Human Metapneumovirus M2-2 Protein Acts as a Negative Regulator of Alpha Interferon Production by Plasmacytoid Dendritic Cells
Human Metapneumovirus M2-2 Protein Acts as a Negative Regulator of Alpha Interferon Production by Plasmacytoid Dendritic Cells
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DOI：
10.1128/jvi.00579-17
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发表时间：
2017-10-01
影响因子：
5.4
通讯作者：
Gotoh, Bin
中科院分区：
文献类型：
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作者：
Kitagawa, Yoshinori;Sakai, Madoka;Gotoh, Bin
人偏肺病毒（HMPV）具有抑制浆细胞样树突状细胞（pDC）产生Toll样受体7（TLR 7）和TLR 9依赖性α干扰素（IFN-α）的能力。然而，其抑制机制在很大程度上仍然未知。为了鉴定负责这种抑制作用的病毒蛋白，我们对HMPV开放阅读框（ORF）进行了筛选，以确定其阻断通过转染髓样分化因子88（MyD 88）、肿瘤坏死因子受体相关因子6（TRAF 6）、IKK α和IFN调节因子7（IRF 7）在HEK 293 T细胞中重建的TLR 7/9依赖性信号传导的能力。该筛选证明M2-2蛋白是TLR 7/9依赖性IFN-α诱导的最有效的抑制剂。其中M2-2 ORF被沉默的重组HMPV确实诱导了比野生型HMPV更高的人pDC产生IFN-α。免疫沉淀实验显示M2-2蛋白与IRF 7的抑制结构域（ID）直接物理关联。作为其自然结果，缺乏ID的IRF 7（组成型活性突变体）的转染导致IFN-α启动子的活化，即使在M2-2存在下也是如此。生物发光共振能量转移试验和裂解海肾荧光素酶互补试验表明，M2-2抑制MyD 88/TRAF 6/IKK α诱导的IRF 7同源二聚化。相比之下，M2-2蛋白的表达不会导致IPS-1诱导的同源二聚化的抑制和IRF 7的最终活化。这表明MyD 88/TRAF 6/IKK α诱导的IRF 7同源二聚化的抑制不是由M2-2结合的空间效应引起的。相反，发现M2-2抑制MyD 88/TRAF 6/IKK α诱导的IRF 7在Ser 477上的磷酸化。这些结果表明，M2-2阻断TLR 7/9依赖性IFN-α的诱导，通过阻止IRF 7同源二聚化，可能通过其对IRF 7的磷酸化状态的影响。重要信息副粘病毒科分为两个亚科，副粘病毒亚科和肺炎病毒亚科。副粘病毒亚科的成员具有抑制TLR 7/9依赖性IFN-α产生的能力，并且已经深入研究了潜在的抑制机制。相反，关于肺炎病毒亚科成员如何调节pDC产生IFN-α的情况知之甚少。我们鉴定了HMPV（肺炎病毒亚科的一个成员）的M2-2蛋白作为pDC产生IFN-α的负调节剂，并揭示了潜在的机制。这项研究部分解释了为什么M2-2敲除重组HMPV是减毒的，并进一步表明M2-2是HMPV治疗的潜在靶点。
Human metapneumovirus (HMPV) has the ability to inhibit Toll-like receptor 7 (TLR7)-and TLR9-dependent alpha interferon (IFN-alpha) production by plasmacytoid dendritic cells (pDCs). However, the inhibition mechanism remains largely unknown. To identify viral proteins responsible for this inhibition, we performed a screening of HMPV open reading frames (ORFs) for the ability to block TLR7/9dependent signaling reconstituted in HEK293T cells by transfection with myeloid differentiation factor 88 (MyD88), tumor necrosis factor receptor-associated factor 6 (TRAF6), IKK alpha, and IFN regulatory factor 7 (IRF7). This screening demonstrated that the M2-2 protein was the most potent inhibitor of TLR7/9-dependent IFN-alpha induction. A recombinant HMPV in which the M2-2 ORF was silenced indeed induced greater IFN-alpha production by human pDCs than wild-type HMPV did. Immunoprecipitation experiments showed direct physical association of the M2-2 protein with the inhibitory domain (ID) of IRF7. As a natural consequence of this, transfection of IRF7 lacking the ID, a constitutively active mutant, resulted in activation of the IFN-alpha promoter even in the presence of M2-2. Bioluminescence resonance energy transfer assays and split Renilla luciferase complementation assays revealed that M2-2 inhibited MyD88/TRAF6/IKK alpha-induced homodimerization of IRF7. In contrast, expression of the M2-2 protein did not result in inhibition of IPS-1-induced homodimerization and resultant activation of IRF7. This indicates that inhibition of MyD88/TRAF6/IKK alpha induced IRF7 homodimerization does not result from a steric effect of M2-2 binding. Instead, it was found that M2-2 inhibited MyD88/TRAF6/IKK alpha -induced phosphorylation of IRF7 on Ser477. These results suggest that M2-2 blocks TLR7/9-dependent IFN-alpha induction by preventing IRF7 homodimerization, possibly through its effects on the phosphorylation status of IRF7.IMPORTANCE The family Paramyxoviridae is divided into two subfamilies, the Paramyxovirinae and the Pneumovirinae. Members of the subfamily Paramyxovirinae have the ability to inhibit TLR7/9-dependent IFN-alpha production, and the underlying inhibition mechanism has been intensively studied. In contrast, little is known about how members of the subfamily Pneumovirinae regulate IFN-alpha production by pDCs. We identified the M2-2 protein of HMPV, a member of the subfamily Pneumovirinae, as a negative regulator of IFN-alpha production by pDCs and uncovered the underlying mechanism. This study explains in part why the M2-2 knockout recombinant HMPV is attenuated and further suggests that M2-2 is a potential target for HMPV therapy.