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Reoviruses are distributed widely in aquatic environments and have been isolated from a wide range of aquatic organisms. Grass carp reovirus (GCRV) is currently one of the most serious pathogens threatening grass carp Ctenopharyngon idellus production with high mortality in China[29]. The virions consist of a double-layered protein capsid containing 11 dsRNA genomic fragments[38]. GCRV was assigned to the genus Aquareovirus of the family Reoviridae by the international committee on Taxonomy of Viruses (ICTV) in 1991[10]. It differed from orthoreovirus in a number of characteristics such as absence of an antigenic relationship and unequal numbers of genomic segments[2]. Grass carp kidney cells (CIK) are sensitive to GCRV infection and have been adapted for viral amplification and so were employed as an ideal in vitro model in the study of grass carp hemorrhagic disease[26, 35] caused by GCRV infection.
Among the defense systems of organisms, RNA interference (RNAi) is a new-found ancient mechanism of gene regulation with important antiviral roles[26]. A core feature of RNA silencing detected in all organisms is the production of 21-to 26-nt small RNAs cleaved by the endoribonuclease Dicer[13] and the role of RNAi as an important antiviral approach has been reported in most multicellular organisms[36]. Fruit fly Drosophila melanogaster is a model system for studying innate immunity, including antiviral host defense[11]. Long dsRNA, emerging during viral infection and replication or after transposition of mobile genetic elements, can efficiently cause gene-silencing in Drosophila through processing to small interfering RNAs (siRNAs) by the trigger nuclease Dicer[24, 33]. siRNAs targeting specific homologous mRNA molecules cause these mRNA to be degraded by ribonucleases[15]. Dicer is the heart of the RNAi molecular machinery and is responsible for gene silencing and micro-RNA (miRNA) processing for gene regulation[9, 27]. Similar to Drosophilar, plants also use Dicer-like proteins as the central enzymes of RNA silencing, which regulate gene expression and mediate defense against viruses[5]. Null alleles of Dicer result in embryo death in fish and mice[12], which signifies the role of Dicer in cellular basic functions besides RNAi. In all these multicellular organisms, RNAi can be efficiently triggered by either long dsRNAs or 21 to 23-nt-long siRNAs[3]. RNAi mechanism has been increasingly explored in invertebrates and mammalian cells for specific gene silencing[22].
Although GCRV can efficiently infect and replicate in CIK cells, recent studies found that chemically-synthesized siRNA targeting the sequence of its dsRNA genome could efficiently inhibit viral replication in CIK cells[23]. Transgenic Gobiocypris rarus bearing specific siRNA toward the viral genome was resistant to GCRV challenge[21]. These results implied that initiation of GCRV infection could be blocked by the effector nuclease of RNAi pathway downstream of initiator endonuclease Dicer. Similar conclusions were drawn from analysis of mammalian reovirus and avian reovirus[17-19]. These studies supported the premise that a siRNA-mediated RNAi pathway could functionally block the infection and replication of reovirus in CIK cells. We thus deduced that there shouldn't be any virus-specific siRNA in cells during efficient reovirus replication. dsRNAs generated from some RNA viruses were shown to be resistant to Dicer digestion due to their specific secondary structure[7, 32], while more viruses survived from RNAi by encoding RNAi suppressors[8, 25]. To define which strategy has been adopted by GCRV to escape the RNAi-mediated defenses, a Northern blot analysis was designed in this study to monitor the GCRV-specific small molecular RNAs in infected CIK cells as well as cells transfected with extracted genomic dsRNA of GCRV.
Although it was believed that input reovirus dsRNA genome might remain within its inner capsid throughout the viral life cycle to evade cellular innate immune response, it could be that minute amounts of accidentally uncoated or packaged genome, as well as secondary structure on mRNA, could be exposed to the cellular dsRNA-dependent enzymes[16]. Using a mouse specific monoclonal antibody, production of naked dsRNA by positive-strand RNA viruses, dsRNA viruses including reoviruses, and DNA viruses have been confirmed in infected cells[37]. It remains a puzzle, in the late phase of virus replication when the cellular viral dsRNA level is high and viral genome has obviously survived from cellular anti-viral immunity, whether the RNAi pathway is still functioning to silence non-viral reporter genes in the infected cells. In the present study, we designed an in vitro EGFP reporter system to monitor the RNAi effect in the context of GCRV replication. Results from this study could help clarify the mechanism of survival of reovirus genomic dsRNA from RNAi pathway of host cells.
Suppression of RNA Interference Pathway in vitro by Grass Carp Reovirus
- Received Date: 15 November 2011
- Accepted Date: 13 February 2012
Abstract: The means of survival of genomic dsRNA of reoviruses from dsRNA-triggered and Dicer-initiated RNAi pathway remains to be defined. The present study aimed to investigate the effect of Grass carp reovirus (GCRV) replication on the RNAi pathway of grass carp kidney cells (CIK). The dsRNA-triggered RNAi pathway was demonstrated unimpaired in CIK cells through RNAi assay. GCRV-specific siRNA was generated in CIK cells transfected with purified GCRV genomic dsRNA in Northern blot analysis; while in GCRV-infected CIK cells, no GCRV-specific siRNA could be detected. Infection and transfection experiments further indicated that replication of GCRV correlated with the increased transcription level of the Dicer gene and functional inhibition of in vitro synthesized egfp-siRNA in silencing the EGFP reporter gene. These data demonstrated that although only the genomic dsRNA of GCRV was sensitive to the cellular RNAi pathway, unidentified RNAi suppressor protein(s) might contribute to the survival of the viral genome and efficient viral replication.