Novel antiviral activity of interferon-gamma against viral replication complex

项目摘要/摘要 具有正义RNA(+RNA)基因组的病毒构成了一大群植物和动物病毒，并有许多 具有医学意义的人类病毒属于这类病毒。所有已知的+RNA病毒都形成并复制 在细胞质中的液泡状结构中，称为复制复合体(RC)。病毒RC是由病毒构成的 通过细胞器膜的重组，为细胞的生长提供了良好的微环境 病毒需要复制。然而，目前还不清楚宿主免疫系统是否以及如何起作用。 如此病毒式的RCS。了解宿主对病毒RC的免疫防御策略可能会让我们开发出 针对+RNA病毒的广泛适用的抗病毒策略。我们最近发现干扰素-γ(IFNG) 在RC形成阶段抑制小鼠诺如病毒(MNV)的复制。有趣的是，这种抗病毒活性 IFNG的表达依赖于参与细胞自噬的蛋白质复合体。自噬是一种进化上保守的 将细胞质物质隔离在双膜结合的自噬小体中并运送它们的途径 到溶酶体进行降解。为了形成球状自噬小体，微管相关蛋白-1-光- 链-3(LC3)共轭体系是必不可少的。我们发现只有自噬的LC_3接合系统，但是 而不是通过自噬的溶酶体降解，是IFNG抑制MNV RC形成所必需的。 有趣的是，IFNG也需要相同的Lc3结合系统，而不是溶酶体的降解，才能破坏 一种含有原生寄生虫弓形虫的胞液液泡。通过比较机制研究 在MNV和弓形虫模型中，我们发现需要LC3接合系统来招募干扰素诱导的 GTP酶，免疫相关GTP酶(IRGS)和鸟氨酸结合蛋白(GBP)，与MNV的RC有关。两者都有 已知IRGS和GBP靶向于含有细菌、原生生物或真菌的液泡的膜。 靶向的膜被泡化，最终空泡破裂，导致暴露的人死亡 病原体。同样，IFNG需要GTP酶来破坏MNV RCS，从而抑制MNV RCS MNV在小鼠和人类系统中的复制。这是一种新颖的、改变范式的抗病毒机制 提示了一种共同的抑制不同病原体在胞浆中复制的效应机制 膜遮蔽物，包括+RNA病毒以及细菌、原生动物和真菌。我们的长期目标是 基于这种抗病毒免疫防御作用机制的医疗效益利用 RCS。这项建议的总体目标，作为追求这一目标的下一步，是确定区域审查委员会如何 MNV被免疫系统检测和破坏。我们的中心假设是MNV RC是由 自噬途径的Lc3结合系统，然后RC的结构/功能被破坏 通过LC3连接系统招募干扰素诱导的GTP酶。中创建的新基础知识 这项研究将对人类健康产生重大的积极影响，因为它将为抗病毒提供新的见解 干扰素使用的机制和潜在的干预病毒疾病的新治疗靶点。

Project Summary/Abstract Viruses with positive-sense RNA (+RNA) genome compose a large group of plant and animal viruses, and many human viruses of medical concerns belong to this group of viruses. All known +RNA viruses form and replicate within vacuole-like structures in the cytoplasm, called replication complex (RC). Viral RC is made by viruses through reorganization of cellular organelle membranes, and it provides a favorable microenvironment for the viruses to replicate. Nevertheless, it has been obscure whether and how the host immune system counteracts such viral RCs. Understanding the host immune defense strategy against viral RC may allow us to develop broadly applicable antiviral strategies against +RNA viruses. We recently found that interferon-gamma (IFNG) inhibits the replication of murine norovirus (MNV) at the stage of RC formation. Intriguingly, this antiviral activity of IFNG depends on a protein complex involved in cellular autophagy. Autophagy is an evolutionarily conserved pathway that sequesters cytoplasmic materials in double-membrane-bound autophagosomes and delivers them to the lysosome for degradation. To form a globular autophagosome, the microtubule-associated-protein-1-light- chain-3 (LC3) conjugation system is essential. We found that only the LC3 conjugation system of autophagy, but not the lysosomal degradation through autophagy, is required for IFNG to inhibit MNV RC formation. Interestingly, IFNG also requires the same LC3 conjugation system, but not the lysosomal degradation, to disrupt a cytosolic vacuole containing a protist parasite Toxoplasma gondii. Through a comparative mechanism study of MNV and T. gondii models, we found that the LC3 conjugation system was required to recruit IFN-inducible GTPases, immunity related GTPases (IRGs) and guanylate binding proteins (GBPs), to the RC of MNV. Both IRGs and GBPs are known to be targeted to the membrane of vacuoles containing bacterium, protist, or fungus. The targeted membranes are vesiculated and eventually the vacuoles rupture, leading to the death of exposed pathogens. Similarly, the GTPases were required for IFNG to disrupt MNV RCs and consequently to inhibit the replication of MNV in both mouse and human systems. This is a novel and paradigm-shifting antiviral mechanism of IFNG, indicating a common effector mechanism against disparate pathogens replicating in cytosolic membranous shelters, including +RNA virus as well as bacterium, protist, and fungus. Our long-term goal is to harness the medical benefits based on the functional mechanism of this antiviral immune defense against viral RCs. The overall objective of this proposal, as the next step to pursue that goal, is to determine how the RC of MNV is detected and disrupted by the immune system. Our central hypothesis is that MNV RC is detected by the LC3 conjugation system of the autophagy pathway and then the structure/function of RC is disrupted by the IFN-inducible GTPases recruited via the LC3 conjugation system. The new fundamental knowledge created in this study will have significant positive impact on human health because it will provide a novel insight into antiviral mechanisms used by interferons and potentially new therapeutic targets of intervention for viral diseases.