The Epstein–Barr virus BRRF2 gene product is involved in viral progeny production

Watanabe, Takahiro; Tsuruoka, Miyuki; Narita, Yohei; Katsuya, Ryotaro; Goshima, Fumi; Kimura, Hiroshi; Murata, Takayuki

doi:10.1016/j.virol.2015.05.010

Cited by 15 publications

(25 citation statements)

References 31 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…3C). Wild-type, mutant, and revertant viruses exhibited comparable levels of expression of E genes (BALF2 and BMRF1), while the protein expression level of L genes (BALF4 [gB] and BRRF2 [8]) in the knockout virusinfected cells was slightly, but appreciably, lower. Additionally, expression of L genes was monitored by reverse transcription-PCR (RT-PCR).…”

mentioning

confidence: 99%

The Epstein-Barr Virus BDLF4 Gene Is Required for Efficient Expression of Viral Late Lytic Genes

Watanabe

Narita

Yoshida

et al. 2015

J Virol

Self Cite

View full text Add to dashboard Cite

Epstein-Barr virus (EBV) is a gammaherpesvirus, associated with infectious mononucleosis and various types of malignancy. We focused here on the BDLF4 gene of EBV and identified it as a lytic gene, expressed with early kinetics. Viral late gene expression of the BDLF4 knockout strain was severely restricted; this could be restored by an exogenous supply of BDLF4. These results indicate that BDLF4 is important for the EBV lytic replication cycle, especially in late gene expression. Epstein-Barr virus (EBV) is a human gammaherpesvirus that is present in Ͼ90% the world's population. EBV is associated with infectious mononucleosis and neoplasms such as Burkitt lymphoma, Hodgkin's lymphoma, posttransplant lymphoproliferative disease, nasopharyngeal carcinoma, and gastric carcinoma (1, 2).The EBV BDLF4 gene is an open reading frame (ORF), located between BGLF1 and BDLF3.5. Sequence similarities suggest that it is conserved among the members of the betaherpesvirus and gammaherpesvirus subfamilies (UL92 of human cytomegalovirus [HCMV] and ORF31 of murine gammaherpesvirus 68 and Kaposi's sarcoma-associated herpesvirus [KSHV]) but that it is not present in the alphaherpesviruses. It has been reported that KSHV ORF31, MHV-68 ORF31, HCMV UL92, and MCMV M92 are required for the efficient transcription of viral true late genes (3-7), but the EBV BDLF4 has not been characterized well.BDLF4 expression in B95-8 cells with early kinetics. We first prepared antiserum against EBV BDLF4 by immunizing rabbits with a glutathione S-transferase (GST)-fused BDLF4 protein expressed in bacteria. This anti-BDLF4 antiserum reacted with a protein of about ϳ22 kDa in the lysate from B95-8 cells, treated with tetradecanoyl phorbol acetate (TPA), A23187, and sodium butyrate (T/A/B) for 48 h (Fig. 1A). The BDLF4 protein could be detected readily by 24 h, and expression was increased at 48 h (Fig. 1B). While BALF4 mRNA expression was clearly blocked by phosphonoacetic acid (PAA), the amount of BDLF4 was decreased only slightly by the inhibitor (Fig. 1C), suggesting that the BDLF4 gene is expressed with E kinetics.Generation of BDLF4 knockout virus by frameshifting. To study the biological role of the BDLF4 gene in EBV lytic replication, we constructed a BDLF4-deficient recombinant virus and its revertant, as depicted in Fig. 2A. The integrity of the viral genomes was examined by restriction enzyme analysis, followed by agarose electrophoresis (Fig. 2B), and sequencing confirmed the intended one-nucleotide (nt) deletion.Knockout of BDLF4 decreased viral L gene expression and progeny production. The recombinant EBV-bacterial artificial chromosome (BAC) DNAs were transfected into HEK293 cells, and cells stably and latently containing EBV DNAs were cloned by hygromycin selection. We found that viral DNA synthesis levels of the wild-type, frameshift mutant, and revertant viruses were comparable (Fig. 3A). Disruption of BDLF4 decreased virus yield to approximately 10% to 20% compared with levels seen with the wild-type and revertant viruses (Fig. 3B)...

show abstract

mentioning

confidence: 99%

The Epstein-Barr Virus BDLF4 Gene Is Required for Efficient Expression of Viral Late Lytic Genes

Watanabe

Narita

Yoshida

et al. 2015

J Virol

Self Cite

View full text Add to dashboard Cite

show abstract

“…The antibody against BRRF1 was prepared by immunizing a rabbit with a fusion protein of GST and full-length BRRF1. Antibodies against BRRF2, BZLF1, BMRF1, BALF2, gB, EBNA1 and LMP1 were used as described previously 14, 21–23 . Antibodies against tubulin, BRLF1 and p53 were purchased from Cell Signaling, Argene Biosoft and Merk, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…The expression vector pcDNABZLF1 22 and pcDNABRRF2 23 were constructed as described previously. The BRRF1 PCR product was cloned into a pcDNA3 vector to generate the BRRF1 expression plasmid.…”

Section: Methodsmentioning

confidence: 99%

“…Eukaryotic 18 S rRNA (Applied Biosystems) was used as an endogenous control. For EBV genome detection, primers and a probe targeting the BALF2-coding region were used as described previously 23 .…”

Section: Methodsmentioning

confidence: 99%

“…B95-8 cells were lytically induced by 200 ng/ml TPA, 0.5 μM A23187 and 5 mM sodium butyrate with or without 400 μg/ml PAA for 48 hours 23 . AGS-Akata cells were lytically induced by 10 ng/ml TPA and 2 mM sodium butyrate with or without 200 μg/ml PAA for 48 hours.…”

Section: Methodsmentioning

confidence: 99%

See 2 more Smart Citations

The Epstein-Barr Virus BRRF1 Gene Is Dispensable for Viral Replication in HEK293 cells and Transformation

Yoshida

Watanabe

Narita

et al. 2017

Sci Rep

Self Cite

View full text Add to dashboard Cite

The Epstein-Barr virus (EBV) is a gamma-herpesvirus associated with several malignancies. It establishes a latent infection in B lymphocytes and is occasionally reactivated to enter the lytic cycle. Here we examined the role of the EBV gene BRRF1, which is expressed in the lytic state. We first confirmed, using a DNA polymerase inhibitor, that the BRRF1 gene is expressed with early kinetics. A BRRF1-deficient recombinant virus was constructed using a bacterial artificial chromosome system. No obvious differences were observed between the wild-type, BRRF1-deficient mutant and the revertant virus in HEK293 cells in terms of viral lytic protein expression, viral DNA synthesis, progeny production, pre-latent abortive lytic gene expression and transformation of primary B cells. However, reporter assays indicated that BRRF1 may activate transcription in promoter- and cell type-dependent manners. Taken together, BRRF1 is dispensable for viral replication in HEK293 cells and transformation of B cells, but it may have effects on transcription.

show abstract

Encyclopedia of EBV-Encoded Lytic Genes: An Update

Murata

2018

Advances in Experimental Medicine and Biology

View full text Add to dashboard Cite

In addition to latent genes, lytic genes of EBV must also be of extreme significance since propagation of the virus can be achieved only through execution of lytic cycle. Research on EBV lytic genes may thus prevent spreading of the virus and alleviate disorders, such as infectious mononucleosis and oral hairy leukoplakia, which are highly associated with EBV lytic infection. Moreover, recent advancements have been demonstrating that at least several lytic genes are expressed to some extent even during latent state. It is also demonstrated now that upon de novo infection, EBV expresses lytic genes in addition to latent genes before establishment of latency (this phase is called "pre-latent abortive lytic state"). In those cases, lytic genes also play important roles in cell proliferation of EBV-positive cells. However, many lytic gene products have not been identified yet nor studied thoroughly, and even worse, some have been misidentified in the literature. Here, I would like to give a detailed up-to-date review on EBV lytic genes.

show abstract

The Epstein–Barr virus BRRF2 gene product is involved in viral progeny production

Cited by 15 publications

References 31 publications

The Epstein-Barr Virus BDLF4 Gene Is Required for Efficient Expression of Viral Late Lytic Genes

The Epstein-Barr Virus BDLF4 Gene Is Required for Efficient Expression of Viral Late Lytic Genes

The Epstein-Barr Virus BRRF1 Gene Is Dispensable for Viral Replication in HEK293 cells and Transformation

Encyclopedia of EBV-Encoded Lytic Genes: An Update

Contact Info

Product

Resources

About