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. 2010 Jan 12;107(2):850-5.
doi: 10.1073/pnas.0911948107. Epub 2009 Dec 22.

AP-1 homolog BZLF1 of Epstein-Barr virus has two essential functions dependent on the epigenetic state of the viral genome

Markus Kalla  1 , Anne Schmeinck, Martin Bergbauer, Dagmar Pich, Wolfgang Hammerschmidt
Affiliations

AP-1 homolog BZLF1 of Epstein-Barr virus has two essential functions dependent on the epigenetic state of the viral genome

Markus Kalla et al. Proc Natl Acad Sci U S A. .

Abstract

EBV, a member of the herpes virus family, is a paradigm for human tumor viruses and a model of viral latency amenable for study in vitro. It induces resting human B lymphocytes to proliferate indefinitely in vitro and initially establishes a strictly latent infection in these cells. BZLF1, related to the cellular activating protein 1 (AP-1) family of transcription factors, is the viral master gene essential and sufficient to mediate the switch to induce the EBV lytic phase in latently infected B cells. Enigmatically, after infection BZLF1 is expressed very early in the majority of primary B cells, but its early expression fails to induce the EBV lytic phase. We show that the early expression of BZLF1 has a critical role in driving the proliferation of quiescent naïve and memory B cells but not of activated germinal center B cells. BZLF1's initial failure to induce the EBV lytic phase relies on the viral DNA at first being unmethylated. We have found that the eventual and inevitable methylation of viral DNA is a prerequisite for productive infection in stably, latently infected B cells which then yield progeny virus lacking cytosine-phosphatidyl-guanosine (CpG) methylation. This progeny virus then can repeat EBV's epigenetically regulated, biphasic life cycle. Our data indicate that the viral BZLF1 protein is crucial both to establish latency and to escape from it. Our data also indicate that EBV has evolved to appropriate its host's mode of methylating DNA for its own epigenetic regulation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
The majority of infected B cells express EBV immediate-early genes. (A) Western blot immunodetection of Zta in primary B cells infected with the B95.8 strain of EBV at different time points after infection. HEK293 cells transiently transfected with a BZLF1 expression plasmid serve as positive control. β-actin signals served as loading control. (B) By semiquantitative RT-PCR analysis, B95.8 EBV-infected B cells detectably express the immediate-early genes BZLF1 (a fully spliced and an unspliced transcript) and BRLF1 and the early gene BMRF1 but do not express the late genes BALF4 and BLLF1 essential for virus synthesis during the first 11 days of infection. (C) Construction of the reporter EBV with the rat CD2 gene expressed from the viral BMRF1 promoter. Shown are parts of the wild-type (2089) and reporter (3875) EBV genomes and their functionally relevant genes (. (D) The majority of GFP-positive B cells infected with the BMRF1 reporter EBV strain express rat CD2 very early after infection.
Fig. 2.
Fig. 2.
EBV-infected primary B cells do not release virus progeny early after infection. (A) Experimental flowchart. (Upper) Primary B cells were infected with wild-type EBV (2089) on day 0 with an MOI of 0.1 and were cultivated for 24 h. One and 2 days p.i., the infected cells were incubated with a neutralizing α-gp350 antibody (ab) (Fig. S2), washed, and cultivated further. Samples were taken beginning on day 3 p.i. (Lower) The infected B cells were placed on a porous sieve above indicator B blasts for 24 h. B-blast proliferation is dependent on either CD40L/IL-4 stimulation or infection with EBV. The B blasts were cultured in 96-well cluster dishes in the absence of CD40L/IL-4. After 5 weeks the number of wells with proliferating B blasts was counted. (B) Detection of infectious progeny virus indicates a failure of early de novo virus synthesis in EBV-infected primary B cells. Progeny virus first became detectable at day 13 p.i. and were detectable for up to 3 months but varied cyclically in the infected B-cell samples from three different donors.
Fig. 3.
Fig. 3.
Virion synthesis is a function of the epigenetic state of EBV DNA. (A) RT-PCR analysis of RNA prepared from HEK293 cells that had been transiently transfected for 3 days with EBV DNA (p2089) prepared from E. coli (unmeth.) or treated in vitro with the methyltransferase M.SssI (meth.). As indicated, p2089 DNA was cotransfected with a BZLF1 expression plasmid. Transcripts of essential viral lytic genes (BRLF1, BMRF1, and BLLF1) are detectable only when p2089 DNA is CpG-methylated before transfection. The genes are indicated on the right; the gene classes are given on the left. E, early; IE, immediate-early L, late; TR–, HEK293 cells stably transfected with EBV, which served as a positive control for the induction of the lytic phase. (B) Immunostaining of transiently transfected HEK293 cells with antibodies detecting Zta, Rta, EA-D, VcAgp125, and gp350/220 encoded by the viral genes BZLF1, BRLF1, BMRF1, BALF4, and BLLF1, respectively. Controls are found in Fig. S5.
Fig. 4.
Fig. 4.
Functional role of BZLF1 expression during the early phase of infection. (A) Expression of BZLF1 provides a proliferative advantage to resting naïve and memory B cells but not to GC B cells. Subpopulations of primary B cells from three different donors were sorted and infected with wild-type EBV or BZLF1-KO EBV and were plated on plastic (no feeder). Proliferation of infected cells was measured by FACS as described (42), and the ratios of the number of viable, GFP-positive cells infected with the BZLF-KO EBV (mut) to the number of cells infected with wild-type EBV were calculated as indicated. Asterisks indicate the statistical significance of the means of three independent experiments in a two-way ANOVA analysis with Bonferroni posttests: ***P < 0.001; ns, not significant. (B) Activation of resting B cells by CD40L feeder cells, and IL-4 for 24 h on day 1 p.i. overcomes the need for BZLF1 expression in memory and naïve B-cells. Proliferating B cells were analyzed as in A. (C) Comparison of the results shown in A and B. The confidence values of untreated versus CD40L/IL-4 activated B cells subpopulations are shown. ***P < 0.001; *P < 0.05; ns, not significant.
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