N7-Methylation of the Coronavirus RNA Cap Is Required for Maximal Virulence by Preventing Innate Immune Recognition
ABSTRACT The ongoing coronavirus (CoV) disease 2019 (COVID-19) pandemic caused by infection with severe acute respiratory syndrome CoV 2 (SARS-CoV-2) is associated with substantial morbidity and mortality. Understanding the immunological and pathological processes of coronavirus diseases is crucial...
| Main Authors: | , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2022-02-01
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| Series: | mBio |
| Subjects: | |
| Online Access: | https://journals.asm.org/doi/10.1128/mbio.03662-21 |
| _version_ | 1818872268248317952 |
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| author | Ruangang Pan Eveline Kindler Liu Cao Yu Zhou Zhen Zhang Qianyun Liu Nadine Ebert Roland Züst Ying Sun Alexander E. Gorbalenya Stanley Perlman Volker Thiel Yu Chen Deyin Guo |
| author_facet | Ruangang Pan Eveline Kindler Liu Cao Yu Zhou Zhen Zhang Qianyun Liu Nadine Ebert Roland Züst Ying Sun Alexander E. Gorbalenya Stanley Perlman Volker Thiel Yu Chen Deyin Guo |
| author_sort | Ruangang Pan |
| collection | DOAJ |
| description | ABSTRACT The ongoing coronavirus (CoV) disease 2019 (COVID-19) pandemic caused by infection with severe acute respiratory syndrome CoV 2 (SARS-CoV-2) is associated with substantial morbidity and mortality. Understanding the immunological and pathological processes of coronavirus diseases is crucial for the rational design of effective vaccines and therapies for COVID-19. Previous studies showed that 2′-O-methylation of the viral RNA cap structure is required to prevent the recognition of viral RNAs by intracellular innate sensors. Here, we demonstrate that the guanine N7-methylation of the 5′ cap mediated by coronavirus nonstructural protein 14 (nsp14) contributes to viral evasion of the type I interferon (IFN-I)-mediated immune response and pathogenesis in mice. A Y414A substitution in nsp14 of the coronavirus mouse hepatitis virus (MHV) significantly decreased N7-methyltransferase activity and reduced guanine N7-methylation of the 5′ cap in vitro. Infection of myeloid cells with recombinant MHV harboring the nsp14-Y414A mutation (rMHVnsp14-Y414A) resulted in upregulated expression of IFN-I and ISG15 mainly via MDA5 signaling and in reduced viral replication compared to that of wild-type rMHV. rMHVnsp14-Y414A replicated to lower titers in livers and brains and exhibited an attenuated phenotype in mice. This attenuated phenotype was IFN-I dependent because the virulence of the rMHVnsp14-Y414A mutant was restored in Ifnar−/− mice. We further found that the comparable mutation (Y420A) in SARS-CoV-2 nsp14 (rSARS-CoV-2nsp14-Y420A) also significantly decreased N7-methyltransferase activity in vitro, and the mutant virus was attenuated in K18-human ACE2 transgenic mice. Moreover, infection with rSARS-CoV-2nsp14-Y420A conferred complete protection against subsequent and otherwise lethal SARS-CoV-2 infection in mice, indicating the vaccine potential of this mutant. IMPORTANCE Coronaviruses (CoVs), including SARS-CoV-2, the cause of COVID-19, use several strategies to evade the host innate immune responses. While the cap structure of RNA, including CoV RNA, is important for translation, previous studies indicate that the cap also contributes to viral evasion from the host immune response. In this study, we demonstrate that the N7-methylated cap structure of CoV RNA is pivotal for virus immunoevasion. Using recombinant MHV and SARS-CoV-2 encoding an inactive N7-methyltransferase, we demonstrate that these mutant viruses are highly attenuated in vivo and that attenuation is apparent at very early times after infection. Virulence is restored in mice lacking interferon signaling. Further, we show that infection with virus defective in N7-methylation protects mice from lethal SARS-CoV-2, suggesting that the N7-methylase might be a useful target in drug and vaccine development. |
| first_indexed | 2024-12-19T12:36:07Z |
| format | Article |
| id | doaj.art-841cb6336f9f44e5a3a759faac211544 |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-12-19T12:36:07Z |
| publishDate | 2022-02-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-841cb6336f9f44e5a3a759faac2115442022-12-21T20:21:09ZengAmerican Society for MicrobiologymBio2150-75112022-02-0113110.1128/mbio.03662-21N7-Methylation of the Coronavirus RNA Cap Is Required for Maximal Virulence by Preventing Innate Immune RecognitionRuangang Pan0Eveline Kindler1Liu Cao2Yu Zhou3Zhen Zhang4Qianyun Liu5Nadine Ebert6Roland Züst7Ying Sun8Alexander E. Gorbalenya9Stanley Perlman10Volker Thiel11Yu Chen12Deyin Guo13State Key Laboratory of Virology, Modern Virology Research Center, Institute for Vaccine Research and RNA Institute, College of Life Sciences, Wuhan University, Wuhan, People’s Republic of ChinaInstitute for Virology and Immunology IVI, Mittelhäusern, SwitzerlandState Key Laboratory of Virology, Modern Virology Research Center, Institute for Vaccine Research and RNA Institute, College of Life Sciences, Wuhan University, Wuhan, People’s Republic of ChinaSchool of Mathematics and Statistics, Wuhan University, Wuhan, ChinaState Key Laboratory of Virology, Modern Virology Research Center, Institute for Vaccine Research and RNA Institute, College of Life Sciences, Wuhan University, Wuhan, People’s Republic of ChinaState Key Laboratory of Virology, Modern Virology Research Center, Institute for Vaccine Research and RNA Institute, College of Life Sciences, Wuhan University, Wuhan, People’s Republic of ChinaInstitute for Virology and Immunology IVI, Mittelhäusern, SwitzerlandSpiez Laboratory, Federal Office for Civil Protection, Spiez, SwitzerlandState Key Laboratory of Virology, Modern Virology Research Center, Institute for Vaccine Research and RNA Institute, College of Life Sciences, Wuhan University, Wuhan, People’s Republic of ChinaDepartment of Medical Microbiology, Leiden University Medical Center, Leiden, The NetherlandsDepartment of Microbiology and Immunology, University of Iowa, Iowa City, Iowa, USAInstitute for Virology and Immunology IVI, Mittelhäusern, SwitzerlandState Key Laboratory of Virology, Modern Virology Research Center, Institute for Vaccine Research and RNA Institute, College of Life Sciences, Wuhan University, Wuhan, People’s Republic of ChinaState Key Laboratory of Virology, Modern Virology Research Center, Institute for Vaccine Research and RNA Institute, College of Life Sciences, Wuhan University, Wuhan, People’s Republic of ChinaABSTRACT The ongoing coronavirus (CoV) disease 2019 (COVID-19) pandemic caused by infection with severe acute respiratory syndrome CoV 2 (SARS-CoV-2) is associated with substantial morbidity and mortality. Understanding the immunological and pathological processes of coronavirus diseases is crucial for the rational design of effective vaccines and therapies for COVID-19. Previous studies showed that 2′-O-methylation of the viral RNA cap structure is required to prevent the recognition of viral RNAs by intracellular innate sensors. Here, we demonstrate that the guanine N7-methylation of the 5′ cap mediated by coronavirus nonstructural protein 14 (nsp14) contributes to viral evasion of the type I interferon (IFN-I)-mediated immune response and pathogenesis in mice. A Y414A substitution in nsp14 of the coronavirus mouse hepatitis virus (MHV) significantly decreased N7-methyltransferase activity and reduced guanine N7-methylation of the 5′ cap in vitro. Infection of myeloid cells with recombinant MHV harboring the nsp14-Y414A mutation (rMHVnsp14-Y414A) resulted in upregulated expression of IFN-I and ISG15 mainly via MDA5 signaling and in reduced viral replication compared to that of wild-type rMHV. rMHVnsp14-Y414A replicated to lower titers in livers and brains and exhibited an attenuated phenotype in mice. This attenuated phenotype was IFN-I dependent because the virulence of the rMHVnsp14-Y414A mutant was restored in Ifnar−/− mice. We further found that the comparable mutation (Y420A) in SARS-CoV-2 nsp14 (rSARS-CoV-2nsp14-Y420A) also significantly decreased N7-methyltransferase activity in vitro, and the mutant virus was attenuated in K18-human ACE2 transgenic mice. Moreover, infection with rSARS-CoV-2nsp14-Y420A conferred complete protection against subsequent and otherwise lethal SARS-CoV-2 infection in mice, indicating the vaccine potential of this mutant. IMPORTANCE Coronaviruses (CoVs), including SARS-CoV-2, the cause of COVID-19, use several strategies to evade the host innate immune responses. While the cap structure of RNA, including CoV RNA, is important for translation, previous studies indicate that the cap also contributes to viral evasion from the host immune response. In this study, we demonstrate that the N7-methylated cap structure of CoV RNA is pivotal for virus immunoevasion. Using recombinant MHV and SARS-CoV-2 encoding an inactive N7-methyltransferase, we demonstrate that these mutant viruses are highly attenuated in vivo and that attenuation is apparent at very early times after infection. Virulence is restored in mice lacking interferon signaling. Further, we show that infection with virus defective in N7-methylation protects mice from lethal SARS-CoV-2, suggesting that the N7-methylase might be a useful target in drug and vaccine development.https://journals.asm.org/doi/10.1128/mbio.03662-21coronavirusnonstructural protein 14 (nsp14)RNA cap structureN7-methylationtype I interferonSARS-CoV-2 |
| spellingShingle | Ruangang Pan Eveline Kindler Liu Cao Yu Zhou Zhen Zhang Qianyun Liu Nadine Ebert Roland Züst Ying Sun Alexander E. Gorbalenya Stanley Perlman Volker Thiel Yu Chen Deyin Guo N7-Methylation of the Coronavirus RNA Cap Is Required for Maximal Virulence by Preventing Innate Immune Recognition mBio coronavirus nonstructural protein 14 (nsp14) RNA cap structure N7-methylation type I interferon SARS-CoV-2 |
| title | N7-Methylation of the Coronavirus RNA Cap Is Required for Maximal Virulence by Preventing Innate Immune Recognition |
| title_full | N7-Methylation of the Coronavirus RNA Cap Is Required for Maximal Virulence by Preventing Innate Immune Recognition |
| title_fullStr | N7-Methylation of the Coronavirus RNA Cap Is Required for Maximal Virulence by Preventing Innate Immune Recognition |
| title_full_unstemmed | N7-Methylation of the Coronavirus RNA Cap Is Required for Maximal Virulence by Preventing Innate Immune Recognition |
| title_short | N7-Methylation of the Coronavirus RNA Cap Is Required for Maximal Virulence by Preventing Innate Immune Recognition |
| title_sort | n7 methylation of the coronavirus rna cap is required for maximal virulence by preventing innate immune recognition |
| topic | coronavirus nonstructural protein 14 (nsp14) RNA cap structure N7-methylation type I interferon SARS-CoV-2 |
| url | https://journals.asm.org/doi/10.1128/mbio.03662-21 |
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