Coronaviruses are positive-sense RNA viruses that generate double-stranded RNA (dsRNA) intermediates during replication, yet evade detection by host innate immune sensors. Here we report that coronavirus nonstructural protein 15 (nsp15), an endoribonuclease, is required for evasion of dsRNA sensors. We evaluated two independent nsp15 mutant mouse coronaviruses, designated N15m1 and N15m3, and found that these viruses replicated poorly and induced rapid cell death in mouse bone marrow-derived macrophages. Infection of macrophages with N15m1, which expresses an unstable nsp15, or N15m3, which expresses a catalysis-deficient nsp15, activated MDA5, PKR, and the OAS/RNase L system, resulting in an early, robust induction of type I IFN, PKR-mediated apoptosis, and RNA degradation. Immunofluorescence imaging of nsp15 mutant virus-infected macrophages revealed significant dispersal of dsRNA early during infection, whereas in WT virus-infected cells, the majority of the dsRNA was associated with replication complexes. The loss of nsp15 activity also resulted in greatly attenuated disease in mice and stimulated a protective immune response. Taken together, our findings demonstrate that coronavirus nsp15 is critical for evasion of host dsRNA sensors in macrophages and reveal that modulating nsp15 stability and activity is a strategy for generating live-attenuated vaccines.
The mechanism by which nsp15 endoribonuclease activity suppresses the activation of dsRNA sensors is unknown. Previously, pestivirus and Lassa virus were shown to encode viral ribonucleases that prevent activation of host sensors by degrading viral dsRNA (59, 60). Given these examples, it seemed logical to evaluate the relative abundance of viral dsRNA in WT- and nsp15 mutant-infected cells (Fig. 6 and Fig. S6). Indeed, while this manuscript was under review, Kindler et al. reported increased accumulation of viral dsRNA in nsp15 mutant virus-infected cells and suggested that nsp15 may be part of a viral RNA decay pathway (61). However, our analysis indicates no difference in the accumulation of dsRNA in cells infected with viruses that express either functional or catalytically-inactive nsp15. We note that the nsp15 catalytic mutant virus reported here (N15m3) encodes a different mutation (H262A) than the virus reported by Kindler et al. (H277A) (61), which may account for the difference in results. Another possibility is that nsp15 may recognize and cleave specific dsRNA targets and that mutations in nsp15 may influence target selection. Bhardwaj et al. found that SARS-CoV nsp15 can bind to a highly conserved, hairpin-structured RNA molecule derived from the 3′ untranslated region of the viral genome (13). This dsRNA-like molecule contains a right-angle turn and a loop with multiple potential cleavage sites. The authors reported that only the site in the right-angle turn could be cleaved, indicating that nsp15 cleavage can be influenced by RNA structure, although it is important to note that these studies were performed in vitro using nonphysiologically relevant manganese concentrations. Future studies are therefore needed to evaluate nsp15-mediated dsRNA cleavage in the context of virus infection.
Another feature of nsp15 is that it colocalizes with membrane-associated viral replication complexes (44, 45). Previous studies suggest that viral dsRNA may be sequestered within membrane-associated replication complexes as a means of protecting it from detection by host sensors (9). It is possible that the functional activity of nsp15 may occur in association with such viral structures. Indeed, the majority of dsRNA was colocalized with replication complex proteins in WT-infected cells. In contrast, in nsp15 mutant-infected cells, we observed early dispersal of dsRNA foci away from replication complexes (Fig. 6), which coincided with early activation of dsRNA sensors. We speculate, therefore, that nsp15 may function as a “gatekeeper” to sequester viral dsRNA within replication complexes and away from host dsRNA sensors. Further studies are needed to fully elucidate the mechanisms used by nsp15 to potentially hide or degrade viral RNA and ultimately prevent activation of host dsRNA sensors.
In summary, this study provides insights into the role of nsp15 as an antagonist of host dsRNA sensors during coronavirus infection in macrophages and in mice and provides new directions for developing live-attenuated vaccines.
Reference & Source information: https://www.pnas.org/
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