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

Abstract

Influenza A virus (IAV) causes annual epidemics and occasional pandemics, and is one of the best-characterized human RNA viral pathogens1. However, a physiologically relevant role for the RNA interference (RNAi) suppressor activity of the IAV non-structural protein 1 (NS1), reported over a decade ago2, remains unknown3. Plant and insect viruses have evolved diverse virulence proteins to suppress RNAi as their hosts produce virus-derived small interfering RNAs (siRNAs) that direct specific antiviral defence4–7 by an RNAi mechanism dependent on the slicing activity of Argonaute proteins (AGOs)8,9. Recent studies have documented induction and suppression of antiviral RNAi in mouse embryonic stem cells and suckling mice10,11. However, it is still under debate whether infection by IAV or any other RNA virus that infects humans induces and/or suppresses antiviral RNAi in mature mammalian somatic cells12–21. Here, we demonstrate that mature human somatic cells produce abundant virus-derived siRNAs co-immunoprecipitated with AGOs in response to IAV infection. We show that the biogenesis of viral siRNAs from IAV double-stranded RNA (dsRNA) precursors in infected cells is mediated by wild-type human Dicer and potently suppressed by both NS1 of IAV as well as virion protein 35 (VP35) of Ebola and Marburg filoviruses. We further demonstrate that the slicing catalytic activity of AGO2 inhibits IAV and other RNA viruses in mature mammalian cells, in an interferon-independent fashion. Altogether, our work shows that IAV infection induces and suppresses antiviral RNAi in differentiated mammalian somatic cells.

Dicer uses distinct modules for recognizing dsRNA termini

“…dmDcr-1 has a degenerate helicase domain and is an ATP-independent enzyme (10), while dmDcr-2, with dedicated antiviral roles (11–13), has a conserved helicase domain that hydrolyzes ATP (14–17). Under certain conditions Homo sapiens Dicer-1 (hsDcr-1) also generates viral siRNAs (18, 19). However, despite conservation of its helicase domain, hsDcr-1 does not hydrolyze ATP in vitro (20), and its helicase domain is not implicated in viral siRNA biogenesis in vivo (19).…”

“…Under certain conditions Homo sapiens Dicer-1 (hsDcr-1) also generates viral siRNAs (18, 19). However, despite conservation of its helicase domain, hsDcr-1 does not hydrolyze ATP in vitro (20), and its helicase domain is not implicated in viral siRNA biogenesis in vivo (19). Differences in activities of the helicase domain of vertebrate and invertebrate Dicers may reflect distinct roles in antiviral defense.…”

“…Unlike dmDcr-2, hsDcr-1 may rely on the Platform-PAZ domain for generating viral siRNAs. Indeed, mutations to the Platform-PAZ domains of hsDcr-1 disrupt viral siRNA biogenesis (19). However, given the conservation of hsDcr-1’s helicase domain, it is intriguing to consider that, under certain conditions, perhaps with additional factors, hsDcr-1 might mediate processive cleavage by threading of dsRNA through the helicase domain.…”

Reply to ‘Questioning antiviral RNAi in mammals’

“…Our study 1 has shown that human HEK-293T, A549 cells and monkey Vero cells produce highly abundant viral siRNAs after infection with either the same PR8 strain of IAV or a related WSN strain, A/WASN/1933(H1N1). Two technical improvements were critical for our success.…”

“…Our article 1 is a follow-up of two published papers in 2013, which provided the first evidence for an antiviral function of RNA interference (RNAi) in mammals 2,3 . The 2013 studies demonstrated production of canonical virus-derived small interfering RNAs (siRNAs) in suckling mice and cultured mouse embryonic stem cells (mESCs) and hamster cells following infection with positive-strand RNA viruses.…”