens and viral capsid antigens in the NPC-KT cells. Moreover, the virus particles were detected in the altered NPC-KT cells by IUDR treatment. The data suggest that the EBV-induced agents activate the EBV productive cycle in a proportion of nasopharyngeal carcinoma cells.