Abstract
Approximately 10,000 New Zealanders die of cancer each year, with breast, lung and colorectal cancers contributing to most of these deaths. Traditional cancer treatments like radiation and chemotherapy often cause off-target damage and long-term toxicities in patients. Oncolytic virotherapy is a promising immunotherapy strategy potentially offering long-term remissions with reduced side-effects. Seneca Valley Virus (SVV) is an oncolytic virus which infects neuroendocrinal cancers expressing tumour endothelial marker 8 (TEM8). TEM8 is ubiquitously expressed in the tumour microenvironment, in both tumour cells and the tumour-associated fibroblasts but is absent in healthy tissue. TEM8 expression has also been implicated in facilitating cancer development and progression of breast, lung, and colorectal cancers. Herein, we attempted to obtain high-resolution structures of the SVV replicative intermediate Protease-Polymerase (3CD) and the polymerase (3DPol) using cryogenic electron microscopy (cryo-EM). Viral proteins were successfully cloned and tagged with His and SUMO tags (HS) for increased protein solubility and to facilitate purification. Subsequently, HS-3CD and HS-3DPol were expressed and purified from a bacterial system. SVV HS- 3DPol was screened via negative staining electron microscopy and sent for cryo-EM data collection. SVV HS-3CD suffered from degradation following purification, potentially resulting from self-directed protease activity. Future work to discern breakdown aetiology, alongside the optimisation of SVV HS-3CD expression and purification is required. Overall, in the future, high-resolution structural data of near-native SVV replicate enzymes can inform rationale engineering efforts to optimize SVV for clinical deployment.