Deciphering the mechanism(s) which limit reovirus spread in resistant lung and HNSCC cancer cells

  • Author / Creator
    James, Kevin
  • Reovirus is a naturally benign virus that preferentially replicates in transformed cells and is currently undergoing clinical trials as a promising oncolytic therapy. In normal mouse fibroblasts, transformation by constitutively activated Ras oncogene is sufficient to promote reovirus infection. However, not all tumour cells containing Ras mutations are susceptible to reovirus, and clinical trials show mixed response to reovirus treatment. Accordingly, we aim to discover additional host-determinants important for conferring susceptibility to reovirus infection. Our first objective was to identify tumorigenic cells that restrict reovirus infection. Reovirus primary infection (at 18 hours post-infection, hpi), and dissemination over two rounds of virus replication (48 hpi) was measured in a panel of lung (H1299, A549, H23, H522, H322) and head and neck (SCC9, A253) carcinoma cells using cell-based ELISA. A549, H322, A253, and SCC9 cells were 8-90 times less permissive to reovirus dissemination relative to highly susceptible H1299 cells. Differences in cell death were observed between cells, notably in H322 and A253 cells which released up to 100-fold less progeny virions within the first 24 hpi. However, it was determined that restricted virion release was not the sole determinant for reduced reovirus cell-cell spread. We next evaluated if antiviral innate signalling contributes a barrier to reovirus dissemination among less-susceptible cells. Multiplex ELISA and qRT-PCR analysis of cell culture media and cellular RNA from reovirus-infected cells showed high levels of IFN-β, IFN-λ, and TNF- among resistant A549, H322, A253, and SCC9 cells. ShRNA-mediated knock-down of IRF3, IRF7, IFN Type I and III receptors, or IFN- neutralization in A549 cells had no effect on reovirus dissemination, suggesting that these factors are singularly insufficient to confer resistance in these cells. It remains to be determined if Type I and III IFN responses act together to promote resistance to reovirus, or if alternative antiviral pathways are involved.

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  • Degree
    Master of Science
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