Evidence for DNA hairpin recognition by Zta at the Epstein-Barr virus origin of lytic replication

Abstract

The Epstein-Barr virus immediate-early protein (Zta) plays an essential role in viral lytic activation and pathogenesis. Zta is a basic zipper (b-Zip) domain-containing protein that binds multiple sites in the viral origin of lytic replication (OriLyt) and is required for lytic-cycle DNA replication. We present evidence that Zta binds to a sequence-specific, imperfect DNA hairpin formed by an inverted repeat within the upstream essential element (UEE) of OriLyt. Mutations in the OriLyt sequence that are predicted to disrupt hairpin formation also disrupt Zta binding in vitro. Restoration of the hairpin rescues the defect. We also show that OriLyt DNA isolated from replicating cells contains a nuclease-sensitive region that overlaps with the inverted-repeat region of the UEE. Furthermore, point mutations in Zta that disrupt specific recognition of the UEE hairpin are defective for activation of lytic replication. These data suggest that Zta acts by inducing and/or stabilizing a DNA hairpin structure during productive infection. The DNA hairpin at OriLyt with which Zta interacts resembles DNA structures formed at other herpesvirus origins and may therefore represent a common secondary structure used by all herpesvirus family members during the initiation of DNA replication.

FIG. 1.

Zta binds specifically to the top strand of OriLyt, where a DNA hairpin is predicted to form. (A) Diagram of the upstream essential element (UEE) of OriLyt_L, which includes the BHLF1 promoter region, consisting of the TATA box, two Zta response elements, and a CCAAT box. (B) The predicted secondary structures of the top (+) and bottom (−) strands of the OriLyt ZRE1/2 sequence were analyzed using the Vienna RNA website program RNAfold with DNA parameters. The predicted free energy (ΔG) of each structure is given, as well as the positional entropy of each nucleotide. (C) Single-stranded (SS) oligonucleotide probes were assayed for their abilities to bind purified Zta using an electromobility gel shift assay. The asterisk indicates a nonspecific band. These data are representative of three independent experiments. (D) Sequences of single-stranded oligonucleotide probes. The percentage of each probe that bound to Zta is given.

FIG. 2.

Zta binds to an OriLyt single-stranded DNA hairpin. (A) Zta was assayed for its ability to bind oligonucleotide substrates with different capacities to form stable hairpins. Oligonucleotide probes 2 and 3 improve the complementarity of the ZRE sites. Probe 5 contains the wild-type sequence but is split into two oligonucleotide molecules that were annealed prior to the EMSA binding reaction. (B) Oligonucleotide probe sequences. (C and D) Zta was assayed for its ability to bind wild-type (probe 1) or mutant (probes 2 to 7) OriLyt hairpin sequences. Mutations 2 and 5 disrupt the hairpin, and mutations 2, 6, and 7 disrupt ZRE recognition. Mutations 3 and 4 alter mostly intervening sequence between the inverted repeats. Mutation 8 is a scrambled sequence that is capable of forming a hairpin but lacks a consensus ZRE. The percentage of each probe bound to Zta is indicated beneath the corresponding lane. The asterisk indicates a nonspecific band. These data are representative of three independent experiments.

FIG. 3.

The OriLyt ZRE1/2 top strand is able to competitively bind Zta. (A) Double-stranded DNA containing the ZRE2 sequence from the EBV R promoter (dsZRE2) was radiolabeled and incubated with or without Zta in the presence of cold competitor double-stranded ZRE1/2 (dsZRE1/2) or single-stranded ZRE1/2 from the top [ssZRE1/2(+)] or bottom [ssZRE1/2(−)] of OriLyt. (B) The percentage of binding of Zta to the radiolabeled dsDNA ZRE2 was quantified for each lane.

FIG. 4.

Zta binding to the ZRE1/2 top strand is dependent on hairpin formation. (A and B) The ZRE1/2 OriLyt sequence was changed to either a ZRE1/AP1 or an AP1/AP1 sequence and was assayed for Zta binding via EMSA. Mutation of the ZRE2 site to an AP1 sequence disrupts hairpin formation, while the AP1/AP1 rescue is predicted to restore hairpin formation. Zta protein was added at 3-fold increasing concentrations ranging from 15 to 135 nM. The percentage of each probe bound to Zta is given beneath the corresponding lane. The asterisk indicates a nonspecific band. These data are representative of three independent experiments.

FIG. 5.

OriLyt is nuclease sensitive during lytic replication. (A) B95-8 cells either were treated with NaB and TPA to induce lytic replication or were left as untreated controls. Forty-eight hours postinduction, viral DNA was isolated, digested with restriction enzymes, purified via BND-cellulose chromatography to enrich for replication intermediates, and treated with S1 nuclease to probe for DNA secondary structure. The amount of DNA isolated during each step of chromatography is given as a percentage of the total DNA input. (B) Cell lysates were analyzed by Western blotting to check for expression of EA-D. (C and D) DNA was either left untreated or treated with 10 U or 100 U of S1 nuclease (C) or with 25 U, 50 U, or 100 U of S1 nuclease (D) and was analyzed via Southern blotting using probes for OriLyt and other regions of the EBV genome, including the R promoter (BRLF1p). Each blot is representative of three independent experiments.

FIG. 6.

Zta mutants that are unable to bind the OriLyt hairpin are also replication incompetent. (A) (Left) Sequences of the basic domains of wild-type and mutant Zta proteins. (Right) Percentages of binding (from the experiment for which results are shown in panel B) and EBV/GAPDH DNA copy numbers (from the experiment for which results are shown in panel C). (B) Wild-type and mutant Zta proteins, expressed in and purified from E. coli, were assayed for their abilities to bind wild-type top-strand OriLyt ZRE1/2 DNA, double-stranded ZRE2 DNA from the viral R promoter (Rp), and methylated ZRE3, also from Rp, in vitro. Filled boxes represent sites of CpG methylation. The asterisk indicates a nonspecific band. These data are representative of three independent experiments. (C) Expression vectors encoding wild-type or mutant Zta proteins were transfected into 293 BAC-EBV Zta knockout (293-ZKO) cells in the presence or absence of an Rta expression vector. Forty-eight hours posttransfection, DNA was isolated and quantified by real-time PCR with primers for the EBV genome or a cellular gene as a control. (D) Transfected-cell lysates were also analyzed for Zta, Rta, and early gene (BALF2) expression via Western blotting. These data are representative of three independent experiments.

FIG. 7.

Model for Zta binding to an OriLyt ZRE1/2 hairpin. (A) The OriLyt ZRE1/2 hairpin (right) contains thymine bases in positions that molecularly mimic methylated ZREs located at other parts of the genome, e.g., methylated Rp-ZRE3 (left). (B) Preferential Zta binding to the top strand of the OriLyt ZRE1/2 DNA hairpin may render the bottom DNA strand accessible to single-stranded DNA nucleases or other replication factors (arrow).