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. 2015 Nov 17;13(7):1456-1466.
doi: 10.1016/j.celrep.2015.10.020. Epub 2015 Nov 5.

Engineered Mammalian RNAi Can Elicit Antiviral Protection that Negates the Requirement for the Interferon Response

Asiel Arturo Benitez  1 , Laura Adrienne Spanko  1 , Mehdi Bouhaddou  2 , David Sachs  3 , Benjamin Robert tenOever  4
Affiliations

Engineered Mammalian RNAi Can Elicit Antiviral Protection that Negates the Requirement for the Interferon Response

Asiel Arturo Benitez et al. Cell Rep. .

Abstract

Although the intrinsic antiviral cell defenses of many kingdoms utilize pathogen-specific small RNAs, the antiviral response of chordates is primarily protein based and not uniquely tailored to the incoming microbe. In an effort to explain this evolutionary bifurcation, we determined whether antiviral RNAi was sufficient to replace the protein-based type I interferon (IFN-I) system of mammals. To this end, we recreated an RNAi-like response in mammals and determined its effectiveness to combat influenza A virus in vivo in the presence and absence of the canonical IFN-I system. Mammalian antiviral RNAi, elicited by either host- or virus-derived small RNAs, effectively attenuated virus and prevented disease independently of the innate immune response. These data find that chordates could have utilized RNAi as their primary antiviral cell defense and suggest that the IFN-I system emerged as a result of natural selection imposed by ancient pathogens.

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Figures

Figure 1
Figure 1. Determining miRNA-mediated silencing potential
(A) Gaussia Luciferase activity from 293T cells co-transfected with a vector expressing miR124 (p124) and a luciferase reporter containing a single miR-124 site with complementarity ranging from 10nts (10×1T) to 20nts (20×1T). Renilla luciferase was used to control for transfection efficiency. Error bars, SD, *p<0.05. (B) As described in (A), where the Gaussia Luciferase construct had two sites of either 16 or 20nts of complementarity. (C) Western blot of protein derived from MDCK or MDCK-124 cells infected with IAV (MOI=0.01) containing two or four target sites described in (A). Top two panels denote NP and actin from MDCK cells, bottom two panels depict the same from MCDK-124 cells. (D) Virus titers derived from multi-cycle growth curves in MDCK or MDCK-124 cells treated with the viruses described in (C). Error bars, SD, *p<0.05. (E) Flow cytometry-based determination of MDCK and MDCK-124 levels following mock infection or treatment with the scrambled (Scbl) or miRNA-targeted (Targeted) virus at 72 hpi.
Figure 2
Figure 2. Self-targeting IAV demonstrates the selective pressure imposed by miRNA-mediated silencing
(A) Schematic depicting a self-targeted virus producing a siRNA against the NP segment (siNP). (B) Northern blot of RNA derived from MDCK cells infected (MOI=1) with IAV-siNP or IAV-si124. Top panel depicts siNP, and lower panel shows U6 as a loading control. (C) Western blot of extracts derived from 293T or NoDice cells infected (MOI=0.1) with IAV-siNP or IAV-si124 for 12 and 24 hpi. Panel depicts NP, NS1, and actin as a loading control. (D) Multi-cycle growth curve in MDCK cells infected with the viruses described in (B). Supernatants were collected at 12, 24, 36, and 48 hpi and plaqued on MDCK cells. Error bars, SD, *p<0.05. (E) Schematic depicting the hairpin in the parental virus (top) and the mutant virus (bottom). (F) Northern blot in MDCK cells infected (MOI=1) with WT IAV and the viruses described in (D). Top panel depicts siNP and lower panel shows U6 as a loading control. (G) Multi-cycle growth in MDCK cells with the viruses in (F). Supernatants were collected and plaqued on MDCK cells. Error bars, SD, *p<0.05.
Figure 3
Figure 3. Determining the role of NS1 during self-targeting
(A) Multi-cycle growth in MDCK cells infected (MOI=0.01) with the corresponding viruses. Supernatants were collected 24 hpi and plaqued on MDCK cells. Error bars, SD, *p<0.05. (B) Schematic depicting the hairpin in the parental virus (top) and the mutant virus (bottom). The siRNA against NP (siNP) is depicted in red. (C) Northern blot of MDCK cells infected with IAV, 16×4-mutant, or 16×4-control viruses. Top panel depicts miR-124 and lower shows U6 as loading control. (D) Virus titers from MDCK cells infected with the viruses in (C). (E) Northern blot of MDCK cells infected with IAV, 20×4-mutant, or 20×4-control viruses. Top panel depicts miR-124 and lower shows U6 as loading control. (F) Virus titers from MDCK cells infected with the viruses in (E). Error bars, SD, *p<0.05. (G) Virus titers from Wt or Irf3−/−/Irf7−/− MEF infected (MOI=1) with IAV, IAV-124, mIAV-124, IAV-124t/wtNS-124, and IAV-124t/mNS-124. Supernatants were collected 24 hpi and plaqued on MDCK cells. Error bars, SD, *p<0.05. (H) Fold titer of the viruses used in (G). Error bars, SD, *p<0.05.
Figure 4
Figure 4. Engineered antiviral RNAi can potently inhibit virus replication in mammals
(A) Western blot of extracts derived from NoDice cells infected (MOI=0.1) with IAV, IAV-Scbl, or with increasing amounts of the NP-targeted viruses (IAV-NPt) for 24hrs. Top panel depicts NS1, and the lower panel shows NP and actin as a loading control. Bracketed values represent egg infectious dose 50 (EID50) units for Scbl and IAV-NPt. (B) Multi-cycle growth curve in A549s cells infected (MOI=0.01) with the viruses used in (A). Supernatants were collected 12, 24, 36, and 48 hpi and plaqued on MDCK cells. LOD denotes the level of detection. (C) Western blot of extracts derived from A549 cells infected (MOI=0.1) with the viruses in (A). Lysates were collected 10, and 20 hpi and blotted for NP and actin.
Figure 5
Figure 5. in vivo demonstration of small RNA-mediated attenuation of virus in both wild type and IFN-defect animals
(A) Graph depicting change in body mass of C57BL mice following intranasal inoculation with PBS, IAV, or IAV-NPt. Error bars, SD, *p<0.05. (B) Histology of lungs from mice infected with IAV, or IAV-NPt. Lungs were harvested 2 and 9 dpi, sectioned and slides were stained with H&E. (C) Graph depicting change in body mass of Ifnar1−/− mice following intranasal inoculation with PBS, IAV, or IAV-NPt. Error bars, SD, *p<0.05.
Figure 6
Figure 6. Attenuation of miRNA-mediated viruses does not require the intrinsic antiviral response
(A and B) Heat maps depicting the transcriptomes of WT or Ifnar1−/− mice infected for 2 and 9 days with IAV or the IAV-NPt viruses based on biological replicates of RNA-Seq data.
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