Teru Kanda

EB virus‐encoded RNAs are recognized by RIG‐I and activate signaling to induce type I IFN

Epstein–Barr virus (EBV)‐encoded small RNAs (EBERs) are nonpolyadenylated, untranslated RNAs, exist most abundantly in latently EBV‐infected cells, and are expected to show secondary structures with many short stem–loops. Retinoic acid‐inducible gene I (RIG‐I) is a cytosolic protein that detects viral double‐stranded RNA (dsRNA) inside the cell and initiates signaling pathways leading to the induction of protective cellular genes, including type I interferons (IFNs). We investigated whether EBERs were recognized by RIG‐I as dsRNA. Transfection of RIG‐I plasmid induced IFNs and IFN‐stimulated genes (ISGs) in EBV‐positive Burkitts lymphoma (BL) cells, but not in their EBV‐negative counterparts or EBER‐knockout EBV‐infected BL cells. Transfection of EBER plasmid or in vitro‐synthesized EBERs induced expression of type I IFNs and ISGs in RIG‐I‐expressing, EBV‐negative BL cells, but not in RIG‐I‐minus counterparts. EBERs activated RIG‐Is substrates, NF‐κB and IFN regulatory factor 3, which were necessary for type I IFN activation. It was also shown that EBERs co‐precipitated with RIG‐I. These results indicate that EBERs are recognized by RIG‐I and activate signaling to induce type I IFN in EBV‐infected cells.

Critical Role of Epstein-Barr Virus (EBV)-Encoded RNA in Efficient EBV-Induced B-Lymphocyte Growth Transformation

ABSTRACT It was demonstrated that Epstein-Barr virus (EBV)-encoded small RNAs (EBERs) were nonessential for B-lymphocyte growth transformation. We revisited this issue by producing a large quantity of EBER-deleted EBV by using an Akata cell system. Although the EBER-deleted virus efficiently infected B lymphocytes, its 50% transforming dose was approximately 100-fold less than that of the EBER-positive EBV. We then engineered the genome of EBER-deleted virus and generated a recombinant virus with the EBER genes reconstituted at their native locus. The resultant EBER-reconstituted EBV exhibited restored transforming ability. In addition, lymphoblastoid cell lines established with the EBER-deleted EBV grew significantly more slowly than those established with wild-type or EBER-reconstituted EBV, and the difference between the growth rates was especially highlighted when the cells were plated at low cell densities. These results clearly demonstrate that EBERs significantly contribute to the efficient growth transformation of B lymphocytes by enhancing the growth potential of transformed lymphocytes.

Epstein-Barr virus nuclear protein EBNA3C is required for cell cycle progression and growth maintenance of lymphoblastoid cells.

Epstein–Barr virus (EBV) infection converts primary human B cells into continuously proliferating lymphoblastoid cell lines (LCLs). To examine the role of EBV nuclear antigen (EBNA) 3C in the proliferation of LCLs, we established LCLs infected with an EBV recombinant that expresses EBNA3C with a C-terminal fusion to a 4-hydroxytamoxifen (4HT)-dependent mutant estrogen receptor, E3C–HT. In the presence of 4HT, LCLs expressed the E3C–HT protein and grew like WT LCLs. When E3C–HT EBV-infected LCLs were transferred to medium without 4HT, E3C–HT protein slowly disappeared, and the LCLs gradually ceased growing. WT EBNA3C expression from an oriP plasmid transfected into E3C–HT LCLs protected the LCLs from growth arrest in medium without 4HT, whereas expression of EBNA3A or EBNA3B did not. The expression of other EBNA proteins and of LMP1, CD21, CD23, and c-myc was unaffected by EBNA3C inactivation. However, EBNA3C inactivation resulted in the accumulation of p16INK4A, a decrease in the hyperphosphorylated form of the retinoblastoma protein, and a decrease in the proportion of cells in S or G2/M phase. These results indicate that EBNA3C has an essential role in cell cycle progression and the growth maintenance of LCLs.

Epstein-Barr Virus BZLF1 Gene, a Switch from Latency to Lytic Infection, Is Expressed as an Immediate-Early Gene after Primary Infection of B Lymphocytes

ABSTRACT We demonstrate here that the Epstein-Barr virus (EBV) BZLF1 gene, a switch from latent infection to lytic infection, is expressed as early as 1.5 h after EBV infection in Burkitts lymphoma-derived, EBV-negative Akata and Daudi cells and primary B lymphocytes. Since BZLF1 mRNA is expressed even when the cells are infected with EBV in the presence of anisomycin, an inhibitor of protein synthesis, its expression does not require prerequisite protein synthesis, indicating that BZLF1 is expressed as an immediate-early gene following primary EBV infection of B lymphocytes.

Production of High-Titer Epstein-Barr Virus Recombinants Derived from Akata Cells by Using a Bacterial Artificial Chromosome System

ABSTRACT An Epstein-Barr virus (EBV) genome in Burkitts lymphoma-derived cell line Akata was cloned into a bacterial artificial chromosome (BAC) vector. The BAC clone, designated AK-BAC, was rapidly and precisely modified by means of efficient homologous recombination in Escherichia coli. This system was used to produce recombinant EBVs with transgenes. An expression cassette of green fluorescent protein (GFP) was inserted into AK-BAC, and the resultant BAC clone, AK-BAC-GFP, was transfected into Akata cells. We found that transfected BAC plasmids efficiently formed episomes in EBV-positive Akata cells. Mixtures of wild-type and AK-BAC-GFP viruses were then produced and used to infect EBV-negative Akata cells. We obtained cell clones that harbored only AK-BAC-GFP but no wild-type episome. These cell clones produced infectious viruses after stimulating virus production, and the recombinant viruses of AK-BAC-GFP efficiently immortalized primary B lymphocytes. We further revised the method so that any kind of cDNA could be rapidly inserted into the unique I-PpoI site that had been artificially introduced into AK-BAC. The AK-BAC system will have a broad range of applications, such as genetic analyses of various viral gene products and development of viral vectors for human gene therapy.

Epstein-Barr Virus (EBV)-Encoded RNA 2 (EBER2) but Not EBER1 Plays a Critical Role in EBV-Induced B-Cell Growth Transformation

ABSTRACT Epstein-Barr virus (EBV)-encoded RNA 1 (EBER1) and EBER2 are untranslated RNAs and the most abundant viral transcripts in latently EBV-infected cells. We previously reported that EBERs play a critical role in efficient EBV-induced growth transformation of primary B cells. To investigate whether EBER1 and EBER2 have distinct roles in B-cell growth transformation, recombinant EBVs carrying either EBER1 or EBER2 were generated. The transforming ability of recombinant EBVs expressing EBER2 was as high as that of EBVs expressing both EBER1 and EBER2. In contrast, the transforming ability of recombinant EBVs carrying EBER1 was impaired and was similar to that of EBV lacking both EBER1 and EBER2. Lymphoblastoid cell lines (LCLs) established with EBVs carrying EBER2 proliferated at low cell densities, while LCLs established with EBVs carrying EBER1 did not. Interleukin 6 (IL-6) production in LCLs expressing EBER2 was more abundant than in those lacking EBER2. The growth of LCLs lacking EBER2 was enhanced by the addition of recombinant IL-6 to the cell culture, while the growth of EBER2-expressing LCLs was inhibited by a neutralizing anti-IL-6 antibody. These results demonstrate that EBER2, but not EBER1, contributes to efficient B-cell growth transformation. We conclude that EBER1 and EBER2, despite their structural similarity, have different functions in latently infected lymphoblastoid cells.

Epigenetic Histone Modification of Epstein-Barr Virus BZLF1 Promoter during Latency and Reactivation in Raji Cells

ABSTRACT The Epstein-Barr virus (EBV) predominantly establishes latent infection in B cells, and the reactivation of the virus from latency is dependent on the expression of the viral BZLF1 protein. The BZLF1 promoter (Zp) normally exhibits only low basal activity but is activated in response to chemical or biological inducers, such as 12-O-tetradecanoylphorbol-13-acetate (TPA), calcium ionophores, or histone deacetylase (HDAC) inhibitors. In some cell lines latently infected with EBV, an HDAC inhibitor alone can induce BZLF1 transcription, while the treatment does not enhance expression in other cell lines, such as B95-8 or Raji cells, suggesting unknown suppressive mechanisms besides histone deacetylation in those cells. Here, we found the epigenetic modification of the BZLF1 promoter in latent Raji cells by histone H3 lysine 27 trimethylation (H3K27me3), H3K9me2/me3, and H4K20me3. Levels of active markers such as histone acetylation and H3K4me3 were low in latent cells but increased upon reactivation. Treatment with 3-deazaneplanocin A (DZNep), an inhibitor of H3K27me3 and H4K20me3, significantly enhanced the BZLF1 transcription in Raji cells when in combination with an HDAC inhibitor, trichostatin A (TSA). The knockdown of Ezh2 or Suv420h1, histone methyltransferases for H3K27me3 or H4K20me3, respectively, further proved the suppression of Zp by the methylations. Taken together, the results indicate that H3K27 methylation and H4K20 methylation are involved, at least partly, in the maintenance of latency, and histone acetylation and H3K4 methylation correlate with the reactivation of the virus in Raji cells.

Epstein-Barr Virus Deubiquitinase Downregulates TRAF6-Mediated NF-κB Signaling during Productive Replication

ABSTRACT Epstein-Barr virus (EBV), a human oncogenic herpesvirus that establishes a lifelong latent infection in the host, occasionally enters lytic infection to produce progeny viruses. The EBV oncogene latent membrane protein 1 (LMP1), which is expressed in both latent and lytic infection, constitutively activates the canonical NF-κB (p65) pathway. Such LMP1-mediated NF-κB activation is necessary for proliferation of latently infected cells and inhibition of viral lytic cycle progression. Actually, canonical NF-κB target gene expression was suppressed upon the onset of lytic infection. TRAF6, which is activated by conjugation of polyubiquitin chains, associates with LMP1 to mediate NF-κB signal transduction. We have found that EBV-encoded BPLF1 interacts with and deubiquitinates TRAF6 to inhibit NF-κB signaling during lytic infection. HEK293 cells with BPLF1-deficient recombinant EBV exhibited poor viral DNA replication compared with the wild type. Furthermore, exogenous expression of BPLF1 or p65 knockdown in cells restored DNA replication of BPLF1-deficient viruses, indicating that EBV BPLF1 deubiquitinates TRAF6 to inhibit NF-κB signal transduction, leading to promotion of viral lytic DNA replication.

Epstein-Barr Virus Transforming Protein LMP1 Plays a Critical Role in Virus Production

ABSTRACT The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), which is critical for EBV-induced B-cell transformation, is also abundantly expressed during the lytic cycle of viral replication. However, the biological significance of this strong LMP1 induction remains unknown. We engineered a bacterial artificial chromosome clone containing the entire genome of Akata strain EBV to specifically disrupt the LMP1 gene. Akata cell clones harboring the episomes of LMP1-deleted EBV were established, and the effect of LMP1 loss on virus production was investigated. We found that the degree of viral DNA amplification and the expression levels of viral late gene products were unaffected by LMP1 loss, demonstrating that the LMP1-deleted EBV entered the lytic replication cycle as efficiently as the wild-type counterpart. This was confirmed by our electron microscopic observation that nucleocapsid formation inside nuclei occurred even in the absence of LMP1. By contrast, loss of LMP1 severely impaired virus release into culture supernatants, resulting in poor infection efficiency. The expression of truncated LMP1 in Akata cells harboring LMP1-deleted EBV rescued the virus release into the culture supernatant and the infectivity, and full-length LMP1 partially rescued the infectivity. These results indicate that inducible expression of LMP1 during the viral lytic cycle plays a critical role in virus production.

The human cytomegalovirus gene products essential for late viral gene expression assemble into prereplication complexes before viral DNA replication.

ABSTRACT The regulation of human cytomegalovirus (HCMV) late gene expression by viral proteins is poorly understood, and these viral proteins could be targets for novel antivirals. HCMV open reading frames (ORFs) UL79, -87, and -95 encode proteins with homology to late gene transcription factors of murine gammaherpesvirus 68 ORFs 18, 24, and 34, respectively. To determine whether these HCMV proteins are also essential for late gene transcription of a betaherpesvirus, we mutated HCMV ORFs UL79, -87, and -95. Cells were infected with the recombinant viruses at high and low multiplicities of infection (MOIs). While viral DNA was detected with the recombinant viruses, infectious virus was not detected unless the wild-type viral proteins were expressed in trans. At a high MOI, mutation of ORF UL79, -87, or -95 had no effect on the level of major immediate-early (MIE) gene expression or viral DNA replication, but late viral gene expression from the UL44, -75, and -99 ORFs was not detected. At a low MOI, preexpression of UL79 or -87, but not UL95, in human fibroblast cells negatively affected the level of MIE viral gene expression and viral DNA replication. The products of ORFs UL79, -87, and -95 were expressed as early viral proteins and recruited to prereplication complexes (pre-RCs), along with UL44, before the initiation of viral DNA replication. All three HCMV ORFs are indispensable for late viral gene expression and viral growth. The roles of UL79, -87, and -95 in pre-RCs for late viral gene expression are discussed.