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1、Coronavirus nonstructural protein 15 mediates evasion of dsRNA sensors and limits apoptosis in macrophages Xufang Denga, Matthew Hackbarta, Robert C. Mettelmana, Amornrat OBriena, Anna M. Mielecha, Guanghui Yib, C. Cheng Kaob, and Susan C. Bakera,1 aDepartment of Microbiology and Immunology, Stritch
2、 School of Medicine, Loyola University Chicago, Maywood, IL 60153; andbDepartment of Molecular and Cellular Biochemistry, Indiana University, Bloomington, IN 47405 Edited by Ralph S. Baric, University of North Carolina at Chapel Hill, Chapel Hill, NC, and accepted by Editorial Board Member Linda J.
3、Saif April 12, 2017 (received for review December 9, 2016) 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
4、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 N
5、15m1, 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. Immu- nofluorescence imaging of nsp15 mutant virus-infe
6、cted macro- phages 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 immun
7、e 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. coronavirus|nsp15|endoribonuclease|dsRNA|interferon
8、 C oronaviruses are a family of positive-sense RNA viruses that infect humans and animals and cause respiratory, gastroin- testinal, or neurologic disease. Coronaviruses can emerge from animal reservoirs to cause significant epidemics in humans, as exemplified by the Severe Acute Respiratory Syndrom
9、e coro- navirus (SARS-CoV) outbreak in 20022003 and Middle East Respiratory Syndrome coronavirus (MERS-CoV), which was recognized as an emerging virus in 2012 (1, 2). A remarkable feature of many coronaviruses is their ability to infect macro- phages and delay triggering of antiviral sensors that wo
10、uld oth- erwise activate production of type I IFN (IFN-/) (35). Coronaviruses encode multiple antagonists that likely impede or delay the activation of type I IFN and IFN-stimulated genes (ISGs), thereby contributing to pathogenesis (6). A recent report using SARS-CoV infection of mice documented th
11、at delayed IFN signaling contributes to disease (7). The goal of this study was to identify viral factors that contribute to the delay in the activation of IFN in response to coronavirus infection. To investigate coronavirus antagonism of the early IFN re- sponse, we use mouse hepatitis virus strain
12、 A59 (MHV-A59). This model coronavirus replicates in multiple murine cell types, including macrophages, and can cause acute hepatitis or lethal encephalitis, depending on the site of injection. The viral geno- mic RNA comprises 32 kb. Two-thirds of the genome encodes a large replicase polyprotein, w
13、hereas the remainder of the ge- nome codes for structural proteins and strain-specific accessory proteins (Fig. 1A). The replicase polyprotein is processed by viral proteases into 16 nonstructural proteins (nsps) that assemble in the host endoplasmic reticulum to generate convoluted mem- branes and
14、double-membrane vesicles (DMVs), which are the sites of viral RNA synthesis (8, 9). Coronavirus RNA replication proceeds via the generation of a nested set of negative-strand RNAs that serve as templates for synthesis of mRNAs and new positive-strand genomes (10). Double-stranded RNA (dsRNA) interme
15、diates, which are potent stimulators of cytoplasmic in- nate sensors, are produced during this process and associate with the DMVs (9). A potential function for coronavirus DMVs may be to sequester viral dsRNA away from host dsRNA sensors; however, it is unclear if DMVs alone are sufficient to preve
16、nt activation of the host innate immune response. Here we report that coronavirus nonstructural protein 15 (nsp15), a highly conserved nidovirus component with endoribonuclease activity, acts in conjunction with the viral replication complex to limit the exposure of viral dsRNA to host dsRNA sensors. Bioinformatic analysis revealed that nsp15 contains a do- main with distant homology to cellular endoribonucleases. The nsp15 endoribonuclease, termed NendoU, is highly conserved among vertebrate ni
