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.2021 Mar 5:12:628364.
doi: 10.3389/fimmu.2021.628364. eCollection 2021.

Human Cytomegalovirus miR-US33as-5p Targets IFNAR1 to Achieve Immune Evasion During Both Lytic and Latent Infection

Affiliations

Human Cytomegalovirus miR-US33as-5p Targets IFNAR1 to Achieve Immune Evasion During Both Lytic and Latent Infection

Qian Zhang et al. Front Immunol..

Abstract

As the first line of antiviral defense, type I interferon (IFN) binds IFN receptor 1 (IFNAR1) and IFNAR2 to activate the Jak-STAT signal transduction pathway, producing IFN-stimulated genes (ISGs) to control viral infection. The mechanisms by which human cytomegalovirus (HCMV) counteracts the IFN pathway are only partially defined. We show that miR-US33as-5p encoded by HCMV is expressed in both lytic and latent infection. By analysis with RNA hybrid and screening with luciferase reporter assays, we identified IFNAR1 as a target of hcmv-miR-US33as-5p, which was further verified by examining the expression of two IFNAR1 mutants and the binding of IFNAR1 to miR-US33as-5p/miR-US33as-5p-M1/miR-US33as-5p-M2. We found that after the transfection of miR-US33as-5p mimics into different cell lines, the phosphorylation of downstream proteins and ISG expression were downregulated. Immunofluorescence showed that the miR-US33as-5p mimics also inhibited STAT1 translocation into the nucleus. Furthermore, we constructed HCMV with mutant miR-US33as-5p and determined that the mutation did not affect HCMV replication. We found that MRC-5/human foreskin fibroblast (HFF) cells infected with ΔmiRNA HCMV exhibited higher IFNAR1 and ISG expression and a reduced viral load in the presence of exogenous IFN than cells infected with WT HCMV did, confirming that the knockout of miR-US33as-5p impaired viral resistance to IFN. Finally, we tested the effect of ΔmiRNA HCMV on THP-1 and d-THP-1 cells, commonin vitro models of latent infection and reactivation, respectively. Again, we found that cells infected with ΔmiRNA HCMV showed a reduced viral load in the presence of IFN than the control cells did, confirming that miR-US33as-5p also affects IFN resistance during both latency and reactivation. These results indicate a new microRNA (miRNA)-based immune evasion mechanism employed by HCMV to achieve lifelong infection.

Keywords: IFNAR1; US33as-5p; cytomegalovirus; immune evasion; viral miRNAs.

Copyright © 2021 Zhang, Song, Ma, Lv, Zhang, Deng and Zhang.

PubMed Disclaimer

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
hcmv-miR-US33as-5p inhibited the expression of IFNAR1 by targeting the 3′-UTR of IFNAR1.(A) Reporter vectors containing the 3′-UTRs of 13 predicted target genes were co-transfected with GV251 blank vector or vector containing US33as-5p into 293 cells. The dual-luciferase reporter assay was used to determine the relative Renilla luciferase activity normalized to firefly luciferase activity in cells in the corresponding wells at 48 h post-transfection (hpi) when cell lysates were harvested.(B) The expression of hcmv-US33as-5p during HCMV infection at different time points was determined by qPCR. U6 was used as a loading control.(C) IFNAR1 protein expression at different time points after the transfection of US33as-5p mimics was examined by western blot analysis. The sample of the control group comes from cells transfected with NC-RNA for 72 h. β-Actin was used as a loading control. The assays were performed in triplicate wells, and data were collected from two different experiments and are represented as the means ± SDs; **p < 0.05.
Figure 2
Figure 2
Main binding site in hcmv-miR-US33as-5p for IFNAR1 mRNA.(A) Schematic of the targeting of IFNAR1 by hcmv-miR-US33as-5p. Mutated nucleotides are shown in color and underlined.(B) Mutation of the IFNAR1-binding site and back mutation of the hcmv-miR-US33as-5p seed region. For IFNAR1 mutagenesis 1, GCA was mutated to CGC, and the miRNA was complementarily mutated to GCG for miR-US33as-5p back mutagenesis 1. For IFNAR1 mutagenesis 2, AUC was mutated to CCG, and the miRNA was complementarily mutated to CGG for miR-US33as-5p back mutagenesis 2. The pmirGLO-IFNAR1-Mutation-UTR vector and GV251-US33as-Mutation-UTR vector were co-transfected into 293 cells cultured in 24-well plates.(C) Dual-luciferase reporter assays to determine relative firefly luciferase activity in 293 cells co-transfected with pmirGLO-IFNAR1-Mutation-UTR vector and GV251-US33as-Mutation-UTR vector were performed, and the calculated data are shown in Figure 1A.(D) Western blot showing the expression of IFNAR1 in 293 cells co-transfected with pmirGLO-IFNAR1(-Mutation)-UTR vector and GV251-US33as(-Mutation)-UTR vector harvested at the same time, with β-actin used as a loading control. The assays were performed in triplicate wells, and data were collected from two different experiments and are represented as the means ± SDs; **p < 0.05.
Figure 3
Figure 3
hcmv-miR-US33as-5p downregulates IFNAR1 and Jak-STAT, limiting the release of ISGs.(A) MRC-5 and HFF cells were cultured in 24-well plates and transiently transfected with hcmv-US33as-5p mimics or a negative control RNA (NC-RNA) at a concentration of 100 nM for 24 h. The cells were treated with 10 μg/mL CHX, followed by stimulation by 1,500 U/mL IFNα for 1 h until harvest. The expression of IFNAR1 and IFNAR2 was determined by qPCR.(B) The mRNA stability of IFNAR1 and IFNAR2 in MRC-5 cells transfected with US33as-5p mimics or NC-RNA and incubated with actinomycin D to inhibit transcription is presented as the amount of mRNA detected at a given time relative to that at 0 h, which was set as 100%.(C) Immunoblotting of lysates from MRC-5 cells underwent treatment similar to that described in Figure 3A and prolonged incubation with 1,500 U/mL IFN-α for 6 h. was performed. Blots were probed for IFNAR1, IFNAR2, STAT1, STAT2, Jak1, Tyk2 and their phosphorylated forms. β-Actin was run as a loading control.(D) The expression of multiple ISGs (Mx1, RSAD2, DDX58, BST2, IFIT2, and ISG20) in MRC-5 cells was determined by qPCR. The RNA harvested from MRC-5 cells underwent treatment similar to that described in Figure 3C. GAPDH was amplified as a loading control. The assays were performed in triplicate wells, and data were collected from two different experiments and are represented as the means ± SDs; **p < 0.05.
Figure 4
Figure 4
hcmv-miR-US33as-5p inhibits the nuclear translocation of STAT1.(A) 293 cells were transfected with GV251 blank vector or vector containing US33as-5p, followed by selection with G418 (0.5 μg/mL) for 3 days. The cells were then transfected with the pDs-RED-STAT1 vector for 24 h and then stimulated with or without IFNα (1,500 IU/mL) for 12 h. STAT1 nuclear localization was detected by indirect immunofluorescence.(B) Percentage of cells with nuclear STAT1 from experiments performed in A. Cells were counted in three random fields and the results are expressed as mean ± SD (**p < 0.01). Fluorescence was visualized with an Olympus FluoView 1000 confocal microscope. All images were captured under a 60× objective lens.
Figure 5
Figure 5
CRISPR-Cas9 introduced mutations in hcmv-US33as-5p and did not obviously affect HCMV virology.(A) The DNA sequence surrounding the hcmv-US33as-5p editing region from each HCMV mutant strain was amplified by PCR and analyzed by DNA sequencing. The PAM sequence and sgRNA-targeting region are indicated with lines. The DNA sequence-editing miRNA bases are highlighted in red. Deletions and insertions are presented by dashed lines or a letter underneath the sequence, respectively. No mutations in the control samples were observed.(B) MRC-5 cells were infected with WT, ΔmiRNA HCMV #1, ΔmiRNA HCMV #2, or ΔmiRNA HCMV #3 at an MOI = 1, and the viral loads from the supernatants at 1d, 2d, 3d, 5d, 7d were determined by qPCR.(C) The expression of several miRNAs encoded by HCMV at 72 hpi was examined by qPCR.
Figure 6
Figure 6
Knockout of hcmv-US33as-5p alleviated blockade of the IFN pathway.(A,B) MRC-5 and HFF cells were infected with WT or ΔmiRNA HCMV for 48 h, followed by treatment with CHX and stimulation with IFN for 24 h. The expression of IFNAR1 and IFNAR2 was determined by qPCR and western blot.(C) The expression of multiple ISGs was examined by qPCR.(D) Total DNA was isolated from the supernatants, and HCMV copy numbers were determined and calculated by qPCR. **p < 0.05.
Figure 7
Figure 7
hcmv-US33as-5p is expressed in both viral latency and reactivation.(A) A schematic diagram depictingin vitro models of latent infection and viral reactivation. THP-1 cells were infected with WT or ΔmiRNA HCMV (MOI = 10) and maintained for 10 days in the culture medium. Then, the THP-1 cells were differentiated into macrophages (d-THP-1) by stimulated with TPA in order to render the cells permissive to HCMV infection and further maintained in the medium for another 5 days.(B) The expression of viral UL122 at 1, 3, 5, 7, and 9 dpi and 1, 3, and 5 days after TPA stimulation was detected by qPCR.(C) The expression of hcmv-US33as-5p at 9 dpi and 3 days after TPA stimulation was determined by qPCR.
Figure 8
Figure 8
A lack of hcmv-US33as-5p impaired viral resistance to the IFN pathway during latency and reactivation.(A) A schematic diagram depicting thein vitro models of latent infection and viral reactivation. For latent infection, THP-1 cells were infected with WT or ΔmiRNA HCMV (MOI = 10) and maintained for 9 days in a culture medium supplemented with or without IFN (500 IU/mL). For viral reactivation, THP-1 cells were infected and maintained for 10 days in the culture medium. Then, the THP-1 cells were stimulated with TPA and further maintained in the medium for another 5 days. The cells were stimulated in a culture medium supplemented with or without IFN (500 IU/mL) beginning at 8 dpi.(B) The expression of miRNA among groups was examined by qPCR.THP-1(C,D) The viral loads at indicated days were determined by qPCR and western blot.(E,F) The expression of IFNAR1 and ISGs (Mx1, RSAD2) among groups were determined by qPCR. **p < 0.05.
Figure 9
Figure 9
Schematic of the mechanism by which hcmv-US33as-5p facilitates the IFN pathway.
See this image and copyright information in PMC

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