Adeno-associated virus (AAV) assembly-activating protein is not an essential requirement for capsid assembly of AAV serotypes 4, 5, and 11

Lauriel F. Earley, John M. Powers, Kei Adachi, Joshua T. Baumgart, Nancy L. Meyer, Qing Xie, Michael Chapman, Hiroyuki Nakai

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

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 serotypespecific 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 serotypedependent heterogeneity of the capsid assembly process and challenge current notions about the role of AAP and the nucleolus in capsid assembly.

Original languageEnglish (US)
Article numbere01980-16
JournalJournal of Virology
Volume91
Issue number3
DOIs
StatePublished - 2017

Fingerprint

Dependovirus
Virus Assembly
capsid
Capsid
serotypes
Proteins
proteins
cell nucleolus
virus assembly
Serogroup
Capsid Proteins
gene therapy
barcoding
DNA barcoding
Transmission Electron Microscopy
Genetic Therapy
Fluorescent Antibody Technique

Keywords

  • Adeno-associated virus
  • Assembly-activating protein
  • Capsid assembly
  • Gene therapy
  • Nucleolus
  • Parvovirus

ASJC Scopus subject areas

  • Immunology
  • Virology

Cite this

Adeno-associated virus (AAV) assembly-activating protein is not an essential requirement for capsid assembly of AAV serotypes 4, 5, and 11. / Earley, Lauriel F.; Powers, John M.; Adachi, Kei; Baumgart, Joshua T.; Meyer, Nancy L.; Xie, Qing; Chapman, Michael; Nakai, Hiroyuki.

In: Journal of Virology, Vol. 91, No. 3, e01980-16, 2017.

Research output: Contribution to journalArticle

Earley, Lauriel F. ; Powers, John M. ; Adachi, Kei ; Baumgart, Joshua T. ; Meyer, Nancy L. ; Xie, Qing ; Chapman, Michael ; Nakai, Hiroyuki. / Adeno-associated virus (AAV) assembly-activating protein is not an essential requirement for capsid assembly of AAV serotypes 4, 5, and 11. In: Journal of Virology. 2017 ; Vol. 91, No. 3.
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AU - Adachi, Kei

AU - Baumgart, Joshua T.

AU - Meyer, Nancy L.

AU - Xie, Qing

AU - Chapman, Michael

AU - Nakai, Hiroyuki

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AB - 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 serotypespecific 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 serotypedependent heterogeneity of the capsid assembly process and challenge current notions about the role of AAP and the nucleolus in capsid assembly.

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