Epstein–Barr virus (EBV) is a causative agent of infectious mononucleosis and several types of cancer. Like other herpesviruses, it establishes an asymptomatic, life-long latent infection, with occasional reactivation and shedding of progeny viruses. During latency, EBV expresses a small number of viral genes, and exists as an episome in the host–cell nucleus. Expression patterns of latency genes are dependent on the cell type, time after infection, and milieu of the cell (e.g., germinal center or peripheral blood). Upon lytic induction, expression of the viral immediate-early genes, BZLF1 and BRLF1, are induced, followed by early gene expression, viral DNA replication, late gene expression, and maturation and egress of progeny virions. Furthermore, EBV reactivation involves more than just progeny production. The EBV life cycle is regulated by signal transduction, transcription factors, promoter sequences, epigenetics, and the 3D structure of the genome. In this article, the molecular basis of EBV latency establishment and reactivation is summarized.

Epstein–Barr virus (EBV) is a ubiquitous virus that causes infectious mononucleosis and several types of cancer, such as Burkitt lymphoma, T/NK-cell lymphoma, and nasopharyngeal carcinoma. As a herpesvirus, it encodes more than 80 genes, many of which have not been characterized. EBV BamHI S rightward reading frame 1 (BSRF1) encodes a tegument protein that, unlike its homologs herpes simplex virus unique long 51 (UL51) and human cytomegalovirus UL71, has not been extensively investigated. To examine the role of BSRF1, we prepared knockout and revertant strains using the bacterial artificial chromosome system. Unexpectedly, the disruption of the gene had little or no effect on EBV lytic replication and the transformation of primary B cells. However, the knockdown of BSRF1 in B95-8 cells decreased progeny production. An immunofluorescence assay revealed that BSRF1 localized to the Golgi apparatus in the cytoplasm, as did its homologs. BSRF1 also associated with BamHI G leftward reading frame 3.5 (BGLF3.5), BamHI B rightward reading frame 2 (BBRF2), and BamHI A leftward reading frame 1 (BALF1), and BALF1 was incorporated into the tegument fraction with BSRF1. Taken together, our results indicate that BSRF1 plays a role in secondary envelopment or virion egress in the cytoplasm, as do its homolog genes.

The Epstein–Barr virus (EBV) is the cause of several malignancies, including diffuse large B cell lymphoma (DLBCL). We recently found that EBV genomes in EBV-positive cancer specimens have various deletions (Okuno et al. Nat Microbiol. 2019). Here, we focus on the deletion of C promoter (Cp), which transcribes EBV nuclear antigen (EBNA) genes in type III latency. The Cp deletion found in a DLBCL patient (332 bp) was introduced into EBV-BAC of the B95-8 strain. Interestingly, the dCp virus transformed B cells more efficiently than WT and revertant strains. Deletion of Cp also promoted tumor formation and severe pathogenicity in a mouse xenograft model. RNA sequencing and qRT–PCR analyses revealed that Cp transcription was undetectable in the dCp cells. Instead, transcription from the W promoter (Wp), an alternative promoter for EBNA, was activated in the dCp mutant. We also found that the expression of latent membrane protein 2A (LMP2A) was somehow induced in the dCp mutant. Double knockout of Cp and LMP2A indicated that LMP2A is crucial for B cell transformation, but the increased transformation induced by Cp deletion cannot be explained by LMP2A alone. We also tested the effect of an anti-apoptotic viral BCL2 homolog, BHRF1, because its expression was reportedly induced more efficiently by that of Wp. However, increased growth transformation via Cp deletion was not due to the BHRF1 gene. Taken together, the results indicated that deletion of a specific region in Cp increased in vitro transformation and the rate of progression of EBV-positive lymphoproliferative disorders in vivo. Our data suggest that genomic alteration not only of the host but also the virus promotes EBV-positive tumor generation and expansion, although the molecular mechanism underlying this phenomenon is still unclear. However, LMP2A and BHRF1 are not involved.

Epstein-Barr virus (EBV) is a member of the gammaherpesvirinae, which causes infectious mononucleosis and several types of cancer. BBRF2 is an uncharacterized gene of EBV and is expressed during the lytic phase. To evaluate its function, BBRF2-knockout EBV was prepared using bacterial artificial chromosome (BAC) technology and the CRISPR/Cas9 system. Although viral gene expression, DNA synthesis, and progeny secretion were not affected, the infectivity of progeny viruses was significantly reduced by the disruption of BBRF2. When expressed alone, BBRF2 protein localized to the nucleus and cytoplasm, while the coexpression of an interacting partner, BSRF1, resulted in its relocalization to the cytoplasm. Interestingly, the coexpression of BBRF2 protected BSRF1 from proteasome/ubiquitin-dependent degradation. Therefore, BBRF2, together with BSRF1, augments viral infectivity.

N6-methyladenosine (m⁶A) is a post-transcriptional modification of RNA involved in transcript transport, degradation, translation, and splicing. We found that HBV RNA is modified by m⁶A predominantly in the coding region of HBx. The mutagenesis of methylation sites reduced the HBV mRNA and HBs protein levels. The suppression of m⁶A by an inhibitor or knockdown in primary hepatocytes decreased the viral RNA and HBs protein levels in the medium. These results suggest that the m⁶A modification of HBV RNA is needed for the efficient replication of HBV in hepatocytes.