Production of functional small interfering RNAs by an amino-terminal deletion mutant of human Dicer

Abstract

Although RNA interference (RNAi) functions as a potent antiviral innate-immune response in plants and invertebrates, mammalian somatic cells appear incapable of mounting an RNAi response and few, if any, small interfering RNAs (siRNAs) can be detected. To examine why siRNA production is inefficient, we have generated double-knockout human cells lacking both Dicer and protein kinase RNA-activated. Using these cells, which tolerate double-stranded RNA expression, we show that a mutant form of human Dicer lacking the amino-terminal helicase domain can process double-stranded RNAs to produce high levels of siRNAs that are readily detectable by Northern blot, are loaded into RNA-induced silencing complexes, and can effectively and specifically inhibit the expression of cognate mRNAs. Remarkably, overexpression of this mutant Dicer, but not wild-type Dicer, also resulted in a partial inhibition of Influenza A virus—but not poliovirus—replication in human cells.

Induction and suppression of antiviral RNA interference by influenza A virus in mammalian cells

“…3d). These findings define a new activity of NS1 in inhibiting the production of its cognate vsiRNAs induced by an authentic virus infection of human somatic cells, and explains why canonical vsiRNAs are largely undetectable during wild-type IAV infection 14,15,25 . Next, we investigated if this assay could be used to identify VSRs capable of suppressing the biogenesis of human vsiRNAs.…”

“…Here, we use this strategy to search for vsiRNAs from IAV-infected human somatic cells. NS1 of IAV suppresses both antiviral RNAi in Drosophila cells and engineered RNAi in plant and mammalian cells 2,22–25 , and shares strong structural similarity 3 in dsRNA binding with the nodaviral VSR B2 proteins, known to inhibit the biogenesis of vsiRNAs in animal cells 10,11,26 . IAV contains a negative-strand RNA genome divided into eight segments, and NS1, encoded by the smallest genome segment, is multifunctional and essential for in vivo infection and virulence 1,3 .…”

The Epstein-Barr virus miR-BHRF1 microRNAs regulate viral gene expression in cis

“…293T cells were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 5% FBS, 50 μg/ml gentamicin, and 1x Antibiotic-Antimycotic. NoDice/ΔPKR cells (Kennedy et al, 2015) and RNaseIII−/− 293T cells (Aguado et al, 2017) were grown in DMEM supplemented with 10% FBS, 50 μg/ml gentamicin, and 1x Antibiotic-Antimycotic.. All cells were grown at 37°C with 5% CO 2 .…”

Reply to ‘Questioning antiviral RNAi in mammals’

“…Indeed, ectopic expression of NS1 can suppress production of siRNAs from either artificial long dsRNA 12 or influenza viral dsRNA replicative intermediates 1 . We have also detected abundant virus-specific small RNAs not associated with RISC in infected human cells, which must be removed before the canonical properties of the viral siRNAs are visible 1 .…”