Supplementary information Human Cytomegalovirus MicroRNA mir-us4-1 Inhibits CD8 + T Cell Response by Targeting ERAP1 Sungchul Kim, Sanghyun Lee, Jinwook Shin, Youngkyun Kim, Irini Evnouchidou, Donghyun Kim, Young-Kook Kim, Young-Eui Kim, Jin-Hyun Ahn, Stanley R. Riddell, Efstratios Stratikos, V. Narry Kim, and Kwangseog Ahn * * To whom correspondence should be addressed: (E-mail) ksahn@snu.ac.kr 1
Supplementary Figure 1 Supplementary Figure 1. Representation of the genomic locations of HCMV mir-us4-1, mir-us5-1, and mir-us5-2 and transfection validation for microarray (a) Schematic representation of the HCMV whole genome and mirnas within the US2-US11 region of the HCMV genome. The HCMV genome consists of 2 linked segments (L and S), each comprising a unique long and a unique short region (UL and US) flanked by inverted repeats (TRL and IRL, TRS and IRS). Three mirnas (mir-us4-1, mir-us5-1, and mir-us5-2) are located within the US2-US11 region. MiR-US4-1 is encoded between the US3 ORF and the US5 ORF with (+) transcriptional orientation. mir-us5-1 and mir-us5-2 are encoded between the US6 ORF and the US7 ORF with (+) orientation, similar to mir-us4-1. (b) Immunoblot analysis to determine the transfection efficiency for microarray analysis. A GFP-targeting sirna was used as control mirna. sirna-pdi targets the coding region of PDI mrna. 2
Supplementary Figure 2 Supplementary Figure 2. Sequences of ERAP1 isoforms and seed-binding region-mutated mir-us4-1 (a) Comparison of ERAP1 isoforms a and b. sierap1, sierap1a, and sierap1b binding sites are indicated in red, green, and blue, respectively. The C-terminal sequence of ERAP1a and ERAP1b is shown in black boxes. (b) The sequence of the wild-type mir-us4-1 sequence and the seed region point mutant, mir-us4-1(m). 3
Supplementary Figure 3 Supplementary Figure 3. PCR efficiency of primers of both ERAP1 isoforms is equal and the expression level of ERAP1b mrna is higher than that of ERAP1a mrna in various cell lines (a) The relative expression levels of ERAP1a and ERAP1b mrna in HEK293T, HeLa-K b, 4
U373MG, HFF, and HFF-TEL cells were analyzed using qrt-pcr and normalized to GAPDH mrna levels. Data are presented as means ± s.d., n = 3, and is representative of 3 independent experiments. (b) 10 pg of pgl3-cmv-erap1a vector DNA and pgl3-cmv-erap1b vector DNA were analyzed by qrt-pcr with primers for ERAP1a, ERAP1b, and firefly luciferase. The fold value of firefly serves as a normalization control. Relative fold value of ERAP1a and ERAP1b are calculated and presented in graph as means ± s.d., n = 3, representative of 2 independent experiments. (c) We transfected 1 µg of pgl3-cmv-erap1a vector DNA and pgl3-cmv-erap1b vector DNA into mouse embryonic fibroblast cell line, NIH3T3. After 24 h post transfection, cells were harvested and total RNA was extracted, followed by cdna synthesis. cdna was analyzed by qrt-pcr with primers for ERAP1a, ERAP1b, and GAPDH. GAPDH serves as a normalization control. Relative fold value of ERAP1a and ERAP1b are calculated and are presented in graph as means ± s.d., n = 3, representative of 2 independent experiments. 5
Supplementary Figure 4 Supplementary Figure 4. Dose-dependent expression of HCMV mir-us4-1 in HEK293T cells (a, b) Expression levels of HCMV mir-us4-1 (a) and mir-16 as a loading control (b) were analyzed using RNA blot analysis. A total of 40 μg total RNA extracted from the indicated cells was resolved on a 12.5% urea-polyacrylamide gel and transferred to a Zetaprobe-GT membrane. Oligonucleotide probes complementary to the mature sequence of each mirna were end-labeled using T4 polynucleotide kinase. Probes were hybridized with membranes and washed. Blots were analyzed using a BAS detector. 6
Supplementary Figure 5 7
Supplementary Figure 5. Experimental validation of RISC IP analysis (a) Expression levels of hsa-mir-21. HEK293T cells were transfected with pcdna3.1(+) mock vector, psuperretro mock vector, pcdna3.1(+)-mir-21 vector, or psuperretro-mir-21 vector. The pcdna3.1(+)-mir-21 vector expressed endogenous human mir-21. After 3 days, total RNA was harvested using Trizol reagent and analyzed using RNA blot analysis. (b) HEK293T cells were transfected with psuperrestro-mir-21, followed by transfection of Mock or FLAGhAGOs vectors. After 48 h, RISC IP was performed, and mirnas were extracted from immunoprecipitates, followed by RPA to detect the mir-21. (c) Immunoblot for expression levels of FLAG-hAGOs. HEK293T cells were transfected with empty (Mock) or N-terminally FLAG-tagged human AGO1, 2, 3, and 4 (FLAG-hAGOs) vectors. To investigate the expression and IP, protein samples from the RISC IP were analyzed by immunoblot analysis with anti- FLAG M2 mab and anti-gapdh Ab. (d, e) qrt-pcr was performed to compare the levels of the indicated mrnas using total RNA samples from RISC IP. The value was normalized to GAPDH (d) or β-actin (e) levels. Data are presented as means ± s.e.m. of 3 independent experiments. 8
Supplementary Figure 6 Supplementary Figure 6. Generation of HCMV ΔUS4 The 6 nt UCGCGA sequence in the hairpin structure of mir-us4-1 was substituted with the GACAAU sequence, disrupting the cropping process of the Drosha complex in the nucleus, and finally resulting in completely diminished pre-mir-us4-1 and subsequent reduction of mature mir-us4-1 production. The HCMV REV used in this study is similar to the HCMV WT and was made using the same method used for the construction of HCMV ΔUS4 with HCMV ΔUS4 as the template. 9
Supplementary Figure 7 Supplementary Figure 7. The mrna level of ERAP1b is downregulated by mir-us4-1 during HCMV infection HFF cells were infected with wild-type HCMV AD169, the ΔUS4 mutant, or the revertant for 1 h at MOI = 5. The total RNA was extracted at 0 h, 24 h, 48 h, and 72 h post infection from infected cells or uninfected cells. qrt-pcr was performed to analyze the level of (a) ERAP1a, ERAP1b, and (b) IE1 mrna. GAPDH served as a normalization control. Data are presented as means ± s.e.m. of 3 independent experiments. 10
Supplementary Figure 8 Supplementary Figure 8. HCMV mir-us4-1 does not affect the expression of components of the MHC class I peptide loading complex Expression levels of Tapasin, MHC class I heavy chain (HC), PDI, ERp57, and GAPDH. Using aliquots from Fig. 6, cells were lysed with 1% NP-40 and analyzed by immunoblot analysis. GAPDH served as loading control. 11
Supplementary Figure 9 Supplementary Figure 9. MHC class I cell surface level of CP, RT, and DK donor fibroblasts infected with HCMV WT, ΔUS4, or RV798 CP, RT, and DK fibroblasts were uninfected or infected with HCMV AD169 wild-type (AD169 WT), HCMV AD169 ΔUS4 mutant (AD169 ΔUS4), or HCMV RV798 (RV798) at MOI = 2 for 1 h. At 48 h post infection, the cells were harvested and flow cytometry was performed using mab W6/32 (HLA-A-, B-, C-specific). Lines in the plots are indicated by short names. No 1st Ab indicates an untreated condition, in which the mab W6/32 was not used in the uninfected cells. 12
Supplementary Figure 10 Supplementary Figure 10. CP, RT, and DK fibroblasts infected with HCMV RV798 express the viral mrna of UL16, UL105, IE1, UL28, and UL23 CP, RT, and DK fibroblasts were infected with HCMV RV798 at MOI = 2 for 1 h. At 0 h and 48 h post infection, we performed RT-PCR to validate the expression of mrnas from UL16, UL105, IE1, UL28, and UL23 genes. GAPDH served as loading control. 13
Supplementary Figure 11 14
Supplementary Figure 11. Validation of ERAP1 knockdown by HCMV mir-us4-1 in fibroblasts (a) CP, (b) RT, and (c) DK fibroblasts were transfected with control mirna, mir-us4-1, or sierap1 RNA oligomer duplexes twice at 36 h intervals. At 24 h after the last transfection, cells were infected with HCMV RV798 at MOI = 2 for 1 h. At 48 h post infection, we performed qrt- PCR to analyze the levels of ERAP1a and ERAP1b mrnas. Relative fold change was normalized to GAPDH level. Data are presented as means ± s.d., n = 3, representative of 2 independent experiments. 15
Supplementary Table 1. Primers or DNA oligomers used in this paper Control mirna Forward GATCCCCGGTTATGTACAGGAACGCATTCAAGA (sigfp) GATGCGTTCCTGTACATAACCTTTTTTTA Reverse AGCTTAAAAAAAGGTTATGTACAGGAACGCATC TCTTGAATGCGTTCCTGTACATAACCGGG mir-us4-1 Forward GATCCCCCGTCTTCGACGTGCAGGGGGATTTCA AGAGAATCCCCCTGCACGTCCATGTCGTTTTTA Reverse AGCTTAAAAA CGACATGGACGTGCAGGGGGATTCTCTTGAAAT CCCCCTGCACGTCCATGTCGGGG mir-us4-1(m) Forward GATCCCC CGACATGGACGTGCAGGGGGATTTCAAGAGAAT CCCCCTGCACGTCCATGTCGTTTTTA Reverse AGCTTAAAAA CGACATGGACGTGCAGGGGGATTCTCTTGAAAT CCCCCTGCACGTCGAAGACGGGG sierap1 Forward GATCCCCAACGTAGTGATGGGACACCATTTTTCA AGAGAAAATGGTGTCCCATCACTACGTTTTTTTA Reverse AGCTTAAAAAAACGTAGTGATGGGACACCATTT TCTCTTGAAAAATGGTGTCCCATCACTACGTTGG G sierap1a Forward GATCCCCCATCTAGCAAATGAGGCACTGTTCTTT CAAGAGAAGAACAGTGCCTCATTTGCTAGATGT TTTTA Reverse AGCTTAAAAACATCTAGCAAATGAGGCACTGTT CTTCTCTTGAAAGAACAGTGCCTCATTTGCTAGA TGGGG 16
sierap1b Forward GATCCCCGGGCTGACTTGTAAAGGCCAAGTCAT TCAAGAGATGACTTGGCCTTTACAAGTCAGCCC TTTTTA Reverse AGCTTAAAAAGGGCTGACTTGTAAAGGCCAAGT CATCTCTTGAATGACTTGGCCTTTACAAGTCAGC CCGGG ERAP1a 3 -UTR Forward CTAGCTAGCAAATCCATCAGAATCTCAGACTACA G Reverse AACTGCAGTCATTTTATGTGTTAATTTTTTATTAA AT ERAP1b 3 -UTR Forward GCTCTAGAAAATTCCTCCCTTGCCAGGTTCCTG Reverse AACTGCAGGGTTTCTGCACATTGTTGTGGCAAT ERAP1 C -term Forward GGAAATTCCATATGAATGAAGTGGAAACTCAATT CAAGGC Reverse CCGCTCGAGCATACGTTCAAGCTTTTCACTTTGC AG US4 mutagenesis Forward GTGCGGATTAGTGGTCGTGGACAACCATGGACG TGCAGGGGG Reverse CCCCCTGCACGTCCATGGTTGTCCACGACCACT AATCCGCAC US4 homologous Forward CGTCAAGAGTCACGTCAGTCGCCCACCCCCATT arm ACACGGCGATATCCCGAGGCCTGGTGATGATGG CGGGATCG Reverse TATTTTTTCTGTCGCGATAGTCGACGATATGTCGC GATAGACAACTCCGGTCAGAAGAACTCGTCAAG AAGGCG UL56 (RT-PCR) Forward CACAAGAGTCTGCCTGTGGA Reverse GCATTTGACCAAATCGTCCT UL28 (RT-PCR) Forward GCCTCGTCAGTGTGTACGAA 17
Reverse GCCCTCTACGATCTCATCCA UL23 (RT-PCR) Forward ACTTCATCACCGTCCCAGAG Reverse AGGTTCCACGTGGTTACTGC UL16 (RT-PCR) Forward TGGGTAGCAAGTCCTCCAAC Reverse CAACCACAGGTCAACCACAG UL105 (RT-PCR) Forward CAAGAGGAGACCCACGACAT Reverse GAAAAATGTCGCGGAAGGTA UL123(IE1) (qrt- Forward CAAGAGGAGACCCACGACAT PCR and RT-PCR) Reverse GAAAAATGTCGCGGAAGGTA GAPDH (qrt- Forward AGAAGGCTGGGGCTCATTTG PCR) Reverse AGGGGCCATCCACAGTCTTC beta-actin (qrt- Forward GAAAAATGTCGCGGAAGGTA PCR) Reverse GAAAAATGTCGCGGAAGGTA pcdna3.1-mir-21 Forward CGGAATTCTGGGGTTCGATCTTAACAGGCCAG Reverse CCGCTCGAGACCAGACAGAAGGACCAGAGTTT CT PDCD4 (qrt-pcr) Forward GAAAATGCTGGGACTGAGGA Reverse TGGACTGGTTGGCACAGTTA Maspin (qrt-pcr) Forward CACAGATGGCCACTTTGAGA Reverse GGCCTCCATGTTCATCATCT TPM1 (qrt-pcr) Forward TCATCATTGAGAGCGACCTG Reverse CTCAGCCTCCTTCAGCTTGT SPRY2 (qrt-pcr) Forward CCCCTCTGTCCAGATCCATA Reverse CCCAAATCTTCCTTGCTCAG RECK (qrt-pcr) Forward GCAGGGGAAGTTGGTTGTTA Reverse TGCCAGCAAAACAAGAACAG PTEN (qrt-pcr) Forward ACCAGGACCAGAGGAAACCT Reverse GCTAGCCTCTGGATTTGACG 18