Understanding how the MERS Coronavirus protein ORF4b interactions with importin alpha modulate innate immunity

中东呼吸综合征冠状病毒(MERS-CoV)是一种与严重急性呼吸综合征冠状病毒(SARS CoV)和SARS-CoV-2相关的高致病性、人畜共患病、非节段性、阳性的RNA病毒，可在人与人之间传播。人畜共患冠状病毒构成的潜在威胁从SARS-CoV-2的出现和随后的新冠肺炎大流行中可见一斑。由于MERS冠状病毒仍然是一种威胁，了解MERS冠状病毒复制的机制，特别是与发病机制和治疗开发相关的机制，仍然至关重要。ORF4b(4b)是MERS冠状病毒辅助蛋白中值得注意的一种，因为尽管病毒复制发生在细胞质中，但它强烈定位于细胞核，并发挥先天免疫逃避功能。这些措施包括抑制干扰素β和干扰素λ的产生，以及抑制依赖于NF-κB的细胞因子的产生。4b蛋白还抑制2‘，5’寡腺苷合成酶-核糖核酸酶L途径，这一活性归因于其C末端的磷酸二酯酶结构域。ORF4b是一种核蛋白，转基因和基于感染的检测都表明，ORF4b核定位信号(NLS)的突变会影响对先天性免疫逃避功能的抑制。一项值得注意的研究发现，4b阻断了NF-ĸB依赖的反应，这与4b竞争NF-ĸB的p65亚单位结合IMPA3的能力有关。我们进行了4b-IMPA相互作用的X射线结晶学研究。我们的初步数据表明，4b唯一地绕过了NLS识别的正则规则，并且在以前被认为对NLS功能至关重要的结合位点上不含有Lys残基。此外，我们的数据并不完全支持4b对IMPA3的特异性。我们发现4b的NLS区域与IMPA2结合，界面比IMPA3更广泛。因此，已经提出的特异性不太可能由这个简单的交互界面来调节。此外，如果4b能够结合比先前提出的更大范围的IMPA亚型，这种MERS-CoV蛋白很可能能够竞争性地抑制其他天然免疫转录因子的核输入，如IRF3和STAT1。与这一模型一致的是，与IMPA2和IMPA3结合的p50 NF-κB NLS的晶体结构的初步数据表明，这些区域与MERSORF4b重叠。基于这些观察结果，我们建议解决MERS-CoV和BAT MerbecVirus ORF4b蛋白与核受体IMPA亚型的复合体中全长和NLS多肽的结构，并确定相互作用界面。这将为ORF4b结合和核进口的特异性提供结构基础。为了比较ORF4b和IMPA的结构数据，我们将确定IMPA异构体与NF-κB的复合体的结构，为免疫逃避奠定结构基础。终于来了。我们将在基于转基因和病毒感染的研究中，通过竞争p50-κ相互作用来检验ORF4b抑制NF-IMPA B信号和干扰素产生的假设。

The Middle East respiratory syndrome coronavirus (MERS-CoV), is a highly pathogenic, zoonotic, non-segmented, positive-sense RNA virus related to the severe acute respiratory syndrome coronavirus (SARS CoV) and SARS-CoV-2 that can spread from person to person. The potential threat posed by zoonotic coronaviruses is demonstrated by the emergence of SARS-CoV-2 and the subsequent COVID-19 pandemic. Because MERS CoV remains a threat, an understanding of mechanisms of MERS CoV replication, particularly as these related to pathogenesis and therapeutic development, remains critical. ORF4b (4b) is notable among the MERS CoV accessory proteins because it strongly localizes to the nucleus, despite virus replication occurring in the cytoplasm, and it exerts innate immune evasion functions. These include inhibition of interferon beta (IFNβ) and IFNλ production and inhibition of NF-κB-dependent cytokine production. The 4b protein also inhibits the 2’, 5’ oligoadenylate synthetase (OAS)-RNase L pathway, an activity attributed to its C-terminal phosphodiesterase domain. ORF4b is a nuclear protein and both transfection- and infection-based assays indicate that mutation of the apparent ORF4b nuclear localization signal (NLS) impairs affects inhibition of innate immune evasion functions. One notable study found that 4b blocks NF-ĸB-dependent responses and this correlated with the capacity of 4b to outcompete the p65 subunit of NF-ĸB for IMPA3 binding. We have undertaken X ray crystallography studies of the 4b-IMPA interaction. Our Preliminary Data demonstrate that 4b has uniquely bypassed canonical rules of NLS recognition and does not contain a Lys residue at a binding site formerly thought to be critical for NLS function. Further, the proposed specificity of 4b for IMPA3 is not fully supported by our data. We found that the NLS region of 4b binds IMPA2 and with an interface that is more extensive than IMPA3. Thus, the specificity that has been proposed is unlikely to be mediated by this simple interaction interface. Furthermore, if 4b is able to bind a greater range of IMPA isoforms than had previously been proposed, this MERS-CoV protein is likely to be able to competitively inhibit the nuclear import of other innate immune transcription factors such as IRF3 and STAT1. Consistent with such a model, Preliminary Data of crystal structures of the p50 NF-κB NLS bound to IMPA2 and IMPA3 demonstrate that these regions overlap with MERS ORF4b. Based on these observations, we propose to solve structures of full-length and NLS peptides of MERS- CoV and bat Merbecovirus ORF4b proteins in complex with nuclear receptor IMPA isoforms and define interaction interfaces. .This will provide a structural basis for the specificity of ORF4b binding and nuclear import. To compare the structural data obtained for ORF4b and IMPAs, we will determine the structures of IMPA isoforms in complex with NF-κB to establish a structural basis for the immune evasion. Finally. we will test the hypothesis that ORF4b inhibits NF-κB signaling and IFN production by competing for p50-IMPA interaction in transfection-based and virus infection studies.