Host defense against DNA virus infection in shrimp is mediated by the siRNA pathway

Abstract

The RNA interference (RNAi) system of eukaryotes using siRNAs has been documented as an immune response against invasion by RNA viruses. However, whether the siRNA pathway can be triggered by the infection with DNA viruses in animals remains to be investigated. In the present study, we show that Marsupenaeus japonicus shrimp can generate an antiviral siRNA (vp28-siRNA) in response to infection by a double-stranded DNA virus, white spot syndrome virus (WSSV). After challenging with WSSV, vp28-siRNA is detected in all the WSSV-infected organs and tissues of shrimp as early as 24 h postinfection (p.i.). The results indicate that the host Dicer2 and Ago2 proteins are required for the biogenesis and function of vp28-siRNA, respectively. We show further that vp28-siRNA predominates in the cytoplasm of shrimp hemocytes at 48 h p.i. Knockdown of Dicer2 by special siRNA or inhibition of vp28-siRNA with locked nucleic acid antisense oligonucleotides both lead to a significant increase in WSSV copy number at 24-48 h p.i. Our study highlights a novel aspect of the siRNA pathway in the immune response of animals against infection by DNA viruses.Keywords: Ago2 r Dicer2 r Shrimp r siRNA r WSSV See accompanying Commentary by Sabin and Cherry IntroductionRNA interference (RNAi) is an evolutionarily conserved mechanism that involves the triggering of adaptive immune responses against RNA viruses by small interfering RNAs (siRNAs) in eukaryotes [1][2][3][4][5]. The double-stranded (ds) siRNAs of approximately 21 base pairs in length are generated from dsRNAs, such as those from RNA viruses, endogenous transposons, or other genomic regions that produce transcripts capable of forming dsRNA structures [6][7][8][9][10][11]. The siRNAs processed from RNA viruses provide antiviral defense in plants and animals through the cleavage and degradation of target viral mRNA [12][13][14][15].The mechanism by which eukaryotic organisms use RNA silencing to combat virus infections was first discovered in plants and invertebrates [16,17]. In plants, either RNA or DNA virus infecCorrespondence: Prof. Xiaobo Zhang e-mail: zxb0812@zju.edu.cn tion can trigger host Dicer-like proteins to produce virus-derived siRNAs (viRNAs) that destroy the RNAs of invading viruses [18][19][20]. To further amplify these antiviral molecules, secondary viRNAs are produced by RNA-dependent RNA polymerases (RdRPs) that generate more dsRNAs from these cleaved transcript fragments, suggesting that the formation of dsRNA is necessary to initiate the synthesis of antiviral siRNA [21,22]. Similar amplification of viRNA with RdRPs was also discovered in Caenorhabditis elegans [5]. However, no RdRP homologs that convert single-stranded (ss) viral transcripts to dsRNAs have been identified thus far in flies or mammals. Recently, DNA viRNA were identified in mosquitoes using high-throughput sequencing of small RNAs, but the underlying mechanism of viral siRNA biogenesis remains unclear [23]. Although the antiviral activities of siRNAs against RNA viruses have been well documented,...

Involvement of Viral MicroRNA in the Regulation of Antiviral Apoptosis in Shrimp

“…Recently, it was revealed that hosts can use virus-derived siRNA or cellular miRNA to restrict viral propagation (18,(21)(22)(23)(24). In turn, a number of viruses, in particular DNA viruses, seem to have evolved mechanisms to express their own miRNAs and employ miRNA-mediated regulations to counter host antiviral responses (25)(26)(27).…”

White spot syndrome virus VP28 specific double-stranded RNA provides protection through a highly focused siRNA population

“…Previously, it has been shown that LvDcr2 and LvAgo2 are directly involved in the RNAi response 17, 18 , while LvSid-1 is a putative dsRNA transport molecule potentially involved in uptake and communication of a dsRNA signal between cells 19 . We show how dsRNA induces upregulation of these genes (Fig.…”

“…As for Drosophila 14–16 , P. vannamei has been found to encode two variants of Dicer and Argonaute, and among these LvDcr2 and LvAgo2 are employed by the siRNA pathway 17–19 . Although Dcr1 and Ago1 are normally associated with the miRNA pathway in Drosophila 14, 20 , a possible involvement in the antiviral response has been shown for homologue genes in white leg shrimp 21–23 .…”

Roles of small RNAs in the immune defense mechanisms of crustaceans

“…In addition, synthesized dsRNA confers protective effects against YHV infection in P. monodon, and the effect lasts for five days (Yodmuang et al, 2006). Continuous injections of vp28-siRNA at different times post-challenge completely eliminate WSSV in M. japonicus shrimp Huang and Zhang, 2013). When administered prophylactically to shrimp before a viral challenge, in vitro-transcribed long dsRNA or siRNA corresponding to viral genes vp28, vp281, and vp19 results in a high survival rate (Kim et al, 2007;Xu et al, 2007).…”