Investigating the role of ORF6 and NSP13 interactions with Cdk5rap2 in SARS-CoV-2 pathogenesis

The continuing COVID-19 pandemic represents one of the most extreme health and economic crises in human history. Excessive release of proinflammatory cytokines, termed 'cytokine storm', is the underlying cause of COVID-19 clinical manifestations. Recently, it was shown that out of 31 proteins produced by the COVID-19 virus, 4 nonstructural (NSP8, 9, 13 & 16), and 3 accessory (ORF3b, 6 & 9b) proteins interact with cyclin-dependent kinase 5 regulatory subunit-associated protein 2 (Cdk5rap2). However, the consequence of these interactions during COVID-19 pathogenesis remains unknown. Interestingly, our laboratory has demonstrated that Cdk5rap2 loss (i) induces cell senescence that accompanies the secretion of high levels of inflammatory cytokines and (ii) stimulates phosphodiesterase 4D (Pde4d), reducing intracellular cAMP concentration, which up- and down-regulates pro- and anti-inflammatory cytokine production, respectively. Since NSP13 and ORF6 of the 7 Cdk5rap2-interacting COVID-19 proteins kills human cells by 30% and 50%, respectively, my HYPOTHESIS is that Cdk5rap2-NSP13/ORF6 interactions stimulate Pde4d, subsequently causing a cytokine storm. I will employ our laboratory's expertise in cell and molecular biological approaches to address my OBJECTIVE to elucidate the role of ORF6 and NSP13 interactions with Cdk5rap2 in COVID-19 pathogenesis. My SPECIFIC AIMS are (i) to investigate whether ORF6 and NSP13 stimulate Pde4d activity via Cdk5rap2 binding and cause downregulation of intracellular cAMP concentration, and (ii) subsequently increase pro-inflammatory cytokine secretion while decreasing anti-inflammatory cytokine release. Results from my studies could provide novel insight into therapeutic strategies to effectively combat COVID-19 infections.