PDF(Sci-hub)
Abstract：
SARS-CoV-2 nonstructural protein 3 (Nsp3) contains a macrodomain that is essential for coronavirus pathogenesis and is thus an attractive target for drug development. This macrodomain is thought to counteract the host interferon (IFN) response, an important antiviral signalling cascade, via the reversal of protein ADP-ribosylation, a posttranslational modification catalyzed by host poly(ADP-ribose) polymerases (PARPs). However, the main cellular targets of the coronavirus macrodomain that mediate this effect are currently unknown. Here, we use a robust immunofluorescence-based assay to show that activation of the IFN response induces ADP-ribosylation of host proteins and that ectopic expression of the SARS-CoV-2 Nsp3 macrodomain reverses this modification in human cells. We further demonstrate that this assay can be used to screen for on-target and cell-active macrodomain inhibitors. This IFN-induced ADP-ribosylation is dependent on PARP9 and its binding partner DTX3L, but surprisingly the expression of the Nsp3 macrodomain or the deletion of either PARP9 or DTX3L does not impair IFN signaling or the induction of IFN-responsive genes. Our results suggest that PARP9/DTX3L-dependent ADP-ribosylation is a downstream effector of the host IFN response and that the cellular function of the SARS-CoV-2 Nsp3 macrodomain is to hydrolyze this end product of IFN signaling, rather than to suppress the IFN response itself.
摘要：
SARS-CoV-2非结构蛋白3（Nsp3）包含一个巨域，对于冠状病毒的发病机理至关重要，因此是药物开发的有吸引力的靶标。这个大域被认为可以抵消宿主干扰素（IFN）反应，一个重要的抗病毒信号级联，通过逆转蛋白质ADP-核糖基化，由宿主聚(ADP-核糖)聚合酶(PARP)催化的翻译后修饰。然而,冠状病毒巨域介导这一效应的主要细胞靶标目前尚不清楚.这里,我们使用了基于免疫荧光的可靠检测方法,证明IFN应答的激活可诱导宿主蛋白的ADP核糖基化,而SARS-CoV-2Nsp3巨域的异位表达可逆转人细胞中的这种修饰.我们进一步证明了该测定可用于筛选中靶和细胞活性巨域抑制剂。这种IFN诱导的ADP核糖基化依赖于PARP9及其结合伴侣DTX3L，但令人惊讶的是,Nsp3巨域的表达或PARP9或DTX3L的缺失不损害IFN信号传导或IFN应答基因的诱导。我们的结果表明，PARP9/DTX3L依赖性ADP核糖基化是宿主IFN应答的下游效应物，SARS-CoV-2Nsp3巨域的细胞功能是水解IFN信号传导的这一最终产物，而不是抑制IFN应答本身。
