SARS-CoV and SARS-CoV-2 Nucleocapsid Function is Affected by Inhibiting Mitochondrial ROS Release by CNP

Abstract

Overexpression of a protein called 2’, 3’-cyclic nucleotide 3’-phosphodiesterase (CNP) inhibits SARS-CoV-2 replication by localizing to and preventing depolarization of mitochondria. SARS-CoV-2 infection causes depolarization to release reactive oxygen species (ROS) from mitochondria, which is thought to aid in the viral nucleocapsid’s (N) function in virion assembly. This project explores two hypothetical mechanisms by which inhibition of ROS release affects the N protein and disrupts virion assembly. The first hypothesis is that cytoplasmic ROS are required for N protein dimerization, which is required for its function. The second hypothesis is that cytoplasmic ROS are required for efficient binding of the N protein to the viral genome. We used transient transfection to express and study the N protein; however, this means there is no infection and no stimulus for ROS release. We therefore analyzed N dimerization status in the presence and absence of hydrogen peroxide (H₂O₂). If the N protein dimerizes in the presence of H₂O₂ but not in its absence, it would suggest that ROS release is required for dimerization, which could affect virion assembly. Using immunoprecipitation (IP) and qPCR, we can isolate the N protein and determine the amount of viral genome bound. We hypothesize that the presence of H2O2 would result in more viral RNA being captured, whereas in its absence, the N protein would have no or significantly less RNA attached. Ultimately, this suggests that inhibition of ROS release disrupts the binding of the viral genome to the N protein. A better understanding of the mechanism by which the later stages of viral replication are hindered can be used to develop more effective therapeutic treatments.