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. 2011 Oct 25;419(2):107-16.
doi: 10.1016/j.virol.2011.08.006. Epub 2011 Sep 1.

Delineation of a core RNA element required for Kaposi's sarcoma-associated herpesvirus ORF57 binding and activity

Emi Sei  1 , Nicholas K Conrad
Affiliations

Delineation of a core RNA element required for Kaposi's sarcoma-associated herpesvirus ORF57 binding and activity

Emi Sei et al. Virology. .

Abstract

The Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 protein is an essential multifunctional regulator of gene expression. ORF57 interaction with RNA is necessary for ORF57-mediated posttranscriptional functions, but little is known about the RNA elements that drive ORF57-RNA specificity. Here, we investigate the cis-acting factors on the KSHV PAN RNA that dictate ORF57 binding and activity. We show that ORF57 binds directly to the 5' end of PAN RNA in KSHV-infected cells. Furthermore, we employ in vitro and cell-based assays to define a 30-nucleotide (nt) core ORF57-responsive element (ORE) that is necessary and sufficient for ORF57 binding and activity. Mutational analysis of the core ORE further suggests that a 9-nt sequence is a specific binding site for ORF57. These studies provide insight into ORF57 specificity determinants and lay a foundation for future analyses of cellular and viral ORF57 targets.

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Figures

Fig. 1
Fig. 1
ORF57 binds the 5′ end of PAN RNA in lytically reactivated cells. (A) Schematic diagram of PAN RNA with approximate positions of qRT-PCR amplicons shown below. Primer set A, B, C, and D amplify PAN RNA base pairs 50–124, 293–372, 642–728, and 994–1064, respectively. The numbering system for PAN RNA in this manuscript is relative to the start site as defined in Zhong et al. (1996). (B) Results from UV cross-linking immunoprecipitation experiments. The y-axis shows the immunoprecipitation efficiencies relative to that for primer set A (see Materials and Methods). The error bars are standard deviation (n=3).
Fig. 2
Fig. 2
ORF57 binds directly to the ORE in vitro. (A) Label transfer and immunoprecipitation assay. Top, Schematic diagram showing the positions of the full-length ORE or control substrate in PAN RNA. Bottom, Results from a representative label transfer assay. Extracts from cells expressing or not expressing flag-tagged ORF57 (Fl-ORF57) were incubated with the indicated substrate as described in the Materials and Methods. The cross-linked, RNase-treated extracts were then immunoprecipitated using anti-flag antibodies; 10% of input is shown. The bottom panels show an anti-flag western blot of the same samples demonstrating expression and immunoprecipitation of Fl-ORF57. (B) Label transfer assays with substrates derived from the ORE region. Top Schematic showing the substrates and their position with respect to the full-length ORE. The bottom panels show the label transfer assay with extract from cells expressing Fl-ORF57 or not as indicated above each lane. The position of ORF57 is indicated by the arrow.
Fig. 3
Fig. 3
The first 79 nt of PAN RNA are sufficient for ORE activity in a heterologous transcript. (A) Top, Schematic diagram of the intronless β-globin reporter. Different portions of the ORE were placed into the 3′ UTR (“PAN Insert”) and tested for ORF57-responsiveness by northern blot. A representative northern blot with β-globin is shown below. The β-globin panels are from the same gel and are shown at the same exposure. The control lanes are probed for a co-transfected loading control. Amounts of co-transfected Fl-ORF57 and the particular insert are given above each lane. (B) Quantification of the northern blot data; error bars are standard deviation (n=3). Each value is relative to the no-insert control with 0.4 μg of ORF57.
Fig. 4
Fig. 4
A 30 nt stem-loop structure is sufficient for ORE activity and binding. (A) Predicted secondary structure of PAN RNA nt 1–79. The three stem loops are labeled SL1, SL2, SL3 and the SL2 “top” (SL2-T) and “bottom” (SL2-B) portions are shown in ovals shaded with blue and red, respectively. (B) Label transfer assay with substrates that delete each of the stem loops (lanes 1–8) in the context of the nt 1–79 fragment. Lanes 11–16 show label transfer with SL2, SL2-T, or SL2-B alone. Cross-linking of each substrate is shown with extract either containing or lacking Fl-ORF57 as indicated. (C) Each of the indicated fragments was inserted into the intronless β-globin construct and examined for ORF57 response by northern blot as described in Figure 3. The dashed lines represent positions where lanes were removed for presentation; the panels displayed are from the same blot at the same exposure. (D) Quantification of the northern blot results was performed as in Figure 3 (n=3).
Fig. 5
Fig. 5
The core ORE is necessary for ORF57 responsiveness in PAN RNA. (A) Schematic representation of the CMV-driven PAN RNA expression constructs. The wild-type (WT) and Δ1 constructs were previously described (Sahin et al., 2010). Constructs that delete nt 23–63, 28–57, and 34–50 were generated to make the CMV-ΔSL2, CMV-ΔSL2-T, and CMV-Δ34-50 constructs, respectively. (B) Representative northern blot data analyzing the effects of the indicated deletions on ORF57 responsiveness. Panels on the left were probed for PAN RNA, while those on the right were the same blot probed for the endogenous 7SK RNA. The PAN RNA panels are from the same gel and are shown at the same exposure. (C) Quantification of the northern blot data. Samples were normalized to the 30 ng ORF57 samples with CMV-WT; error bars represent standard deviation (n=4).
Fig. 6
Fig. 6
Point mutations in the loop portion of SL2-T abrogate ORF57 binding and response. (A) Schematic diagram of triple and double point mutations generated in SL2-T. The mutations introduced are shown in red adjacent to the name of the mutant. (B) Label transfer assays with mutant substrates. The mutant substrates were generated in the context of the SL2-T sequence, which is used as a positive control (lanes 1, 2, 9, 10). Presence of Fl-ORF57 in the extract is indicated above each lane and the position of ORF57 is given by the arrow. (C) RNA immunoprecipitation of SL2-T and UUU→AAA45–47 substrates. Five percent of Input and 100% of pellets are shown at the same exposure from the same gel of an immunoprecipitation experiment with anti-flag agarose beads. The presence or absence of Fl-ORF57 is indicated above each lane as is the substrate. (D) Representative northern blot data analyzing the effects of the indicated mutations on ORF57 responsiveness probed for PAN RNA or a co-transfected loading control. The PAN RNA panels are from the same gel and are from the same exposure. (E) Quantification of northern blots. Values are relative to the CMV-WT at 100 ng transfection. The error bars are standard deviation (n=3). The p-values are derived from a two-tailed unpaired Student’s t-test comparing each sample to the WT.
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