Investigation of severe coronavirus infection with the host

Abstract

Over the last two decades, three zoonotic viruses, SARS-CoV, MERS-CoV, and SARS-CoV-2 have emerged and posed significant threats to human health. After the release of the viral genome in the cytoplasm, 16 NSPs are cleaved and released from the ORF1a and ORF1ab essential for forming the replication-transcription complex to replicate the viral genome. Virus replication relies on the interaction with host proteins for the synthesis of the viral genome, and this process could introduce mutations into the viral genome, but the interactome profile and functionality are undetermined. Also, no developed tool for studying mutations, especially recombination, for the MERS-CoV and SARS-CoV-2 allows for sequencing long amplicons. Here, this thesis demonstrated that an amplicon-sequencing approach, named Rapid Sequencing Long Amplicons (RSLAs), combined with Oxford Nanopore technology can generate data from long amplicons from MERS-CoV and SARS-CoV- 2 genomes in clinical samples suitable for indel and recombination studies. Also, this thesis showed that NSP9 and NSP12, the main components of RTC, interacted with host proteins essential for virus replication. The functioning of SARS-CoV-2 NSP9 was dependent on eEF1A1 and MTHFD1, and NSP12 binding to TRiC/CCT complex was critical to the viral replication and NSP12 stability. Furthermore, NSP12 variants exhibited distinct associations with components of a phosphatase complex, including PP2A and STRN3. The virus that carried NSP12L323 demonstrated reduced susceptibility to disruption in PP2A. These findings allow the scientific communities to investigate mutational profiles of coronavirus for a better understanding of any potential evolution of coronavirus in the future using RSLA. They also showed the dependency of SARS-CoV-2 replication on host proteins revealing new targets for therapeutic interventions.