Nuclear translocation of adeno-associated virus type 2 capsid proteins for virion assembly

Popa-Wagner, Ruth; Sonntag, Florian; Schmidt, Kristin; King, James G.; Kleinschmidt, Jürgen A.

doi:10.1099/vir.0.043232-0

Cited by 20 publications

(19 citation statements)

References 33 publications

(83 reference statements)

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“…The N termini of both VP1 and VP2 incorporate the so-called basic region 3 (BR3; PKRKKART) representing conservative nuclear localization sequence (NLS) motif, which is necessary for AAV to deliver its genome within the nucleus and subsequently transduce the cells. [27][28][29] It is thus not surprising that a 3-fold increase of each VP1 and VP2 increases the yield of a more infectious virus.…”

Section: Discussionmentioning

confidence: 99%

Direct Head-to-Head Evaluation of Recombinant Adeno-associated Viral Vectors Manufactured in Human versus Insect Cells

et al. 2017

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The major drawback of the Baculovirus/Sf9 system for recombinant adeno-associated viral (rAAV) manufacturing is that most of the Bac-derived rAAV vector serotypes, with few exceptions, demonstrate altered capsid compositions and lower biological potencies. Here, we describe a new insect cell-based production platform utilizing attenuated Kozak sequence and a leaky ribosome scanning to achieve a serotype-specific modulation of AAV capsid proteins stoichiometry. By way of example, rAAV5 and rAAV9 were produced and comprehensively characterized side by side with HEK293-derived vectors. A mass spectrometry analysis documented a 3-fold increase in both viral protein (VP)1 and VP2 capsid protein content compared with human cell-derived vectors. Furthermore, we conducted an extensive analysis of encapsidated single-stranded viral DNA using next-generation sequencing and show a 6-fold reduction in collaterally packaged contaminating DNA for rAAV5 produced in insect cells. Consequently, the re-designed rAAVs demonstrated significantly higher biological potencies, even in a comparison with HEK293-manufactured rAAVs mediating, in the case of rAAV5, 4-fold higher transduction of brain tissues in mice. Thus, the described system yields rAAV vectors of superior infectivity and higher genetic identity providing a scalable platform for good manufacturing practice (GMP)-grade vector production.

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Section: Discussionmentioning

confidence: 99%

Direct Head-to-Head Evaluation of Recombinant Adeno-associated Viral Vectors Manufactured in Human versus Insect Cells

et al. 2017

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“…It has to be determined whether these mutant VPs are also defective in AAP binding. A very interesting VP mutation in this respect is K688A/K692A in the protein's C terminus, which produces a strong assembly defect (18). Although AAP is able to bind to the mutated VP (data not shown) and accumulates in the nucleus, it does not accumulate in the nucleoli and is deposited in nuclear speckles.…”

Section: Discussionmentioning

confidence: 99%

“…Although coprecipitation of VP with antibodies against the AAP-fused AU1 tag was reproducible, the recovery of AAP in the immunoprecipitate was (3,26) are shown in blue. Mutant constructs described in this study and by Popa-Wagner et al (18) are shown in red, while the B1 antibody epitope is shown in green. Note the clustering of assembly mutant constructs at the 2-fold symmetry axes.…”

Section: Discussionmentioning

confidence: 99%

Properties of the Adeno-Associated Virus Assembly-Activating Protein

Naumer

Sonntag

Schmidt

et al. 2012

J Virol

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b Adeno-associated virus (AAV) capsid assembly requires expression of the assembly-activating protein (AAP) together with capsid proteins VP1, VP2, and VP3. AAP is encoded by an alternative open reading frame of the cap gene. Sequence analysis and site-directed mutagenesis revealed that AAP contains two hydrophobic domains in the N-terminal part of the molecule that are essential for its assembly-promoting activity. Mutation of these sequences reduced the interaction of AAP with the capsid proteins. Deletions and a point mutation in the capsid protein C terminus also abolished capsid assembly and strongly reduced the interaction with AAP. Interpretation of these observations on a structural basis suggests an interaction of AAP with the VP C terminus, which forms the capsid protein interface at the 2-fold symmetry axis. This interpretation is supported by a decrease in the interaction of monoclonal antibody B1 with VP3 under nondenaturing conditions in the presence of AAP, indicative of steric hindrance of B1 binding to its C-terminal epitope by AAP. In addition, AAP forms high-molecular-weight oligomers and changes the conformation of nonassembled VP molecules as detected by conformation-sensitive monoclonal antibodies A20 and C37. Combined, these observations suggest a possible scaffolding activity of AAP in the AAV capsid assembly reaction.A deno-associated virus (AAV) is a nonenveloped singlestranded DNA virus of the Parvoviridae family (15). To date, 13 distinct human or nonhuman primate AAV serotypes have been described and numerous recombinant species have been isolated (10). The AAV assembly pathway proposed by Myers and Carter suggests the rapid formation of empty capsids into which the single-stranded genome is inserted in a slow reaction (16). While the process of genome replication has been elucidated in great detail (15,22), molecular events underlying capsid formation and genome encapsidation are less well understood (12).Capsid assembly occurs in the nuclei of infected cells, where capsids are first detectable in the nucleoli but are spread throughout the nucleus at later stages of infection (23). Expression of the cap gene is sufficient for capsid formation. Besides the three capsid proteins, VP1, VP2, and VP3, known to be expressed from open reading frame 1 (ORF1), the cap gene encodes an assembly factor, the assembly-activating protein (AAP), from a second ORF, ORF2 (21). AAP is essential for capsid assembly. It targets newly synthesized capsid proteins to the nucleolus and promotes capsid formation in a still unknown way. AAPs of some, but not all, AAV serotypes can cross-complement each other in the assembly reaction (20). AAP is a rather unstable protein but becomes stabilized upon the coexpression of capsid protein VP3. However, this stabilizing effect depends very much on the serotype of the coexpressed capsid protein, indicating specific AAP-VP protein interactions (20). AAP amino acid sequence alignment of serotypes 1 to 13 shows a high degree of homology. Only AAPs from serotypes 4, 5, 1...

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“…As shown in Fig. 3h, it is an adeno-associated viral capsid consisting of VP1 (full-length VP protein), VP2 (lacking uVP1), and VP3 (lacking nuclear localization signal, NLS), but not VP4, with a ratio of around 1:1:10 [148,149]. Why do all VPs contain the same receptor-binding site?…”

Section: Parvoviridaementioning

confidence: 99%

Sialoglycovirology of Lectins: Sialyl Glycan Binding of Enveloped and Non-enveloped Viruses

Sriwilaijaroen

Suzuki

2020

Methods in Molecular Biology

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On the cell sur "face", sialoglycoconjugates act as receptionists that have an important role in the first step of various cellular processes that bridge communication between the cell and its environment. Loss of Sia production can cause the developmental of defects and lethality in most animals; hence, animal cells are less prone to evolution of resistance to interactions by rapidly evolved Sia-binding viruses. Obligative intracellular viruses mostly have rapid evolution that allows escape from host immunity, leading to an epidemic variant, and that allows emergence of a novel strain, occasionally leading to pandemics that cause healthsocial-economic problems. Recently, much attention has been given to the mutual recognition systems via sialosugar chains between viruses and their host cells and there has been rapid growth of the research field "sialoglycovirology." In this chapter, the structural diversity of sialoglycoconjugates is overviewed, and enveloped and non-enveloped viruses that bind to Sia are reviewed. Also, interactions of viral lectins-host Sia receptors, which determine viral transmission, host range, and pathogenesis, are presented. The future direction of new therapeutic routes targeting viral lectins, development of easy-to-use detection methods for diagnosis and monitoring changes in virus binding specificity, and challenges in the development of suitable viruses to use in virus-based therapies for genetic disorders and cancer are discussed.

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Nuclear translocation of adeno-associated virus type 2 capsid proteins for virion assembly

Cited by 20 publications

References 33 publications

Direct Head-to-Head Evaluation of Recombinant Adeno-associated Viral Vectors Manufactured in Human versus Insect Cells

Direct Head-to-Head Evaluation of Recombinant Adeno-associated Viral Vectors Manufactured in Human versus Insect Cells

Properties of the Adeno-Associated Virus Assembly-Activating Protein

Sialoglycovirology of Lectins: Sialyl Glycan Binding of Enveloped and Non-enveloped Viruses

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