Studies in poxvirus host range genes and tropism Project Summary In this proposal we will uncover the fundamental mechanisms by which Myxoma virus (MYXV) host interactive proteins modulate cellular and tissue functions that mediate host range and cell tropism. 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 will examine the molecular mechanisms used by MYXV host range protein, M029, which interacts with multiple host proteins and signaling pathways that mediate host anti-viral responses. M029 is a member of the poxvirus E3 family of dsRNA Binding Domain (dsRBD) containing proteins, but unlike the vaccinia virus (VACV) E3 version, M029 lacks the entire N terminal Z-DNA binding domain. We have reported that M029 is essential for both in vitro and in vivo host and cellular tropism of MYXV, particularly in human cells. Indeed, the M029- knockout MYXV has the most pronounced host range defect we have ever observed, and this virus cannot replicate in any human cell tested to date, indicating that the M029 protein is a potent modulator of cellular factors and pathways designed to protect human cells from virus infections. Thus, deconstructing M029 targets in human cells is important not only for the development of MYXV as a therapeutic oncolytic virus, but also as a tool to investigate innate and intrinsic cellular restriction factors that have evolved to protect human cells from viral pathogens. We have recently reported that the M029 protein binds and unexpectedly activates a key cellular RNA helicase, RHA/DHX9, to mediate expanded cellular tropism, particularly in human cells. We have also identified several additional human DDX/DHX RNA helicases as protein-interacting partners of M029, suggesting that RNA helicases may have a much broader role in governing viral host and cellular tropism. In addition to RNA helicases, M029 also binds and inhibits protein kinase R (PKR) activation during MYXV infection. MYXV thus regulates both PKR and multiple cellular RNA helicases via a single immunomodulatory protein, and we propose to uncover novel molecular mechanisms that are crucial for successful MYXV replication in diverse cell types, particularly human cancer cells, and also how this modulator mediates pathogenesis in its permissive rabbit host. Based on our observations we propose to investigate the followings:
Aim 1. Investigate the role of RHA/DHX9 and PKR in MYXV replication, pathogenesis and cellular tropism: We will examine the molecular mechanisms by which M029 modulates DHX9 and PKR functions. In addition, we will investigate how the co-regulation of PKR and DHX9 by M029 also affects DHX9-mediated tropism of MYXV. We will construct mutants of M029 within recombinant MYXV that alter these interactions and/or localization of M029, and examine the consequences of these specific M029-interactive alterations in both cultured cells and in virus-infected rabbits. We will also specifically dissect the mechanisms by which DHX9 and PKR regulate MYXV tropism and oncolysis of human cancer cells.
Aim 2. Elucidate the role of other RNA helicases in MYXV replication and expanding cellular tropism: Using an siRNA library for all the human DDX/DHX RNA helicases, and specific M029 mutants, we identified several other RNA helicases that are required for optimum viral gene expression and replication in diverse cell types. Additionally, we have identified new RNA helicases that, in contrast, inhibit MYXV replication in human cells. We will study how these cellular helicases mediate the expanded host tropism of MYXV, particularly in human cancer cells. Some of these RNA helicases interact directly with M029, either in a dsRNA dependent or independent manner. We will use M029 mutants to investigate the significance of these interactions in MYXV tropism. We will specifically test the hypothesis that some of the helicases play role in titrating dsRNA levels and determine whether specific helicase members function as poxvirus sensors in a cell type specific manner.
This project addresses several important central issues that have broad application in the areas of virus-host interactions, cellular transformation, and viral oncolysis of cancer cells. Those include: how viral proteins modulate cellular and tissue functions that mediate host range and cell tropism, how and why related but distinct cell signaling pathways are regulated by viruses, and why understanding the molecular mechanisms of cellular transformation are important for oncolytic virotherapy. The proposed studies will demonstrate the role of a key and potent viral modulator of cellular factors of the RNA helicase superfamily that mediate expanded cellular tropism, particularly in human cells, how modulation of multiple signaling pathways benefit the virus replication and expand the cellular tropism of the virus and their potential oncolysis of cancer cells. The model virus proposed here is Myxoma virus (MYXV), which infects only rabbits in nature, and has significant potential for the treatment of multiple human cancers. The knowledge which will be gained from this project will have broader implications in the treatment of human cancers using MYXV and can be applicable for other oncolytic viruses as well.
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