Mechanism and Function of Antiviral RNA Interference in Mice

Abstract

Distinct mammalian RNA viruses trigger Dicer-mediated production of virus-derived small-interfering RNAs (vsiRNA) and encode unrelated proteins to suppress vsiRNA biogenesis. However, the mechanism and function of the mammalian RNA interference (RNAi) response are poorly understood. Here, we characterized antiviral RNAi in a mouse model of infection with Nodamura virus (NoV), a mosquito-transmissible positive-strand RNA virus encoding a known double-stranded RNA (dsRNA)-binding viral suppressor of RNAi (VSR), the B2 protein. We show that inhibition of NoV RNA replication by antiviral RNAi in mouse embryonic fibroblasts (MEFs) requires Dicer-dependent vsiRNA biogenesis and Argonaute-2 slicer activity. We found that VSR-B2 of NoV enhances viral RNA replication in wild-type but not RNAi-defective MEFs such as Argonaute-2 catalytic-dead MEFs and Dicer or Argonaute-2 knockout MEFs, indicating that VSR-B2 acts mainly by suppressing antiviral RNAi in the differentiated murine cells. Consistently, VSR-B2 expression in MEFs has no detectable effect on the induction of interferon-stimulated genes or the activation of global RNA cleavages by RNase L. Moreover, we demonstrate that NoV infection of adult mice induces production of abundant vsiRNA active to guide RNA slicing by Argonaute-2. Notably, VSR-B2 suppresses the biogenesis of both vsiRNA and the slicing-competent vsiRNA-Argonaute-2 complex without detectable inhibition of Argonaute-2 slicing guided by endogenous microRNA, which dramatically enhances viral load and promotes lethal NoV infection in adult mice either intact or defective in the signaling by type I, II, and III interferons. Together, our findings suggest that the mouse RNAi response confers essential protective antiviral immunity in both the presence and absence of the interferon response. IMPORTANCE Innate immune sensing of viral nucleic acids in mammals triggers potent antiviral responses regulated by interferons known to antagonize the induction of RNA interference (RNAi) by synthetic long double-stranded RNA (dsRNA). Here, we show that Nodamura virus (NoV) infection in adult mice activates processing of the viral dsRNA replicative intermediates into small interfering RNAs (siRNAs) active to guide RNA slicing by Argonaute-2. Genetic studies demonstrate that NoV RNA replication in mouse embryonic fibroblasts is inhibited by the RNAi pathway and enhanced by the B2 viral RNAi suppressor only in RNAi-competent cells. When B2 is rendered nonexpressing or nonfunctional, the resulting mutant viruses become nonpathogenic and are cleared in adult mice either intact or defective in the signaling by type I, II, and III interferons. Our findings suggest that mouse antiviral RNAi is active and necessary for the in vivo defense against viral infection in both the presence and absence of the interferon response.

Zebrafish as an animal model for the antiviral RNA interference pathway

“…Notably, after normalization of vsiRNAs by viral abundance, we observed that greater abundance of vsiRNA was induced by NoVΔB2 compared to NoV WT ( Fig. 2f, Table 1), indicating that NoV B2 protein potentially counteracted the production of vsiRNAs in zebrafish as in mammals [9,13]. These results suggest that NoV could be used to dissect antiviral RNAi in zebrafish.…”

“…The virion of NoV contains a bipartite positive-sense viral genome designated RNA1 and RNA2, which is responsible for the production of RNA-dependent RNA polymerase (RdRp) and viral capsid protein, respectively [33]. Non-structural protein B2 produced from subgenomic RNA3 has been clearly demonstrated as VSR [9,13,34,35]. The available evidence suggests that some members of Batenodavirus, another subgroup of nodaviruses, can robustly infect a myriad of fish species and have a devastating effect on the aquaculture industry [36,37].…”

“…The underlined mechanism needs to be further investigated. NoV is another positive-sense RNA virus that performs excellently in the investigation of antiviral RNAi in mammals, and the non-structural protein B2 is a well-documented suppressor to significantly inhibit production of vsiRNAs [9,13]. In fact, previous studies have suggested that Betanodavirus is capable of infecting fish and B2 can act as a suppressor to inhibit antiviral RNAi in a similar mode of action to Alphanodavirus [36,37,44].…”

“…established an in vitro slicing assay to illuminate the role of mammalian Ago2 in antiviral RNAi [13]. However, there has a remarkable limitation in the evaluation of Ago2 activity in vivo, given that knockout of Ago2 produces an embryoniclethal phenotype that restricts direct proof to demonstrate antiviral RNAi in mammals [17,48].…”

“…Progress in the area of mammalian antiviral RNAi has fostered a debate about the relationship between the interferon response and antiviral RNAi [4]. Although some studies suggest that the interferon response may mask or inhibit antiviral RNAi in differentiated mammalian cells [15, 38, 39], we and others have provided evidence that AGO-loaded, Dicer-dependent vsiRNAs can not only be detected, but also execute the antiviral activity in the IFN-competent cells [10, 11, 13]. Given this cross-interaction between innate immunity and antiviral RNAi [40], the interferon pathway-related genes of zebrafish larvae have also been examined.…”