Prediction,Identification And Function Of MicroRNAs Encoded By Hepatitis A Virus

MicroRNAs (MiRNA) are a class of small non-coding RNA transcribed from the genomes of all multicellular organisms and some viruses, approximately19-25nucleotide (nt) in length, that posttranscriptionally regulate gene expression via inhibition of translation, degradation of target mRNA. MiRNAs play an important role in many biologic processes such as development, virus defense, hematopoiesis, organogenesis, proliferation, apoptosis and fat metabolism.In addition to miRNAs derived from animals and plants, many viruses encode miRNAs for regulating both viral and cellular genes involved in host-virus interaction. There are436virally encoded miRNAs in miRBase database19.0version. DNA viruses account for the majority of known virus-encoded miRNAs with the herpesvirus family which encode well-known viral miRNAs, but naturally expressed by RNA viruses are not widely accepted. To date, only three RNA viruses, including Human Immunodeficiency virus (HIV), Bovine Leukemia virus (BLV) and West Nile virus (WNV), have been accepted to encode virally miRNAs. It is considered that a retrovirus can encode miRNAs, due to a DNA stage in a viral life cycle. However, it is commonly believed that RNA virus that replicates in the cytoplasm would not produce viral miRNAs due to theoretical obstacles. Nevertheless, it has been demonstrated that functional cellular/viral miRNAs can be produced by cytoplasmic RNA virus when the pre-miRNA sequence is incorporated into the virus genome. Hepatitis A virus (HAV) is a typical cytoplasmic plus-trand RNA virus and belongs to the Picornaviridae family, the genus Hepatovirus. HAV has several unique biological characteristics that distinguish it from other members of this family which include slow replication, persistent infection in most HAV/cell culture system without a cytopathic effect. It suggests that HAV most likely encodes functional miRNAs for regulating viral replication in infected cells like West Nile virus.In this study, we investigated whether Hepatitis A virus, a cytoplasmic plus-strand RNA virus, can encode miRNAs. We further characterized the processing pathway of HAV-derived miRNAs and revealed the potentially biological function of miRNAs. In the first part of this study, we founded four potential pre-miRNA stem-loops in the HAV genome and three potential pre-miRNA stem-loops in antigenom RNA by VMir software. As well as, we obtained twenty-eight miRNAs from the splicing of predicted pre-miRNAs by homologous gene searching. In the second part of this study, we identified and characterized the miR’NAs which were predicted by bioinformatics in the first part by experimental approaches. Firstly, we confirmed expression of nine HAV-derived miRNAs in virus-infected KMB17cells by stem-loop RT-PCR. These sequences amplicated by stem-loop RT-PCR were deemed candidate miRNAs and naming:hav-miR-N1-5p, hav-miR-N1-3p, hav-miR-D1-5p, hav-miR-D2-5p, hav-miR-N2-3p, hav-miR-D3a, hav-miR-D3b, hav-miR-D4-3p, hav-miR-D4-5p. Meanwhile, we cloned the nine candidate HAV-derived miRNAs based on RT-PCR method. The sequencing results confirmed the existence of the sequence identity between the PCR products and the predicted miRNAs. After obtaining the exact sequence, we detected expression the of nine candidate HAV-derived miRNAs by other strategies. There was no hybrid signal in Northern blot, but poly (A)-tailed RT-PCR. Nine candidate HAV-derived miRNAs were amplicated specifically by poly (A)-tailed RT-PCR. To determine the interrelation between the predicted pre-miRNAs and mature miRNA, miRNA expression plasmids pcDNATM6.2-GW/mGFP-HAV-miR were constructed and transfected into human cell strain embryonic lung diploid fibroblast KMB17cells and human embryonic kidney293T cells. Expression of candidate miRNAs from pcDNATM6.2-GW/mGFP-HAV-miR were detected by qRT-PCR. The results showed nine candidate miRNAs were expressed by pcDNATM6.2-GW/mGFP-HAV-miR plasmids. It suggested that the cloned miRNAs were derived from the predicted pre-miRNAs, instead of random degradation products of the HAV genome or antigenome. To characterize the processing pathway of HAV-derived miRNAs, we knocked down the key proteins, including the Drosha, the Dicer, the Exportin-5and the AG02proteins using siRNA and monitored the effects of such knockdowns on the expression of the HAV-derived miRNAs. Knockdown of expression of the key proteins were confirmed by qRT-PCR and Western blot. The results showed that levels of HAV-derived miRNAs were reduced when down-regulated expression of the Drosha, the Dicer, the Exportin-5or the AGO2protein, but unaffected when down-regulated the Exportin-5. It suggested that HAV encodes miRNAs in a Drosha-, Dicer-, AGO2-dependent, but Exportin-5independent manner. Therefore, we characterized a noncanonical cytoplasmic processing pathway of viral miRNA, especially RNA virus-derived miRNAs. To determine the PTGS functionality of HAV-derived miRNAs, we constructed and transfected a dual luciferas reporter plasmids containing antisense DNA oligo into human embryonic kidney293T cells. Relative luciferase activity was analyzed at48hours posttransfection. The results demonstrated that candidate HAV-derived miRNAs except for hav-miR-N1-3p were capable of inducing PTGS (Post-transcriptional Gene Silencing). It strongly indicated HAV-derived miRNAs were fully functional bonafide miRNAs and capable of mediating PTGS. Time course expression analysis of HAV-derived miRNAs indicated that peak expression of miRNAs emerged at four to eight hours postinfection, followed by low level of persistent expression.In the third part of this study, we investigated the potentially biological function of HAV-derived miRNAs and the exact molecular mechanism of the biological function. When predicted targets of HAV-derived miRNAs using RNAhybrid, we found the perfectly complementarity between HAV-derived miRNAs and viral genome or antigenome. It suggested that the interaction between HAV-derived miRNAs and viral genome or antigenome likely to occur. Next, we constructed a luciferase reporter plasmid, named as pmirGLO-HAV-miRNA-Twt inserting two copies of the perfect inverse complement of HAV miRNAs, and pmirGLO-HAV-miRNA-Tmut, inserting two copies of the mutational inverse complement of HAV miRNAs. Relative luciferase activity analysis demonstrated that HAV-derived miRNAs were able to strongly inhibit targets expression through perfectly complementary sequences located miRNA sensors. At the same time, we demonstrated the inhibitor effect of HAV-derived miRNAs on viral replication in subgenome and genome levels by "gain of function" and "loss of function" strategy in HAV-infected KMB17cells system. We up-regulated the level of HAV-derived miRNAs by miRNA mimics and down-regulate the level of HAV-derived miRNAs by miRNA inhibitors. Furthermore, we revealed the exact molecular mechanism of the inhibitor effect. We demonstrated that HAV-derived miRNAs are antisense complementarity to genome or antigenome of HAV and act as a siRNA to direct cis cleavage of the genome or antigenome, thus inhibiting viral replication and resulting in a slow replication cycle.In this study, we discovered a novel class of miRNAs derived from the coding region and antigenome of the HAV, a typical cytoplasmic RNA virus. Moreover we revealed a noncanonical cytoplasmic processing pathway of viral miRNAs. Furthermore, we confirmed the inhibitor effect of miRNAs on viral replication and reveal the exact molecular mechanism of the inhibitor effect. We also provide a reason that slow replication cycle of HAV in cell culture.