Loss of IKK subunits limits NF-?B signaling in reovirus infected cells

Andrew J. McNamara; Pranav Danthi

our RNA-seq analyses, we measured the capacity of vgRNA and TNFα to induce the 158 expression of an NF-κB target gene in reovirus infected cells using RT-qPCR. For these 159 experiments we monitored the transcript levels of IκBα, an NF-κB target gene. Because 160 the IκBα protein inhibits NF-κB nuclear translocation, its expression serves as a 161 feedback inhibitor of NF-κB activity (20) . Consistent with our RNA-seq data, we found 162 that reovirus inhibits IκBα expression to a significant extent following treatment with 163 either agonist (Fig. 3A, 3B ). Because the effect of reovirus on both NF-κB agonists was 164 equivalent, we used TNFα for the remainder of our experiments. TNFα treatment of 165 cells should promote nuclear translocation of p65. We measured nuclear p65 levels in 166 mock-infected and reovirus-infected cells treated with TNFα. As expected, TNFα 167 treatment of mock infected cells resulted in an accumulation of p65 in the nucleus within 168 1 h (Fig. 3C ). Prior infection with T3A prevented TNFα driven accumulation of p65 in the 169 nucleus. These data agree with previous evidence indicating that degradation of the 170 IκBα protein is blocked in reovirus infected cells (21). IκBα degradation is initiated by the 171 phosphorylation of IκBα by the IκB Kinase (IKK) complex, which leads to 172 . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/843680 doi: bioRxiv preprint polyubiquitination and subsequent degradation of IκBα by the proteasome (9). Thus, the 173 reduction in nuclear p65 levels in T3A infected cells treated with TNFα may be due to an 174 absence of sufficient levels of active IKK. In addition to IκBα, the IKK complex also 175 phosphorylates p65 at Ser536 prior to nuclear translocation (10). IKK-mediated p65 176