Human Cytomegalovirus MiRNA-US33-5p Targeted Regulation Human Cytomegalovirus UL16 Gene Expression

Background: Human cytomegalovirus(HCMV),also called Human herpesvirus beta subfamily,is the only beta herpesvirus which has been reported can encode micro RNAs(miRNA).Studies have shown that these miRNAs that play a post-transcriptional regulatory role are closely related to the processes of HCMV virus replication,immune evasion,and latent infection.This study is focus on HCMV encoded miRNA-US33-5p.We found that HCMV UL16 is one of HCMV miR-US33-5p potiently targets by Hybrid PCR,and demonstrated the luciferase activity of HCMV UL16 3'-UTR can be down regulated by HCMV miR-US33-5p.HCMV UL16 gene encodes a glucose protein,which interacts with major histocompatibility complex class I-related chain B(MICB)and UL16 binding protein(ULBP),block the activity of nature killer cells(NK cells)to achieve immune evasion.Objective: This study aims to investigate HCMV miR-US33-5p targeted regulation of the protein level of UL16 expression,to provide a basis for further explore the biological functions of miR-US33-5p through regulating UL16 gene expression and understand the molecular mechanism of HCMV infection and anti-infective targeted therapy.Methods: 1.Obtain the HCMV UL16 gene with c-Myc tag by PCR and construct it into the pBI-CMV2 vector.Construct pBI-UL16 recombinant plasmid;2.Transfect the pBI-UL16 recombinant plasmid with miRNA-US33-5p mimics and miRNA-US33-5p mimics NC into HEK-293 cells respectively using lipofectamine 3000 transfection reagent;3.Detect the expression level of miRNA-US33-5p in HEK-293 cells after transfection of pBI-UL16 recombinant plasmid and miRNA-US33-5p mimics,miRNA-US33-5p mimics NC using real-time fluorescence quantitative PCR(q RT-PCR).4.Detect the changes of UL16 gene protein expression in HEK-293 cells after transfection miRNA-US33-5p mimics by c-Myc antibody using Western blot;5.Construction of miR-US33-5p gene deletion strain: prepare miR-US33-5p specific Kan resistance gene fragment and competent E.coli DY380-Han-BAC,electrotransform Kan resistance gene fragment into competent E.coli DY380-Han-BAC,the Han-?miR-US33-5p-BAC plasmid was extracted from the colonies that were positive by PCR,electrotransformed with a suspension of human embryonic lung fibroblast(HELF),and cultured routinely.Results: 1.pBI-UL16 recombinant plasmid be successfully constructed,expression of UL16 gene in vitro;2.The expression of miRNA-US33-5p transfected with miRNA-US33-5p mimics 50 n M/100 n M both increased significantly(P<0.0001),miRNA-US33-5p mimics and pBI-UL16 recombinant plasmid were successfully transfected into HEK-293 cells;3.The results of Western blot showed that the expression of c-Myc-UL16 protein in transfected mimics 50 n M and mimics 100 n M groups both decreased significantly compared with the mimics NC group(P<0.05,P<0.01);4.The miR-US33 gene deletion strain did not produce green fluorescent protein.Conclusion: Overexpression of human cytomegalovirus miR-US33-5p reduces the expression of HCMV UL16 protein.