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

Kuo-Feng Weng Chuan-Tien Hung Po-Ting Hsieh Mei-Ling Li Guang-Wu Chen Yu-An Kung Peng-Nien Huang Rei-Lin Kuo Li-Lien Chen Jing-Yi Lin Robert Yung-Liang Wang Shu-Jen Chen Petrus Tang Jim-Tong Horng Hsing-I Huang Jen-Ren Wang David M. Ojcius Gary Brewer Shin-Ru Shih C The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. - 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 nextgeneration 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. Cells produce small RNAs, which are noncoding RNAs 20 30 nucleotides (nt) in length (1). These small RNAs can finetune the biological functions of cells by modulating gene expression and modifying the genome (2,3). For example, endogenous microRNAs (miRNAs) regulate specific gene expression and control the associated downstream activities (2). Another type of cellular small RNAs, PIWI-interacting RNAs (piRNAs), maintain genomic integrity by preventing the invasion of transposable elements (3). Mammalian cells produce numerous small RNAs via a canonical miRNA biogenesis pathway that involves nuclear processing by an RNase III-type protein, Drosha, and sub*To whom correspondence should be addressed. Tel: +886 3 2118800 (Ext 5497); Fax: +886 3 2118174; Email: The authors wish it to be known that, in their opinion, the first three authors should be regarded as joint First Authors. sequent cytoplasmic processing by another RNase III-type protein, Dicer (1). Viruses that replicate in the nucleus, such as deoxyribonucleic acid (DNA) viruses and retroviruses, can produce their own small RNAs through the canonical miRNA biogenesis pathway. These virus-derived small RNAs (vsRNAs) either fine-tune viral replication or inhibit antiviral mechanisms in infected cells (46). Alternatively, Dicer enzymes in plant and insect cells process the genome of a cytoplasmic RNA virus into small RNAs. Infected cells use these vsRNAs as an antiviral defence mechanism to reduce viral replication through RNA interference (7). Conversely, West Nile virus uses Dicer in mosquito cells to produce miRNA-like vsRNAs for the benefit of the virus (8). However, similar mechanisms for generating vsRNA and RNA-based defences against cytoplasmic RNA viruses in mammals require further research (2,910). Recent studies have shown that cytoplasmic RNA viruses can induce non-canonical cytoplasmic miRNA biogenesis pathways in mammalian cells (11,12). For example, an engineered Sindbis virus with a primary miRNA hairpin in its subgenomic RNA generated functional miRNA through a Dicer-dependent, DGCR8-independent pathway (12,13). These studies have suggested that a cytoplasmic RNA virus containing a primary miRNA-like hairpin may be capable of producing vsRNA through its own structured RNA in infected mammalian cells. In addition, deep sequencing techniques have been used to identify vsRNAs and siRNAs in mammalian cells infected with cytoplasmic RNA viruses (1416). However, the functions of these vsRNAs are still debated (17). Similar to poliovirus, enterovirus 71 (EV71) is a positivestranded RNA virus that replicates in the cytoplasm. EV71 outbreaks have occurred worldwide, and EV71 infection is associated with severe neurological diseases and high mortality rates (18,19). The 5 untranslated region (5 UTR) of the EV71 genomic RNA is highly structured (20,21); it contains a cloverleaf structure that is essential for viral RNA replication and an internal ribosomal entry site (IRES) that is responsible for viral translation (22,23). Because positivestranded viruses use the same RNA template for both translation and replication, viruses must regulate their translation (or IRES activity) (24,25). Recently discovered proteins called IRES trans-acting factors (ITAFs) can regulate EV71 IRES activity (19,2628). In this study, we showed that a cytoplasmic positivestranded RNA virus generated functional vsRNAs in mammalian cells. One vsRNA (vsRNA1) down-regulated viral translation by targeting the stem-loop II region of the viral IRES. This study demonstrated a novel mechanism by which virus self-regulates its translation by generating a RNA-based ITAF. MATERIALS AND METHODS Deep sequencing and data analysis SF268 (human glioblastoma) cells were mock-infected or virus-infected with Enterovirus 71 strain Tainan/4643/98 (GenBank accession number: AF304458.1) at a moi of 40. After 6 h post-infection (p.i.), the total RNA was extracted with a TRIzol reagent (Invitrogen) according to manufacturer instructions. The integrity and quality of the total RNA was evaluated using an Agilent 2100 BioAnalyzer (Agilent Technologies). Forty micrograms of RNA were sent to the Beijing Genomics Institute (BGI) for Solexa analysis. Small RNAs under 50 bases were PAGE-purified and ligated with a pair of Solexa adaptors to their 5 and 3 ends. The small RNAs were then reverse transcribed and amplified by PCR using a pair of adaptor primers. The resulting cDNA library then underwent cluster generation and sequencing analysis using the Illumina HiSeq 2000 sequencing system (Illumina). Raw sequencing data were analyzed using a CLC Genomics Workbench 4.7 (CLC Bio). Raw reads were filtered by discarding low-quality reads and removing adaptor sequences to generate clean and usable reads with sizes 15 nt and 50 nt. To discard unique sequences originating from cellular miRNA and other noncoding RNA, including rRNA, tRNA, small nuclear RNA (snRNA) and small nucleolar RNA (snoRNA), clean reads were first mapped to the miRBase version 18 (www.mirbase. org) and Homo sapiens.GRCh37.65.ncrna (www.ensembl. org). The remaining reads were then mapped to the EV71 geno (...truncated)