title: Identification and validation of a novel microRNA-like molecule derived from a cytoplasmic RNA virus antigenome by bioinformatics and experimental approaches.
abstract: BACKGROUND
  It is generally believed that RNA virus replicating in the cell cytoplasm would not encode microRNAs (miRNAs) due to nucleus inaccessibility. Recent studies have described cytoplasmic RNA virus genome-derived miRNAs in West Nile virus (WNV) and Dengue virus (DENV). However, naturally occurring miRNAs derived from the antigenome of a cytoplasmic RNA virus have not been described.
  
  
  METHODS
  Hepatitis A virus (HAV) was served as a model virus to investigate whether the antigenome of a cytoplasmic RNA virus would be processed into miRNAs or miRNA-like small RNAs upon infection. HAV antigenome was queried for putative miRNA precursors (pre-miRNA) with the VMir analyzer program. Mature miRNA prediction was performed using MatureBayes and Bayes-SVM-MiRNA web server v1.0. Finally, multiple experimental approaches, including cloning and sequencing-, RNAi-, plasmid-based miRNA expression- and luciferase reporter assays, were performed to identify and validate naturally occurring viral antigenome-derived miRNAs.
  
  
  RESULTS
  Using human HAV genotype IA (isolate H2) (HAVH2), a virally encoded miRNA-like small RNA was detected on the antigenome and named hav-miR-N1-3p. Transcription of viral pre-miRNA in KMB17 and HEK293T cells led to mature hav-miR-N1-3p production. In addition, silencing of the miRNA-processing enzyme Dicer or Drosha caused a dramatic reduction in miRNA levels. Furthermore, artificial target of hav-miR-N1-3p was silenced by synthesized viral miRNA mimics and the HAVH2 naturally-derived hav-miR-N1-3p.
  
  
  CONCLUSIONS
  These results suggested that the antigenome of a cytoplasmic RNA virus could be processed into functional miRNAs. Our findings provide new evidence supporting the hypothesis that cytoplasmic RNA viruses naturally encode miRNAs through cellular miRNA processing machinery.
authors: Jiandong
authors: J
authors: Shi
authors: Zhiqing
authors: Z
authors: Duan
authors: Jing
authors: J
authors: Sun
authors: Meini
authors: M
authors: Wu
authors: Bin
authors: B
authors: Wang
authors: Jing
authors: J
authors: Zhang
authors: Haixuan
authors: H
authors: Wang
authors: Ningzhu
authors: N
authors: Hu
authors: Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Diseases, Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, China. huyunzhangym@126.com.
authors: Yunzhang
authors: Y
authors: Hu
mesh_terms: D002460:Cell Line; D019295:Computational Biology; D016679:Genome, Viral; D030041:Hepatitis A virus / Q000235:genetics*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism
chemical_list: D035683:MicroRNAs; D012367:RNA, Viral