Structure and Function of Wild Type and Alanine Mutated SARS-CoV-2 Orf6

The ongoing COVID-19 pandemic has cause global economic disruption, increased hospitalizations, and death. The virus responsible for this disease, SARS-CoV-2, is now known to have several mechanisms for directly inhibiting or otherwise bypassing the natural innate immune responses to viral infection, including the Orf6 accessory protein, recently described as antagonizing the Type-1 IFN pathway: a critical aspect of the antiviral response. Orf6 is also of interest due to it being a homologous protein to Orf6 in SARS-CoV-1, which also was involved in antagonizing the Type-1 IFN response. To further explore the structure and function of SARS2 Orf6 several experiments were performed using both wild type SARS1 and SARS2 Orf6 as well as 3 Orf6 mutants, with stretches of the protein having their normal amino acids replaced with alanine. The function of the WT and mutant proteins were quantified using an ISRE reporter assay which measured the extent to which ISRE expression was inhibited by Orf6, and thus the degree to which specific alanine mutations affected its function. These same test cases were also visualized using GFP-tagged STAT and HA-tagged Orf6 incubated in cells either exposed or not exposed to IFN-u. Overall it was displayed that alanine mutants that disrupted AA59-61 and AA54-58 seemed to retain no function whereas the AA49-53 alanine mutant retained a reduced functionality. These discoveries display that the C-terminal of SARS2 Orf6 is critical to its function, and justifies research into whether this domain is involved in the molecular mechanism of Orf6.