Tuning VSV-G Expression Improves Baculovirus Integrity, Stability and Mammalian Cell Transduction Efficiency
Baculoviral vectors (BVs) derived from <i>Autographa californica</i> multiple nucleopolyhedrovirus (AcMNPV) are an attractive tool for multigene delivery in mammalian cells, which is particularly relevant for CRISPR technologies. Most applications in mammalian cells rely on BVs that are...
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| author | Martina Mattioli Renata A. Raele Gunjan Gautam Ufuk Borucu Christiane Schaffitzel Francesco Aulicino Imre Berger |
| author_facet | Martina Mattioli Renata A. Raele Gunjan Gautam Ufuk Borucu Christiane Schaffitzel Francesco Aulicino Imre Berger |
| author_sort | Martina Mattioli |
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| description | Baculoviral vectors (BVs) derived from <i>Autographa californica</i> multiple nucleopolyhedrovirus (AcMNPV) are an attractive tool for multigene delivery in mammalian cells, which is particularly relevant for CRISPR technologies. Most applications in mammalian cells rely on BVs that are pseudotyped with vesicular stomatitis virus G-protein (VSV-G) to promote efficient endosomal release. VSV-G expression typically occurs under the control of the hyperactive polH promoter. In this study, we demonstrate that polH-driven VSV-G expression results in BVs characterised by reduced stability, impaired morphology, and VSV-G induced toxicity at high multiplicities of transduction (MOTs) in target mammalian cells. To overcome these drawbacks, we explored five alternative viral promoters with the aim of optimising VSV-G levels displayed on the pseudotyped BVs. We report that Orf-13 and Orf-81 promoters reduce VSV-G expression to less than 5% of polH, rescuing BV morphology and stability. In a panel of human cell lines, we elucidate that BVs with reduced VSV-G support efficient gene delivery and CRISPR-mediated gene editing, at levels comparable to those obtained previously with polH VSV-G-pseudotyped BVs (polH VSV-G BV). These results demonstrate that VSV-G hyperexpression is not required for efficient transduction of mammalian cells. By contrast, reduced VSV-G expression confers similar transduction dynamics while substantially improving BV integrity, structure, and stability. |
| format | Article |
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| institution | OA Journals |
| issn | 1999-4915 |
| language | English |
| publishDate | 2024-09-01 |
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| spelling | doaj-art-db963da6dc9e487ca3ca339ecea236842025-08-20T01:56:13ZengMDPI AGViruses1999-49152024-09-01169147510.3390/v16091475Tuning VSV-G Expression Improves Baculovirus Integrity, Stability and Mammalian Cell Transduction EfficiencyMartina Mattioli0Renata A. Raele1Gunjan Gautam2Ufuk Borucu3Christiane Schaffitzel4Francesco Aulicino5Imre Berger6School of Biochemistry, University of Bristol, 1 Tankard’s Close, Bristol BS8 1TD, UKSchool of Biochemistry, University of Bristol, 1 Tankard’s Close, Bristol BS8 1TD, UKSchool of Biochemistry, University of Bristol, 1 Tankard’s Close, Bristol BS8 1TD, UKGW4 Cryo-EM Facility, University of Bristol, Life Sciences Building, Bristol BS8 1TQ, UKSchool of Biochemistry, University of Bristol, 1 Tankard’s Close, Bristol BS8 1TD, UKSchool of Biochemistry, University of Bristol, 1 Tankard’s Close, Bristol BS8 1TD, UKSchool of Biochemistry, University of Bristol, 1 Tankard’s Close, Bristol BS8 1TD, UKBaculoviral vectors (BVs) derived from <i>Autographa californica</i> multiple nucleopolyhedrovirus (AcMNPV) are an attractive tool for multigene delivery in mammalian cells, which is particularly relevant for CRISPR technologies. Most applications in mammalian cells rely on BVs that are pseudotyped with vesicular stomatitis virus G-protein (VSV-G) to promote efficient endosomal release. VSV-G expression typically occurs under the control of the hyperactive polH promoter. In this study, we demonstrate that polH-driven VSV-G expression results in BVs characterised by reduced stability, impaired morphology, and VSV-G induced toxicity at high multiplicities of transduction (MOTs) in target mammalian cells. To overcome these drawbacks, we explored five alternative viral promoters with the aim of optimising VSV-G levels displayed on the pseudotyped BVs. We report that Orf-13 and Orf-81 promoters reduce VSV-G expression to less than 5% of polH, rescuing BV morphology and stability. In a panel of human cell lines, we elucidate that BVs with reduced VSV-G support efficient gene delivery and CRISPR-mediated gene editing, at levels comparable to those obtained previously with polH VSV-G-pseudotyped BVs (polH VSV-G BV). These results demonstrate that VSV-G hyperexpression is not required for efficient transduction of mammalian cells. By contrast, reduced VSV-G expression confers similar transduction dynamics while substantially improving BV integrity, structure, and stability.https://www.mdpi.com/1999-4915/16/9/1475baculoviruspseudotypingVSV-GAcMNPVgene deliveryviral vector |
| spellingShingle | Martina Mattioli Renata A. Raele Gunjan Gautam Ufuk Borucu Christiane Schaffitzel Francesco Aulicino Imre Berger Tuning VSV-G Expression Improves Baculovirus Integrity, Stability and Mammalian Cell Transduction Efficiency Viruses baculovirus pseudotyping VSV-G AcMNPV gene delivery viral vector |
| title | Tuning VSV-G Expression Improves Baculovirus Integrity, Stability and Mammalian Cell Transduction Efficiency |
| title_full | Tuning VSV-G Expression Improves Baculovirus Integrity, Stability and Mammalian Cell Transduction Efficiency |
| title_fullStr | Tuning VSV-G Expression Improves Baculovirus Integrity, Stability and Mammalian Cell Transduction Efficiency |
| title_full_unstemmed | Tuning VSV-G Expression Improves Baculovirus Integrity, Stability and Mammalian Cell Transduction Efficiency |
| title_short | Tuning VSV-G Expression Improves Baculovirus Integrity, Stability and Mammalian Cell Transduction Efficiency |
| title_sort | tuning vsv g expression improves baculovirus integrity stability and mammalian cell transduction efficiency |
| topic | baculovirus pseudotyping VSV-G AcMNPV gene delivery viral vector |
| url | https://www.mdpi.com/1999-4915/16/9/1475 |
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