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. 2021 Aug 3;17(8):e1009790. doi: 10.1371/journal.ppat.1009790

Fig 1. Efficient Dicer processing of IAV derived-dsRNA in human 293T cells active in type I IFN response.

Fig 1

A. Induction of type I IFN response in NoDice cells. NoDice cells were mock-inoculated or infected with PR8/delNS1 at 6 hours post-transfection with the plasmid vector, an hDcr-expressing plasmid alone or together another plasmid to express IAV-NS1 or ZIKV-NS1. The accumulation levels of IFN-β, RIG-I, MAVS, ISG15, ISG54, and ISG56 mRNAs were determined by RT-qPCR at 24 hours post-infection. Each experiment was repeated at least three times independently and error bars indicate the standard deviation. The mRNA level of mock-inoculated cells without plasmid transfection was set as 1. ** indicates p<0.01, *** indicates p<0.001, **** indicates p<0.0001 (Student’s t-test). ns indicates no significance. B. Western blotting detection of the ectopically expressed human Dicer and/or IAV-NS1 /hDcr+ZIKV-NS1 as described in (A). Endogenous β-actin was detected as a loading control. C. Relative abundance of cellular miRNAs and IAV-derived 21- to 23-nt small RNAs per million total 18- to 28-nt reads in the individual small RNA libraries prepared from the NoDice cells at 24 hours post-infection as described in (A). D-F. Size distribution of IAV-derived 18- to 28-nt small RNAs (left) and duplex pattern of 22-nt IAV vsiRNAs (right) in the 3 libraries (no. 3, 4 and 5) presented in (C) from PR8/delNS1-infected NoDice cells ectopically expressing hDcr alone (D) or with IAV-NS1 (E) or ZIKV-NS1 (F). Data information: Reads were shown as per million total 18- to 28-nt reads. The 5’ terminal nucleotide of 18- to 28-nt viral small RNAs was indicated by color and 1U % of 21- to 23-nt vsiRNA given in parenthesis. The “-2” peak corresponded to the pair of canonical 22-nt vsiRNAs with a 20-nt duplex region plus 2-nt 3’ overhangs, calculated by an algorithm described previously counting pairs of complementary 22-nt vsiRNAs in each distance category (in nucleotides) between 5’ and 3’ ends of each pair. Length (nt) indicates x-axis for size distribution. Distance category indicates x-axis for duplex pattern. G. In vitro dicing of a synthetic 200 bp dsRNA by FLAG-tagged wild-type (lanes 3 & 4) or mutant (lane 2) human Dicer immune-precipitated from NoDice cells with or without PR8/delNS1. The Dicer substrate and product RNAs were fractionated by 15% PAGE and detected by GelRed staining. Lane 1: 22bp marker. Each experiment was repeated at least three times independently with one represented image shown.