Unfolding of viral protein 1 N-termini facilitates genome ejection from recombinant adeno-associated virus serotype 8
The role of viral protein (VP) 1 and VP2, which comprise the recombinant adeno-associated virus (rAAV) capsid, in heat-induced genome release was investigated using rAAV serotype 8 (rAAV8) samples with a high VP1/VP2 to VP3 ratio, a low VP1/VP2 to VP3 ratio, and VP3 only. The thermal unfolding of th...
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Elsevier
2025-06-01
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| Series: | Molecular Therapy: Methods & Clinical Development |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2329050125000750 |
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| author | Yuki Yamaguchi Saki Shimojo Tomohiko Ikeda Mitsuko Fukuhara Yasuo Tsunaka Risa Shibuya Mark Allen Vergara Rocafort Ryoji Nakatsuka Kiichi Hirohata Tetsuo Torisu Susumu Uchiyama |
| author_facet | Yuki Yamaguchi Saki Shimojo Tomohiko Ikeda Mitsuko Fukuhara Yasuo Tsunaka Risa Shibuya Mark Allen Vergara Rocafort Ryoji Nakatsuka Kiichi Hirohata Tetsuo Torisu Susumu Uchiyama |
| author_sort | Yuki Yamaguchi |
| collection | DOAJ |
| description | The role of viral protein (VP) 1 and VP2, which comprise the recombinant adeno-associated virus (rAAV) capsid, in heat-induced genome release was investigated using rAAV serotype 8 (rAAV8) samples with a high VP1/VP2 to VP3 ratio, a low VP1/VP2 to VP3 ratio, and VP3 only. The thermal unfolding of the VP1 N-termini was closely monitored by nano-differential scanning fluorimetry with an onset temperature (Tonset1) of ∼55°C and a melting temperature of ∼60°C (which was below the onset temperature of capsid disassembly [Tonset2] >70°C), which is related to genome release upon heating. The folded VP1 N-termini prevented release of the full-length genome at temperatures below 60°C, whereas unfolding of the VP1 N-termini facilitated genome release above 60°C. Above Tonset1 and below Tonset2, most rAAV8 particles remained as monomeric particles in three states: capsids encapsidating their single-stranded DNA (ssDNA), capsids that had fully released their genome, and capsids that had fully ejected the genome while tethering the genome on the capsid surface as evidenced by large frictional ratios in analytical ultracentrifugation. The ratio of VP1 and/or VP2 to total VPs had little effect on the extent of genome release. These findings provide new insights into heat-induced genome release from rAAV at the molecular level. |
| format | Article |
| id | doaj-art-7ce85e20fc3f4fdfafa7d0899c5ef87a |
| institution | OA Journals |
| issn | 2329-0501 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj-art-7ce85e20fc3f4fdfafa7d0899c5ef87a2025-08-20T02:28:38ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012025-06-0133210148010.1016/j.omtm.2025.101480Unfolding of viral protein 1 N-termini facilitates genome ejection from recombinant adeno-associated virus serotype 8Yuki Yamaguchi0Saki Shimojo1Tomohiko Ikeda2Mitsuko Fukuhara3Yasuo Tsunaka4Risa Shibuya5Mark Allen Vergara Rocafort6Ryoji Nakatsuka7Kiichi Hirohata8Tetsuo Torisu9Susumu Uchiyama10Department of Biotechnology, Graduate School of Engineering, The University of Osaka, 2-1 Yamadaoka, Suita, Osaka 565-0871, JapanDepartment of Biotechnology, Graduate School of Engineering, The University of Osaka, 2-1 Yamadaoka, Suita, Osaka 565-0871, JapanDepartment of Biotechnology, Graduate School of Engineering, The University of Osaka, 2-1 Yamadaoka, Suita, Osaka 565-0871, JapanDepartment of Biotechnology, Graduate School of Engineering, The University of Osaka, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; U-Medico Inc., 2-1 Yamadaoka, Suita, Osaka 565-0871, JapanDepartment of Biotechnology, Graduate School of Engineering, The University of Osaka, 2-1 Yamadaoka, Suita, Osaka 565-0871, JapanDepartment of Biotechnology, Graduate School of Engineering, The University of Osaka, 2-1 Yamadaoka, Suita, Osaka 565-0871, JapanDepartment of Biotechnology, Graduate School of Engineering, The University of Osaka, 2-1 Yamadaoka, Suita, Osaka 565-0871, JapanDepartment of Biotechnology, Graduate School of Engineering, The University of Osaka, 2-1 Yamadaoka, Suita, Osaka 565-0871, JapanDepartment of Biotechnology, Graduate School of Engineering, The University of Osaka, 2-1 Yamadaoka, Suita, Osaka 565-0871, JapanDepartment of Biotechnology, Graduate School of Engineering, The University of Osaka, 2-1 Yamadaoka, Suita, Osaka 565-0871, JapanDepartment of Biotechnology, Graduate School of Engineering, The University of Osaka, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan; Corresponding author: Susumu Uchiyama, PhD, Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.The role of viral protein (VP) 1 and VP2, which comprise the recombinant adeno-associated virus (rAAV) capsid, in heat-induced genome release was investigated using rAAV serotype 8 (rAAV8) samples with a high VP1/VP2 to VP3 ratio, a low VP1/VP2 to VP3 ratio, and VP3 only. The thermal unfolding of the VP1 N-termini was closely monitored by nano-differential scanning fluorimetry with an onset temperature (Tonset1) of ∼55°C and a melting temperature of ∼60°C (which was below the onset temperature of capsid disassembly [Tonset2] >70°C), which is related to genome release upon heating. The folded VP1 N-termini prevented release of the full-length genome at temperatures below 60°C, whereas unfolding of the VP1 N-termini facilitated genome release above 60°C. Above Tonset1 and below Tonset2, most rAAV8 particles remained as monomeric particles in three states: capsids encapsidating their single-stranded DNA (ssDNA), capsids that had fully released their genome, and capsids that had fully ejected the genome while tethering the genome on the capsid surface as evidenced by large frictional ratios in analytical ultracentrifugation. The ratio of VP1 and/or VP2 to total VPs had little effect on the extent of genome release. These findings provide new insights into heat-induced genome release from rAAV at the molecular level.http://www.sciencedirect.com/science/article/pii/S2329050125000750gene therapyadeno-associated virusgenome releasethermal unfoldingmass photometrynano-differential scanning fluorimetry |
| spellingShingle | Yuki Yamaguchi Saki Shimojo Tomohiko Ikeda Mitsuko Fukuhara Yasuo Tsunaka Risa Shibuya Mark Allen Vergara Rocafort Ryoji Nakatsuka Kiichi Hirohata Tetsuo Torisu Susumu Uchiyama Unfolding of viral protein 1 N-termini facilitates genome ejection from recombinant adeno-associated virus serotype 8 Molecular Therapy: Methods & Clinical Development gene therapy adeno-associated virus genome release thermal unfolding mass photometry nano-differential scanning fluorimetry |
| title | Unfolding of viral protein 1 N-termini facilitates genome ejection from recombinant adeno-associated virus serotype 8 |
| title_full | Unfolding of viral protein 1 N-termini facilitates genome ejection from recombinant adeno-associated virus serotype 8 |
| title_fullStr | Unfolding of viral protein 1 N-termini facilitates genome ejection from recombinant adeno-associated virus serotype 8 |
| title_full_unstemmed | Unfolding of viral protein 1 N-termini facilitates genome ejection from recombinant adeno-associated virus serotype 8 |
| title_short | Unfolding of viral protein 1 N-termini facilitates genome ejection from recombinant adeno-associated virus serotype 8 |
| title_sort | unfolding of viral protein 1 n termini facilitates genome ejection from recombinant adeno associated virus serotype 8 |
| topic | gene therapy adeno-associated virus genome release thermal unfolding mass photometry nano-differential scanning fluorimetry |
| url | http://www.sciencedirect.com/science/article/pii/S2329050125000750 |
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