Leveraging Transcriptomics and Proteomics to Identify Novel Coronavirus Genomic Elements and Host Kinase Determinants of Replication

The emergence of severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2 has demonstrated that coronaviruses (CoVs) pose global threat for zoonotic spillover and severe disease to human populations. Development of vaccines and therapeutics for CoV disease has been accelerated by the COVID-19 pandemic, however there remains a need to study the replication and pathogenesis of CoVs to understand viral and host determinants of disease severity and to identify broad acting targets for future CoV emergence. CoVs employ discontinuous transcription to generate subgenomic RNA (sgmRNA) for expression of structural and accessory proteins. We investigated the hypothesis that discontinuous transcription in MERS-CoV not only generated canonical sgmRNA but also non-canonical sgmRNA species that encoded novel viral proteins. By employing Illumina and Oxford nanopore sequencing, a complex transcriptional landscape of non-canonical sgmRNA species was identified during MERS-CoV replication. By mining preexistent proteomics datasets, we identified new open reading frames in the viral genome, one of which was supported by ribosome profiling data. We verified expression of the novel protein, ORF8c, via targeted proteomic analyses and linked potential expression to a non-canonical sgmRNA that was encoded near the 3’ end of the MERS-CoV genome.COVID-19 pathogenesis includes acute, subacute, and chronic phases suggesting that host directed treatment modalities may provide novel opportunities to decrease viral replication and immunopathology. Kinases represent a large group of proteins for which there are already clinically approved inhibitors and treatments that may be rapidly repurposed to combat CoV disease. To address this question, we profiled the host kinase landscape, or kinome, following MERS-CoV and SARS-CoV-2 infection and identified potential pro-viral host kinases that could be targeted to attenuate CoV replication. To measure host kinome responses, multiplex inhibitory bead mass spectrometry and phosphoproteomic analyses were applied to identify key kinase signaling pathways, like the activation of the PI3K/AKT/mTOR pathway following CoV infection, as therapeutic targets. Treatment of cells with PI3K/mTOR inhibitors exhibited potent antiviral effect on both MERS-CoV and SARS-CoV-2 replication at similar concentrations to Remdesivir. Our data support use of PI3K/mTOR kinase inhibitors as potential broad-acting host-directed betacoronavirus antivirals for zoonotic CoV outbreaks.