Novel antiviral activity of interferon-gamma against viral replication complex

项目总结/摘要 具有正义RNA（+RNA）基因组的病毒组成了植物和动物病毒的一大类，并且许多 医学上关注的人类病毒属于这类病毒。所有已知的+RNA病毒都会形成并复制 在细胞质中的空泡样结构内，称为复制复合物（RC）。病毒RC是由病毒 通过细胞器膜的重组，它提供了一个有利的微环境， 病毒复制。然而，宿主免疫系统是否以及如何抵消 这样的病毒RC。了解宿主对病毒RC的免疫防御策略可能使我们能够开发 针对+RNA病毒的广泛适用抗病毒策略。我们最近发现干扰素-γ（IFNG） 在RC形成阶段抑制鼠诺如病毒（MNV）的复制。有趣的是，这种抗病毒活性 IFNG依赖于一种参与细胞自噬的蛋白质复合物。自噬是进化上保守的 在双膜结合的自噬体中隔离细胞质物质并将其递送的途径 到溶酶体进行降解。为了形成球状自噬体，微管相关蛋白-1-轻- 链-3（LC 3）缀合系统是必需的。我们发现只有LC 3结合系统的自噬，但 而不是通过自噬的溶酶体降解是IFNG抑制MNVRC形成所必需的。 有趣的是，IFNG也需要相同的LC 3缀合系统，但不需要溶酶体降解，以破坏 含有原生生物寄生虫刚地弓形虫的胞质空泡。通过比较机制研究 MNV和T.在弓形虫模型中，我们发现LC 3结合系统需要募集IFN-γ诱导的 GTPases，免疫相关的GTPases（IRGs）和鸟苷酸结合蛋白（GBP），对MNV的RC。两 已知IRG和GBP靶向含有细菌、原生生物或真菌的空泡的膜。 靶向的细胞膜被囊泡化，最终空泡破裂，导致暴露的细胞死亡。 病原体类似地，GTP酶是IFNG破坏MNV RC并因此抑制MNV RC所必需的。 MNV在小鼠和人类系统中的复制。这是一种新的和范式转移的抗病毒机制 IFNG，表明一个共同的效应机制，对不同的病原体复制在胞质 膜状庇护所，包括+RNA病毒以及细菌、原生生物和真菌。我们的长期目标是 利用基于这种抗病毒免疫防御病毒的功能机制的医疗益处， RC。作为实现这一目标的下一步，本建议的总体目标是确定 MNV被免疫系统检测并破坏。我们的中心假设是，MNV RC是由 自噬途径的LC 3缀合系统，然后RC的结构/功能被自噬途径的LC 3缀合系统破坏。 通过LC 3缀合系统募集的IFN诱导型GTP酶。新的基础知识创造于 这项研究将对人类健康产生重大的积极影响，因为它将为抗病毒药物提供新的见解。 干扰素使用的机制和潜在的新的治疗目标的干预病毒性疾病。

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.