Abstract：
The production of type I interferon (IFN) is the hallmark of the innate immune response. Most, if not all, mammalian viruses have a way to circumvent this response. Fundamental knowledge on viral evasion of innate immune responses may facilitate the design of novel antiviral therapies. To investigate how human metapneumovirus (HMPV) interacts with the innate immune response, recombinant viruses lacking G, short hydrophobic (SH), or M2-2 protein expression were assessed for IFN induction in A549 cells. HMPV lacking G or SH protein expression induced similarly low levels of IFN, compared to the wild-type virus, whereas HMPV lacking M2-2 expression induced significantly more IFN than the wild-type virus. However, sequence analysis of the genomes of M2-2 mutant viruses revealed large numbers of mutations throughout the genome. Over 70% of these nucleotide substitutions were A-to-G and T-to-C mutations, consistent with the properties of the adenosine deaminase acting on RNA (ADAR) protein family. Knockdown of ADAR1 by CRISPR interference confirmed the role of ADAR1 in the editing of M2-2 deletion mutant virus genomes. More importantly, Northern blot analyses revealed the presence of defective interfering RNAs (DIs) in M2-2 mutant viruses and not in the wild-type virus or G and SH deletion mutant viruses. DIs are known to be potent inducers of the IFN response. The presence of DIs in M2-2 mutant virus stocks and hypermutated virus genomes interfere with studies on HMPV and the innate immune response and should be addressed in future studies. IMPORTANCE Understanding the interaction between viruses and the innate immune response is one of the barriers to the design of antiviral therapies. Here, we investigated the role of the G, SH, and M2-2 proteins of HMPV as type I IFN antagonists. In contrast to other studies, no IFN-antagonistic functions could be observed for the G and SH proteins. HMPV with a deletion of the M2-2 protein did induce type I IFN production upon infection of airway epithelial cells. However, during generation of virus stocks, these viruses rapidly accumulated DIs, which are strong activators of the type I IFN response. Additionally, the genomes of these viruses were hypermutated, which was prevented by generating stocks in ADAR knockdown cells, confirming a role for ADAR in hypermutation of HMPV genomes or DIs. These data indicate that a role of the HMPV M2-2 protein as a bona fide IFN antagonist remains elusive.
摘要：
I型干扰素(IFN)的产生是先天免疫应答的标志。大多数,如果不是全部,哺乳动物病毒有办法规避这种反应。关于病毒逃避先天免疫反应的基本知识可能有助于设计新型抗病毒疗法。为了研究人类偏肺病毒（HMPV）如何与先天免疫反应相互作用，缺乏G的重组病毒，短疏水性（SH），在A549细胞中评估IFN诱导的或M2-2蛋白表达。缺乏G或SH蛋白表达的HMPV诱导类似低水平的IFN，与野生型病毒相比，而缺乏M2-2表达的HMPV比野生型病毒诱导更多的IFN。然而,对M2-2突变病毒基因组的序列分析揭示了整个基因组中的大量突变.超过70％的这些核苷酸取代是A到G和T到C突变，与腺苷脱氨酶作用于RNA（ADAR）蛋白家族的特性一致。通过CRISPR干扰敲除ADAR1证实了ADAR1在编辑M2-2缺失突变病毒基因组中的作用。更重要的是,Northern印迹分析显示在M2-2突变病毒中存在缺陷干扰RNA(DI),而在野生型病毒或G和SH缺失突变病毒中不存在。已知DI是IFN应答的有效诱导物。M2-2突变病毒原种和超突变病毒基因组中DI的存在会干扰HMPV和先天免疫应答的研究，应在未来的研究中解决。重要性了解病毒与先天免疫反应之间的相互作用是抗病毒疗法设计的障碍之一。这里,我们研究了G的作用，SH,和作为I型IFN拮抗剂的HMPV的M2-2蛋白。与其他研究相比，对于G和SH蛋白没有观察到IFN拮抗功能。缺失M2-2蛋白的HMPV在气道上皮细胞感染后确实诱导了I型IFN的产生。然而,在病毒库存的产生过程中，这些病毒迅速积累了DI，是I型IFN应答的强激活剂。此外,这些病毒的基因组发生了过度突变，通过在ADAR击倒细胞中产生股票来阻止，证实ADAR在HMPV基因组或DI超突变中的作用。这些数据表明HMPVM2-2蛋白作为真正的IFN拮抗剂的作用仍然难以捉摸。
