Adeno-Associated Virus Type 2 VP2 Capsid Protein Is Nonessential and Can Tolerate Large Peptide Insertions at Its N Terminus

Warrington, Kenneth H.; Gorbatyuk, Oleg S.; Harrison, Jeffrey K.; Opie, Shaun R.; Zolotukhin, Sergei; Muzyczka, Nicholas

doi:10.1128/jvi.78.12.6595-6609.2004

Cited by 205Virus-based gene therapy researchRNA Interference and Gene DeliveryViral Infectious Diseases and Gene Expression in Insects

Adeno-Associated Virus Type 2 VP2 Capsid Protein Is Nonessential and Can Tolerate Large Peptide Insertions at Its N Terminus

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Warrington, Kenneth H., Gorbatyuk, Oleg S., Harrison, Jeffrey K., Opie, Shaun R., Zolotukhin, Sergei, Muzyczka, Nicholas·2004·Journal of Virology

DOI 10.1128/jvi.78.12.6595-6609.2004PMC PMC416546

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Abstract

Direct insertion of amino acid sequences into the adeno-associated virus type 2 (AAV) capsid open reading frame ( cap ORF) is one strategy currently being developed for retargeting this prototypical gene therapy vector. While this approach has successfully resulted in the formation of AAV particles that have expanded or retargeted viral tropism, the inserted sequences have been relatively short, linear receptor binding ligands. Since many receptor-ligand interactions involve nonlinear, conformation-dependent binding domains, we investigated the insertion of full-length peptides into the AAV cap ORF. To minimize disruption of critical VP3 structural domains, we confined the insertions to residue 138 within the VP1-VP2 overlap, which has been shown to be on the surface of the particle following insertion of smaller epitopes. The insertion of coding sequences for the 8-kDa chemokine binding domain of rat fractalkine (CX3CL1), the 18-kDa human hormone leptin, and the 30-kDa green fluorescent protein (GFP) after residue 138 failed to lead to formation of particles due to the loss of VP3 expression. To test the ability to complement these insertions with the missing capsid proteins in trans , we designed a system for producing AAV vectors in which expression of one capsid protein is isolated and combined with the remaining two capsid proteins expressed separately. Such an approach allows for genetic modification of a specific capsid protein across its entire coding sequence leaving the remaining capsid proteins unaffected. An examination of particle formation from the individual components of the system revealed that genome-containing particles formed as long as the VP3 capsid protein was present and demonstrated that the VP2 capsid protein is nonessential for viral infectivity. Viable particles composed of all three capsid proteins were obtained from the capsid complementation groups regardless of which capsid proteins were supplied separately in trans . Significant overexpression of VP2 resulted in the formation of particles with altered capsid protein stoichiometry. The key finding was that by using this system we successfully obtained nearly wild-type levels of recombinant AAV-like particles with large ligands inserted after residue 138 in VP1 and VP2 or in VP2 exclusively. While insertions at residue 138 in VP1 significantly decreased infectivity, insertions at residue 138 that were exclusively in VP2 had a minimal effect on viral assembly or infectivity. Finally, insertion of GFP into VP1 and VP2 resulted in a particle whose trafficking could be temporally monitored by using confocal microscopy. Thus, we have demonstrated a method that can be used to insert large (up to 30-kDa) peptide ligands into the AAV particle. This system allows greater flexibility than current approaches in genetically manipulating the composition of the AAV particle and, in particular, may allow vector retargeting to alternative receptors requiring interaction with full-length conformation-dependent peptide ligands.

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Vp1 is required for infectivity, while Vp2 has been shown to be nonessential.
“While Vp1 is required for infectivity, Vp2 has been shown to be nonessential (44).”
From Packaging Capacity of Adeno-Associated Virus Serotypes: Impact of Larger Genomes on Infectivity and Postentry Steps (2005) · cites this paper as [44]
The Discussion says VP2 is nonessential and VP1 is required for *efficient* viral infectivity. But it also reports that particles lacking VP1 have low infectivity, so the unqualified statement that VP1 is required for infectivity overstates the finding. (LLM, conf=0.88)
Experimental — accuracy not yet measured
Mutational studies on the entire cap gene showed that the VP3 common sequence is enough to assemble AAV capsids by knocking out the start codons of VP1 and VP2.
“Mutational studies on the entire cap gene showed that the VP3 common sequence is enough to assemble AAV capsids by knocking out the start codons of VP1 and VP2 [31–34].”
From Understanding Capsid Assembly and Genome Packaging for Adeno-Associated Viruses (2017) · cites this paper as [34]

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Adeno-associated virus vector as a platform for gene therapy delivery
Wang, D., Tai, P. W. L., & Gao, G. (2019) · Nature Reviews Drug Discovery · doi:10.1038/s41573-019-0012-9 · Cited by 2,444 · see citing works
Adeno-Associated Virus (AAV) as a Vector for Gene Therapy
Naso, M. F., Tomkowicz, B., Perry, W. L., & Strohl, W. R. (2017) · BioDrugs · doi:10.1007/s40259-017-0234-5 · Cited by 1,362 · see citing works
Packaging Capacity of Adeno-Associated Virus Serotypes: Impact of Larger Genomes on Infectivity and Postentry Steps
Grieger, J. C., & Samulski, R. J. (2005) · Journal of Virology · doi:10.1128/jvi.79.15.9933-9944.2005 · Cited by 419 · see citing works
Recombinant Adeno-Associated Virus Gene Therapy in Light of Luxturna (and Zolgensma and Glybera): Where Are We, and How Did We Get Here?
Keeler, A. M., & Flotte, T. R. (2019) · Annual Review of Virology · doi:10.1146/annurev-virology-092818-015530 · Cited by 322 · see citing works
Engineering and Selection of Shuffled AAV Genomes: A New Strategy for Producing Targeted Biological Nanoparticles
Li, W., Asokan, A., Wu, Z., Van Dyke, T., DiPrimio, N., Johnson, J. S., Govindaswamy, L., Agbandje-McKenna, M., Leichtle, S., Eugene Redmond Jr, D., et al. (2008) · Molecular Therapy · doi:10.1038/mt.2008.100 · Cited by 261 · see citing works
Capsid modifications for targeting and improving the efficacy of AAV vectors
Büning, H., & Srivastava, A. (2019) · Molecular Therapy - Methods & Clinical Development · doi:10.1016/j.omtm.2019.01.008 · Cited by 260 · see citing works
Intracellular trafficking of adeno-associated viral vectors
Ding, W., Zhang, L., Yan, Z., & Engelhardt, J. F. (2005) · Gene Therapy · doi:10.1038/sj.gt.3302527 · Cited by 251 · see citing works
AAV Capsid Structure and Cell Interactions
Agbandje-McKenna, M., & Kleinschmidt, J. (2012) · Methods in Molecular Biology · doi:10.1007/978-1-61779-370-7_3 · Cited by 215 · see citing works
Mutational analysis of narrow pores at the fivefold symmetry axes of adeno-associated virus type 2 capsids reveals a dual role in genome packaging and activation of phospholipase A2 activity
Bleker, S., Sonntag, F., & Kleinschmidt, J. A. (2005) · Journal of Virology · doi:10.1128/jvi.79.4.2528-2540.2005 · Cited by 200 · see citing works
A conformational change in the adeno-associated virus type 2 capsid leads to the exposure of hidden VP1 N termini
Kronenberg, S., Böttcher, B., von der Lieth, C. W., Bleker, S., & Kleinschmidt, J. A. (2005) · Journal of Virology · doi:10.1128/jvi.79.9.5296-5303.2005 · Cited by 198 · see citing works
Mutations on the External Surfaces of Adeno-AssociatedVirus Type 2 Capsids That Affect Transduction andNeutralization
Lochrie, M. A., Tatsuno, G. P., Christie, B., McDonnell, J. W., Zhou, S., Surosky, R., Pierce, G. F., & Colosi, P. (2006) · Journal of Virology · doi:10.1128/jvi.80.2.821-834.2006 · Cited by 201 · see citing works
AAV hybrid serotypes: improved vectors for gene delivery
Choi, V., McCarty, D., & Samulski, R. (2005) · Current Gene Therapy · doi:10.2174/1566523054064968 · Cited by 191 · see citing works
Separate basic region motifs within the adeno-associated virus capsid proteins are essential for infectivity and assembly
Grieger, J. C., Snowdy, S., & Samulski, R. J. (2006) · Journal of Virology · doi:10.1128/jvi.02723-05 · Cited by 172 · see citing works
Controlling AAV Tropism in the Nervous System with Natural and Engineered Capsids
Castle, M. J., Turunen, H. T., Vandenberghe, L. H., & Wolfe, J. H. (2016) · Methods in Molecular Biology · doi:10.1007/978-1-4939-3271-9_10 · Cited by 151 · see citing works
“Although initial experiments focused on the insertion of small peptides (5–15 amino acids), later studies have shown that insertions of full length proteins, such as GFP or mCherry, can also be tolerated without loss of virus function [78, 80].”
Background
Adeno-Associated Virus Gene Therapy for Liver Disease
Kattenhorn, L. M., Tipper, C. H., Stoica, L., Geraghty, D. S., Wright, T. L., Clark, K. R., & Wadsworth, S. C. (2016) · Human Gene Therapy · doi:10.1089/hum.2016.160 · Cited by 138 · see citing works
Structural insights into adeno-associated virus serotype 5
Govindasamy, L., DiMattia, M. A., Gurda, B. L., Halder, S., McKenna, R., Chiorini, J. A., Muzyczka, N., Zolotukhin, S., & Agbandje-McKenna, M. (2013) · Journal of Virology · doi:10.1128/jvi.00867-13 · Cited by 95 · see citing works
Self-citation
Surface-exposed adeno-associated virus Vp1-NLS capsid fusion protein rescues infectivity of noninfectious wild-type Vp2/Vp3 and Vp3-only capsids but not that of fivefold pore mutant virions
Grieger, J. C., Johnson, J. S., Gurda-Whitaker, B., Agbandje-McKenna, M., & Samulski, R. J. (2007) · Journal of Virology · doi:10.1128/jvi.00580-07 · Cited by 83 · see citing works
Adeno-associated virus (AAV) vectors: Rational design strategies for capsid engineering
Lee, E. J., Guenther, C. M., & Suh, J. (2018) · Current Opinion in Biomedical Engineering · doi:10.1016/j.cobme.2018.09.004 · Cited by 83 · see citing works
Understanding Capsid Assembly and Genome Packaging for Adeno-Associated Viruses
Bennett, A., Mietzsch, M., & Agbandje-McKenna, M. (2017) · Future Virology · doi:10.2217/fvl-2017-0011 · Cited by 82 · see citing works
Modulation of CD8+ T cell responses to AAV vectors with IgG-derived MHC class II epitopes
Hui, D. J., Basner-Tschakarjan, E., Chen, Y., Davidson, R. J., Buchlis, G., Yazicioglu, M., Pien, G. C., Finn, J. D., Haurigot, V., Tai, A., et al. (2013) · Molecular Therapy · doi:10.1038/mt.2013.166 · Cited by 47 · see citing works
Cytotoxic-t-lymphocyte-mediated elimination of target cells transduced with engineered adeno-associated virus type 2 vector in vivo
Li, C., Hirsch, M., DiPrimio, N., Asokan, A., Goudy, K., Tisch, R., & Samulski, R. J. (2009) · Journal of Virology · doi:10.1128/jvi.00278-09 · Cited by 42 · see citing works
Synthetic Intron Improves Transduction Efficiency of <i>Trans</i> -Splicing Adeno-Associated Viral Vectors
Lai, Y., Yue, Y., Liu, M., & Duan, D. (2006) · Human Gene Therapy · doi:10.1089/hum.2006.17.1036 · Cited by 37 · see citing works

Authors: Warrington, K. H., Gorbatyuk, O. S., Harrison, J. K., Opie, S. R., Zolotukhin, S., & Muzyczka, N.
Affiliations: University of Florida (US)
Department of Pharmacology and Therapeutics
Department of Molecular Genetics and Microbiology
ORCID: Gorbatyuk, Oleg S.
Harrison, Jeffrey K.
Venue: Journal of Virology
Publisher: American Society for Microbiology
Published: 2004-06-15
Volume: 78
Issue: 12
Pages: 6595-6609
ISSN: 0022-538X (ISSN-L)
1098-5514
DOI: 10.1128/jvi.78.12.6595-6609.2004
PMC: PMC416546
OpenAlex: W1964959966
Cited by: 205
References: 67
Citations by year: 2026: 5 · 2025: 16 · 2024: 11 · 2023: 8 · 2022: 7 · 2021: 7 · 2020: 7 · 2019: 13 · 2018: 6 · 2017: 8 · 2016: 5 · 2015: 10 · 2014: 5 · 2013: 4 · 2012: 8
FWCI: 8.21
Type: journal article
Language: en
Open access: green
www.ncbi.nlm.nih.gov
Licence: journals.asm.org/non-commercial-tdm-license (tdm)
Locations: Journal of Virology (publishedVersion)
PubMed Central (publishedVersion)
PubMed (publishedVersion)
Funders: National Institutes of Health
National Heart, Lung, and Blood Institute (P50-HL59412, P01-HL51811, P01HL051811, P50HL059412)
National Institute of Allergy and Infectious Diseases (T32 AI 7110, T32AI007110)
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 type 2, AAV capsid, VP2 capsid protein, VP3 capsid protein, peptide insertion, fractalkine, leptin, green fluorescent protein, viral assembly, infectivity, viral tropism, gene therapy vectors
MeSH terms: Dependovirus, Base Sequence, HeLa Cells, Humans, Molecular Sequence Data, Mutation, Peptides, Plasmids, Virion, Microscopy, Confocal, Virus Assembly, Capsid Proteins

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Adeno-Associated Virus Type 2 VP2 Capsid Protein Is Nonessential and Can Tolerate Large Peptide Insertions at Its N Terminus

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