RNA interference functions as an antiviral immunity mechanism in mammals

Abstract

Diverse eukaryotic hosts produce virus-derived small interfering RNAs (siRNAs) to direct antiviral immunity by RNA interference (RNAi). However, it remains unknown whether the mammalian RNAi pathway has a natural antiviral function. Here, we show that infection of hamster cells and suckling mice by Nodamura virus (NoV), a mosquito-transmissible RNA virus, requires RNAi suppression by its B2 protein. Loss of B2 expression or its suppressor activity leads to abundant production of viral siRNAs and rapid clearance of the mutant viruses in mice. However, viral small RNAs detected during virulent infection by NoV do not have the properties of canonical siRNAs. These findings have parallels with the induction and suppression of antiviral RNAi by the related Flock house virus in fruit flies and nematodes and reveal a mammalian antiviral immunity mechanism mediated by RNAi.

Fig. 1. siRNA properties of vsRNAs in BHK-21 cells

(A) Length distribution and abundance of positive- or negative-strand vsRNAs from cells 2 or 3 dpi with NoV or NoVΔB2. (B) Total counts of pairs of complementary 22-nt vsRNAs of NoV or NoVΔB2 in each distance category (in nucleotides) between 5′ and 3′ ends of a complementary vsRNA pair, defined as 0 for perfect base-paired 22-nt vsRNAs with blunt ends, –2 for pairs with 2-nt overhang at the 3′-end of each strand (α and β), or 20 for pairs with 20-nt overhang at the 5′-ends (α and γ).

Fig. 2. NoV infection requires RNAi suppression

(A) BHK-21 cells or BHK cells stably expressing B2 or VP35 were mock-infected or infected by NoVΔB2 or NoV of the same titer. Every 12 hours postinfection (hpi), the viral genomic RNA1 levels were determined by quantitative RT-PCR with the accumulation level of NoVΔB2 in BHK-21 cells at 12 hpi set as 1. Error bars indicate standard deviation of three replicates. (B) Accumulation of NoV and NoVΔB2 RNAs 1 to 3 in the infected cells detected by Northern blotting. RNA1 signal quantified by phosphorimaging was shown with that of NoVΔB2 in BHK-21 cells (lanes 4) set as 1. Detection of B2 transgene mRNA (arrow) was visible. 18S rRNA staining served as loading control.

Fig. 3. In vivo virus clearance associated with production of viral siRNAs

(A and B) Accumulation of NoV, NoVΔB2, and NoVmB2 in mouse fore- (F) and hind- (H) limb tissues detected by quantitative RT-PCR of the viral RNA1 and Northern blotting, respectively. NoVΔB2 level in hind limb at 1 dpi was set as 1, and error bars indicate standard deviation of three replicates (A). NoV RNAs 1 and 2 (arrows) were visible after rRNA staining to show equal loading (B). (C) Suckling mice remained as healthy 4 weeks post-infection with either NoVΔB2 (right) or NoVmB2 (not shown) as mock-inoculated mice (left), whereas all of the five NoV-inoculated mice died by 5 dpi (not shown). (D) Northern blot detection of negative-strand viral siRNAs in mice infected with NoVΔB2 (middle) or NoVmB2 (left) and of vsRNAs from NoV-infected mice (right). The hybridizing positions of four siRNA probes were given in Fig. 4B, and size markers were synthetic 21- and 25-nt RNAs. The same filters were probed for mouse microRNA 127 (miR-127) and U6 RNA as loading controls. At least three independent repeats with reproducible results were performed with each experiment.

Fig. 4. Properties of mouse viral siRNAs produced in vivo

(A) Length distribution and abundance of positive- or negative-strand vsRNAs from mice 1 or 2 dpi with NoVΔB2 or with NoV at 4 dpi. (B) Total counts of pairs of complementary 22-nt vsRNAs of NoVΔB2 and NoV in each distance category as defined in Fig. 1B. (C) Virus genome distribution of 21-to 23-nt viral siRNAs sequenced from either sucking mice (top two panels) or BHK-21 cells (bottom two panels) after infection by NoVΔB2. The functional proteins encoded by the viral bipartite RNA genome and transcription of B2 mRNA (RNA3) from RNA1 are shown. Arrows indicate the positions of the four locked nucleic acid probes used to detect negative-strand viral siRNAs in mice.