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

Andrew J. McNamara; Pranav Danthi

Ser536 phosphorylation is critical for NF-κB dependent gene expression and is 177 considered to be a marker for IKK activity (10). To determine if IKK activity is 178 compromised in T3A-infected cells, we assessed the capacity of TNFα to promote p65 179 phosphorylation at Ser536. While TNFα potently induced p65 phosphorylation in mock 180 infected cells, both basal and TNFα induced p65 phosphorylation was dramatically 181 reduced in T3A-infected cells (Fig. 3D) of the reovirus replication cycle that is required for the loss of the IKK complex and the 199 inhibition of NF-κB, we treated cells with ribavirin, which diminishes viral gene 200 expression (13, 22) . We found that ribavirin treatment prevented T3A mediated loss of 201 IKKβ (Fig. 5A) . Consistent with this, in cells treated with ribavirin, T3A was no longer 202 able to prevent TNFα-driven nuclear accumulation of p65 (Fig. 5B ). Further, ribavirin 203 treatment also reduced the capacity of T3A to block NF-κB dependent gene expression 204 The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. In this study, we sought to evaluate if reovirus infection alters the cellular response to 225 pathogen invasion. Using RNA-seq, which allowed us to examine changes in the global 226 transcriptional landscape, we found that target genes of IRF were induced to a similar 227 extent in cells infected with reovirus and cells transfected with vgRNA. NF-κB target 228 genes, however, were expressed to a much lower extent in infected cells in comparison 229 to cells transfected with vgRNA. Moreover, exogenous NF-κB agonists failed to induce 230 a NF-κB dependent gene expression program in infected cells. These data indicate that 231 NF-κB activity is blocked in infected cells. We found that this blockade of NF-κB 232 dependent gene expression is caused by a loss of IKKβ and NEMO, two critical 233 components of the IKK complex. We propose that reovirus inhibits NF-κB to counter its 234 antiviral effects and produce a cellular environment that is conducive for replication. 235