Sullivan Owen M, Nesbitt Daniel J, Schaack Grace A, Feltman Elizabeth M, Nipper Thomas, Kongsomros Supasek, Reed Sevilla G, Nelson Sarah L, King Cason R, Shishkova Evgenia, Coon Joshua J, Mehle Andrew
Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin, USA.
J Virol. 2025 Jul 22;99(7):e0028625. doi: 10.1128/jvi.00286-25. Epub 2025 Jun 11.
Host cells produce a vast network of antiviral factors in response to viral infection. The interferon-induced proteins with tetratricopeptide repeats (IFITs) are important effectors of a broad-spectrum antiviral response. In contrast to their canonical roles, we previously identified IFIT2 and IFIT3 as pro-viral host factors during influenza A virus (IAV) infection. During IAV infection, IFIT2 binds and enhances translation of AU-rich cellular mRNAs, including many IFN-stimulated gene products, establishing a model for its broad antiviral activity. However, IFIT2 also binds viral mRNAs and enhances their translation, resulting in increased viral replication. The ability of IFIT3 to bind RNA and whether this is important for its function was not known. Here, we validate direct interactions between IFIT3 and RNA using electrophoretic mobility shift assays. RNA-binding site identification experiments then identified an RNA-binding surface composed of residues conserved in IFIT3 orthologs and IFIT2 paralogs. Mutation of the RNA-binding site reduced the ability of IFIT3 to promote IAV gene expression and translation efficiency compared to wild-type IFIT3. The functional units of IFIT2 and IFIT3 are homo- and heterodimers; however, the RNA-binding surfaces are located near the dimerization interface. Using co-immunoprecipitation, we showed that mutations to these sites do not affect dimerization. Together, these data establish the link between IFIT3 RNA binding and its ability to modulate translation of viral mRNAs during IAV infection.IMPORTANCEInfluenza A viruses (IAVs) cause considerable morbidity and mortality through sporadic pandemics as well as annual epidemics. Zoonotic IAV strains pose an additional risk of spillover into a naive human population where prior immunity may have minimal effect. In this case, the first line of defense in the host is the innate immune response. Interferon-stimulated genes (ISGs) produce a suite of proteins that are front-line effectors of innate immune responses. While ISGs are typically considered antiviral, new research has revealed an emerging trend where viruses co-opt ISGs for pro-viral function. Here, we determine how the ISG IFIT3 is used by IAV as a pro-viral factor, advancing our understanding of IFIT3 function generally and specifically in the context of IAV infection.
宿主细胞在受到病毒感染时会产生一个庞大的抗病毒因子网络。干扰素诱导的具有四肽重复序列的蛋白（IFITs）是广谱抗病毒反应的重要效应分子。与它们的经典作用相反，我们之前发现IFIT2和IFIT3在甲型流感病毒（IAV）感染期间是病毒宿主因子。在IAV感染期间，IFIT2结合并增强富含AU的细胞mRNA的翻译，包括许多干扰素刺激的基因产物，从而建立了其广泛抗病毒活性的模型。然而，IFIT2也结合病毒mRNA并增强其翻译，导致病毒复制增加。IFIT3与RNA结合的能力以及这对其功能是否重要尚不清楚。在这里，我们使用电泳迁移率变动分析验证了IFIT3与RNA之间的直接相互作用。RNA结合位点鉴定实验随后确定了一个由IFIT3直系同源物和IFIT2旁系同源物中保守的残基组成的RNA结合表面。与野生型IFIT3相比，RNA结合位点的突变降低了IFIT3促进IAV基因表达和翻译效率的能力。IFIT2和IFIT3的功能单位是同二聚体和异二聚体；然而，RNA结合表面位于二聚化界面附近。通过免疫共沉淀，我们表明这些位点的突变不影响二聚化。总之，这些数据建立了IFIT3 RNA结合与其在IAV感染期间调节病毒mRNA翻译能力之间的联系。
重要性
甲型流感病毒（IAVs）通过偶发的大流行以及每年的流行导致相当高的发病率和死亡率。人畜共患的IAV毒株还存在传播到未接触过该病毒的人群中的额外风险，在这些人群中先前的免疫力可能影响极小。在这种情况下，宿主的第一道防线是先天免疫反应。干扰素刺激基因（ISGs）产生一系列蛋白，它们是先天免疫反应的一线效应分子。虽然ISGs通常被认为具有抗病毒作用，但新的研究揭示了一种新趋势，即病毒会利用ISGs发挥病毒促进功能。在这里，我们确定了IAV如何将ISG IFIT3用作病毒促进因子，这有助于我们全面且具体地了解IFIT3在IAV感染背景下的功能。
