Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, Florida, United States of America.
PLoS Pathog. 2013;9(7):e1003465. doi: 10.1371/journal.ppat.1003465. Epub 2013 Jul 4.
Myxoma virus (MYXV)-encoded protein M029 is a member of the poxvirus E3 family of dsRNA-binding proteins that antagonize the cellular interferon signaling pathways. In order to investigate additional functions of M029, we have constructed a series of targeted M029-minus (vMyx-M029KO and vMyx-M029ID) and V5-tagged M029 MYXV. We found that M029 plays a pivotal role in determining the cellular tropism of MYXV in all mammalian cells tested. The M029-minus viruses were able to replicate only in engineered cell lines that stably express a complementing protein, such as vaccinia E3, but underwent abortive or abated infection in all other tested mammalian cell lines. The M029-minus viruses were dramatically attenuated in susceptible host European rabbits and caused no observable signs of myxomatosis. Using V5-tagged M029 virus, we observed that M029 expressed as an early viral protein is localized in both the nuclear and cytosolic compartments in virus-infected cells, and is also incorporated into virions. Using proteomic approaches, we have identified Protein Kinase R (PKR) and RNA helicase A (RHA)/DHX9 as two cellular binding partners of M029 protein. In virus-infected cells, M029 interacts with PKR in a dsRNA-dependent manner, while binding with DHX9 was not dependent on dsRNA. Significantly, PKR knockdown in human cells rescued the replication defect of the M029-knockout viruses. Unexpectedly, this rescue of M029-minus virus replication by PKR depletion could then be reversed by RHA/DHX9 knockdown in human monocytic THP1 cells. This indicates that M029 not only inhibits generic PKR anti-viral pathways, but also binds and conscripts RHA/DHX9 as a pro-viral effector to promote virus replication in THP1 cells. Thus, M029 is a critical host range and virulence factor for MYXV that is required for replication in all mammalian cells by antagonizing PKR-mediated anti-viral functions, and also conscripts pro-viral RHA/DHX9 to promote viral replication specifically in myeloid cells.
粘液瘤病毒（MYXV）编码的蛋白 M029 是痘病毒 E3 家族的 dsRNA 结合蛋白成员之一，可拮抗细胞干扰素信号通路。为了研究 M029 的其他功能，我们构建了一系列靶向 M029 缺失（vMyx-M029KO 和 vMyx-M029ID）和 V5 标记的 M029 MYXV。我们发现 M029 在决定 MYXV 在所有测试的哺乳动物细胞中的细胞嗜性方面起着关键作用。缺失 M029 的病毒仅在稳定表达互补蛋白（如牛痘 E3）的工程细胞系中能够复制，但在所有其他测试的哺乳动物细胞系中均发生了流产或减弱感染。缺失 M029 的病毒在易感宿主欧洲兔中明显减弱，并且不会引起明显的粘液瘤病迹象。使用 V5 标记的 M029 病毒，我们观察到作为早期病毒蛋白表达的 M029 定位于病毒感染细胞的核和胞质区室中，并且还被整合到病毒粒子中。使用蛋白质组学方法，我们已经鉴定出蛋白激酶 R（PKR）和 RNA 解旋酶 A（RHA）/DHX9 作为 M029 蛋白的两种细胞结合伴侣。在病毒感染的细胞中，M029 以 dsRNA 依赖性方式与 PKR 相互作用，而与 DHX9 的结合不依赖于 dsRNA。重要的是，在人细胞中敲低 PKR 可挽救 M029 缺失病毒的复制缺陷。出乎意料的是，在人单核细胞 THP1 细胞中敲低 RHA/DHX9 可逆转 PKR 耗竭对 M029 缺失病毒复制的挽救。这表明 M029 不仅抑制通用的 PKR 抗病毒途径，而且还结合并征募 RHA/DHX9 作为促病毒效应子，以促进 THP1 细胞中的病毒复制。因此，M029 是 MYXV 的关键宿主范围和毒力因子，通过拮抗 PKR 介导的抗病毒功能，在所有哺乳动物细胞中复制均需要该因子，并且还征募促病毒的 RHA/DHX9 来特异性促进髓样细胞中的病毒复制。
