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）是一种高致病性、人畜共患、非分节性、正义RNA病毒，与严重急性呼吸综合征冠状病毒（SARS -CoV）和SARS-CoV-2相关，可在人与人之间传播。SARS-CoV-2的出现和随后的COVID-19大流行证明了人畜共患冠状病毒构成的潜在威胁。由于中东呼吸综合征冠状病毒仍然是一种威胁，因此了解中东呼吸综合征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）在MERS冠状病毒附属蛋白中是值得注意的，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。这些包括抑制干扰素β (IFNβ)和IFNλ的产生以及抑制NF-κ b依赖性细胞因子的产生。4b蛋白还抑制2 ‘,5 ’寡聚腺苷酸合成酶(OAS)-RNase L途径，这种活性归因于其c端磷酸二酯酶结构域。ORF4b是一种核蛋白，转染和感染实验均表明，ORF4b核定位信号（NLS）的突变会影响先天免疫逃避功能的抑制。一项值得注意的研究发现，4b阻断NF-ĸB-dependent反应，这与4b与NF-ĸB的p65亚基竞争IMPA3结合的能力相关。我们进行了4b-IMPA相互作用的X射线晶体学研究。我们的初步数据表明，4b独特地绕过了NLS识别的规范规则，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区与IMPA2结合，并且具有比IMPA3更广泛的界面。因此，已经提出的特异性不太可能由这个简单的相互作用界面介导。此外，如果4b能够结合比先前提出的更大范围的IMPA异构体，那么这种MERS-CoV蛋白可能能够竞争性地抑制其他先天性免疫转录因子（如IRF3和STAT1）的核输入。与该模型一致，结合IMPA2和IMPA3的p50 NF-κB NLS的晶体结构的初步数据表明，这些区域与MERS ORF4b重叠。基于这些观察结果，我们建议解决MERS- CoV和蝙蝠Merbecovirus ORF4b蛋白与核受体IMPA亚型复合物的全长和NLS肽结构，并确定相互作用界面。这将为ORF4b结合特异性和核导入提供结构基础。为了比较ORF4b和IMPAs的结构数据，我们将确定与NF-κB复合物的IMPA亚型的结构，为免疫逃避建立结构基础。最后。我们将在基于转染和病毒感染的研究中验证ORF4b通过竞争p50-IMPA相互作用抑制NF-κB信号传导和IFN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.