A cytoplasmic RNA virus generates functional viral small RNAs and regulates viral IRES activity in mammalian cells

Abstract

The roles of virus-derived small RNAs (vsRNAs) have been studied in plants and insects. However, the generation and function of small RNAs from cytoplasmic RNA viruses in mammalian cells remain unexplored. This study describes four vsRNAs that were detected in enterovirus 71-infected cells using next-generation sequencing and northern blots. Viral infection produced substantial levels (>105 copy numbers per cell) of vsRNA1, one of the four vsRNAs. We also demonstrated that Dicer is involved in vsRNA1 generation in infected cells. vsRNA1 overexpression inhibited viral translation and internal ribosomal entry site (IRES) activity in infected cells. Conversely, blocking vsRNA1 enhanced viral yield and viral protein synthesis. We also present evidence that vsRNA1 targets stem-loop II of the viral 5′ untranslated region and inhibits the activity of the IRES through this sequence-specific targeting. Our study demonstrates the ability of a cytoplasmic RNA virus to generate functional vsRNA in mammalian cells. In addition, we also demonstrate a potential novel mechanism for a positive-stranded RNA virus to regulate viral translation: generating a vsRNA that targets the IRES.

Diverse Strategies Used by Picornaviruses to Escape Host RNA Decay Pathways

“…Recent studies using deep sequencing techniques have revealed that viral small RNAs (vsRNAs) are produced from HAV, EMCV and EV71 RNA in a Dicer-dependent manner [153,154,155]. While the roles of the HAV and EMCV vsRNAs during infection have not been elucidated, the study of EV71 vsRNAs has revealed interesting new ways in which the virus re-purposes yet another potentially antiviral cellular pathway.…”

“…While the roles of the HAV and EMCV vsRNAs during infection have not been elucidated, the study of EV71 vsRNAs has revealed interesting new ways in which the virus re-purposes yet another potentially antiviral cellular pathway. One of the EV71 vsRNAs, vsRNA-1, is derived from the second stem loop (SL-II) of the 5′ NCR and negatively regulates viral translation and replication [155]. Instead of inhibiting virus replication via canonical miRNA mechanisms, it was discovered that vsRNA-1 may regulate EV71 IRES-driven translation by promoting the binding of both positive and negative ITAFs to SL-II of the 5′ NCR.…”

Mammalian RNA virus-derived small RNA: biogenesis and functional activity

“…These findings indicate that mammalian cells can process small RNAs from the structured region of viral RNAs. Our recent identification of a vsRNA generated from the 5 0 -structured region of a cytoplasmic RNA virus, enterovirus 71 (EV71), through Dicer-dependent activity supports these findings [12].…”

“…Deep sequencing detected only "vanishing levels" of certain vsRNAs in infected mammalian cells [8]. However, other groups have detected these "vanishing levels" of vsRNAs, which are theoretically undetectable, by using northern blot assays [12,18,25,35]. These observations suggest that a bias could occur in vsRNA measurement by using deep sequencing or other detection methods.…”

“…Specifically, vsRNAs generated in influenza-Avirus-infected cells can regulate viral transcriptionreplication switching by associating with the viral RdRp [19,21]. In addition, we identified a vsRNA generated from the 5 0 -structured region of EV71 genomic RNA and determined that this vsRNA can regulate viral translation by targeting the sequence of the viral internal ribosomal entry site [12]. Although this vsRNA can be enriched by Ago-2 immunoprecipitation, our results suggest that Ago-2 knockdown cannot affect its ability to regulate internal ribosomal entry site function.…”