Human cytomegalovirus microRNA miR-US4-1 inhibits CD8(+) T cell responses by targeting the aminopeptidase ERAP1

Abstract

Major histocompatibility complex (MHC) class I molecules present peptides on the cell surface to CD8(+) T cells, which is critical for the killing of virus-infected or transformed cells. Precursors of MHC class I-presented peptides are trimmed to mature epitopes by the aminopeptidase ERAP1. The US2-US11 genomic region of human cytomegalovirus (HCMV) is dispensable for viral replication and encodes three microRNAs (miRNAs). We show here that HCMV miR-US4-1 specifically downregulated ERAP1 expression during viral infection. Accordingly, the trimming of HCMV-derived peptides was inhibited, which led to less susceptibility of infected cells to HCMV-specific cytotoxic T lymphocytes (CTLs). Our findings identify a previously unknown viral miRNA-based CTL-evasion mechanism that targets a key step in the MHC class I antigen-processing pathway.

Figure 1. The mRNA level and protein of ERAP1 isoform b but not ERAP1 isoform a are reduced by HCMV miR-US4-1 expression

(a, b, c) Relative change in cellular mRNA levels by viral miRNAs (a, miR-US4-1; b, miR-US5-1; c, miR-US5-2) compared to control miRNA (siRNA-GFP). The expression level of ERAP1 is indicated with a red dot. (d, e) The expression levels of (d) ERAP1a and (e) ERAP1b mRNA were analyzed using qRT-PCR and normalized to GAPDH (glyceraldehyde 3-phosphate dehydrogenase). *P < 0.05, two-tailed Student’s t-test compared with control miRNA. Data are presented “relative” to the value of GAPDH as means ± s.d., n = 3. (f) HEK293T cells were transfected with 10 µg of pSuperRetro vectors, siGFP as control miRNA, miR-US4-1, siERAP1, and miR-US4-1(M), followed by selection with 2 µg puromycin for 1 week. (g) HEK293T cells were transfected with different concentrations of pSuperRetro-miR-US4-1 vector (2 µg, 4 µg, and 10 µg in lanes 2, 3, and 4, respectively). (f, g) The expression level of the ERAP1 protein was analyzed by immunoblot, with GAPDH as a protein loading control. The expression level of miR-US4-1 was analyzed by (f) RNase protection assay (RPA) or (g) RNA blot analysis. Hsa-miR-16 served as an RNA loading control. Data are representative of 3 independent experiments. C, control miRNA.

Figure 2. HCMV miR-US4-1 targets the 3′-UTR of ERAP1 isoform b and physically binds to ERAP1 isoform b mRNA within the RISC

Predicted binding site of the 3′-UTR of ERAP1a mRNA (a) and ERAP1b mRNA (b) by miR-US4-1. Bold characters indicate the expected seed region interaction site. The nucleotide sequence in red was replaced with the sequence indicated by the arrow in mutant 3′-UTRs. The HCMV miR-US4-1 sequence is shown in blue. (c) Dual luciferase assays were performed in HEK293T cells using 5 µg of the miRNA-expressing vector, 10 ng of the pGL3-3′-UTR-containing vector, and 5 ng of the Renilla control vector. (d) A dual luciferase assay was performed under the same conditions as in (c), except for the use of 1 µg, 2 µg, or 5 µg of the miR-US4-1-expressing vector and 5 µg of the control miRNA-expressing vector. C, control miRNA. (c, d) *P < 0.05, two-tailed Student’s t-test compared with control miRNA. Data are presented by the luminescence of firefly luciferase “relative” to the luminescence of Renilla luciferase as means ± s.d., n = 3, and are representative of 3 independent experiments. (e) HEK293T cells were transfected with the mix of pSuperRetro vectors indicated and empty (Mock) or N-terminally FLAG-tagged human AGO1, 2, 3, and 4 (AGO) vectors. RISC IP was performed at 48 h after transfection. Total RNA was extracted from RISC IP samples, and an RPA was performed to detect miR-US4-1. NT-NR indicates no target-no RNase that the sample did not contain either extracted total RNA or RNase A/T1. NT indicates no target that the sample did not contain extracted total RNA. *Undigested probe, **HCMV miR-US4-1. (f) Aliquots of RISC immunoprecipitates and cell lysates were probed with anti-FLAG Ab to confirm the IP and expression of FLAG-AGOs. (g, h) Using RNA extracted from immunoprecipitated RISC or total sample, qRT-PCR was performed to compare the levels of ERAP1a (g) and ERAP1b mRNA (h). The level of ERAP1 mRNA was normalized to the GAPDH level. *P < 0.05, **P < 0.01, two-tailed Student’s t-test compared with control miRNA. Data are presented “relative” to the value of GAPDH as means ± s.d., n = 3, and are representative of 3 independent experiments.

Figure 3. ERAP1 is downregulated in HCMV-infected cells

(a) 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. An RPA was performed to analyze the level of miR-US4-1. NT-NR, no target-no RNase that the sample did not contain either extracted total RNA or RNase A/T1. NT, no target that the sample did not contain extracted total RNA. *Undigested probe, **HCMV miR-US4-1. (b) Using the cell aliquots from the samples in (a) immunoblot analysis to determine changes in the protein amount of ERAP1, IE1/2, and GAPDH at each post infection time. GAPDH served as protein loading control. Data are representative of 3 independent experiments. U, uninfected. WT, wild type. Mut, mutant. Rev, revertant.

Figure 4. HCMV miR-US4 inhibits the trimming of Ova_257–264 peptide from ovalbumin precursor peptide by ERAP1

(a) H-2K^b-expressing HeLa cells were transfected with control miRNA, miR-US4-1, siERAP1, or miR-US4-1(M). After 1 week of selection with puromycin, the expression levels of ERAP1a and ERAP1b mRNA were analyzed using qRT-PCR and normalized to GAPDH. *P < 0.05, two-tailed Student’s t-test compared with control miRNA. Data are presented “relative” to the value of GAPDH as means ± s.d., n = 3, and are representative of 3 independent experiments. (b, c) H-2K^b-expressing HeLa cells were transfected with control miRNA, miR-US4-1, siERAP1, or miR-US4-1(M). After 1 week of selection with puromycin, the pUG1-N5OVA8 vector (b) or the pUG1-OVA8 vector (c) were transfected. After 48 h, 1.0 × 10⁴ transfected H-2K^b-expressing HeLa cells were co-cultured with 1.0 × 10⁴ (1), 3.0 × 10⁴ (3), or 9.0 × 10⁴ (9) B3Z cells. After 16 h incubation, cells were harvested. The lacZ activity was calculated by measuring β-galactosidase production with the lacZ substrate CPRG and presented in graphs as the “relative” levels compared to control miRNA. *P < 0.001, two-tailed Student’s t-test compared with control miRNA. Data are presented as means ± s.e.m. of 3 independent experiments.

Figure 5. HCMV miR-US4-1 inhibits the generation of HCMV-derived antigenic peptides and the CD8⁺ CTL responses

(a) Characteristic chromatograms indicating epitope production in vitro by ERAP1. For each epitope precursor 3 chromatograms are depicted: Top, precursor alone; Middle, precursor incubated with a moderate amount of enzyme; and Bottom, precursor incubated with a larger amount of enzyme. In each case, the peak that corresponds to the mature epitope is indicated by an asterisk. Reaction conditions are indicated next to each chromatogram. The N-terminally extended 2 amino acid sequence within each precursor is underlined. (b) For the Chromium release assay, autologous fibroblasts were transfected twice with control miRNA, miR-US4-1, or siERAP1 RNA duplex. After 24 h, cells were infected with HCMV RV798 at MOI = 2 for 1 h. At 48 h post infection, cells were collected and incubated with ⁵¹Cr for 2 h in a 37°C CO₂ incubator. These cells were washed and used as target cells. 1.0 × 10⁴ target cells were co-incubated with 1.0 × 10⁵ (10), 5.0 × 10⁴ (5), 2.5 × 10⁴ (2.5), or 1.25 × 10⁴ (1.25) CTLs, of which the clone names are presented above each panel, as effector cells for 6 h in a 37°C CO₂ incubator. The released ⁵¹Cr level was measured by γ-irradiation counting. The level of specific CTL lysis was calculated by the following formula: ([specific release - spontaneous release] / [total release - spontaneous release]) × 100 (%). A spontaneous release of less than about 5% of the total release was observed in all assays. *P < 0.001, two-tailed Student’s t-test compared with control miRNA. Data are presented as means ± s.e.m. of 3 independent experiments.