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. 2009 Sep 1;391(2):221-31.
doi: 10.1016/j.virol.2009.06.014. Epub 2009 Jul 9.

The zinc finger DNA-binding domain of K-RBP plays an important role in regulating Kaposi's sarcoma-associated herpesvirus RTA-mediated gene expression

Zhilong Yang  1 , Hui-Ju Wen, Veenu Minhas, Charles Wood
Affiliations

The zinc finger DNA-binding domain of K-RBP plays an important role in regulating Kaposi's sarcoma-associated herpesvirus RTA-mediated gene expression

Zhilong Yang et al. Virology. .

Abstract

K-RBP is a KRAB-containing zinc finger protein with multiple zinc finger motifs and represses Kaposi's sarcoma-associated herpesvirus (KSHV) transactivator RTA-mediated transactivation of several viral lytic gene promoters, including the ORF57 promoter. Whether K-RBP binds DNA through its zinc fingers and the role of zinc finger domain in repressing gene expression are unclear. Here we report that K-RBP binds DNA through its zinc finger domain and the target DNA sequences contain high GC content. Furthermore, K-RBP binds to KSHV ORF57 promoter, which contains a GC-rich motif. K-RBP suppresses the basal ORF57 promoter activity as well as RTA-mediated activation. The zinc finger domain of K-RBP is sufficient for the suppression of ORF57 promoter activation mediated by the viral transactivator RTA. Finally, we show that K-RBP inhibits RTA binding to ORF57 promoter. These findings suggest that the DNA-binding activity of K-RBP plays an important role in repressing viral promoter activity.

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Figures

FIG. 1
FIG. 1
(A) Schematic representation of K-RBP protein showing the KRAB domain and 12 zinc finger motifs. Amino acid alignment of the zinc finger motifs in K-RBP showing the amino acids involved in Zn2+ binding (in bold). The −1, +3 and +6 indicate the amino acids in the α-helical region of a zinc finger motif that determine its DNA-binding specificity. (B) Coomassie blue staining of cell lysates from E. coli expressing K-RBP protein. Lane 1: protein marker; Lane 2: Lysate from E. coli cells containing K-RBP expression plasmid without IPTG induction; Lane 3: soluble fraction of Lysate from E. coli cells containing K-RBP expression plasmid with IPTG induction; Lane 4: insoluble fraction of the lysate from E. coli cells harboring K-RBP expression plasmid with IPTG induction. Arrow indicates K-RBP protein. (C) Coomassie blue staining and Western blot analysis of His-tagged K-RBP protein after purification, urea-solubilization and refolding. Lane 1: Coomassie blue staining of K-RBP protein after purification; Lane 2: Coomassie blue staining of K-RBP protein after refolding; Lane 3: Western blot analysis of protein using anti-His antibody after refolding. Arrow indicates K-RBP protein.
FIG. 2
FIG. 2
DNA-binding activity of K-RBP protein. (A) The bacterially expressed K-RBP was refolded and incubated with cellulose without DNA-conjugation or dsDNA-cellulose. The bound (B) and non-bound (NB) fractions were separated by centrifugation. The bound protein was washed and eluted by increasing concentrations of NaCl in the binding buffer. The K-RBP protein in different fractions was separated by SDS-PAGE and analyzed by Western blot using anti-K-RBP antibody. (B) K-RBP was incubated with dsDNA cellulose in the presence of ZnSO4 and EDTA. DNA cellulose bound protein was eluted with 1.25% SDS. (C) Zinc finger domain, but not KRAB domain of K-RBP binds DNA. The E. coli expressed His-tagged KRAB domain (1–98), zinc finger domain (141–554), full-length K-RBP and E. coli lysate were resolved on SDS-PAGE, transferred to PVDF membrane and analyzed by Western blot using anti-His antibody (left panel). The second membrane with the same samples was incubated in K-RBP refolding buffer overnight at 4°C and hybridized to the random dsDNA probe labeled with [α-32P] dATP (right panel).
FIG. 3
FIG. 3
Selection of K-RBP binding sequences. (A) Schematic representation of the procedure used to identify K-RBP binding sequences. (B) Consensus (Con) sequences for K-RBP binding. Numbers indicate the frequencies of the specific nucleotide at each position (Pos). The most abundant nucleotides at each position were used to generate the consensus sequence shown in the bottom. The underlined CGG motif was pre-synthesized in the pool of the second round selection oligomers. (C) K-RBP binds clone #14 probe in EMSA. The probe was labeled with [α-32P]dATP. The clone #14 and Oct-1 probe were used as unlabeled competitors. Unlabeled competitors were added at an 80–100-fold molar excess over the labeled probe. Various amounts of Ni-NTA agarose beads (5 and 15 μl) were used to remove the His-tagged K-RBP protein in EMSA. GST beads (15 μl) were used as a control. Mouse anti-His or Flag antibodies were added in the reaction prior to adding the probe as shown in lanes 11 and 12. The arrow indicates the bound complex. One μl K-RBP protein contains 5 ng purified K-RBP protein approximately.
FIG. 4
FIG. 4
K-RBP binds ORF57 promoter. (A) Schematic representation of RRE in KSHV ORF57 promoter on which the locations of various putative promoter-regulatory elements are indicated. The whole 57RRE region was boxed. The GC-rich region is in gray letters. The RBP-Jkappa binding element is underlined. The bold lines below the sequence indicate the ORF57 promoter probes that were used for EMSA. (B) EMSA of the clone #14, 57R, 57RRE2 and 57P probes with K-RBP protein. Purified His-tagged K-RBP was incubated with 32P labeled probes. Arrow indicates the weak binding between K-RBP and 57RRE2. (C) K-RBP binds 57R probe in EMSA. Experiment was performed as described in Fig. 3C except that different unlabeled competitors were used as indicated. Arrow indicates the binding complex between K-RBP and 57R. One μl K-RBP protein contains about 5 ng purified K-RBP protein.
FIG. 5
FIG. 5
K-RBP suppresses KSHV ORF57 promoter is dependent on the GC-rich element. (A) Schematic representation of the luciferase promoter reporters used. (B-F) 293T cells were transfected with 100ng of pGL3-basic as shown in (B), p57Pluc1 in (C), p57-3RRE in (D), p57-3RRE4 in (E), p57-3RRE2 in (F) using the indicated amounts of K-RBP plasmid pcDNAK-RBP. The luciferase activities were detected at 24 h post-transfection. For all transfections, total DNA amounts used in each transfection were normalized by the addition of control plasmid. The percentages of inhibition by K-RBP were indicated. The error bars indicate standard deviations of at least 3 independent experiments. Transfection efficiency for each experiment was normalized using a β-Gal expression plasmid as internal control. The basal levels of the promoter activity were normalized to 100%.
FIG. 6
FIG. 6
Effect of RTA-mediated transactivation of ORF57 promoter (p57Pluc1) by K-RBP and its mutants in BJAB cells. (A–C) BJAB cells were transfected with 1 μg of p57Pluc1, expression plasmids of RTA and GAL4BD-tagged K-RBP in (A), K-RBP99-554 (zinc fingers) in (B), K-RBP1-98 (KRAB) in (C). The luciferase activities were detected at 48 h post-transfection. In all transfections, the total DNA amounts used in each transfection were normalized by the addition of control expression plasmid. The basic promoter activity was normalized as 1 fold. The error bars indicate standard deviations of at least 3 independent experiments. Transfection efficiency for each experiment was normalized using a β-Gal expression plasmid as internal control. (D) The expression of K-RBP and its mutants. The indicated proteins expressed from plasmids were immunoblotted with rabbit anti-GAL4BD antibody. The higher molecular band from the control plasmid in the first lane is probably a GAL4BD fusion protein translated from a read-through transcript from the vector. Tubulin was used as the loading control. (E) Schematic diagrams of K-RBP deletion mutants used and their effects on RTA-mediated ORF57 transactivation. “+” and “−” indicate the ability of the various deletion clones to repress RTA-mediated transactivation.
FIG. 7
FIG. 7
Inhibition of RTA binding to ORF57 promoter by K-RBP. (A) The zinc finger domain of K-RBP binds ORF57 promoter. The E. coli expressed His-tagged zinc finger domain, full-length K-RBP and E. coli lysate were resolved on SDS-PAGE, transferred to PVDF membrane and analyzed by Coomassie blue staining (left panel). The second membrane with the same samples was refolded and hybridized to the 57P probe labeled with [α-32P] dATP (right panel). (B) Baculovirus expressed RTA, E. coli expressed K-RBP and [α-32P] dATP labeled 57P probe were used in EMSA. The EMSA was performed as described in Fig. 3C except that different unlabeled competitors and proteins were used as indicated in the figure. Open arrows indicate specific binding of RTA to probes. Filled arrows indicate the binding of K-RBP to probes. Numbers below the bands indicate the relative intensities of the bands measured by Image J.
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