Murine Hepatitis Virus nsp14 Exoribonuclease Activity Is Required for Resistance to Innate Immunity

Abstract

Coronaviruses (CoVs) are positive-sense RNA viruses that infect numerous mammalian and avian species and are capable of causing severe and lethal disease in humans. CoVs encode several innate immune antagonists that counteract the host innate immune response to facilitate efficient viral replication. CoV nonstructural protein 14 (nsp14) encodes 3'-to-5' exoribonuclease activity (ExoN), which performs a proofreading function and is required for high-fidelity replication. Outside of the order , arenaviruses are the only RNA viruses that encode an ExoN, which functions to degrade double-stranded RNA (dsRNA) replication intermediates. In this study, we tested the hypothesis that CoV ExoN also functions to antagonize the innate immune response. We demonstrate that viruses lacking ExoN activity [ExoN(-)] are sensitive to cellular pretreatment with interferon beta (IFN-β) in a dose-dependent manner. In addition, ExoN(-) virus replication was attenuated in wild-type bone marrow-derived macrophages (BMMs) and partially restored in interferon alpha/beta receptor-deficient (IFNAR) BMMs. ExoN(-) virus replication did not result in IFN-β gene expression, and in the presence of an IFN-β-mediated antiviral state, ExoN(-) viral RNA levels were not substantially reduced relative to those of untreated samples. However, ExoN(-) virus generated from IFN-β-pretreated cells had reduced specific infectivity and decreased relative fitness, suggesting that ExoN(-) virus generated during an antiviral state is less viable to establish a subsequent infection. Overall, our data suggest murine hepatitis virus (MHV) ExoN activity is required for resistance to the innate immune response, and antiviral mechanisms affecting the viral RNA sequence and/or an RNA modification act on viruses lacking ExoN activity. CoVs encode multiple antagonists that prevent or disrupt an efficient innate immune response. Additionally, no specific antiviral therapies or vaccines currently exist for human CoV infections. Therefore, the study of CoV innate immune antagonists is essential for understanding how CoVs overcome host defenses and to maximize potential therapeutic interventions. Here, we sought to determine the contributions of nsp14 ExoN activity in the induction of and resistance to the innate immune response. We show that viruses lacking nsp14 ExoN activity are more sensitive than wild-type MHV to restriction by exogenous IFN-β and that viruses produced in the presence of an antiviral state are less capable of establishing a subsequent viral infection. Our results support the hypothesis that murine hepatitis virus ExoN activity is required for resistance to the innate immune response.

Fitness Barriers Limit Reversion of a Proofreading-Deficient Coronavirus

“…The apparent increased fitness of ExoN-AE resulted from all lineages reverting to WT-ExoN-DE during the experiment. Finally, our previous studies have shown that adaptation of ExoN-AA includes partial compensation for the replication fidelity defect, as measured by reduced susceptibility to the mutagen 5-fluorouracil (5-FU) (7)(8)(9)(10)(11)25). None of the intermediate variants demonstrated statistically significant differences in 5-FU sensitivity compared to that of ExoN-AA ( Fig.…”

“…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 (5). All CoVs encode a proofreading exoribonuclease within nonstructural protein 14 (nsp14-ExoN) that is critical for replication, fidelity, fitness, and virulence, and ExoN inactivation has been proposed as a strategy for live attenuated vaccine development (6)(7)(8)(9)(10)(11)(12)(13)(14)(15). As members of the DEDDh superfamily of exonucleases, CoV ExoNs hydrolyze nucleotides using four metal-coordinating amino acids arranged in three motifs: I (DE), II (E), and III (D) (16,17).…”

“…Alanine substitution in ExoN motif I (DE-to-AA) disrupts ExoN biochemical activity in both severe acute respiratory syndrome (SARS)-CoV and human CoV 229E (hCoV-229E) (16,18,19). The betacoronaviruses murine hepatitis virus (MHV) and SARS-CoV tolerate disruption of ExoN activity [ExoN(Ϫ)] but display mutator phenotypes accompanied by defects in replication, competitive fitness, and evasion of innate immune responses (10,13,14). ExoN active-site mutants in alphacoronaviruses, including transmissible gastroenteritis virus and hCoV-229E, have yet to be recovered and are proposed to be lethal for replication (19,20).…”

PEDV and PDCoV Pathogenesis: The Interplay Between Host Innate Immune Responses and Porcine Enteric Coronaviruses

“…The coronavirus nsps have been shown to be involved mainly in viral RNA synthesis (123)(124)(125)(126)(127). Nevertheless, nsp1, 3, 5, 7, 14, 15, and 16 have been observed to play additional roles in host immune modulatory functions (50,60,(128)(129)(130)(131)(132)(133)(134)(135)(136).…”

Differential induction of type I interferon by type I and type II feline coronaviruses in vitro

“…The mechanism of inhibition of type I IFN response by IFNβ has been closely investigated in several viruses. For example, in TGEV, PEDV, murine hepatitis virus, severe acute respiratory syndrome (SARS)-CoV, and middle east respiratory syndrome (MERS)-CoV, it has been reported that structural proteins, excluding non-structural proteins and spike protein, inhibit IFNβ production (Case et al, 2018(Case et al, , 2016Ding et al, 2017;Hu et al, 2017;Li et al, 2016;Lu et al, 2011;Lui et al, 2016;Zhang et al, 2016). However, it is unclear whether type I IFN responses are inhibited by FCoV.…”