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、非节段、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。人畜共患冠状病毒构成的潜在威胁由SARS-CoV-2的出现和随后的COVID-19大流行证明。由于MERS CoV仍然是一种威胁，因此了解MERS CoV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。ORF 4 b（4 b）在MERS CoV辅助蛋白中是值得注意的，因为它强烈定位于细胞核，尽管病毒复制发生在细胞质中，并且它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。4 b蛋白还抑制2 '，5'寡腺苷酸合成酶（OAS）-RNase L途径，该活性归因于其C-末端磷酸二酯酶结构域。ORF 4 b是一种核蛋白，基于转染和感染的测定表明，表观ORF 4 b核定位信号（NLS）损伤的突变影响先天免疫逃避功能的抑制。一项值得注意的研究发现，4 b阻断NF-KB依赖性应答，这与4 b竞争超过NF-KB的p65亚基与IMPA 3结合的能力相关。我们进行了4 b-IMPA相互作用的X射线晶体学研究。我们的初步数据表明，4 b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处不含Lys残基。此外，我们的数据并不完全支持4 b对IMPA 3的特异性。我们发现4 b的NLS区域结合IMPA 2，并且具有比IMPA 3更广泛的界面。因此，已经提出的特异性不太可能通过这种简单的相互作用界面来介导。此外，如果4 b能够结合比先前提出的更大范围的IMPA同种型，则该MERS-CoV蛋白可能能够竞争性地抑制其他先天免疫转录因子如IRF 3和STAT 1的核输入。与该模型一致，与IMPA 2和IMPA 3结合的p50 NF-κB NLS的晶体结构的初步数据表明这些区域与MERS ORF 4 B重叠。基于这些观察结果，我们建议解决MERS-CoV和蝙蝠默贝科病毒ORF 4 b蛋白与核受体IMPA亚型复合的全长和NLS肽的结构，并定义相互作用界面。这将为ORF 4 b结合和核输入的特异性提供结构基础。为了比较ORF 4 B和IMPA获得的结构数据，我们将确定与NF-κB复合的IMPA同种型的结构，以建立免疫逃避的结构基础。终于来了我们将在基于转染和病毒感染的研究中检验ORF 4 B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.