Abstract
BackgroundIt 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.MethodsHepatitis 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.ResultsUsing 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.ConclusionThese 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.
Highlights
It is generally believed that RNA virus replicating in the cell cytoplasm would not encode microRNAs due to nucleus inaccessibility
The rationale is that viruses with DNA genomes that replicate in the nucleus have access to cellular miRNA processing machinery; on the contrary, RNA viruses replicate in the cytoplasm and would not encode miRNAs due to inaccessible miRNA processing machinery [10,11]
VMir analyzer program prediction revealed that MR50 was derived from the cell-adapted HAVH2K5 passage
Summary
It is generally believed that RNA virus replicating in the cell cytoplasm would not encode microRNAs (miRNAs) due to nucleus inaccessibility. Naturally occurring miRNAs derived from the antigenome of a cytoplasmic RNA virus have not been described. It is noteworthy that the majority of known viral miRNAs are encoded by DNA viruses and only a few derived from RNA viruses. DNA virusencoded miRNAs are generally accepted, while miRNAs from RNA viruses, especially those that replicate in the cytoplasm, are controversial [10,11]. The rationale is that viruses with DNA genomes that replicate in the nucleus have access to cellular miRNA processing machinery; on the contrary, RNA viruses replicate in the cytoplasm and would not encode miRNAs due to inaccessible miRNA processing machinery [10,11]. Naturally occurring miRNAs derived from RNA viruses have not been widely acknowledged
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