Immune evasion by SARS-CoV-2: the role of HLA class I

摘要 严重急性呼吸综合征冠状病毒-2(SARS-CoV-2)是目前大流行的罪魁祸首 冠状病毒引起的疾病2019年(新冠肺炎)。自2019年12月以来，协调一致的研究努力 提供了丰富的关键数据，增强了我们诊断和治疗新冠肺炎的能力。到目前为止， 然而，新冠肺炎发病的许多分子和细胞机制仍然存在。 难以捉摸。SARS-CoV-2基因组包含9个独立的开放阅读框架(ORF)，编码 对病毒复制不重要但似乎在调节宿主方面发挥重要作用的蛋白质 抗病毒免疫。在这些辅助蛋白中，ORF8蛋白因其独特的特性而脱颖而出。 ORF8是SARS相关冠状病毒中的一种高度可变的蛋白质。ORF8似乎也参与了一个网络 感染细胞内宿主与病原体的相互作用：ORF8损害抗原等免疫途径 目前，干扰素I型和核因子-kB，以及可能的激活生长途径。一个中环 问题是ORF8如何调节与多个宿主蛋白靶标的相互作用。为了实现这个目标，我们正在 研究ORF8与主要组织相容性I类(MHC I)分子的相互作用。 最近发现SARS-CoV-2 ORF8通过下调MHC抑制CD8+T细胞反应 I分子，包括体外和体内。我们假设ORF8抑制了人类白细胞抗原-A的表面表达和 人类白细胞抗原B分子保护感染细胞不被CD8+T细胞识别，但不包括人类白细胞抗原C和人类白细胞抗原E 避免自然杀伤(NK)细胞的激活。ORF8对MHC I显示基因座特异性的可能性 将为SARS-CoV-2提供一种机制，在适应性免疫和先天免疫之间保持微妙的平衡。 具体地说，我们将通过使用一个人类白细胞抗原-8小组来分析ORF8和MHC I之间的相互作用。 A、-B、-C和-E分子，并确定ORF8是否选择性地下调相关细胞中的MHC I 系统(目标1)。我们将把这些研究扩展到ORF8变种，这些变种已经在 并评估这些突变是否以及如何影响MHC I结合和下调 (目标2)。这些目标的成功完成预计将揭示ORF8和ORF8的未知特征 阐明它在抑制抗原提呈中的作用，并告诉我们MHC I的选择压力 对SARS-CoV-2病毒的作用。我们提议的研究的直接和长期影响都很高。这是第一次 研究：(1)表征ORF8与宿主蛋白的分子相互作用；(2)临床评估 相关的ORF8变种。通过研究ORF8介导的基因下调背后的免疫相互作用 MHC I，我们将增加对SARS-CoV-2如何破坏细胞免疫的理解。重要的是，我们的 这项研究将促进对其他ORF8相互作用因子的类似研究，为 针对ORF8或其宿主靶点的治疗药物的发展。

ABSTRACT Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the current pandemic of coronavirus-induced disease 2019 (COVID-19). Since December 2019, coordinated research efforts have provided a wealth of critical data that have enhanced our ability to diagnose and treat COVID-19. To date, however, much of the molecular and cellular mechanisms underlying the pathogenesis of COVID-19 remain elusive. The genome of SARS-CoV-2 contains nine independent open reading frames (ORFs) coding for proteins that are not essential for viral replication but seem to exert important functions in modulating host antiviral immunity. Of these accessory proteins, the ORF8 protein stands out for its unique characteristics. ORF8 is a highly variable protein among SARS-related CoVs. ORF8 also appears to be involved in a network of host-pathogen interactions inside infected cells: ORF8 impairs immune pathways such as antigen presentation, interferon type I, and nuclear factor-kB, as well as possibly activate growth pathways. A central question is how ORF8 mediates interactions with multiple host protein targets. Towards this goal, we are investigating ORF8 interaction with major histocompatibility class I (MHC I) molecules. It was shown recently that SARS-CoV-2 ORF8 suppresses CD8+ T cell responses by downregulating MHC I molecules, both in vitro and in vivo. We hypothesize that ORF8 suppresses surface expression of HLA-A and HLA-B molecules to protect infected cells from recognition by CD8+ T cells, but spares HLA-C and HLA-E to avoid activation of natural killer (NK) cells. The possibility that ORF8 displays a locus specificity toward MHC I would provide a mechanism for SARS-CoV-2 to walk a fine line between adaptive and innate immunity. Specifically, we will undertake an analysis of interactions between ORF8 and MHC I by using a panel of HLA- A, -B, -C, and -E molecules, and also determine if ORF8 selectively downregulates MHC I in relevant cell systems (Aim 1). We will extend these studies to ORF8 variants that have been positively selected during the course of the pandemic and evaluate if and how these mutations affect MHC I binding and downregulation (Aim 2). The successful completion of these Aims is expected to uncover unknown features of ORF8 and elucidate its role in suppressing antigen presentation, as well as inform us on the selective pressure that MHC I exerts on SARS-CoV-2. The immediate and long-term impact of our proposed research is high. This is the first study that: (1) characterizes molecular interaction of ORF8 with a host protein; and (2) evaluates clinically relevant ORF8 variants. By studying the immune interactions underlying ORF8-mediated downregulation of MHC I, we will increase our understanding of how SARS-CoV-2 derails cellular immunity. Importantly, our study will stimulate similar investigations of other ORF8-interactors, providing novel opportunities for the development of therapeutics directed against ORF8 or its host targets.