Genetic evidence that EBNA-1 is needed for efficient, stable latent infection by Epstein-Barr virus

Abstract

Replication and maintenance of the 170-kb circular chromosome of Epstein-Barr virus (EBV) during latent infection are generally believed to depend upon a single viral gene product, the nuclear protein EBNA-1. EBNA-1 binds to two clusters of sites at oriP, an 1, 800-bp sequence on the EBV genome which can support replication and maintenance of artificial plasmids introduced into cell lines that contain EBNA-1. To investigate the importance of EBNA-1 to latent infection by EBV, we introduced a frameshift mutation into the EBNA-1 gene of EBV by recombination along with a flanking selectable marker. EBV genomes carrying the frameshift mutation could be isolated readily after superinfecting EBV-positive cell lines, but not if recombinant virus was used to infect EBV-negative B-cell lines or to immortalize peripheral blood B cells. EBV mutants lacking almost all of internal repeat 3, which encode a repetitive glycine and alanine domain of EBNA-1, were generated in the same way and found to immortalize B cells normally. An EBNA-1-deficient mutant of EBV was isolated and found to be incapable of establishing a latent infection of the cell line BL30 at a detectable frequency, indicating that the mutant was less than 1% as efficient as an isogenic, EBNA-1-positive strain in this assay. The data indicate that EBNA-1 is required for efficient and stable latent infection by EBV under the conditions tested. Evidence from other studies now indicates that autonomous maintenance of the EBV chromosome during latent infection does not depend on the replication initiation function of oriP. It is therefore likely that the viral chromosome maintenance (segregation) function of oriP and EBNA-1 is what is required.

FIG. 1

Insertion of a selective gene, CMVIE-neo, next to the EBNA-1 gene without disrupting the adjacent gene, BKRF2. (Upper) Part of the EBV genome, from the SalI site at 105,296 to the EcoRV site at 116,865. The EBNA-1 coding sequence (BKRF1) and an adjacent gene, BKRF2, are indicated. The dark portion of the EBNA-1 coding region is IR3, which encodes the Gly-Gly-Ala repeats. The indicated restriction enzyme sites are as follows: E, EcoRV; P, PvuII; Nc, NcoI; Ns, NsiI; Sa, SalI; Sf, SfiI; Ss, SstII; X, XbaI. Only the most relevant sites are shown for NcoI, PvuII, and SstII. (Lower) Relevant portion of the plasmid p652. The CMVIE-neo gene was inserted along with a duplication of nearly 400 bp of EBV sequence, from an SstII site to a PvuII site, placing the duplicated sequence on each side of the insertion. The duplicated sequence includes the 3′ end of the EBNA-1 gene (BKRF1) and continues through much of the adjacent gene, BKRF2. Thus a complete copy of each gene together with its 3′ and 5′ regulatory regions remains intact. Restriction sites that were destroyed where sequences were joined are indicated with a slash. Ns∗ represents filling in of an NcoI site to create an NsiI site and a frameshift mutation in the EBNA-1 gene. The zig-zag line at the right end indicates plasmid vector sequences.

FIG. 2

Southern analysis of EBV genomes carrying the inserted CMVIE-neo gene and a deletion of IR3 in immortalized B-cell clones. (A) B cells were immortalized with recombinant EBV that was generated by using each indicated plasmid. Five micrograms of total cellular DNA was digested with EcoRV plus SstII. The blot was probed with the CMVIE-neo gene. To the left, 294 pg of digested p652 DNA was used, corresponding to 9 DNA molecules per cell. For p652, the CMVIE-neo gene is on an 11.5-kb SstII-EcoRV fragment; its position is indicated. (B) The blot was stripped and reprobed with a BamHI-XbaI fragment spanning the EBNA-1 gene. DF1 is an EBV-immortalized B-cell line that carries between 5 and 10 EBV genomes per cell. Cytosine methylation at several SstII sites can account for the large size of the major band and for the partial digestion seen with DF1. (C) Southern analysis of two secondary B-cell clones (2°) that were obtained by using virus released from each of the primary isolates (1°), clones 1, 2, and 3, that were analyzed for panels A and B. DNA was digested with EcoRV plus SstII as for panels A and B and probed with the BamHI-XbaI 3.2-kb fragment. DF2 is an EBV-immortalized B-cell line that contains approximately 100 EBV genomes per cell. Marks to the left of A, B, and C indicate size markers (bacteriophage lambda DNA cut with HindIII). (D) Restriction maps of the relevant portion of the EBV genome for strain B95-8 and for B652. Cleavage sites for SstII (Ss) and EcoRV (E) and the sizes of fragments from the double digest, in kilobase pairs, are indicated; see the legend to Fig. 1 for details. DNAs used to make hybridization probes are shown below.

FIG. 3

Detection of EBNA-1 in B-cell clones carrying recombinant EBV. (A and B) Immunoblot analysis of protein extracted from 100,000 cells of different B-cell clones that were immortalized with EBV that had recombined with the indicated plasmids. The numbers above the lanes of the gels designate specific clones, most of which were also analyzed for viral DNA in Fig. 2 and 4. EBNA-1 of the B95-8 size and of the dl7 size are indicated by EBNA1 and dl7, respectively, to the left. Louckes is an EBV-negative Burkitt’s lymphoma cell line. EBV of Raji has fewer IR3 repeats than B95-8 EBV, so its EBNA-1 is correspondingly smaller. DF1 is a human B-cell clone immortalized with B95-8 EBV.

FIG. 4

Failure to transfer the InNco frameshift mutation to recombinant EBV genomes recovered in immortalized B cells. Five micrograms of DNA from each clone was digested with EcoRV plus NsiI and probed with the CMVIE-neo gene in panel A. For panel B, the membrane was stripped and reprobed with the 2.2 kb of DNA extending from the NsiI site at the frameshift mutation (InNco) leftward to the next NsiI site (Fig. 1). DF2 is a human B-cell clone that carries approximately 100 EBV genomes (strain B95-8) per cell. To the left, digested p652 DNA was loaded at 294 and 589 pg per lane, corresponding to 9 and 18 copies per cell. An 11.8-kb NsiI-EcoRV fragment from the plasmid is indicated. The other indicated bands are described in the text. (C) Restriction maps of the relevant portion of the EBV genome for strains B95-8, B652, and B652InNco. Cleavage sites for NsiI (Ns) and EcoRV (E) and the sizes of fragments from the double digest, in kilobase pairs, are indicated; see Fig. 1 for details. DNAs used to make hybridization probes are shown below.

FIG. 5

BL30 clones, obtained by selecting for stable infection by EBNA-1-deficient virus, all contain EBNA-1 protein. Immunoblot of protein extracted from 100,000 cells. Nine G418-resistant clones of BL30, labeled a through j, were obtained by infection with EBV released from B95-8 cells superinfected with B652InNco. The arrow indicates B95-8 EBNA-1.

FIG. 6

Stable infection of BL30 cells with B652InNco EBV selects for recombinants with the EBNA-1 gene restored. (A) Southern analysis of 0.5 μg of DNA from superinfected B95-8 clones carrying either B652InNco virus or B652 virus and of 5 μg of DNA from four G418-resistant clones of BL30 infected with virus released from B95/B652InNco superinfected cells. The DNA was cut with a combination of NsiI and BglII (left) or with NcoI (right). Approximate sizes are given for bands of interest. DNA fragments that resulted from recombination between the two viruses, B652InNco and B95-8, are marked with an asterisk. (B) Restriction site maps of the relevant part of the B95-8 and B652InNco viral genomes and their recombination products. B, BglII; Nc, NcoI; Ns, NsiI. An asterisk marks the NsiI site created by the frameshift mutation. The probe that was used contained the sequences 3′ of the EBNA-1 gene that were duplicated (small filled rectangle) and are present on both sides of the selective marker. Note that one of the reciprocal recombinants is identical to B652. (The probe contained, in addition to the region shown, about 100 bp of EBV sequence to the left of oriP, so EBV DNA fragments of 5.1 kb in the NcoI digestion and 0.6 kb in the NsiI-BglII digestion [not shown] were also detected weakly.)