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. 1998 Nov;72(11):8559-67.
doi: 10.1128/JVI.72.11.8559-8567.1998.

The Epstein-Barr virus lytic transactivator Zta interacts with the helicase-primase replication proteins

Z Gao  1 , A Krithivas, J E Finan, O J Semmes, S Zhou, Y Wang, S D Hayward
Affiliations

The Epstein-Barr virus lytic transactivator Zta interacts with the helicase-primase replication proteins

Z Gao et al. J Virol. 1998 Nov.

Abstract

The Epstein-Barr virus transactivator Zta triggers lytic gene expression and is essential for replication of the lytic origin, oriLyt. Previous analysis indicated that the Zta activation domain contributed a replication-specific function. We now show that the Zta activation domain interacts with components of the EBV helicase-primase complex. The three helicase-primase proteins BBLF4 (helicase), BSLF1 (primase), and BBLF2/3 (primase-associated factor) were expressed fused to the Myc epitope. When expression plasmids for BBLF4 or BBLF2/3 plus BSLF1 (primase subcomplex) were separately transfected, the proteins localized to the cytoplasm. Interaction between Zta and the components of the helicase-primase complex was tested by examining the ability of Zta to alter the intracellular localization of these proteins. Cotransfection of Zta with Myc-BBLF4 resulted in nuclear translocation of Myc-BBLF4; similarly, cotransfection of Zta with the primase subcomplex led to nuclear translocation of the Myc-BSLF1 and Myc-BBLF2/3 proteins. This relocalization provides evidence for an interaction between Zta and the helicase and Zta and the primase subcomplex. An affinity assay using glutathione S-transferase-Zta fusion proteins demonstrated that Myc-BBLF4 and Myc-BBLF2/3 plus BSLF1 bound to the Zta activation domain (amino acids 1 to 133). In the nuclear relocalization assay, the amino-terminal 25 amino acids of Zta were required for efficient interaction with the primase subcomplex but not for interaction with BBLF4. Evidence for interaction between oriLyt bound Zta and the helicase-primase complex was obtained in a superactivation assay using an oriLyt-chloramphenicol acetyltransferase (CAT) reporter. Zta activated expression from a CAT reporter containing the complete oriLyt region and regulated by the oriLyt BHLF1 promoter. Cotransfection of the helicase-primase proteins, one of which was fused to a heterologous activation domain, led to Zta-dependent superactivation of CAT expression. This assay also provided evidence for an interaction between the single-stranded DNA binding protein, BALF2, and the Zta-tethered helicase-primase complex. The helicase-primase interaction is consistent with a role for Zta in stabilizing the formation of an origin-bound replication complex.

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Figures

FIG. 1
FIG. 1
Immunofluorescence assay showing the intracellular localization of the proteins of the helicase-primase complex. Vero cells were transfected with Myc-tagged BSLF1, BBLF4, and BBLF2/3 and a control, BALF2. Transfected proteins were visualized with anti-Myc antibody and FITC-conjugated anti-mouse IgG secondary antibody.
FIG. 2
FIG. 2
Expression of the Myc-tagged helicase-primase proteins. Expression vectors for Myc-BBLF2/3, Myc-BBLF4, and Myc-BSLF1 and for a control replication protein, Myc-BALF5, were individually transfected into Cos cells, and protein expression was analyzed by Western blotting using anti-Myc antibody and visualization by chemiluminescence. Positions of the helicase-primase protein bands are indicated with arrowheads; positions of the 97- and 68-kDa molecular size markers are indicated on the right.
FIG. 3
FIG. 3
Immunofluorescence assay showing the ability of BBLF4, BSLF1, and BBLF2/3 to form a tripartite complex that localizes to the nucleus in cotransfected Vero cells. Cotransfection of any two of the helicase-primase proteins did not confer nuclear localization. Transfected proteins were visualized with anti-Myc antibody and FITC-conjugated anti-mouse IgG secondary antibody.
FIG. 4
FIG. 4
Immunofluorescence assay demonstrating relocalization of Myc-BBLF4, Myc-BBLF2/3 plus BSLF1, and Myc-BSLF1 plus BBLF2/3 in the presence of cotransfected Zta. Myc-tagged proteins were visualized with anti-Myc antibody and FITC-conjugated anti-mouse IgG secondary antibody. Zta was detected with mouse monoclonal antibody. The ratio of cotransfected Zta to Myc-tagged expression vector was 2:1. Both nuclear and cytoplasmic Myc-BBLF4 were detected at this ratio.
FIG. 5
FIG. 5
Immunofluorescence assay showing that cotransfected cells in which Myc-BBLF4 or Myc-BBLF2/3 plus BSLF1 localize to the nucleus express Zta. Vero cells were cotransfected with Zta plus Myc-BBLF4 or with Zta plus Myc-BBLF2/3 and BSLF1 and stained with anti-Zta (a, c, and e) and anti-Myc (b, d, and f) primary antibodies. Secondary antibodies: (a and f) FITC-conjugated donkey anti-rabbit; (b and e) rhodamine-conjugated goat anti-mouse; (c) rhodamine-conjugated donkey anti-rabbit; (d) FITC-conjugated goat anti-mouse. Not all cells express both cotransfected proteins, but cells expressing nuclear Myc-tagged proteins always also express Zta.
FIG. 6
FIG. 6
BBLF4 and BBLF2/3 plus BSLF1 interact with the Zta activation domain, as determined by GST affinity assay in which extracts of 293T cells transfected with Myc-BBLF4 (lanes 2, 4, and 6) or Myc-BBLF2/3 plus BSLF1 (lanes 1, 3, and 5) were incubated with GST-Zta(1-133) and control GST proteins. Bound protein was separated by SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with anti-Myc antibody. Reactive proteins were visualized by chemiluminescence. The input lanes were loaded with 1/15 of the amount of extract incubated with the GST beads. The position of the 97-kDa molecular size marker is indicated on the right.
FIG. 7
FIG. 7
Immunofluorescence assay showing the intracellular localization in transfected Vero cells of the three helicase-primase proteins and BALF2 expressed as fusions with E2TANLS. Transfected proteins were visualized with anti-EBNA2 antibody and FITC-conjugated anti-mouse IgG secondary antibody. Addition of the tag sequences resulted in nuclear localization of BBLF4 and BBLF2/3 but was insufficient to relocate individually transfected BSLF1. The localization of the nuclear BALF2 protein was not affected by the addition of the tag.
FIG. 8
FIG. 8
Transient expression assay using Zta-dependent superactivation of expression from an oriLyt-CAT reporter as a measure of Zta-helicase-primase interaction. The results shown are representative of results obtained in three different experiments. Vero cells were cotransfected with oriLyt-CAT plus or minus Zta, and the indicated individual replication genes were expressed as E2TANLS fusions (indicated by an asterisk) or groups of replication proteins, one of which was expressed as an E2TANLS fusion. Cotransfection of the three helicase-primase proteins with one member tagged with the E2TANLS consistently resulted in Zta-dependent superactivation of CAT expression.
FIG. 9
FIG. 9
Representation of the initial stages of core replication protein assembly as deduced from this study. OriLyt bound dimers of Zta interact through the activation domain with both the helicase (BBLF4) and primase subcomplex (BSLF1 plus BBLF2/3) to provide a stabilizing tether for the core replication complex. The single-stranded DNA binding protein BALF2 may in turn contact the helicase-primase complex.
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