Coronavirus nonstructural protein 15 mediates evasion of dsRNA sensors and limits apoptosis in macrophages
Sunday, 2017/05/28 | 05:47:01
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Xufang Deng, Matthew Hackbart, Robert C. Mettelman, Amornrat O’Brien, Anna M. Mielech, Guanghui Yi, C. Cheng Kao, and Susan C. Baker MICROBIOLOGYSignificanceMacrophages are immune cells equipped with multiple double-stranded RNA (dsRNA) sensors designed to detect viral infection and amplify innate antiviral immunity. However, many coronaviruses can infect and propagate in macrophages without activating dsRNA sensors. Here we present a function of murine coronavirus nonstructural protein 15 in preventing detection of viral dsRNA by host sensors. We show that coronaviruses expressing a mutant form of nonstructural protein 15 allow for activation of dsRNA sensors, resulting in an early induction of interferon, rapid apoptosis of macrophages, and a protective immune response in mice. Identifying the strategies used by viruses to evade detection provides us with new approaches for generating vaccines that elicit robust innate immune responses and protective immunity. AbstractCoronaviruses 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.
See: http://www.pnas.org/content/114/21/E4251.abstract.html?etoc PNAS May 23 2017; vol.114. no.21: E4251–E4260
Fig. 2. Nsp15 mutant viruses trigger an early induction of type I IFN in macrophages. (A–C) B6 BMDMs were infected with WT or mutant MHV at an MOI of 0.1. At indicated time points, total RNA was extracted and analyzed for (A) the mRNA levels of IFN-α11 or (B) N gene by qPCR. The levels of mRNA relative to β-actin mRNA were expressed as 2-ΔCT [ΔCT = CT(gene of interest) − CT(β-actin)]. (C) The cell supernatant was collected for the detection of secreted IFN-α by quantitative ELISA. (D) ifih1−/− BMDMs were infected with WT or mutant MHV at an MOI of 0.1. At 16 hpi, the cell supernatant was collected for ELISA. Values were analyzed using a two-way ANOVA test by time in A–C or an unpaired t test in D. *P < 0.05; n.s., not significant. **P < 0.01; ***P < 0.001; ****P < 0.0001. Data are representative of two to three independent experiments and presented as the mean ± SD. Red dashed line is limit of detection. |
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