Distinct modes of RNA degradation by the structurally related coronavirus and arenavirus exoribonucleases.

Coronaviruses and arenaviruses encode evolutionarily related DEDDh-family 3'-5' exoribonuclease (ExoN) domains, which support the replication of their RNA genomes and evasion of the host's antiviral immunity. Here, we report comparative biochemical analyses of SARS-CoV-2 and Lassa virus (LASV) ExoN, which show that the two enzymes have distinct characteristics despite structural conservation and their shared preference for double-stranded RNA (dsRNA) substrates. SARS-CoV-2 nsp14/10 exhibits a highly processive behavior and tends to remove many nucleotides upon each binding event. By contrast, LASV NP-ExoN exhibits a less processive behavior and removes fewer nucleotides upon each binding event. The two enzymes also show a difference in the minimal dsRNA length for efficient degradation. We show that nsp14/10 associates with a dsRNA substrate at a faster rate than NP-ExoN and correspondingly has a higher affinity for dsRNA, which is likely to account for the higher processivity. These observations help to understand the biological functions of the viral ExoN enzymes and underscore roles for nsp14/10 beyond proofreading. IMPORTANCE: Coronaviruses such as SARS-CoV-2 and arenaviruses such as Lassa virus (LASV) share a structurally similar exoribonuclease (ExoN) domain as part of the viral non-structural protein 14 (nsp14) and nucleoprotein (NP), respectively, which degrades RNA strands from the 3' terminus. The ExoN activity of coronavirus nsp14 is known for its role in proofreading, where it removes nucleotides misincorporated during viral RNA synthesis, whereas that of arenavirus NP is known for its importance in suppressing the host's immune response by degrading immunostimulatory RNAs. In this study, we show that SARS-CoV-2 ExoN is highly processive and removes many nucleotides upon each binding event, whereas LASV NP ExoN is less processive and removes fewer nucleotides at a time. We further show that the contrasting behaviors are attributable to distinct RNA-binding kinetics of these two viral enzymes. These observations help to understand the biological roles of the viral ExoN enzymes and suggest their pleiotropic functions.