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.