Generation of HiBiT-tagged recombinant murine coronaviruses to quantitatively study early events during virus replication

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Wilson, Natalie
Sterling, Cade
Smith, Everett Clinton
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University of the South , Biology Department , Senior Honor Theses 2022 , coronavirus , mouse hepatitis virus , transformation-associated recombination
Coronaviruses (CoVs) are a large family of single-stranded, positive-sense RNA viruses that infect a wide range of vertebrate hosts. Along with SARS-CoV-2, the etiological agent of the COVID-19 pandemic, there are six other characterized human CoVs (HCoVs). Infection by the four endemic HCoVs (e.g., -OC43, -NL63, -229E, -HKU1) can cause cold-like illness, while infection with epidemic HCoVs (e.g., SARS-CoV and MERS-CoV) can result in severe disease with high mortality. A comprehensive understanding of viral biology is required to develop effective therapeutics, such as vaccines and antiviral drugs. Early steps during viral replication, such as cell-surface binding and entry, are challenging to study in a quantitative manner. To circumvent some of these technical challenges, we developed tagged recombinant murine coronaviruses expressing a small, N-terminal portion of the luciferase enzyme (NanoLuc) encoded by the deep-sea shrimp Oplophorus gracilirostris. The small size of this tag (HiBiT, 11 amino acids), relative to the remaining catalytic portion of NanoLuc (LgBiT, ~170 amino acids), reduces the risk that the tag interferes with viral protein function. Complementation of the viral protein-HiBiT fusion with LgBiT results in a catalytically active complex referred to as NanoBiT. Because even small amino acid tags can disrupt protein function, we generated constructs that tagged either the N- or C-terminus of each CoV structural protein (N, E, and M), excepting Spike (S), which was only C-terminally tagged. Three of the seven recombinant viruses were recovered, and two showed evidence of replication but were unable to be propagated further. Serial dilution of these viral stocks demonstrated several log10 of linear signal, indicating that they will be useful tools to quantitatively investigate early events during viral replication.