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

了解 MERS 冠状病毒蛋白 ORF4b 与 importin alpha 的相互作用如何调节先天免疫

• 批准号: 10536332
• 负责人: Christopher F Basler
• 金额: $ 18.67万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10536332
• 关键词: Understanding; how; MERS; Coronavirus; protein

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.

中东呼吸道综合征冠状病毒（MERS-COV）是一种高度致病的，人畜共患病，非细分，阳性的RNA病毒，与严重的急性呼吸综合征冠状病毒（SARS COV）和SARS-COV-2有关，可以从人传播到人。 SARS-COV-2的出现和随后的Covid-19-19大流行证明了潜在的威胁。由于MERS COV仍然是一个威胁，因此对MERS COV复制机制的理解，特别是与发病机理和治疗性发育相关的机制仍然至关重要。 ORF4B（4B）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，发生的目标病毒复制发生在细胞质中，并且具有先天的免疫进化功能。这些包括抑制干扰素β（IFNβ）和IFNλ产生以及抑制NF-κB依赖性细胞因子的产生。 4B蛋白还抑制2'，5'寡腺苷酸合成酶（OAS） - RNase L途径，这是归因于其C-末端磷酸二酯酶结构域的活性。 ORF4B是一种核蛋白，基于转化和感染的测定法表明，明显的ORF4B核定位信号（NLS）的突变会损害先天免疫驱虫功能。一项值得注意的研究发现，4B阻断了NF-ĸB依赖性响应，这与4B的能力相关，以使Impa3结合的NF-ĸB的P65亚基占据p65亚基。我们已经对4B-IMPA相互作用进行了X射线晶体学研究。我们的初步数据表明，4B具有NLS识别的规范规则，并且不包含在以前被认为对NLS功能至关重要的绑定位置的LYS住所。此外，我们的数据并未完全支持4B对IMPA3的特异性。我们发现4B的NLS区域结合Impa2，并且界面比Impa3更广泛。这就是提出的特异性不太可能由这种简单的相互作用接口介导。此外，如果4B能够结合比以前提出的更大范围的IMPA同工型，则该MERS-COV蛋白可能能够竞争地抑制其他先天免疫转录因子（例如IRF3和STAT1）的核进口。与这样的模型一致，P50 NF-κBNLS的晶体结构的初步数据与Impa2和Impa3结合在一起表明，这些区域与MERS ORF4B重叠。基于这些观察结果，我们建议求解Mers-CoV和BAT Merbecovirus ORF4B蛋白的全长和NLS辣椒的结构，该结构与核接收器IMPA同工型复合物，并定义相互作用界面。 。这将为ORF4B结合和核进口的特异性提供结构性基础。为了比较ORF4B和IMPAS获得的结构数据，我们将确定与NF-κB复杂的IMPA同工型的结构，以建立免疫进化的结构基础。最后。我们将检验以下假设：ORF4B通过在基于翻译和病毒感染研究中竞争P50-IMPA相互作用来抑制NF-κB信号传导和IFN产生。