Abstract
Attenuating the expression of immediate early (IE) proteins is essential for controlling the lytic replication of human cytomegalovirus (HCMV). The human microRNAs (hsa-miRs), miR-200b-3p and miR-200c-3p, have been identified to bind the 3′-untranslated region (3′-UTR) of the mRNA encoding IE proteins. However, whether hsa-miRs can reduce IE72 expression and HCMV viral load or exhibit a crosstalk with the host cellular signaling machinery, most importantly the NF-κB cascade, has not been evaluated. In this study, argonaute-crosslinking and immunoprecipitation-seq revealed that miR-200b-3p and miR-200c-3p bind the 3′-UTR of UL123, which is a gene that encodes IE72. The binding of these miRNAs to the 3′-UTR of UL123 was verified in transfected cells stably expressing GFP. We used miR-200b-3p/miR-200c-3p mimics to counteract the downregulation of these miRNA after acute HCMV infection. This resulted in reduced IE72/IE86 expression and HCMV VL during lytic infection. We determined that IE72/IE86 alone can inhibit the phosphorylation of RelA/p65 at the Ser536 residue and that p-Ser536 RelA/p65 binds to the major IE promoter/enhancer (MIEP). The upregulation of miR-200b-3p and miR-200c-3p resulted in the phosphorylation of RelA/p65 at Ser536 through the downregulation of IE, and the binding of the resultant p-Ser536 RelA/p65 to MIEP resulted in a decreased production of pro-inflammatory cytokines. Overall, miR-200b-3p and miR-200c-3p—together with p-Ser536 RelA/p65—can prevent lytic HCMV replication during acute and latent infection
Highlights
Introduction iationsLifelong latent infection with human cytomegalovirus (HCMV) can alter the host immune system through the excessive clonal inflation of highly differentiated HCMV-specificCD45RA+ CD8+ effector memory T lymphocytes [1,2]
We searched for hsa-miRs that could bind to the 30 -untranslated region (30 -UTR) of UL123 using bioinformatic analysis
We evaluated the binding of miRNAs to the 30 -UTR of mRNA by using an assay that employed the intracellular expression of green fluorescent protein (GFP) as an indicator of binding
Summary
Introduction iationsLifelong latent infection with human cytomegalovirus (HCMV) can alter the host immune system through the excessive clonal inflation of highly differentiated HCMV-specificCD45RA+ CD8+ effector memory T lymphocytes [1,2]. Lifelong latent infection with human cytomegalovirus (HCMV) can alter the host immune system through the excessive clonal inflation of highly differentiated HCMV-specific. Subclinical immune senescence caused by the intermittent reactivation of latent HCMV infection can induce various chronic inflammatory morbidities and aging in individuals, causing difficulties in making a good diagnosis and that metagenomics can help in the diagnostic process [1–7]. Lytic HCMV replication can be—directly as well as indirectly—detrimental to the health of hematopoietic stem cell or solid organ transplant (SOT) recipients [8,9]. Such crucial effects of the HCMV latency on various populations indicate that primary infection should be controlled or antiviral strategies should be developed based on a thorough understanding of the pathophysiologic mechanisms [10–13].
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