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            Tagged nsp15 Reveals Interactions with Coronavirus Replication/Transcription Complex-Associated Proteins

Athmer, Jeremiah; Fehr, Anthony R.; Grunewald, Matthew; Smith, Everett Clinton; Denison, Mark R.; Perlman, Stanley

doi:10.1128/mbio.02320-16

Cited by 58 publications

(60 citation statements)

References 51 publications

(76 reference statements)

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“…Remarkably, Nsp15 binds to the Nsp7/Nsp8 complex with a stronger affinity than to Nsp8. In contrast to previous IP results (20), both the pulldown and MST assays could not detect an interaction between Nsp15 and Nsp12, possibly due to extremely weak interaction. Moreover, given that Nsp12 can complex with Nsp8 and Nsp7 (20), another possible explanation might be that the detected interaction between Nsp12 and Nsp15 by IP was a result of the indirect binding of Nsp12 with Nsp8 instead of the direct binding with Nsp15.…”

Section: Discussioncontrasting

confidence: 99%

Structural and Biochemical Characterization of Endoribonuclease Nsp15 Encoded by Middle East Respiratory Syndrome Coronavirus

Zhang

Yan

et al. 2018

J Virol

114

127

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Nonstructural protein 15 (Nsp15) encoded by coronavirus (CoV) is a nidoviral uridylate-specific endoribonuclease (NendoU) that plays an essential role in the life cycle of the virus. Structural information on this crucial protein from the Middle East respiratory syndrome CoV (MERS-CoV), which is lethally pathogenic and has caused severe respiratory diseases worldwide, is lacking. Here, we determined the crystal structure of MERS-CoV Nsp15 at a 2.7-Å resolution and performed the relevant biochemical assays to study how NendoU activity is regulated. Although the overall structure is conserved, MERS-CoV Nsp15 shows unique and novel features compared to its homologs. Serine substitution of residue F285, which harbors an aromatic side chain that disturbs RNA binding compared with that of other homologs, increases catalytic activity. Mutations of residues residing on the oligomerization interfaces that distort hexamerization, namely, N38A, Y58A, and N157A, decrease thermostability, decrease affinity of binding with RNA, and reduce the NendoU activity of Nsp15. In contrast, mutant D39A exhibits increased activity and a higher substrate binding capacity. Importantly, Nsp8 was found to interact with both monomeric and hexameric Nsp15. The Nsp7/Nsp8 complex displays a higher binding affinity for Nsp15. Furthermore, Nsp8 and the Nsp7/Nsp8 complex also enhance the NendoU activity of hexameric Nsp15 Taking the findings together, this work first provides evidence on how the activity of Nsp15 may be functionally mediated by catalytic residues, oligomeric assembly, RNA binding efficiency, or the possible association with other nonstructural proteins. The lethally pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV) and the severe acute respiratory syndrome coronavirus (SARS-CoV) pose serious threats to humans. Endoribonuclease Nsp15 encoded by coronavirus plays an important role in viral infection and pathogenesis. This study determines the structure of MERS-CoV Nsp15 and demonstrates how the catalytic activity of this protein is potentially mediated, thereby providing structural and functional evidence for developing antiviral drugs. We also hypothesize that the primase-like protein Nsp8 and the Nsp7/Nsp8 complex may interact with Nsp15 and affect enzymatic activity. This contributes to the understanding of the association of Nsp15 with the viral replication and transcription machinery.

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Section: Discussioncontrasting

confidence: 99%

Structural and Biochemical Characterization of Endoribonuclease Nsp15 Encoded by Middle East Respiratory Syndrome Coronavirus

Zhang

Yan

et al. 2018

J Virol

114

127

View full text Add to dashboard Cite

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“…Previous studies reported that coronavirus dsRNA associates with replication complexes early during infection and is buried in DMVs, which are thought to protect viral RNA from host sensors (8,9,43). Additionally, our group and others have shown that nsp15 also associates with replication complexes (44,45) and interacts with single-strand RNA and dsRNA (13). Therefore, we hypothesized that that nsp15 may function to maintain the asso-ciation of dsRNA with replication complexes.…”

Section: Mhv Nsp15 Mutant Viruses Trigger Rapid Apoptotic Cell Death Inmentioning

confidence: 94%

Coronavirus nonstructural protein 15 mediates evasion of dsRNA sensors and limits apoptosis in macrophages

Deng

Hackbart

Mettelman

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

324

411

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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 liveattenuated vaccines.

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“…The RNA was transcribed into cDNA using Moloney murine leukemia virus reverse transcriptase (MMLV RT) (Thermo Fisher Scientific). Subgenomic RNA levels were measured on a QuantStudio qPCR 3 system (Thermo Fisher Scientific) using previously described subgenomic RNA primers (41). The levels of subgenomic RNA were normalized to hypoxanthine-guanine phosphoribosyltransferase (HPRT) by the following threshold cycle (C T ) equation: ΔC T ϭ C T of gene of interest Ϫ C T of HPRT.…”

Section: Methodsmentioning

confidence: 99%

Murine Olfactory Bulb Interneurons Survive Infection with a Neurotropic Coronavirus

Wheeler

Athmer

Meyerholz

et al. 2017

J Virol

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Viral infection of the central nervous system is complicated by the mostly irreplaceable nature of neurons, as the loss of neurons has the potential to result in permanent damage to brain function. However, whether neurons or other cells in the CNS sometimes survive infection and the effects of infection on neuronal function are largely unknown. To address this question, we used the rJHM strain (rJ) of mouse hepatitis virus, (MHV), a neurotropic coronavirus, which causes acute encephalitis in susceptible strains of mice. To determine whether neurons or other CNS cells survive acute infection with this virulent virus, we developed a recombinant JHMV that expresses Cre recombinase (rJ-Cre) and infected mice that universally expressed a silent (floxed) version of tdTomato. Infection of these mice with rJ-Cre resulted in expression of tdTomato in host cells. The results showed that some cells were able to survive the infection, as demonstrated by continued tdTomato expression after virus antigen could no longer be detected. Most notably, interneurons in the olfactory bulb, which are known to be inhibitory, represented a large fraction of the surviving cells. In conclusion, our results indicated that some neurons are resistant to virus-mediated cell death and provide a framework for studying the effects of prior coronavirus infection on neuron function. We developed a novel recombinant virus that allows for the study of cells that survive an infection by a central nervous system-specific strain of murine coronavirus. Using this virus, we identified neurons and to a lesser extent, non-neuronal cells in the brain that were infected during the acute phase of the infection and survived for approximately two weeks until the mice succumbed to the infection. We focused on neurons and glial cells within the olfactory bulb because the virus enters the brain at this site. Our results show that interneurons of the olfactory bulb were the primary cell type able to survive infection. Further, these results indicate that this system will be useful for functional and gene expression studies of cells in the brain that survive acute infection.

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In Situ Tagged nsp15 Reveals Interactions with Coronavirus Replication/Transcription Complex-Associated Proteins

Cited by 58 publications

References 51 publications

Structural and Biochemical Characterization of Endoribonuclease Nsp15 Encoded by Middle East Respiratory Syndrome Coronavirus

Structural and Biochemical Characterization of Endoribonuclease Nsp15 Encoded by Middle East Respiratory Syndrome Coronavirus

Coronavirus nonstructural protein 15 mediates evasion of dsRNA sensors and limits apoptosis in macrophages

Murine Olfactory Bulb Interneurons Survive Infection with a Neurotropic Coronavirus

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