Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus Entry
ABSTRACT The lipid composition of the host cell membrane is one of the key determinants of the entry of enveloped viruses into cells. To elucidate the detailed mechanisms behind the cell entry of rubella virus (RuV), one of the enveloped viruses, we searched for host factors involved in such entry b...
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2022-12-01
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| Series: | mBio |
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| Online Access: | https://journals.asm.org/doi/10.1128/mbio.01698-22 |
| _version_ | 1798004722923208704 |
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| author | Yoshio Mori Masafumi Sakata Shota Sakai Toru Okamoto Yuichiro Nakatsu Shuhei Taguwa Noriyuki Otsuki Yusuke Maeda Kentaro Hanada Yoshiharu Matsuura Makoto Takeda |
| author_facet | Yoshio Mori Masafumi Sakata Shota Sakai Toru Okamoto Yuichiro Nakatsu Shuhei Taguwa Noriyuki Otsuki Yusuke Maeda Kentaro Hanada Yoshiharu Matsuura Makoto Takeda |
| author_sort | Yoshio Mori |
| collection | DOAJ |
| description | ABSTRACT The lipid composition of the host cell membrane is one of the key determinants of the entry of enveloped viruses into cells. To elucidate the detailed mechanisms behind the cell entry of rubella virus (RuV), one of the enveloped viruses, we searched for host factors involved in such entry by using CRISPR/Cas9 genome-wide knockout screening, and we found sphingomyelin synthase 1 (SMS1), encoded by the SGMS1 gene, as a candidate. RuV growth was strictly suppressed in SGMS1-knockout cells and was completely recovered by the overexpression of enzymatically active SMS1 and partially recovered by that of SMS2, another member of the SMS family, but not by that of enzymatically inactive SMS1. An entry assay using pseudotyped vesicular stomatitis virus possessing RuV envelope proteins revealed that sphingomyelin generated by SMSs is crucial for at least RuV entry. In SGMS1-knockout cells, lipid mixing between the RuV envelope membrane and the membrane of host cells occurred, but entry of the RuV genome from the viral particles into the cytoplasm was strongly inhibited. This indicates that sphingomyelin produced by SMSs is essential for the formation of membrane pores after hemifusion occurs during RuV entry. IMPORTANCE Infection with rubella virus during pregnancy causes congenital rubella syndrome in infants. Despite its importance in public health, the detailed mechanisms of rubella virus cell entry have only recently become somewhat clearer. The E1 protein of rubella virus is classified as a class II fusion protein based on its structural similarity, but it has the unique feature that its activity is dependent on calcium ion binding in the fusion loops. In this study, we found another unique feature, as cellular sphingomyelin plays a critical role in the penetration of the nucleocapsid into the cytoplasm after hemifusion by rubella virus. This provides important insight into the entry mechanism of rubella virus. This study also presents a model of hemifusion arrest during cell entry by an intact virus, providing a useful tool for analyzing membrane fusion, a biologically important phenomenon. |
| first_indexed | 2024-04-11T12:27:56Z |
| format | Article |
| id | doaj.art-613778f4c98645988313fa92ca64cef8 |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-04-11T12:27:56Z |
| publishDate | 2022-12-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-613778f4c98645988313fa92ca64cef82022-12-22T04:23:52ZengAmerican Society for MicrobiologymBio2150-75112022-12-0113610.1128/mbio.01698-22Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus EntryYoshio Mori0Masafumi Sakata1Shota Sakai2Toru Okamoto3Yuichiro Nakatsu4Shuhei Taguwa5Noriyuki Otsuki6Yusuke Maeda7Kentaro Hanada8Yoshiharu Matsuura9Makoto Takeda10Department of Virology, National Institute of Infectious Diseases, Musashimurayama, Tokyo, JapanDepartment of Virology, National Institute of Infectious Diseases, Musashimurayama, Tokyo, JapanDepartment of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Shinjuku, Tokyo, JapanInstitute for Advanced Co-Creation Studies, Osaka University, Suita, Osaka, JapanDepartment of Virology, National Institute of Infectious Diseases, Musashimurayama, Tokyo, JapanCenter for Infectious Disease Education and Research, Osaka University, Suita, Osaka, JapanDepartment of Virology, National Institute of Infectious Diseases, Musashimurayama, Tokyo, JapanLaboratory of Viral Dynamism Research, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, JapanDepartment of Biochemistry and Cell Biology, National Institute of Infectious Diseases, Shinjuku, Tokyo, JapanCenter for Infectious Disease Education and Research, Osaka University, Suita, Osaka, JapanDepartment of Virology, National Institute of Infectious Diseases, Musashimurayama, Tokyo, JapanABSTRACT The lipid composition of the host cell membrane is one of the key determinants of the entry of enveloped viruses into cells. To elucidate the detailed mechanisms behind the cell entry of rubella virus (RuV), one of the enveloped viruses, we searched for host factors involved in such entry by using CRISPR/Cas9 genome-wide knockout screening, and we found sphingomyelin synthase 1 (SMS1), encoded by the SGMS1 gene, as a candidate. RuV growth was strictly suppressed in SGMS1-knockout cells and was completely recovered by the overexpression of enzymatically active SMS1 and partially recovered by that of SMS2, another member of the SMS family, but not by that of enzymatically inactive SMS1. An entry assay using pseudotyped vesicular stomatitis virus possessing RuV envelope proteins revealed that sphingomyelin generated by SMSs is crucial for at least RuV entry. In SGMS1-knockout cells, lipid mixing between the RuV envelope membrane and the membrane of host cells occurred, but entry of the RuV genome from the viral particles into the cytoplasm was strongly inhibited. This indicates that sphingomyelin produced by SMSs is essential for the formation of membrane pores after hemifusion occurs during RuV entry. IMPORTANCE Infection with rubella virus during pregnancy causes congenital rubella syndrome in infants. Despite its importance in public health, the detailed mechanisms of rubella virus cell entry have only recently become somewhat clearer. The E1 protein of rubella virus is classified as a class II fusion protein based on its structural similarity, but it has the unique feature that its activity is dependent on calcium ion binding in the fusion loops. In this study, we found another unique feature, as cellular sphingomyelin plays a critical role in the penetration of the nucleocapsid into the cytoplasm after hemifusion by rubella virus. This provides important insight into the entry mechanism of rubella virus. This study also presents a model of hemifusion arrest during cell entry by an intact virus, providing a useful tool for analyzing membrane fusion, a biologically important phenomenon.https://journals.asm.org/doi/10.1128/mbio.01698-22rubella virusfusionhemifusionsphingomyelinCRISPR/Cas9membrane fusion |
| spellingShingle | Yoshio Mori Masafumi Sakata Shota Sakai Toru Okamoto Yuichiro Nakatsu Shuhei Taguwa Noriyuki Otsuki Yusuke Maeda Kentaro Hanada Yoshiharu Matsuura Makoto Takeda Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus Entry mBio rubella virus fusion hemifusion sphingomyelin CRISPR/Cas9 membrane fusion |
| title | Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus Entry |
| title_full | Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus Entry |
| title_fullStr | Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus Entry |
| title_full_unstemmed | Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus Entry |
| title_short | Membrane Sphingomyelin in Host Cells Is Essential for Nucleocapsid Penetration into the Cytoplasm after Hemifusion during Rubella Virus Entry |
| title_sort | membrane sphingomyelin in host cells is essential for nucleocapsid penetration into the cytoplasm after hemifusion during rubella virus entry |
| topic | rubella virus fusion hemifusion sphingomyelin CRISPR/Cas9 membrane fusion |
| url | https://journals.asm.org/doi/10.1128/mbio.01698-22 |
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