Adeno-associated Virus (AAV) Assembly-Activating Protein Is Not an Essential Requirement for Capsid Assembly of AAV Serotypes 4, 5, and 11
Open accessAbstract
Adeno-associated virus (AAV) vectors have made great progress in their use for gene therapy; however, fundamental aspects of AAV's capsid assembly remain poorly characterized. In this regard, the discovery of assembly-activating protein (AAP) sheds new light on this crucial part of AAV biology and vector production. Previous studies have shown that AAP is essential for assembly; however, how its mechanistic roles in assembly might differ among AAV serotypes remains uncharacterized. Here, we show that biological properties of AAPs and capsid assembly processes are surprisingly distinct among AAV serotypes 1 to 12. In the study, we investigated subcellular localizations and assembly-promoting functions of AAP1 to -12 (i.e., AAPs derived from AAV1 to -12, respectively) and examined the AAP dependence of capsid assembly processes of these 12 serotypes using combinatorial approaches that involved immunofluorescence and transmission electron microscopy, barcode-Seq (i. e., a high-throughput quantitative method using DNA barcodes and a next-generation sequencing technology), and quantitative dot blot assays. This study revealed that AAP1 to -12 are all localized in the nucleus with serotype-specific differential patterns of nucleolar association; AAPs and assembled capsids do not necessarily colocalize; AAPs are promiscuous in promoting capsid assembly of other serotypes, with the exception of AAP4, -5, -11, and -12; assembled AAV5, -8, and -9 capsids are excluded from the nucleolus, in contrast to the nucleolar enrichment of assembled AAV2 capsids; and, surprisingly, AAV4, -5, and -11 capsids are not dependent on AAP for assembly. These observations highlight the serotype-dependent heterogeneity of the capsid assembly process and challenge current notions about the role of AAP and the nucleolus in capsid assembly. IMPORTANCE Assembly-activating protein (AAP) is a recently discovered adeno-associated virus (AAV) protein that promotes capsid assembly and provides new opportunities for research in assembly. Previous studies on AAV serotype 2 (AAV2) showed that assembly takes place in the nucleolus and is dependent on AAP and that capsids colocalize with AAP in the nucleolus during the assembly process. However, through the investigation of 12 different AAV serotypes (AAV1 to -12), we find that AAP is not an essential requirement for capsid assembly of AAV4, -5, and -11, and AAP, assembled capsids, and the nucleolus do not colocalize for all the serotypes. In addition, we find that there are both serotype-restricted and serotype-promiscuous AAPs in their assembly roles. These findings challenge widely held beliefs about the importance of the nucleolus and AAP in AAV assembly and show the heterogeneous nature of the assembly process within the AAV family.
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All serotypes tested, with the exception of AAV4, AAV5 and AAV11, require AAP in order to assemble their capsid.
“In the most recent studies, it has been shown that all serotypes tested, with the exception of AAV4, AAV5 and AAV11, require AAP in order to assemble their capsid [35,39, 45–47].”
From Understanding Capsid Assembly and Genome Packaging for Adeno-Associated Viruses (2017) · cites this paper as [45]
The small packaging capacity of AAV vectors makes AAV vector design for diseases involving larger genes challenging or impossible.
“This limitation makes AAV vector design for diseases involving larger genes challenging or impossible.”
From Viral Vector Systems for Gene Therapy: A Comprehensive Literature Review of Progress and Biosafety Challenges (2020) · cites this paper as [38]
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- Authors
- Earley, L. F., Powers, J. M., Adachi, K., Baumgart, J. T., Meyer, N. L., Xie, Q., Chapman, M. S., & Nakai, H.
- Affiliations
- Oregon Health & Science University (US)
- Oregon National Primate Research Center (US)
- Venue
- Journal of Virology
- Publisher
- American Society for Microbiology
- Published
- 2017-02
- Volume
- 91
- Issue
- 3
- ISSN
- 0022-538X (ISSN-L)
- 1098-5514
- PMC
- PMC5244341
- OpenAlex
- W2550119581
- Cited by
- 108
- References
- 30
- Citations by year
- 2026: 5 · 2025: 9 · 2024: 20 · 2023: 13 · 2022: 5 · 2021: 16 · 2020: 12 · 2019: 4 · 2018: 15 · 2017: 9
- FWCI
- 7.54
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Journal of Virology (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Funders
- HHS | NIH | National Institute of Allergy and Infectious Diseases (T32 AI007472)
- HHS | NIH | National Institute of General Medical Sciences (R01 GM066875, T32 GM071338)
- HHS | NIH | National Institute of Diabetes and Digestive and Kidney Diseases (R01 DK078388)
- HHS | NIH | National Institute of Neurological Disorders and Stroke (R01 NS088399)
- HHS | NIH | National Eye Institute (T32 EY232113)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Viral Infectious Diseases and Gene Expression in Insects
- Keywords
- adeno-associated virus, capsid assembly, AAV serotypes, nucleolus, AAV5, AAV2, AAV8, AAV9, subcellular localization, adeno-associated virus vectors, gene therapy vectors
- MeSH terms
- Dependovirus, Amino Acid Sequence, Animals, Capsid, Genetic Complementation Test, Genetic Vectors, Humans, Viral Proteins, Virion, Virus Replication, Gene Expression, Virus Assembly, Capsid Proteins, Serogroup
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