The Interaction of Influenza A NS1 and Cellular TRBP Protein Modulates the Function of RNA Interference Machinery

Abstract

Influenza A virus (IAV), one of the most prevalent respiratory diseases, causes pandemics around the world. The multifunctional non-structural protein 1 (NS1) of IAV is a viral antagonist that suppresses host antiviral response. However, the mechanism by which NS1 modulates the RNA interference (RNAi) pathway remains unclear. Here, we identified interactions between NS1 proteins of Influenza A/PR8/34 (H1N1; IAV-PR8) and Influenza A/WSN/1/33 (H1N1; IAV-WSN) and Dicer’s cofactor TAR-RNA binding protein (TRBP). We found that the N-terminal RNA binding domain (RBD) of NS1 and the first two domains of TRBP protein mediated this interaction. Furthermore, two amino acid residues (Arg at position 38 and Lys at position 41) in NS1 were essential for the interaction. We generated TRBP knockout cells and found that NS1 instead of NS1 mutants (two-point mutations within NS1, R38A/K41A) inhibited the process of microRNA (miRNA) maturation by binding with TRBP. PR8-infected cells showed masking of short hairpin RNA (shRNA)-mediated RNAi, which was not observed after mutant virus-containing NS1 mutation (R38A/K41A, termed PR8/3841) infection. Moreover, abundant viral small interfering RNAs (vsiRNAs) were detected in vitro and in vivo upon PR8/3841 infection. We identify, for the first time, the interaction between NS1 and TRBP that affects host RNAi machinery.

Impact of the Transboundary Interference Inhibitor on RNAi and the Baculovirus Expression System in Insect Cells

“…It also indicates that VSRs in insect cells share characteristics similar to those found in plant or animal cells, such as the P19 protein’s ability to bind with newly formed 21 nt and 22 nt RNA in plant cells, thereby hindering RISC assembly [ 26 , 44 ]. This regulatory mechanism is not limited to single-stranded RNA viruses but is also observed with FHV-B2 protein, NS1 protein of influenza A virus, Tomato aspermy virus (TAV)-2b protein, and other similar proteins [ 45 , 46 , 47 ]. Despite changes in gene regulation, there was no significant alteration observed within 24 h regarding the rate of apoptosis in Sf9 cells.…”

Research progress on the nonstructural protein 1 (NS1) of influenza a virus

“… 293T cells. A/PR/8/34 (H1N1), A/WSN/33 (H1N1) [ 47 ] 2023 Mutations in the NS1 alter its function in regulating RIG-I-like receptor-mediated antiviral responses. 293T, A549, and MDCK cells.…”

Current advances in antiviral RNA interference in mammals

“…Several studies including ours have now found that vsiRNAs were produced using VSR‐deficient mutant viruses (e.g. B2‐deficient mutant of NoV, NS1‐deficient mutant of IAV, 3A‐deficient mutant of HEV71 and NS2A‐deficient mutant of DENV2) in mammalian cells or suckling mice [19–21,24,25,30,34,57] as well as using wild‐type viruses [e.g. encephalomyocarditis virus (EMCV), ZIKV, Sindbis virus (SINV) and NoV] in undifferentiated cells or somatic cells [22,26–29].…”