Structure and role of the terminal repeats of Epstein-Barr virus in processing and packaging of virion DNA

Zimmermann, Jürgen; Hammerschmidt, Wolfgang

doi:10.1128/jvi.69.5.3147-3155.1995

Cited by 77 publications

(34 citation statements)

References 26 publications

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“…Virtually nothing is known regarding the mechanism of packaging VV DNA. Unlike the situation with bacteriophage lambda (8) and herpesviruses (29,30,54), the processing of VV DNA to unit-length genomes is not linked to particle formation since it occurs even when assembly is blocked with rifampin (33). As morphogenesis was interrupted at an even later stage when A32 expression was repressed, the finding of normal DNA processing was not surprising.…”

Section: Discussionmentioning

confidence: 96%

DNA Packaging Mutant: Repression of the Vaccinia Virus A32 Gene Results in Noninfectious, DNA-Deficient, Spherical, Enveloped Particles

Cassetti

Merchlinsky

Wolffe

et al. 1998

J Virol

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The vaccinia virus A32 open reading frame was predicted to encode a protein with a nucleoside triphosphatebinding motif and a mass of 34 kDa. To investigate the role of this protein, we constructed a mutant in which the original A32 gene was replaced by an inducible copy. The recombinant virus, vA32i, has a conditional lethal phenotype: infectious virus formation was dependent on isopropyl-␤-D-thiogalactopyranoside (IPTG). Under nonpermissive conditions, the mutant synthesized early-and late-stage viral proteins, as well as viral DNA that was processed into unit-length genomes. Electron microscopy of cells infected in the absence of IPTG revealed normal-appearing crescents and immature virus particles but very few with nucleoids. Instead of brick-shaped mature particles with defined core structures, there were numerous electron-dense, spherical particles. Some of these spherical particles were wrapped with cisternal membranes, analogous to intracellular and extracellular enveloped virions. Mutant viral particles, purified by sucrose density gradient centrifugation, had low infectivity and transcriptional activity, and the majority were spherical and lacked DNA. Nevertheless, the particle preparation contained representative membrane proteins, cleaved and uncleaved core proteins, the viral RNA polymerase, the early transcription factor and several enzymes, suggesting that incorporation of these components is not strictly coupled to DNA packaging.Vaccinia virus (VV), the prototype poxvirus, replicates within the cytoplasm and has a linear double-stranded DNA genome of 185 kbp with inverted terminal repeats (16, 50) and covalently linked ends (2,17). Approximately 185 open reading frames (ORFs) are likely to encode proteins, though the functions of less than half of these are known (21,36). Some insights into protein function have come from comparative sequence analysis. Eight VV ORFs (A18, A32, A48, D5, D6, D11, I8, and J2) have predicted purine nucleoside triphosphate binding motifs (18,27,28), and there is evidence that most of the corresponding proteins have roles in transcription, replication, alteration of DNA topology, or nucleotide metabolism. Of these proteins, least is known about the A32 gene product. Based on limited sequence similarity to the products of gene I of filamentous, single-stranded DNA bacteriophages and to the Iva2 gene of adenovirus, Koonin et al. (28) suggested that the A32 gene product may be an ATPase involved in DNA packaging. In addition, a highly conserved function was predicted from the degree of similarity of the deduced amino acid sequences of the VV A32 protein to its homolog in the distantly related molluscum contagiosum virus (43,44).In the absence of any experimental data regarding the expression or role of the VV A32 gene, we have taken an in vivo genetic approach to the subject. Here we describe the construction and properties of a conditional lethal mutant of VV with an inducible A32 gene. MATERIALS AND METHODS Cells and viruses. BS-C-1 (ATCC CCL6) and HeLa S3 cells (ATCC C...

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Section: Discussionmentioning

confidence: 96%

DNA Packaging Mutant: Repression of the Vaccinia Virus A32 Gene Results in Noninfectious, DNA-Deficient, Spherical, Enveloped Particles

Cassetti

Merchlinsky

Wolffe

et al. 1998

J Virol

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“…This EBV DNA fragment also contains the lytic origin of DNA replication, oriLyt, together with the BHRF1 and BHLF1 loci, which flank it. The plasmid p554 and p613 DNAs can be packaged into infectious EBV particles, because p554 and p613 also contain TRs, the essential DNA packaging elements of EBV (Hammerschmidt and Sugden, 1989;Zimmermann and Hammerschmidt, 1995). p554 and p613 differ in that p613 is incapable of expressing EBNA2 (Hammerschmidt and Sugden, 1989).…”

Section: A Minimal Set Of Ebv Genes Is Sufficient To Support B Cell Amentioning

confidence: 99%

The first days in the life of naïve human B-lymphocytes infected with Epstein-Barr virus

Pich

Mrozek-Górska

Bouvet

et al. 2019

Preprint

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Epstein-Barr virus (EBV) infects and activates resting human B-lymphocytes, reprograms them, induces their proliferation, and establishes a latent infection in them. In established EBV-infected cell lines many viral latent genes are expressed. Their roles in supporting the continuous proliferation of EBV-infected B cells in vitro are known, but their functions in the early, pre-latent phase of infection have not been investigated systematically. In studies during the first eight days of infection using derivatives of EBV with mutations in single genes of EBVs we found only EBNA2 to be essential for activating naïve human B-lymphocytes, inducing their growth in cell volume, driving them into rapid cell divisions, and preventing cell death in a subset of infected cells. EBNA-LP, LMP2A and the viral microRNAs have supportive, auxiliary functions, but mutants of LMP1, EBNA3A, EBNA3C, and the noncoding EBER RNAs had no discernable phenotype compared with wild-type EBV. B cells infected with a double mutant of EBNA3A and 3C had an unexpected proliferative advantage and did not regulate the DNA damage response (DDR) of the infected host cell in the pre-latent phase. Even EBNA1 which has very critical longterm functions in maintaining and replicating the viral genomic DNA in established cell lines, was dispensable for the early activation of infected cells. Our findings document that the virus dose is a critical parameter and indicate that EBNA2 governs the infected cells initially and implements a strictly controlled temporal program independent of other viral latent genes. It thus appears that EBNA2 is sufficient to control all requirements for clonal cellular expansion and to reprogram human B-lymphocytes from energetically quiescent to activated cells. Author summaryThe preferred target of Epstein-Barr virus (EBV) are human resting B-lymphocytes. We found that their infection induces a well-coordinated, time-driven program that starts with a substantial 3 increase in cell volume followed by cellular DNA synthesis after three days and subsequent rapid rounds of cell divisions on the next day accompanied by some DNA replication stress (DRS). Two to three days later the cells decelerate and turn into stably proliferating lymphoblast cell lines. With the aid of 16 different recombinant EBV strains we investigated the individual contributions of EBV's multiple latent genes during early B-cell infection and found that many do not exert a detectable phenotype or contribute little to EBV's pre-latent phase. The exception is EBNA2 that is essential in governing all aspects of B-cell reprogramming. EBV relies on EBNA2 to turn the infected B-lymphocytes into proliferating lymphoblasts preparing the infected host cell for the ensuing stable, latent phase of viral infection. In the early steps of B-cell reprogramming viral latent genes other than EBNA2 are dispensable but some, EBNA-LP for example, support the viral program and presumably stabilize the infected cells once viral latency is established.

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“…The EBV terminal repeat unit varies between approximately 500 and 1,000 nucleotides in length [Gilligan et al, 1990], and variation in the number of EBV terminal repeat units arises during productive EBV replication as a consequence of variable-site cleavage of the concatemeric viral genome during virion packaging [Sato et al, 1990;Zimmermann and Hammerschmidt, 1995]. After infection of an individual, a single EBV genotype may generate numerous EBV termini variants that infect multiple different B-lymphocytes within that individual.…”

Section: Discussionmentioning

confidence: 99%

Molecular markers of clonality and identity in epstein–barr virus‐associated B‐cell lymphoproliferative disease

Walling

Andritsos

Etienne

et al. 2004

Journal of Medical Virology

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Epstein-Barr virus (EBV)-associated B-cell lymphoproliferative disease may be polyclonal, oligoclonal, or monoclonal. The degree of tumor clonality reflects the disease pathogenesis and may have implications for disease diagnosis, prognosis, and treatment. In this study, specimens of EBV-associated B-cell lymphoproliferative disease obtained from immunocompromised hosts were analyzed for molecular markers of cellular and virologic clonality and virologic identity. Each tumor specimen was assessed for immunoglobulin gene JH region rearrangement, the structure of the EBV genome termini, and the EBV genotype(s) present using a new EBV genotyping assay based upon LMP-1 gene sequence variation. The results of the JH rearrangement and EBV termini assays were generally concordant in their assessment of tumor specimen clonality, and both assays contributed to establishing clonal identity between different tumor specimens. The EBV genotyping assay did not significantly contribute to the assessment of tumor clonality but did established clear virologic identity between different tumor specimens obtained from the same individual. In one individual, these three assays together characterized a multi-focal, monoclonal tumor that may have arisen through clonal selection after sequential infections with two different EBV genotypes. In summary, the JH rearrangement and EBV termini assays each provided different but complementary information on tumor clonality, while the EBV genotyping assay proved most useful for establishing virologic identity among tumors. Utilization of these three assays together may provide new insight into the pathogenesis of EBV-associated B-cell lymphoproliferative disease.

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Structure and role of the terminal repeats of Epstein-Barr virus in processing and packaging of virion DNA

Cited by 77 publications

References 26 publications

DNA Packaging Mutant: Repression of the Vaccinia Virus A32 Gene Results in Noninfectious, DNA-Deficient, Spherical, Enveloped Particles

DNA Packaging Mutant: Repression of the Vaccinia Virus A32 Gene Results in Noninfectious, DNA-Deficient, Spherical, Enveloped Particles

The first days in the life of naïve human B-lymphocytes infected with Epstein-Barr virus

Molecular markers of clonality and identity in epstein–barr virus‐associated B‐cell lymphoproliferative disease

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