The interaction of influenza A NS1 and cellular TRBP protein modulates the function of RNA interference machinery. The interaction of influenza A NS1 and cellular TRBP protein modulates the function of RNA interference machinery

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. Overall design: 5 virus-derived small RNA from 293T cells or mice lung infected with PR8/3841 or PR8-WT was detected by small RNA-seq.

甲型流感病毒（Influenza A virus, IAV）是全球范围内最流行的呼吸道传染病病原体之一，可引发世界性大流行。IAV的多功能非结构蛋白1（non-structural protein 1, NS1）是一类病毒拮抗因子，能够抑制宿主的抗病毒免疫应答。然而，NS1调控RNA干扰（RNA interference, RNAi）通路的具体分子机制仍不明确。本研究鉴定了甲型流感病毒A/PR8/34（H1N1；IAV-PR8）与A/WSN/1/33（H1N1；IAV-WSN）的NS1蛋白与Dicer的辅助因子TAR RNA结合蛋白（TAR-RNA binding protein, TRBP）之间的相互作用。研究发现，NS1的N端RNA结合结构域（RNA binding domain, RBD）以及TRBP蛋白的前两个结构域介导了该相互作用。此外，NS1中两个氨基酸残基（第38位精氨酸与第41位赖氨酸）对该相互作用至关重要。我们构建了TRBP基因敲除细胞系，并发现野生型NS1（而非携带R38A/K41A双点突变的NS1突变体）可通过结合TRBP，抑制微小RNA（microRNA, miRNA）的成熟过程。感染PR8野生型病毒的细胞会出现短发夹RNA（short hairpin RNA, shRNA）介导的RNAi通路被遮蔽的现象，而感染携带NS1 R38A/K41A突变的重组病毒（命名为PR8/3841）的细胞则未观察到此现象。进一步研究发现，在体外与体内感染PR8/3841病毒后，均可检测到大量病毒小干扰RNA（viral small interfering RNAs, vsiRNAs）。本研究首次揭示了NS1与TRBP的相互作用可影响宿主RNAi通路的功能。总体实验设计：通过小RNA测序（small RNA-seq），检测了PR8/3841或PR8野生型病毒感染的293T细胞及小鼠肺组织中的5份病毒源性小RNA样本。