Tracing adaptive pathways in a proofreading-deficient coronavirus

Abstract

Coronaviruses (CoVs) are a family of positive-sense RNA viruses that cause human illnesses ranging from the common cold to severe and lethal respiratory disease. Since 2002, two CoVs (SARS- and MERS-CoV) have emerged as zoonoses with pandemic potential, and closely-related viruses continue to circulate in animal populations. CoVs are distinguished from other RNA viruses by the complexity of their replication machinery, including the presence of a 3'-5' exoribonuclease (ExoN) within nonstructural protein 14 (nsp14-ExoN). The CoV-nsp14-ExoN is the first and, to date, only proofreading enzyme identified in an RNA virus and mediates high-fidelity replication. ExoN activity is critical for CoV biology, as proofreading-deficient CoVs with disrupted ExoN activity [ExoN(-)] are either nonviable or have significant defects in replication, RNA synthesis, fidelity, and in vivo virulence. Remarkably, despite these fitness costs, ExoN(-) CoVs do not revert the engineered mutations under diverse selective environments. In this dissertation, I use experimental evolution to examine the adaptive landscape of an ExoN(-) CoV, murine hepatitis virus (MHV). I show that the lack of reversion of MHV-ExoN(-) is driven by the limitations and opportunities of the adaptive landscape, which favors compensation over direct reversion. These results reveal a remarkable capacity for MHV to compensate for a disrupted ExoN, support the proposed link between CoV fidelity and fitness, illuminate complex functional and evolutionary relationships between CoV replicase proteins, and identify potential mechanisms for stabilization of attenuated ExoN(-) CoVs. New assays for measuring CoV fidelity and fitness are also discussed.