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

Qian Zhang,Xin Song,Yangyang Zhang,Yanyu Zhang,Ping Ma,Jiang Deng,Liping Lv

doi:10.3389/fimmu.2021.628364

Qian Zhang, Xin Song + Show 5 more

Open Access

https://doi.org/10.3389/fimmu.2021.628364

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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, common in 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.

Highlights

Human cytomegalovirus (HCMV), a prevalent human pathogen, is a member of the subfamily of β-herpesviruses, which are enveloped, double-stranded DNA viruses that maintains persistent latent infection for the duration of the host’s lifetime [1]
The ability of hcmv-miR-US33as-5p to bind the 3′-untranslated region (3′-UTR) of each putative mRNA was verified by dual-luciferase reporter assay
From the 13 predictive targets, two targets were confirmed, and the relative luciferase activity in cells co-transfected with pmirGLO-STAT5AUTR or pmirGLO-interferon alpha and beta receptor subunit 1 (IFNAR1)-UTR and hcmv-miR-US33as-5p mimics was obviously downregulated by 14.2% (p = 0.072) and 27.3% (p < 0.05), respectively, compared with that in cells co-transfected with negative control miRNA (Figure 1A)

Summary

Introduction

Human cytomegalovirus (HCMV), a prevalent human pathogen, is a member of the subfamily of β-herpesviruses, which are enveloped, double-stranded (ds) DNA viruses that maintains persistent latent infection for the duration of the host’s lifetime [1]. MicroRNAs (miRNAs) are short non-coding RNAs (19–22 nucleotides in length) that post-transcriptionally regulate gene expression, causing the degradation and translational inhibition of target mRNAs by base-pairing with the 3′-untranslated region (3′-UTR) through the RNA-induced silencing complex (RISC) [4, 5]. HCMV has a large genome of 230–250 kb and is currently known to encode 26 miRNAs from 16 precursors. We and other researchers found that miR-US33as-5p is encoded by HCMV and expressed in both lytic and latent infection [7]

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