Alternative Capture of Noncoding RNAs or Protein-Coding Genes by Herpesviruses to Alter Host T Cell Function  Yang Eric Guo, Kasandra J. Riley, Akiko Iwasaki, Joan A. Steitz  Molecular Cell  Volume 54, Issue 1, Pages 67-79 (April 2014) DOI: 10.1016/j.molcel.2014.03.025 Copyright © 2014 Elsevier Inc. Terms and Conditions

Molecular Cell 2014 54, 67-79DOI: (10.1016/j.molcel.2014.03.025) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 1 Identification of miR-27 Targets by HITS-CLIP (A) Distribution of Ago-bound mRNA fragments correlates with miR-27 seed (7-mer) binding sites. Ago-bound mRNA fragments and miR-27 (7-mer) binding sites were plotted relative to the centers (set to 0 nt) of 2279 miR-27-Ago clusters (PH ≥ 2). (B) Enrichment of miR-27 binding sites around Ago-mRNA crosslinking sites. MiR-27 7-mer seed binding sites were plotted relative to statistically significant (FDR < 0.001) CIMS including deletion and insertion sites. Insertions are not induced by Ago-mRNA crosslinks (Zhang and Darnell, 2011), serving as a control. (C) Genomic locations of the 67 statistically significant miR-27-Ago clusters (p value ≲ 0.05, BC ≥ 3, and PH ≥ 10). 5′ UTR/CDS indicates clusters mapping to the junctions of 5′ UTRs and CDSs. 3′ UTR/CDS clusters mapped to the junctions of 3′ UTRs and CDSs. (D) Peak height (PH) values for miR-27-Ago clusters were plotted against transcript levels. See also Figure S1. Molecular Cell 2014 54, 67-79DOI: (10.1016/j.molcel.2014.03.025) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 2 MiR-27 Regulates TCR Signaling Components and Effector Molecules of T Cell Activation (A) The TCR signaling pathway is significantly enriched among HITS-CLIP-identified miR-27 targets. Percent of miR-27 mRNA targets in each GO pathway is indicated. FDR, false discovery rate. (B) Genes containing the ten most robust miR-27-Ago clusters in their 3′ UTRs. ∗Genes of unknown biological function. Genes studied further are highlighted in red. (C) Schematic depicting miR-27 as a repressor of three key modulators or effectors of T cell activation, SEMA7A, GRB2, and IFN-γ. Diagram drawn based on (Suzuki et al., 2008). ? denotes a possible interaction between SEMA7A and integrin (Liu et al., 2010). Images of IFN-γ and TCR/MHC/CD8 were adapted from David S. Goodsell (RCSB PDB). See also Figures S2 and S3. Molecular Cell 2014 54, 67-79DOI: (10.1016/j.molcel.2014.03.025) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 3 MiR-27 Attenuates Activation of JNKs and p38, as well as Induction of CD69 (A) Jurkat T cells transfected with miR-27 or a scrambled control were stimulated by anti-CD3 antibody for the indicated times. The JNK activation profile was determined by western blot analysis (WB) for phosphorylated JNKs (P-JNK) and total JNKs (JNK). Relative quantity = (phosphorylated kinase signal intensity)/(total kinase signal intensity). Nonstimulated sample (i.e., 0 min time point) was set to 1. In repeat experiments, the p54 isoform of JNKs was only slightly activated, and no significant difference in activation was observed between miR-27 and the scrambled control (data not shown). (B) The p38 activation profile was determined as described in (A). (C) FACS data and histogram show CD69 expression in Jurkat cells transfected with miR-27 or the scrambled control with (Act) and without (Non) activation by anti-CD3 and anti-CD28. MFI, median fluorescence intensity. Values are means ± SD in three experiments; p values were determined by Student’s t test. ∗p < 0.05; ∗∗p < 0.01. See also Figure S4. Molecular Cell 2014 54, 67-79DOI: (10.1016/j.molcel.2014.03.025) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 4 HSUR 1 Regulates SEMA7A through miR-27 Degradation (A) Ago-bound mRNA fragments from four HITS-CLIP replicates (different colors), mRNA-Seq reads, and predicted miRNA target sites are mapped on the marmoset SEMA7A 3′ UTR. Base-pairing interactions between miR-27 or EBV BART-13 and the WT (red) or mutant (Mut, blue) target sites in the reporters used in (B) are shown. A gap in the marmoset reference genome (gray bar) was sequenced. (B) Luciferase reporter assays performed with full-length WT or Mut SEMA7A 3′ UTR in HEK293T cells transfected with synthetic WT, scrambled miR-27, or EBV BART-13. RLU, relative luciferase units. (C) WB of SEMA7A in Jurkat cells transfected with WT or scrambled miR-27. (D) WB of SEMA7A in Δ2A cells transfected with a miR-27 LNA inhibitor or control. (E) WT cells transfected with an ASO against HSUR 1 (α-H1), HSUR 2 (α-H2), or GFP (α-GFP) were subjected to WB for SEMA7A and northern blot analysis (NB) for miRNAs and HSURs. Values are means ± SD in three experiments; p values were determined by Student’s t test. See also Figure S5. Molecular Cell 2014 54, 67-79DOI: (10.1016/j.molcel.2014.03.025) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 5 GRB2 and IFN-γ Are Regulated by HSUR 1 via miR-27 (A) Ago-bound mRNA fragments, mRNA-Seq reads, and predicted miRNA binding sites are mapped on the marmoset GRB2 3′ UTR as in Figure 4. Base-pairing interactions between miR-27 or EBV BART-13 and WT or a Mut 8-mer target site are shown. (B) Luciferase reporter assays were performed with the full-length WT or Mut GRB2 3′ UTR as described in Figure 4. (C) WB of GRB2 after transfection of WT or scrambled miR-27 into Jurkat cells. (D) WB of GRB2 in Δ2A cells transfected with a miR-27 LNA inhibitor or control. (E) WB of GRB2 in WT cells after transfection with α-H1, α-H2, or α-GFP ASO. (F) Ago-bound mRNA fragments, mRNA-Seq reads, and predicted miRNA binding sites are mapped on the marmoset IFNG 3′ UTR as in Figure 4. Base-pairing interactions between miR-27 or EBV BART-13 and the WT or a Mut 7-mer target site are shown. (G) Luciferase reporter assays were performed with the full-length WT or Mut IFNG 3′ UTR as described in Figure 4. (H) Enzyme-linked immunosorbent assay (ELISA) measured extracellular IFN-γ concentration after knockdown of HSUR 1 with α-H1 compared to α-H2 ASO. The cell number was determined before harvesting. The 6-mer miR-27 sites (red ●) in both the GRB2 and IFNG 3′ UTRs were not active enough to be detected in luciferase reporter assays (data not shown). Values are means ± SD in at least three experiments; p values were determined by Student’s t test. See also Figure S5. Molecular Cell 2014 54, 67-79DOI: (10.1016/j.molcel.2014.03.025) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 6 Wild-Type, but Not a miR-27 Binding Site-Mutated HSUR 1, Rescues Levels of miR-27 Target Proteins in Δ2A Cells (A) Sequence of the WT and Mut HSUR 1, with nucleotides conserved between HVS strains in bold. Mutations introduced into the miR-27 binding site are highlighted in red; the miR-27 seed is in yellow. (B) NB shows that Jurkat T cells infected with lentiviruses carrying WT HSUR 1 have lower levels of miR-27 compared to cells infected with lentiviruses carrying Mut HSUR 1. Quantifications of miR-27 levels relative to miR-16 are given below. (C) NB shows levels of WT and Mut HSUR 1 expressed by lentiviruses in Δ2A cells. (D) Levels of miR-27 and a control, miR-181a, in Δ2A cells infected with lentiviruses carrying WT or Mut HSUR 1, determined by Q-PCR. Endogenous U6 snRNA was the normalization control for the quantifications. (E) WB of SEMA7A in Δ2A cells infected with lentiviruses carrying WT or Mut HSUR 1. zsGREEN is a marker protein expressed by the pAGM lentiviral transfer vector. GAPDH provided a loading control for the quantifications below. (F) GRB2 levels in Δ2A cells rescued with WT or Mut HSUR 1 as described above. Values are means ± SD in two experiments. Molecular Cell 2014 54, 67-79DOI: (10.1016/j.molcel.2014.03.025) Copyright © 2014 Elsevier Inc. Terms and Conditions

Figure 7 Gene Maps and Phylogenetic Tree of T-Lymphotropic γ-Herpesviruses (A) Syntenic regions flanked by H-DNA and ORF3 (formylglycineamide ribotide amidotransferase, FGARAT) are shown (Ensser and Fleckenstein, 2005; Russell et al., 2009). STP-A11, Saimiri transformation protein of HVS strain A11; DHFR, dihydrofolate reductase; Tio, two-in-one protein of HVA; HSUR and HAUR, HVS- and HVA-encoded URNA, respectively; A1, A2, A3, and A4, AlHV-1 ORFs; Ov2, Ov2.5, Ov3, and Ov3.5, OvHV-2 ORFs; v-ATF3, v-IL10, and v-SEMA7A, viral homologs of cellular proteins. (B) Phylogenetic tree of commonly studied γ-herpesviruses with T-lymphotropic viruses in red. EBV, Epstein-Barr virus; KSHV, Kaposi’s sarcoma-associated herpesvirus; MHV-68, Murid herpesvirus 68. See also Figures S6 and S7. Molecular Cell 2014 54, 67-79DOI: (10.1016/j.molcel.2014.03.025) Copyright © 2014 Elsevier Inc. Terms and Conditions