Intramolecular interaction of NEP regulated by CRM1 ensures the unidirectional transport of M1 for the nuclear export of influenza viral ribonucleoprotein
IntroductionThe influenza virus genome consists of single-stranded RNAs and forms viral ribonucleoprotein (RNP) complexes. After viral genome replication in the nucleus, the viral RNP interacts with viral protein M1. The M1-viral RNP complex is exported to the cytoplasm via the CRM1-dependent pathwa...
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Frontiers Media S.A.
2023-08-01
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| Series: | Frontiers in Virology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fviro.2023.1232906/full |
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| author | Mikako Hirohama Shun Yamashita Masamitsu N. Asaka Takahiro Kuroki Atsushi Kawaguchi Atsushi Kawaguchi Atsushi Kawaguchi Atsushi Kawaguchi |
| author_facet | Mikako Hirohama Shun Yamashita Masamitsu N. Asaka Takahiro Kuroki Atsushi Kawaguchi Atsushi Kawaguchi Atsushi Kawaguchi Atsushi Kawaguchi |
| author_sort | Mikako Hirohama |
| collection | DOAJ |
| description | IntroductionThe influenza virus genome consists of single-stranded RNAs and forms viral ribonucleoprotein (RNP) complexes. After viral genome replication in the nucleus, the viral RNP interacts with viral protein M1. The M1-viral RNP complex is exported to the cytoplasm via the CRM1-dependent pathway using NS2/NEP as an export adaptor protein. NEP is a 14 kDa protein and diffusely localizes in the nucleus and cytoplasm. Upon binding to the NLS motif of M1, NEP inhibits the nuclear accumulation of M1 and promotes the nuclear export of M1-viral RNP complex. However, the detail mechanism by which NEP binds to M1 only in the nucleus remains unclear.MethodsTo visualize the interaction of NEP with M1 in the formation of vRNP export complexes, we performed in situ proximity ligation assays. The close proximity of N-terminal and C-terminal domains of NEP was tested by split Renilla luciferase complementation assays in which the N-terminal and C-terminal fragments of Renilla luciferase were fused to the N-terminus and C-terminus of NEP, respectively.Results and discussionWe found that the intramolecular interaction of NEP inhibits the interaction of NEP with M1. The intramolecular interaction of NEP was mediated through the interaction of the N-terminal NES motif with the M1-binding domain at the C-terminus. By adding leptomycin B, a potent inhibitor of CRM1, the interaction of NEP with M1 was impaired. These results suggest that CRM1 disrupts the intramolecular interaction of NEP by recognizing the NES motif at the N-terminus of NEP, thereby promoting the interaction of NEP with M1. We also found that NEP mutant deficient in the intramolecular interaction was co-localized with M1 at the plasma membrane and did not show nuclear localization with M1. Based on these results, we propose that the intramolecular interaction of NEP regulated by CRM1 ensures the unidirectional transport of M1. |
| first_indexed | 2024-03-12T14:26:48Z |
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| id | doaj.art-e7a687dafaea4fbeb7706bc544e2ed7e |
| institution | Directory Open Access Journal |
| issn | 2673-818X |
| language | English |
| last_indexed | 2024-03-12T14:26:48Z |
| publishDate | 2023-08-01 |
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| spelling | doaj.art-e7a687dafaea4fbeb7706bc544e2ed7e2023-08-18T07:01:42ZengFrontiers Media S.A.Frontiers in Virology2673-818X2023-08-01310.3389/fviro.2023.12329061232906Intramolecular interaction of NEP regulated by CRM1 ensures the unidirectional transport of M1 for the nuclear export of influenza viral ribonucleoproteinMikako Hirohama0Shun Yamashita1Masamitsu N. Asaka2Takahiro Kuroki3Atsushi Kawaguchi4Atsushi Kawaguchi5Atsushi Kawaguchi6Atsushi Kawaguchi7Department of Infection Biology, Faculty of Medicine, University of Tsukuba, Tsukuba, JapanGraduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, JapanDepartment of Infection Biology, Faculty of Medicine, University of Tsukuba, Tsukuba, JapanGraduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, JapanDepartment of Infection Biology, Faculty of Medicine, University of Tsukuba, Tsukuba, JapanGraduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, JapanTransborder Medical Research Center, University of Tsukuba, Tsukuba, JapanMicrobiology Research Center for Sustainability, University of Tsukuba, Tsukuba, JapanIntroductionThe influenza virus genome consists of single-stranded RNAs and forms viral ribonucleoprotein (RNP) complexes. After viral genome replication in the nucleus, the viral RNP interacts with viral protein M1. The M1-viral RNP complex is exported to the cytoplasm via the CRM1-dependent pathway using NS2/NEP as an export adaptor protein. NEP is a 14 kDa protein and diffusely localizes in the nucleus and cytoplasm. Upon binding to the NLS motif of M1, NEP inhibits the nuclear accumulation of M1 and promotes the nuclear export of M1-viral RNP complex. However, the detail mechanism by which NEP binds to M1 only in the nucleus remains unclear.MethodsTo visualize the interaction of NEP with M1 in the formation of vRNP export complexes, we performed in situ proximity ligation assays. The close proximity of N-terminal and C-terminal domains of NEP was tested by split Renilla luciferase complementation assays in which the N-terminal and C-terminal fragments of Renilla luciferase were fused to the N-terminus and C-terminus of NEP, respectively.Results and discussionWe found that the intramolecular interaction of NEP inhibits the interaction of NEP with M1. The intramolecular interaction of NEP was mediated through the interaction of the N-terminal NES motif with the M1-binding domain at the C-terminus. By adding leptomycin B, a potent inhibitor of CRM1, the interaction of NEP with M1 was impaired. These results suggest that CRM1 disrupts the intramolecular interaction of NEP by recognizing the NES motif at the N-terminus of NEP, thereby promoting the interaction of NEP with M1. We also found that NEP mutant deficient in the intramolecular interaction was co-localized with M1 at the plasma membrane and did not show nuclear localization with M1. Based on these results, we propose that the intramolecular interaction of NEP regulated by CRM1 ensures the unidirectional transport of M1.https://www.frontiersin.org/articles/10.3389/fviro.2023.1232906/fullCRM1nuclear exportNEPintramolecular interactioninfluenza A virus |
| spellingShingle | Mikako Hirohama Shun Yamashita Masamitsu N. Asaka Takahiro Kuroki Atsushi Kawaguchi Atsushi Kawaguchi Atsushi Kawaguchi Atsushi Kawaguchi Intramolecular interaction of NEP regulated by CRM1 ensures the unidirectional transport of M1 for the nuclear export of influenza viral ribonucleoprotein Frontiers in Virology CRM1 nuclear export NEP intramolecular interaction influenza A virus |
| title | Intramolecular interaction of NEP regulated by CRM1 ensures the unidirectional transport of M1 for the nuclear export of influenza viral ribonucleoprotein |
| title_full | Intramolecular interaction of NEP regulated by CRM1 ensures the unidirectional transport of M1 for the nuclear export of influenza viral ribonucleoprotein |
| title_fullStr | Intramolecular interaction of NEP regulated by CRM1 ensures the unidirectional transport of M1 for the nuclear export of influenza viral ribonucleoprotein |
| title_full_unstemmed | Intramolecular interaction of NEP regulated by CRM1 ensures the unidirectional transport of M1 for the nuclear export of influenza viral ribonucleoprotein |
| title_short | Intramolecular interaction of NEP regulated by CRM1 ensures the unidirectional transport of M1 for the nuclear export of influenza viral ribonucleoprotein |
| title_sort | intramolecular interaction of nep regulated by crm1 ensures the unidirectional transport of m1 for the nuclear export of influenza viral ribonucleoprotein |
| topic | CRM1 nuclear export NEP intramolecular interaction influenza A virus |
| url | https://www.frontiersin.org/articles/10.3389/fviro.2023.1232906/full |
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