Major HBV splice variant encoding a novel protein important for infection
Copyright © 2024 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved..
BACKGROUND & AIMS: HBV expresses more than 10 spliced RNAs from the viral pregenomic RNA, but their functions remain elusive and controversial. To address the function of HBV spliced RNAs, we generated splicing-deficient HBV mutants and conducted experiments to assess the impact of these mutants on HBV infection.
METHODS: HepG2-NTCP cells, human hepatocyte chimeric FRG mice (hu-FRG mice), and serum from patients with chronic hepatitis B were used for experiments on HBV infection. Additionally, SHifter assays and cryo-electron microscopy were performed.
RESULTS: We found the infectivity of splicing-deficient HBV was decreased 100-1,000-fold compared with that of wild-type HBV in hu-FRG mice. Another mutant, A487C, which loses the most abundant spliced RNA (SP1), also exhibits severely impaired infectivity. SP1 hypothetically encodes a novel protein HBcSP1 (HBc-Cys) that lacks the C-terminal cysteine from full-length HBc. In the SHifter assay, HBcSP1 was detected in wild-type viral particles at a ratio of about 20-100% vs. conventional HBc, as well as in the serum of patients with chronic hepatitis B, but not in A487C particles. When infection was conducted with a shorter incubation time of 4-8 h at lower PEG concentrations in HepG2-NTCP cells, the entry of the A487C mutant was significantly slower. SP1 cDNA complementation of the A487C mutant succeeded in rescuing its infectivity in hu-FRG mice and HepG2-NTCP cells. Moreover, cryo-electron microscopy revealed a disulfide bond between HBc cysteine 183 and 48 in the HBc intradimer of the A487C capsid, leading to a locked conformation that disfavored viral entry in contrast to the wild-type capsid.
CONCLUSIONS: Prior studies unveiled the potential integration of the HBc-Cys protein into the HBV capsid. We confirmed the proposal and validated its identity and function during infection.
IMPACT AND IMPLICATIONS: HBV SP1 RNA encodes a novel HBc protein (HBcSP1) that lacks the C-terminal cysteine from conventional HBc (HBc-Cys). HBcSP1 was detected in cell culture-derived HBV and confirmed in patients with chronic infection by both immunological and chemical modification assays at 10-50% of capsid. The splicing-deficient mutant HBV (A487C) impaired infectivity in human hepatocyte chimeric mice and viral entry in the HepG2-NTCP cell line. Furthermore, these deficiencies of the splicing-deficient mutant could be rescued by complementation with the SP1-encoded protein HBcSP1. We confirmed and validated the identity and function of HBcSP1 during infection, building on the current model of HBV particles.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2024 |
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| Erschienen: |
2024 |
| Enthalten in: |
Zur Gesamtaufnahme - year:2024 |
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| Enthalten in: |
Journal of hepatology - (2024) vom: 07. Feb. |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Chung, Chen-Yen [VerfasserIn] |
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| Links: |
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| Themen: |
Disulfide bond |
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| Anmerkungen: |
Date Revised 21.03.2024 published: Print-Electronic Citation Status Publisher |
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| doi: |
10.1016/j.jhep.2024.01.037 |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM368258424 |
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| 245 | 1 | 0 | |a Major HBV splice variant encoding a novel protein important for infection |
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| 500 | |a Date Revised 21.03.2024 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status Publisher | ||
| 520 | |a Copyright © 2024 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved. | ||
| 520 | |a BACKGROUND & AIMS: HBV expresses more than 10 spliced RNAs from the viral pregenomic RNA, but their functions remain elusive and controversial. To address the function of HBV spliced RNAs, we generated splicing-deficient HBV mutants and conducted experiments to assess the impact of these mutants on HBV infection | ||
| 520 | |a METHODS: HepG2-NTCP cells, human hepatocyte chimeric FRG mice (hu-FRG mice), and serum from patients with chronic hepatitis B were used for experiments on HBV infection. Additionally, SHifter assays and cryo-electron microscopy were performed | ||
| 520 | |a RESULTS: We found the infectivity of splicing-deficient HBV was decreased 100-1,000-fold compared with that of wild-type HBV in hu-FRG mice. Another mutant, A487C, which loses the most abundant spliced RNA (SP1), also exhibits severely impaired infectivity. SP1 hypothetically encodes a novel protein HBcSP1 (HBc-Cys) that lacks the C-terminal cysteine from full-length HBc. In the SHifter assay, HBcSP1 was detected in wild-type viral particles at a ratio of about 20-100% vs. conventional HBc, as well as in the serum of patients with chronic hepatitis B, but not in A487C particles. When infection was conducted with a shorter incubation time of 4-8 h at lower PEG concentrations in HepG2-NTCP cells, the entry of the A487C mutant was significantly slower. SP1 cDNA complementation of the A487C mutant succeeded in rescuing its infectivity in hu-FRG mice and HepG2-NTCP cells. Moreover, cryo-electron microscopy revealed a disulfide bond between HBc cysteine 183 and 48 in the HBc intradimer of the A487C capsid, leading to a locked conformation that disfavored viral entry in contrast to the wild-type capsid | ||
| 520 | |a CONCLUSIONS: Prior studies unveiled the potential integration of the HBc-Cys protein into the HBV capsid. We confirmed the proposal and validated its identity and function during infection | ||
| 520 | |a IMPACT AND IMPLICATIONS: HBV SP1 RNA encodes a novel HBc protein (HBcSP1) that lacks the C-terminal cysteine from conventional HBc (HBc-Cys). HBcSP1 was detected in cell culture-derived HBV and confirmed in patients with chronic infection by both immunological and chemical modification assays at 10-50% of capsid. The splicing-deficient mutant HBV (A487C) impaired infectivity in human hepatocyte chimeric mice and viral entry in the HepG2-NTCP cell line. Furthermore, these deficiencies of the splicing-deficient mutant could be rescued by complementation with the SP1-encoded protein HBcSP1. We confirmed and validated the identity and function of HBcSP1 during infection, building on the current model of HBV particles | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a HBV | |
| 650 | 4 | |a HBV in vitro infection | |
| 650 | 4 | |a HBV in vivo infection | |
| 650 | 4 | |a HBV spliced RNA | |
| 650 | 4 | |a HBc(-Cys) | |
| 650 | 4 | |a HBc(SP1) | |
| 650 | 4 | |a HepG2-NTCP cells | |
| 650 | 4 | |a disulfide bond | |
| 650 | 4 | |a hu-FRG mice | |
| 700 | 1 | |a Sun, Cheng-Pu |e verfasserin |4 aut | |
| 700 | 1 | |a Tao, Mi-Hua |e verfasserin |4 aut | |
| 700 | 1 | |a Wu, Hui-Lin |e verfasserin |4 aut | |
| 700 | 1 | |a Wang, Sheng-Han |e verfasserin |4 aut | |
| 700 | 1 | |a Yeh, Shiou-Hwei |e verfasserin |4 aut | |
| 700 | 1 | |a Zheng, Qing-Bing |e verfasserin |4 aut | |
| 700 | 1 | |a Yuan, Quan |e verfasserin |4 aut | |
| 700 | 1 | |a Xia, Ning-Shao |e verfasserin |4 aut | |
| 700 | 1 | |a Ogawa, Kenji |e verfasserin |4 aut | |
| 700 | 1 | |a Nakashima, Kenji |e verfasserin |4 aut | |
| 700 | 1 | |a Suzuki, Tetsuro |e verfasserin |4 aut | |
| 700 | 1 | |a Chen, Pei-Jer |e verfasserin |4 aut | |
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