Rep*: a viral element that can partially replace the origin of plasmid DNA synthesis of Epstein-Barr virus

Abstract

Replication of the Epstein-Barr viral (EBV) genome occurs once per cell cycle during latent infection. Similarly, plasmids containing EBV's plasmid origin of replication, oriP, are replicated once per cell cycle. Replication from oriP requires EBV nuclear antigen 1 (EBNA-1) in trans; however, its contributions to this replication are unknown. oriP contains 24 EBNA-1 binding sites; 20 are located within the family of repeats, and 4 are found within the dyad symmetry element. The site of initiation of DNA replication within oriP is at or near the dyad symmetry element. We have identified a plasmid that contains the family of repeats but lacks the dyad symmetry element whose replication can be detected for a limited number of cell cycles. The detection of short-term replication of this plasmid requires EBNA-1 and can be inhibited by a dominant-negative inhibitor of EBNA-1. We have identified two regions within this plasmid which can independently contribute to this replication in the absence of the dyad symmetry element of oriP. One region contains native EBV sequences within the BamHI C fragment of the B95-8 genome of EBV; the other contains sequences within the simian virus 40 genome. We have mapped the region contributing to replication within the EBV sequences to a 298-bp fragment, Rep*. Plasmids which contain three copies of Rep* plus the family of repeats support replication more efficiently than those with one copy, consistent with a stochastic model for the initiation of DNA synthesis. Plasmids with three copies of Rep* also support long-term replication in the presence of EBNA-1. These observations together indicate that the latent origin of replication of EBV is more complex than formerly appreciated; it is a multicomponent origin of which the dyad symmetry element is one efficient component. The experimental approach described here could be used to identify eukaryotic sequences which mediate DNA synthesis, albeit inefficiently.

FIG. 1

Wild-type EBNA-1 and its derivatives. The DNA linking domains (grey boxes) (18, 36), the DNA binding and dimerization domain (hatched box) (3, 6, 28, 38, 49), the internal repeated sequence consisting entirely of glycine and alanine residues (Gly-Gly-Ala), and the nuclear localization sequence (NLS) (2) of EBNA-1 are noted. Regions rich in basic (+) and acidic (−) residues are indicated. The protein products of vectors encoding wild-type EBNA-1 and its derivatives are shown. EBNA-1 contains amino acids (aa) 1 to 641 and is wild-type EBNA-1 of the B95-8 strain of EBV (5). NΔ330-641 contains aa 331 to 641 of EBNA-1 (29), and NΔ450-641 contains the nuclear localization sequence (aa 379 to 386) fused in frame to aa 451 to 641 of EBNA-1 (40).

FIG. 2

Sequences within nt 9132 to 10905 of the EBV genome and within the t-antigen intron and the T-antigen poly(A) addition signal of SV40 contribute to short-term replication of FR-BamHI C-Luc by EBNA-1. The amount of DpnI-resistant DNA of each derivative of oriP-BamHI C-Luc detected at 96 h postelectroporation was compared to that of FR-BamHI C-Luc, using quantitative competitive PCR as described in Materials and Methods. Maps of reporter plasmids tested are shown on the left. N, number of replicates (a subset of the data from Table 3 and Fig. 3 is included). The efficiency of replication of each reporter by EBNA-1 is expressed as a percentage of DpnI-resistant DNA relative to FR-BamHI C-Luc, which is set to 100% (100% = 3.4 ± 1.2 copies of DpnI-resistant DNA per transfected cell). In these experiments, FR-BamHI C-Luc replicates 14% as efficiently as does oriP-BamHI C-Luc. Data were analyzed by using the Wilcoxon rank sum test (26) with Mstat (version 1.3, by Norman Drinkwater) and the P value comparing each derivative to FR-BamHI C-Luc is noted.

FIG. 3

Sequences within nt 9132 to 10905 of the EBV genome and within the SV40 t-antigen intron and the SV40 T-antigen poly(A) addition signal can rescue short-term replication of FR-Backbone by EBNA-1. Maps of reporter plasmids FR-BamHI C-Luc, FR- Backbone, FR-Δ, and FR-Δ2 are shown on the left. N, number of replicates (a subset of the data from Table 3 and Fig. 2 is included). The efficiency of replication of each reporter by EBNA-1 is expressed as a percentage of DpnI-resistant DNA relative to FR-BamHI C-Luc, which is set to 100% (100% = 4.5 ± 0.1.8 copies of DpnI-resistant DNA per transfected cell). In these experiments, FR-BamHI C-Luc replicates 13% as efficiently as does oriP-BamHI C-Luc. Data were analyzed by using the Wilcoxon rank sum test (26) with Mstat (version 1.3, by Norman Drinkwater), and the P value comparing each derivative to FR-BamHI C-Luc or FR-Backbone is noted.

FIG. 4

A 298-bp region of the EBV genome supports DNA replication in the absence of the DS. Maps of reporter plasmids oriP-BamHI C-Luc, oriP-Backbone, FR-Backbone, FR-Δ2, 1859, 1860, 1858, 1861, and 1862 are shown on the left. N, number of replicates. The efficiency of replication of each reporter by EBNA-1 is expressed as a percentage of DpnI-resistant DNA relative to oriP-Backbone at 96 h, which is set to 100% (100% = 7.9 ± 2.9 copies of DpnI-resistant DNA per transfected cell). Data were analyzed by using the Wilcoxon rank sum test (26) with Mstat (version 1.3, by Norman Drinkwater), and the P value comparing each derivative to FR-Δ2 is noted.