Hepatitis E Virus Encoded MicroRNA-A6Regulate Virus Replication

Hepatitis E virus (Hepatitis E Virus, HEV) is a major pathogens that seriously harm to human health. Transmittion of HEV occurs by the fecal-oral route. HEV, the sole member of the genus Hepevirus in the family Hepeviridae, is a single-stranded, positive-sense RNA, non-enveloped virus. The genome of HEV contains three open reading frames (ORF). HEV is a zoonosis, recent studies have indicated that zoonic transmission of HEV from a varity of many animal species to humans. HEV frequently causes epidemics in developing coutries, high mortality and morbidity rate can reach up to25%in pregnant women.The molecular mechanism of HEV replication is unknown nowdays. MicroRNAs (miR) are small RNAs that evolutionarily conserved, which play an important roles in regulation for gene expression. MiR encoded by virus is a protential, small, non-antigenic regulators in gene expression, which can create a suitable environment for viral replication via modulating the host cell immune system. MicroRNA-A6, one of miRs encoded by HEV, enhances expression of HEV antigen in vivo. Our study focus on how miR-A6regulate HEV replication, and provides the foundation for the mechanisms of HEV. The study includes three parts:1. Screening of miR-A6target genewe obtain three candidate target genes:SIRP-a (signal-regulatory protein alpha),1NG5(inhibitor of growth family, member5) and ZAP (zinc-finger antiviral protein) by TaregetScan5.1database combined with "microRNA-target genes" statistical analysis. The3’UTR of the target genes are respectively inserted pMIR-REPORT vector, building candidate target genes reporter vector. The three candidate target gene reporter vector and miR-A6mimic were co-transfected A549cells24h after transfection, luciferase experiments confirmed miR-A6has interactions with SIRP-a. ING5and ZAP.2. Regulation of miR-A6to candidate target genesConstruction vector of target gene combaied with enhanced green fluorescent protein (EGFP) reporter gene fusion expression, named pcDNA3.1(+)-SIRP/EGFP, pcDNA3.1(+)-ING5/EGFP, pcDNA3.1(+)-ZAP/EGFP. These recombinated plasmids were transfected into HepG2cells by fluorescence to confirme them expressed in eukaryotic cell. pcDNA3.1(+)-SIRP/EGFP, pcDNA3.1(+)-ING5/EGFP, pcDNA3.1(+)-ZAP/EGFP eukaryotic expression plasmids and miR-A6mimic were co-transfected HEK293T cells, respectively. Regulation of miR-A6to candidate target genes in mRNA level using Real-time qPCR; Regulation of miR-A6to candidate target genes in protein level via Western Blot. Our results show that miR-A6up-regulated ZAP, also down-regulated SIRP and ING5.3. Effect of HEV replication by miR-A6interacted with HEVHEV positive samples were inoculated on A549and HepG2cells that transfected PGC-A6plasmid, respectively. Cells were inoculated with DMEM culture containing2%new-born calf serum for4days. Distribution of HEV in cells deteced by immunofluorescence method; in addition, after miR-A6mimic and anti-A6co-transfected A549and HepG2cells24h, HEV positive samples were inoculated on cells cultrued with DMEM containing2%new-born calf serum for6days. Regulation of miR-A6to HEV positive-strand and negtive-strand in mRNA level using Real-time qPCR; Regulation of miR-A6to HEV ORF2in protein level via Western Blot. Our results indicated that miR-A6could increase HEV either mRNA or protein.This study screened three candidate target genes, interaction of miR-A6to target genes deteced by luciferase assay. Effect of miR-A6interacted with HEV by Real-time qPCR and Western Blot test. Our results show that miR-A6enhance HEV mRNA and ORF2expression levels in vitro. Moreover, we think HEV replication is possibly depedent on regulation of miR-A6to target genes and host innate immunity response. This research HEV culture in vitro, provides a basis for mechanism of HEV replication.