Replication of Epstein-Barr viral DNA

Abstract

Epstein-Barr virus (EBV) is a paradigm for human tumor viruses: it is the first virus recognized to cause cancer in people; it causes both lymphomas and carcinomas; yet these tumors arise infrequently given that most people in the world are infected with the virus. EBV is maintained extrachromosomally in infected normal and tumor cells. Eighty-four percent of these viral plasmids replicate each S phase, are licensed, require a single viral protein for their synthesis, and can use two functionally distinct origins of DNA replication, oriP, and Raji ori. Eighty-eight percent of newly synthesized plasmids are segregated faithfully to the daughter cells. Infectious viral particles are not synthesized under these conditions of latent infection. This plasmid replication is consistent with survival of EBV's host cells. Rare cells in an infected population either spontaneously or following exogenous induction support EBV's lytic cycle, which is lethal for the cell. In this case, the viral DNA replicates 100-fold or more, uses a third kind of viral origin of DNA replication, oriLyt, and many viral proteins. Here we shall describe the three modes of EBV's replication as a function of the viral origins used and the viral and cellular proteins that mediate the DNA synthesis from these origins focusing, where practical, on recent advances in our understanding.

Figure 1.

A physical map of the B95-8 laboratory strain of EBV DNA of 165 kbp is shown. The letters on the inner surface of the circle denote the fragments generated by digestion of this DNA with the BamH1 endonuclease and used to mark its transcripts (Baer et al. 1984). The primary transcript for EBNA1 is denoted by the dashed line. The white box marked TRs indicates the terminal repeats of linear virion DNA that mediate circularization of the viral DNA on infection of cells. The sites for oriP and oriLyt (L, left) are shown with the approximate location of DNA deleted from the B95-8 strain, present in all other analyzed strains, that contains a second copy of oriLyt (R, right). Raji ori, a region that is defined only approximately maps to sequences spanning the deletion found in the B95-8 strain.

Figure 2.

Shown are depictions of oriP and EBNA1. (A) oriP consists of multiple sets of binding sites for EBNA1. The family of repeats in the B95-8 strain of EBV has 20 binding sites for EBNA1, which together mediate maintenance of oriP plasmids. DS has two pairs of binding sites and is the site or close to the site at which DNA synthesis initiates within oriP. Rep* can substitute for DS functionally but inefficiently. It too contains a pair of EBNA1-binding sites with the same spacing as each pair in DS. When multimerized eightfold, Rep* functions as efficiently as does DS to support initiation of DNA synthesis. (B) The sequence and spacing of EBNA1-binding sites in DS of oriP are shown. The two sites of each pair are separated by 21 bps allowing similar contacts on one face of helical DNA. The nonomer repeats that can bind telomere-associated proteins are underlined and denoted a, b, and c. (C) The domains of EBNA1 from the B95-8 strain of EBV are represented and are composed of 641 residues. LR1 and LR2 are linking regions rich in Gly-, Arg residues, which when fused to protein DNA-binding domains, can link the DNAs presumably by the AT-hook activities intrinsic to LR1 and LR2. LR1 and LR2 each contain unique sequences, UR1 and UR2. UR1 is involved in transcription regulated by EBNA1; UR2 does not have a known function. The Gly, Ala repeats have various activities ascribed to them but little or no effect on EBNA1’s functions in cell culture. EBNA1 has one identified nuclear localization sequence (NLS) (Yates 1996). EBNA1’s carboxy-terminal one-third comprises a dimerization and DNA-binding domain.

Figure 3.

oriLyt and its expanded core domain are shown. oriLyt spans about 7700 bp of the B95-8 strain of EBV including two genes BHLF1 and BHRF1 and their promoters. Sequences in these genes contribute to but are not essential for the function of oriLyt. The essential or core domain of oriLyt includes the promoters for and intergenic region of these genes. Two essential components (upstream and downstream) of oriLyt bind viral (BZLF1) at BZLF1-responsive elements (ZRE) and cellular factors (ZBP-89, Sp1, and likely additional unknown proteins) are required for oriLyt’s function. The BZLF1 protein also binds to promoter elements of the BHRF1 gene to enhance oriLyt’s activity. This domain (ZRE sites 5, 6, and 7) can be replaced by a heterologous enhancer to support oriLyt’s function. The boxed element marked R binds the viral BRLF1 transactivator, which does not contribute directly to EBV’s lytic DNA replication.