Unexpected Instability of Family of Repeats (FR), the Critical cis-Acting Sequence Required for EBV Latent Infection, in EBV-BAC Systems

A group of repetitive sequences, known as the Family of Repeats (FR), is a critical cis-acting sequence required for EBV latent infection. The FR sequences are heterogeneous among EBV strains, and they are sometimes subject to partial deletion when subcloned in E. coli-based cloning vectors. However, the FR stability in EBV-BAC (bacterial artificial chromosome) system has never been investigated. We found that the full length FR of the Akata strain EBV was not stably maintained in a BAC vector. By contrast, newly obtained BAC clones of the B95-8 strain of EBV stably maintained the full length FR during recombinant virus production and B-cell transformation. Investigation of primary DNA sequences of Akata–derived EBV-BAC clones indicates that the FR instability is most likely due to a putative secondary structure of the FR region. We conclude that the FR instability in EBV-BAC clones can be a pitfall in E. coli-mediated EBV genetics

Efficient production of infectious viruses requires enzymatic activity of Epstein-Barr virus protein kinase

AbstractThe Epstein-Barr virus (EBV) BGLF4 gene product is the only protein kinase encoded by the virus genome. In order to elucidate its physiological roles in viral productive replication, we here established a BGLF4-knockout mutant and a revertant virus. While the levels of viral DNA replication of the deficient mutant were equivalent to those of the wild-type and the revertant, virus production was significantly impaired. Expression of the BGLF4 protein in trans fully complemented the low yield of the mutant virus, while expression of a kinase-dead (K102I) form of the protein failed to restore the virus titer. These results demonstrate that BGLF4 plays a significant role in production of infectious viruses and that the kinase activity is crucial

Epstein-Barr Virus genome deletions in Epstein-Barr Virus-positive T/NK cell lymphoproliferative diseases

The main target cells for Epstein-Barr virus (EBV) infection and persistence are B lymphocytes, although T and NK cells can also become infected. In this paper, we characterize the EBV present in 21 pediatric and adult patients who were treated in France for a range of diseases that involve infection of T or NK cells. Of these 21 cases, 5 pediatric patients (21%) and 11 adult patients (52%) were of Caucasian origin. In about 30% of the cases, some of the EBV genomes contain a large deletion. The deletions are different in every patient but tend to cluster near the BART region of the viral genome. Detailed investigation of a family in which several members have persistent T or NK cell infection by EBV indicates that the virus genome deletions arise or are selected independently in each individual patient. Genome sequence polymorphisms in the EBV in these T or NK cell diseases reflect the geographic origin of the patient and not a distinct type of EBV (the 21 cases studied included examples of both type 1 and type 2 EBV infection). Using virus produced from type 1 or type 2 EBV genomes cloned in bacterial artificial chromosome (BAC) vectors, we demonstrate infection of T cells in cord blood from healthy donors. Our results are consistent with transient infection of some T cells being part of normal asymptomatic infection by EBV in young children. IMPORTANCE EBV contributes to several types of human cancer. Some cancers and nonmalignant lymphoproliferative diseases involving T or NK cells contain EBV. These diseases are relatively frequent in Japan and China and have been shown sometimes to have deletions in the EBV genome in the disease cells. We identify further examples of deletions within the EBV genome associated with T or NK cell diseases, and we provide evidence that the virus genomes with these deletions are most likely selected in the individual cases, rather than being transmitted between people during infection. We demonstrate EBV infection of cord blood T cells by highly characterized, cloned EBV genomes and suggest that transient infection of T cells may be part of normal asymptomatic infection by EBV in young children

The Human Cytomegalovirus UL76 Gene Regulates the Level of Expression of the UL77 Gene

Human cytomegalovirus (HCMV) can be reactivated under immunosuppressive conditions causing several fatal pneumonitis, hepatitis, retinitis, and gastrointestinal diseases. HCMV also causes deafness and mental retardation in neonates when primary infection has occurred during pregnancy. In the genome of HCMV at least 194 known open reading frames (ORFs) have been predicted, and approximately one-quarter, or 41 ORFs, are required for viral replication in cell culture. In contrast, the majority of the predicted ORFs are nonessential for viral replication in cell culture. However, it is also possible that these ORFs are required for the efficient viral replication in the host. The UL77 gene of HCMV is essential for viral replication and has a role in viral DNA packaging. The function of the upstream UL76 gene in the HCMV-infected cells is not understood. UL76 and UL77 are cistons on the same viral mRNA and a conventional 5' mRNA for UL77 has not been detected. The vast majority of eukaryotic mRNAs are monocistronic, i.e., they encode only a single protein.To determine whether the UL76 ORF affects UL77 gene expression, we mutated UL76 by ORF frame-shifts, stop codons or deletion of the viral gene. The effect on UL77 protein expression was determined by either transfection of expression plasmids or infection with recombinant viruses. Mutation of UL76 ORF significantly increased the level of UL77 protein expression. However, deletion of UL76 upstream of the UL77 ORF had only marginal effects on viral growth.While UL76 is not essential for viral replication, the UL76 ORF is involved in regulation of the level of UL77 protein expression in a manner dependent on the translation re-initiation. UL76 may fine-tune the UL77 expression for the efficient viral replication in the HCMV- infected cells

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

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

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

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

Rapid CRISPR/Cas9-Mediated Cloning of Full-Length Epstein-Barr Virus Genomes from Latently Infected Cells

Herpesviruses have relatively large DNA genomes of more than 150 kb that are difficult to clone and sequence. Bacterial artificial chromosome (BAC) cloning of herpesvirus genomes is a powerful technique that greatly facilitates whole viral genome sequencing as well as functional characterization of reconstituted viruses. We describe recently invented technologies for rapid BAC cloning of herpesvirus genomes using CRISPR/Cas9-mediated homology-directed repair. We focus on recent BAC cloning techniques of Epstein-Barr virus (EBV) genomes and discuss the possible advantages of a CRISPR/Cas9-mediated strategy comparatively with precedent EBV-BAC cloning strategies. We also describe the design decisions of this technology as well as possible pitfalls and points to be improved in the future. The obtained EBV-BAC clones are subjected to long-read sequencing analysis to determine complete EBV genome sequence including repetitive regions. Rapid cloning and sequence determination of various EBV strains will greatly contribute to the understanding of their global geographical distribution. This technology can also be used to clone disease-associated EBV strains and test the hypothesis that they have special features that distinguish them from strains that infect asymptomatically

Epstein-Barr virus genome packaging factors accumulate in BMRF1-cores within viral replication compartments.

Productive replication of Epstein-Barr virus (EBV) during the lytic cycle occurs in discrete sites within nuclei, termed replication compartments. We previously proposed that replication compartments consist of two subnuclear domains: "ongoing replication foci" and "BMRF1-cores". Viral genome replication takes place in ongoing replication foci, which are enriched with viral replication proteins, such as BALF5 and BALF2. Amplified DNA and BMRF1 protein accumulate in BMRF1-cores, which are surrounded by ongoing replication foci. We here determined the locations of procapsid and genome-packaging proteins of EBV via three-dimensional (3D) surface reconstruction and correlative fluorescence microscopy-electron microscopy (FM-EM). The results revealed that viral factors required for DNA packaging, such as BGLF1, BVRF1, and BFLF1 proteins, are located in the innermost subdomains of the BMRF1-cores. In contrast, capsid structural proteins, such as BBRF1, BORF1, BDLF1, and BVRF2, were found both outside and inside the BMRF1-cores. Based on these observations, we propose a model in which viral procapsids are assembled outside the BMRF1-cores and subsequently migrate therein, where viral DNA encapsidation occurs. To our knowledge, this is the first report describing capsid assembly sites in relation to EBV replication compartments