1. Diverse Herpesvirus MicroRNAs Target the Stress-Induced Immune Ligand MICB to Escape Recognition by Natural Killer Cells 
Daphna Nachmani, Noam Stern-Ginossar, Ronit Sarid, Ofer Mandelboim 
Cell Host & Microbe 
Volume 5, Issue 4, Pages (April 2009)
DOI: /j.chom
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1 KSHV and EBV MiRNA-Mediated Translational Repression of MICB
(A) FACS analysis of ULBP3 and of MICB expression by RKO cells transduced with lentivirus encoding either the KSHV miRNA cluster or the EBV pri-miR-BART2. The gray empty histogram represents the staining of ULBP3 or of MICB in the viral miRNA-transduced cells. The black empty histogram represents the staining of cells transduced with a control miRNA (miR-K12-12). The filled gray histogram represents the staining of the secondary mAb only.
(B) Western blot analysis of MICB protein levels expressed by the cells stained in (A). Quantification of relative intensity was performed by ImageJ software.
(C) Quantitative real-time PCR analysis of the MICB mRNA levels in the transduced RKO cells. Relative mRNA abundance is shown as a percentage of the level of HPRT mRNA (encoding hypoxanthine guanine phosphoribosyltransferase), and error bars (SD) are derived from triplicates. MiR-K12-12 served as negative control.
Cell Host & Microbe 2009 5, DOI: ( /j.chom )
Copyright © 2009 Elsevier Inc. Terms and Conditions

3. Figure 2
MiR-K12-7 and MiR-BART2-5p Are the Viral MiRNAs that Mediate MICB Downregulation
(A) FACS analysis of MICA expression by 293T cells transduced with lentivirus encoding the above indicated KSHV miRNA. The gray empty histogram represents the staining of MICA expression by the transduced cell. The black empty histogram represents the staining of miR-K12-1, which was used as a control. The filled gray histogram represents the staining of the secondary mAb only.
(B) FACS analysis of MICB expression by RKO cells transduced with lentivirus encoding the indicated KSHV miRNA. The gray empty histogram represents the staining of MICB expression by the transduced cell. The black empty histogram represents the staining of miR-K12-1 or miR-K12-2 miRNAs that were used as controls. The filled gray histogram represents the staining of the secondary mAb only. The red histogram shows the MICB downregulation mediated by miR-K12-7.
(C) FACS analysis of MICB and MICA expression in HeLa cells transduced with miR-BART2-5p. The gray empty histogram represents the staining of miR-BART2-5p, the red histogram shows the MICB downregulation, the black empty represents the control miRNA, and the filled gray represents the secondary mAb only.
(D) FACS analysis of RKO cells transfected with RNA duplexes of the mature viral miRNAs. The gray empty histogram represents the staining of cells transfected with the RNA duplex. The black empty histogram represents the staining of mock transfection. The filled gray histogram represents the staining of the secondary mAb only.
Cell Host & Microbe 2009 5, DOI: ( /j.chom )
Copyright © 2009 Elsevier Inc. Terms and Conditions

4. Figure 3 MiR-K12-7 and MiR-BART2-5p Directly Bind to the 3′UTR of MICB
(A) Alignments of the predicted binding site of miR-K12-7 to the MICB 3′UTR (upper) and of the mutated site in the MICB 3′UTR (named mut-K, lower). The seed region is underlined, and the location of the site is noted in brackets.
(B) The translation repression of luciferase reporter gene activity mediated by miR-K12-7. The figure demonstrates relative luciferase activity after transfection of the indicated reporter plasmids into RKO cells expressing either miR-K12-7 (white) or GFP as control (gray). Firefly luciferase activity was normalized to Renilla luciferase activity and then normalized to the average activity of the control reporter. Shown are mean values ± SD. Statistically significant differences are indicated (∗p < 0.015, ∗∗p < , by one-tailed t test). Error bars (SD) are derived from triplicates.
(C) Alignments of two potential binding sites of miR-BART2-5p to the 3′UTR of MICB (named E1 and E2). The seed region is underlined, and their location is noted in brackets.
(D) Alignments of miR-BART2-5p to the two mutated sites in the 3′UTR of MICB (named mut-E1 and mut-E2).
(E) The translation repression of luciferase reporter gene activity mediated by miR-BART2-5p. The figure demonstrates relative luciferase activity after transfection of the indicated reporter plasmids into RKO cells expressing either miR-BART2-5p (white) or GFP as control (gray). Firefly luciferase activity was normalized to Renilla luciferase activity and then normalized to the average activity of the control reporter. Shown are mean values ± SD. Statistically significant differences are indicated (∗p < 0.003, ∗∗p < , by one-tailed t test). Error bars (SD) are derived from triplicates.
Cell Host & Microbe 2009 5, DOI: ( /j.chom )
Copyright © 2009 Elsevier Inc. Terms and Conditions

5. Figure 4
The MICB Downregulation by MiR-K12-7 and MiR-BART2-5p Results in Reduced NKG2D-Mediated Killing
(A and B) Cells expressing miR-K12-7 or miR-BART2-5p exhibit reduced NK killing. Bulk NK cells were preincubated either with anti-NKG2D mAb (white) or with isotype control mAb (gray). Labeled RKO cells expressing miR-K12-7 (A), miR-BART2-5p (B), or control miRNA (miR-K12-12) (A and B) were then added and incubated for 5 hr. Shown are mean values ± SD. Statistically significant differences are indicated (∗p ≤ 0.03, by one-tailed t test). Error bars (SD) are derived from triplicates.
Cell Host & Microbe 2009 5, DOI: ( /j.chom )
Copyright © 2009 Elsevier Inc. Terms and Conditions

6. Figure 5
MiR-K12-7 and MiR-BART2-5p Are Expressed and Are Functional during Authentic Viral Infection
(A) Quantitative real-time PCR analysis of viral miRNA expression using custom-designed Taqman RT primer and real-time PCR probe. The KSHV-infected BCBL1 cell line was assayed for the expression of miR-K12-7. The EBV-transformed cell line was assayed for the expression of miR-BART2-5p. The cell lines served as negative controls for each other. RKO cells transduced with the relevant lentivirus were used as positive controls. Error bars (SD) are derived from triplicates.
(B) FACS analysis of the expression of MICA and MICB by KSHV-infected and EBV-infected cell lines. Left side shows staining of BCBL1 cells transduced with an anti-miR-K12-7-sponge (gray empty histogram) or with anti-miR-BART2-5p sponge as control (black empty histogram). The filled gray histogram is of secondary mAb staining only. Right side shows staining of cells transduced with an anti-miR-BART2-5p sponge (gray empty histogram) or with anti-miR-K12-7 sponge as control (black empty histogram). The filled dark gray histogram is the staining of MICB expression by parental The filled gray histogram is of secondary mAb staining only.
(C) The specific effect of the antiviral miRNA sponges was tested in a dual luciferase reporter assay. 293T cells were transduced with miR-K12-7 (left) or with miR-BART2-5p (right). In addition, each 293T cell was transduced with either a relevant sponge (white) or an irrelevant sponge (gray). Firefly luciferase activity was normalized to Renilla luciferase activity and then normalized to the average activity of the control reporter. Shown are mean values ± SD. Statistically significant differences are indicated (∗p < 0.006, by one-tailed t test). Error bars (SD) are derived from triplicates.
Cell Host & Microbe 2009 5, DOI: ( /j.chom )
Copyright © 2009 Elsevier Inc. Terms and Conditions

7. Figure 6
Functional Conservation of the EBV and KSHV Viral MiRNAs during Authentic Infection
(A and B) The NK cell killing of the sponge-transduced BCBL1 (A) and (B) cell lines. Open circle indicates cells transduced with a control vector; filled triangles indicate cells transduced with the relevant antiviral miRNA sponge. Both BCBL1 and cells transduced with the antiviral miRNA sponge exhibit an increase in NK killing. Shown are mean values ± SD. Statistically significant differences are indicated (∗p ≤ 0.03, ∗∗p ≤ 0.003, by one-tailed t test). Error bars (SD) are derived from triplicates.
Cell Host & Microbe 2009 5, DOI: ( /j.chom )
Copyright © 2009 Elsevier Inc. Terms and Conditions

8. Figure 7
MICA Escaped Viral MiRNA Targeting by Mutating and Severing Its 3′UTR
(A) The alignments between each of the three viral miRNAs and the 3′UTR of MICB (left) or of MICA (right). Noted in brackets is the location of the binding site with respect to the beginning of the 3′UTR, and in red are the nucleotides, which appear in the MICA binding site but not in the MICB binding site.
(B) When mutated to the miR-BART2-5p-MICB binding site, MICA 3′UTR is repressed. The figure demonstrates the relative activity of a Firefly luciferase when fused to a mutated MICA 3′UTR. Firefly activity was normalized as stated. Shown are mean values ± SD. Statistically significant differences are indicated (∗p ≤ 0.002, by one-tailed t test). Error bars (SD) are derived from triplicates.
(C) A schematic representation of the 3′UTR of MICA (upper) and MICB (lower) and of the target sites of the three herpesvirus miRNAs: HCMV (miR-UL112) (orange), EBV (miR-BART2-5p) (green), and KSHV (miR-K12-7) (red).
Cell Host & Microbe 2009 5, DOI: ( /j.chom )
Copyright © 2009 Elsevier Inc. Terms and Conditions