Studies in Poxvirus Host Range Genes and Tropism

在本研究中，我们将揭示RNA解旋酶超家族的细胞因子和病毒编码的宿主范围因子相互调节功能的基本机制，这是病毒-宿主相互作用、先天免疫、细胞转化和溶瘤病毒用于癌症治疗的关键。黏液瘤病毒（MYXV）是一种兔特异性痘病毒，也显示出感染广泛的人类癌症和转化细胞的能力。MYXV目前正被开发为一种溶瘤病毒治疗药物，用于治疗各种类型的癌症。我们最近报道了含有RNA解旋酶的细胞DEAD-box成员的鉴定，这些RNA解旋酶在人癌细胞中严格调节MYXV的趋向性。我们的目标之一是发现新的病毒-宿主相互作用，并解剖人类细胞中选定的关键RNA解旋酶的亲抗病毒功能。此外，我们发现阻断exportin1-nuclear export通路不仅可以增强病毒复制，还可以协同促进人类癌细胞的杀伤，而不影响正常的人类细胞。我们的目标是研究这种核输出途径在MYXV溶瘤病毒治疗中的体外和体内影响。我们将继续努力了解两个关键的MYXV宿主范围蛋白M029和M-T5的功能。M029是含有dsRNA结合域（dsRBD）蛋白的poxvirus E3家族的成员，在人类癌细胞中调节关键抗病毒RNA解旋酶（如DHX9和PKR）的功能。M-T5是ANK蛋白家族的成员，我们新的初步数据表明，当核输出途径被抑制时，人类癌细胞中MYXV复制的增强需要M-T5。因此，我们的目标是进一步了解M029和M-T5的宿主范围和免疫调节功能。根据我们的观察，我们提出以下调查：1。阐明选定的RNA解旋酶在MYXV复制和细胞趋向性中的作用。我们拟研究在人类癌细胞和原代免疫细胞中调控MYXV趋向性的关键细胞RNA解旋酶的抗病毒和抗病毒功能。2. 探讨M029在细胞趋向性和先天免疫应答中的作用。我们将剖析M029调节癌症和先天免疫细胞中依赖于宿主限制因子的核/细胞质穿梭的不同细胞途径的分子机制。我们将确定M029与宿主的其他相互作用，并剖析M029相互作用蛋白DHX9和PKR调节人类癌细胞的MYXV趋向性和溶瘤的机制。3. 探讨核输出抑制剂和溶瘤性MYXV联合使用如何增强病毒复制、癌细胞杀伤和溶瘤性病毒治疗。我们将进行体外和体内研究，以了解抑制核输出通路如何增强MYXV复制和肿瘤细胞中MYXV复制受到限制的溶瘤活性的机制。此外，我们将研究myxv编码的ANK蛋白M-T5的作用，当核输出途径受到限制时，这种增强的病毒复制是必需的。

In this proposal we will uncover the fundamental mechanisms by which cellular factors of the RNA helicase superfamily and virus encoded host range factors modulate each other’s function, which is key for virus-host interactions, innate immunity, cellular transformation and use of oncolytic viruses for cancer treatment. Myxoma virus (MYXV) is a rabbit specific poxvirus that also exhibits the capacity to infect a wide spectrum of human cancer and transformed cells. MYXV is currently being developed as an oncolytic virotherapeutic to treat various classes of cancer. We have recently reported the identification of members of cellular DEAD-box containing RNA helicases that tightly regulate the tropism of MYXV in human cancer cells. One of our goals is to discover the novel virus-host interactions and dissect the pro- vs anti-viral functions of selected key RNA helicases in human cells. In addition, we discovered that blocking the exportin1-nuclear export pathway not only enhances viral replication but also synergistically promotes killing of human cancer cells without affecting normal human cells. Our goal is to study the in vitro and in vivo impact of this nuclear export pathway in MYXV oncolytic virotherapy. We will continue our effort on understanding the function of two key MYXV host range proteins M029 and M-T5. M029 is a member of the poxvirus E3 family of dsRNA Binding Domain (dsRBD) containing proteins, which modulates the functions of key anti-viral RNA helicases such as DHX9 and PKR in human cancer cells. M-T5 is a member of the ANK family of proteins, and our new preliminary data suggests that M-T5 is required for the enhanced MYXV replication in human cancer cells when nuclear export pathway is inhibited. Thus, our goal is to further understand the host range and immune regulatory functions of M029 and M-T5. Based on our observations we propose to investigate the followings: 1. Elucidate the role of select RNA helicases in MYXV replication and cellular tropism. We propose to investigate the anti-viral and pro-viral functions of key cellular RNA helicases that regulate MYXV tropism in human cancer cells and primary immune cells. 2. Investigate the role of M029 in cellular tropism and innate immune responses. We will dissect the molecular mechanisms by which M029 modulates different cellular pathways in cancer and innate immune cells that depend upon nuclear/cytoplasmic shuttling of host restriction factors. We will identify additional M029 and host interactions and dissect the mechanisms by which the M029 interacting proteins DHX9 and PKR regulate MYXV tropism and oncolysis of human cancer cells. 3. Investigate how combination of nuclear export inhibitor and oncolytic MYXV enhances virus replication, cancer cell killing, and oncolytic virotherapy. We will perform in vitro and in vivo studies to understand the mechanisms how inhibition of nuclear export pathway enhances MYXV replication and oncolytic activity in cancer cell, where MYXV replication is restricted. Additionally, we will investigate the role of MYXV-encoded ANK protein M-T5, which is required for this enhanced virus replication when nuclear export pathway is restricted.