Novel Translational Control Mechanisms in Host Range Restriction of Poxvirus

Project Summary/Abstract The recent global emergence of mpox virus (MPXV) from unidentified animal reservoirs underscores the significant public health threat posed by orthopoxviruses (OPXVs). This event highlights critical knowledge gaps in understanding poxvirus host tropism, complicating efforts to predict, monitor, and prevent OPXV outbreaks. Over the past two decades, our research has focused on elucidating the molecular determinants of poxvirus host specificity, leading to the discovery of two interferon-stimulated genes, SAMD9 and SAMD9L (collectively SAMD9/9L), which serve as key host restriction factors against poxvirus infections. SAMD9/9L have been under positive selection across mammals, particularly in rodents, which are primary OPXV reservoirs. Species-specific differences in SAMD9/9L act as barriers to cross-species poxvirus infection, while all OPXVs encode at least one of three host-range factors (K1, C7, CP77) that inhibit SAMD9/9L and promote viral spread. Our previous studies revealed SAMD9 as a poxvirus-activatable tRNA anticodon nuclease that selectively depletes tRNAPhe, leading to ribosomal stalling and a proteotoxic stress response. We identified the tRNase domain in SAMD9/9L and found that its activity underlies the known cellular functions of SAMD9/9L, including the inhibitions of poxvirus replication, cellular protein synthesis, and cellular proliferation. Preliminary data indicate that SAMD9/9L constitute a family of tRNases with distinct tRNA specificities, cleaving multiple tRNAs, generating tRNA fragments, and inducing ribotoxic stress responses (RSR). Poxviruses counter these activities through the C7 family of proteins, which specifically target SAMD9/9L's tRNA-binding and catalytic sites, driving SAMD9/9L evolution in response. This renewal proposal builds on our initial discovery to elucidate the novel molecular mechanisms by which SAMD9/9L restrict viral replication and the viral strategies that counteract these defenses. In Aim 1, we will define the tRNA specificity of SAMD9/9L, assess the impact of tRNA cleavage and fragmentation on poxvirus replication, and characterize the downstream signaling pathway activated by ribosomal stalling. Aim 2 focuses on defining how SAMD9 is activated by poxvirus infection, hypothesizing that viral uncoating releases specific pathogen-associated molecular patterns (PAMPs) that activate SAMD9. Aim 3 will investigate the molecular "arms race" between SAMD9/9L tRNase and poxvirus inhibitors, to understand how these viral proteins inhibit SAMD9/9L function and shed light on the co-evolutionary dynamics between host restriction factors and viral inhibitors. This research will provide fundamental insights into SAMD9/9L-mediated antiviral mechanisms and poxvirus evasion strategies, establishing a foundation for novel therapeutic approaches to combat poxvirus infections. Additionally, it will enhance our understanding of pattern recognition mechanisms in eukaryotes and reveal translational control mechanisms that link tRNA modification, cleavage, and cellular stress responses.