Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Faculty of Advanced Life Science, Hokkaido University, Sapporo, Japan.
J Virol. 2020 Aug 17;94(17). doi: 10.1128/JVI.01169-20.
Measles virus (MeV) is a highly immunotropic and contagious pathogen that can even diminish preexisting antibodies and remains a major cause of childhood morbidity and mortality worldwide despite the availability of effective vaccines. MeV is one of the most extensively studied viruses with respect to the mechanisms of JAK-STAT antagonism. Of the three proteins translated from the MeV gene, P and V are essential for inactivation of this pathway. However, the lack of data from direct analyses of the underlying interactions means that the detailed molecular mechanism of antagonism remains unresolved. Here, we prepared recombinant MeV V protein, which is responsible for human JAK-STAT antagonism, and a panel of variants, enabling the biophysical characterization of V protein, including direct V/STAT1 and V/STAT2 interaction assays. Unambiguous direct interactions between the host and viral factors, in the absence of other factors such as Jak1 or Tyk2, were observed, and the dissociation constants were quantified for the first time. Our data indicate that interactions between the C-terminal region of V and STAT2 is 1 order of magnitude stronger than that of the N-terminal region of V and STAT1. We also clarified that these interactions are completely independent of each other. Moreover, results of size exclusion chromatography demonstrated that addition of MeV-V displaces STAT2-core, a rigid region of STAT2 lacking the N- and C-terminal domains, from preformed complexes of STAT2-core/IRF-associated domain (IRF9). These results provide a novel model whereby MeV-V can not only inhibit the STAT2/IRF9 interaction but also disrupt preassembled interferon-stimulated gene factor 3. To evade host immunity, many pathogenic viruses inactivate host Janus kinase signal transducer and activator of transcription (STAT) signaling pathways using diverse strategies. Measles virus utilizes P and V proteins to counteract this signaling pathway. Data derived largely from cell-based assays have indicated several amino acid residues of P and V proteins as important. However, biophysical properties of V protein or its direct interaction with STAT molecules using purified proteins have not been studied. We have developed novel molecular tools enabling us to identify a novel molecular mechanism for immune evasion whereby V protein disrupts critical immune complexes, providing a clear strategy by which measles virus can suppress interferon-mediated antiviral gene expression.
麻疹病毒（Measles virus，MeV）是一种高度免疫原性和传染性病原体，即使在有效疫苗存在的情况下，它仍能降低先前存在的抗体，并仍是全球儿童发病率和死亡率的主要原因。就 JAK-STAT 拮抗作用的机制而言，MeV 是研究最多的病毒之一。在从 MeV 基因翻译的三种蛋白中，P 和 V 对于该途径的失活至关重要。然而，由于缺乏对潜在相互作用的直接分析数据，拮抗的详细分子机制仍未解决。在这里，我们制备了负责人类 JAK-STAT 拮抗作用的重组 MeV V 蛋白及其变体，从而能够对 V 蛋白进行生物物理特性分析，包括直接的 V/STAT1 和 V/STAT2 相互作用测定。在没有 Jak1 或 Tyk2 等其他因素的情况下，观察到宿主和病毒因子之间的明确直接相互作用，并首次对解离常数进行了量化。我们的数据表明，V 蛋白的 C 末端与 STAT2 之间的相互作用比 V 蛋白的 N 末端与 STAT1 之间的相互作用强一个数量级。我们还澄清了这些相互作用是完全相互独立的。此外，排阻层析的结果表明，MeV-V 的加入会将 STAT2 核心（缺乏 N 端和 C 端结构域的 STAT2 刚性区域）从 STAT2 核心/干扰素相关结构域（IRF9）形成的复合物中置换出来。这些结果提供了一种新模型，其中 MeV-V 不仅可以抑制 STAT2/IRF9 相互作用，还可以破坏预组装的干扰素刺激基因因子 3。为了逃避宿主免疫，许多致病性病毒使用多种策略使宿主 Janus 激酶信号转导和转录激活因子（STAT）信号通路失活。麻疹病毒利用 P 和 V 蛋白来对抗这种信号通路。很大程度上基于细胞的测定数据表明，P 和 V 蛋白的几个氨基酸残基很重要。然而，V 蛋白的生物物理特性或其与 STAT 分子的直接相互作用尚未使用纯化蛋白进行研究。我们开发了新的分子工具，使我们能够确定一种新的免疫逃避分子机制，其中 V 蛋白破坏关键的免疫复合物，为麻疹病毒抑制干扰素介导的抗病毒基因表达提供了明确的策略。
