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Intermediates of adeno-associated virus type 2 assembly: identification of soluble complexes containing Rep and Cap proteins
Open accessAbstract
The proteins encoded by the adeno-associated virus type 2 (AAV-2) rep and cap genes obtained during a productive infection of HeLa cells with AAV-2 and adenovirus type 2 were fractionated according to solubility, cellular localization, and sedimentation properties. The majority of Rep and Cap proteins accumulated in the nucleus, where they distributed into a soluble and an insoluble fraction. Analysis of the soluble nuclear fraction of capsid proteins by sucrose density gradients showed that they formed at least three steady-state pools: a monomer pool sedimenting at about 6S, a pool of oligomeric intermediates sedimenting between 10 and 15S, and a broad pool of assembly products with a peak between 60 and 110S, the known sedimentation positions of empty and full capsids. While the soluble nuclear monomer and oligomer pool contained predominantly only two capsid proteins, the 30 to 180S assembly products contained VP1, VP2, and VP3 in a stoichiometry similar to that of purified virions. They probably represent different intermediates in capsid assembly, DNA encapsidation, and capsid maturation. In contrast, the cytoplasmic fraction of capsid proteins showed a pattern of oligomers continuously increasing in size without a defined peak, suggesting that assembly of 60S particles occurs in the nucleus. Soluble nuclear Rep proteins were distributed over the whole sedimentation range, probably as a result of association with AAV DNA. Subfractions of the Rep proteins with defined sedimentation values were obtained in the soluble nuclear and cytoplasmic fractions. We were able to coimmunoprecipitate capsid proteins sedimenting between 60 and 110S with antibodies against Rep proteins, suggesting that they exist in common complexes possibly involved in AAV DNA packaging. Antibodies against the capsid proteins, however, precipitated Rep78 and Rep68 predominantly with a peak around 30S representing a second complex containing Rep and Cap proteins.
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“Sedimentation sucrose gradient analysis of nuclear and cytoplasmic fractions of an AAV infection demonstrated that the cytoplasmic fraction contains a population of VP monomers and oligomers ranging in size from 10 to 15S consistent with the sedimentation coefficients for trimers and pentamers, respectively, of VP1, VP2 and VP3.”
From Understanding Capsid Assembly and Genome Packaging for Adeno-Associated Viruses (2017) · cites this paper as [27]
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“The nuclear fraction contained VP oligomers with sedimentation coefficients between 60 and 110S consistent with empty and full capsids, respectively [27].”
From Understanding Capsid Assembly and Genome Packaging for Adeno-Associated Viruses (2017) · cites this paper as [27]
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“Immunofluorescence analyses were performed as previously described (39), using the anti-Rep monoclonal antibody 303.9, which was raised to an amino-terminally truncated AAV2 Rep78 ( 52 ) (American Research Products, Inc., Belmont, Mass.), and a fluorescein isothiocyanate-conjugated anti-mouse secondary antibody (ICN B…”
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“…important key steps of rAAV particle production including vDNA replication (Li et al., 1997), vDNA targeting to capsids, vDNA packaging into capsids (Dubielzig et al., 1999; King et al., 2001; Wistuba et al., 1995), and Rep/Cap protein expression (Pereira et al., 1997; Trempe & Carter, 1988).”
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Metadata
- Authors
- Wistuba, A., Weger, S., Kern, A., & Kleinschmidt, J. A.
- Affiliations
- German Cancer Research Center (DE)
- ORCID
- Weger, S
- Venue
- Journal of Virology
- Publisher
- American Society for Microbiology
- Published
- 1995-09
- Volume
- 69
- Issue
- 9
- Pages
- 5311-5319
- ISSN
- 0022-538X (ISSN-L)
- 1098-5514
- PMC
- PMC189369
- OpenAlex
- W2172045470
- Cited by
- 147
- References
- 44
- Citations by year
- 2026: 3 · 2025: 4 · 2024: 5 · 2023: 6 · 2022: 2 · 2021: 3 · 2020: 3 · 2019: 3 · 2018: 6 · 2017: 4 · 2016: 2 · 2015: 1 · 2014: 3 · 2013: 2 · 2012: 7
- FWCI
- 3.75
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Locations
- Journal of Virology (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
- Health Sciences › Medicine › Infectious Diseases › Parvovirus B19 Infection Studies
- Health Sciences › Medicine › Epidemiology › Virology and Viral Diseases
- Keywords
- adeno-associated virus type 2, capsid assembly, Rep protein, capsid protein, capsid maturation, DNA encapsidation, Rep78, Rep68, co-immunoprecipitation, sedimentation analysis
- MeSH terms
- Dependovirus, Capsid, Cell Fractionation, Cell Nucleus, Cytoplasm, Electrophoresis, Polyacrylamide Gel, Genes, Viral, HeLa Cells, Humans, Virus Replication, Immunoblotting, Viral Nonstructural Proteins
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