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Impact of capsid conformation and Rep-capsid interactions on adeno-associated virus type 2 genome packaging.

ABSTRACT: Single-stranded genomes of adeno-associated virus (AAV) are packaged into preformed capsids. It has been proposed that packaging is initiated by interaction of genome-bound Rep proteins to the capsid, thereby targeting the genome to the portal of encapsidation. Here we describe a panel of mutants with amino acid exchanges in the pores at the fivefold axes of symmetry on AAV2 capsids with reduced packaging and reduced Rep-capsid interaction. Mutation of two threonines at the rim of the fivefold pore nearly completely abolished Rep-capsid interaction and packaging. This suggests a Rep-binding site at the highly conserved amino acids at or close to the pores formed by the capsid protein pentamers. A different mutant (P. Wu, W. Xiao, T. Conlon, J. Hughes, M. Agbandje-McKenna, T. Ferkol, T. Flotte, and N. Muzyczka, J. Virol. 74:8635-8647, 2000) with an amino acid exchange at the interface of capsid protein pentamers led to a complete block of DNA encapsidation. Analysis of the capsid conformation of this mutant revealed that the pores at the fivefold axes were occupied by VP1/VP2 N termini, thereby preventing DNA introduction into the capsid. Nevertheless, the corresponding capsids had more Rep proteins bound than wild-type AAV, showing that correct Rep interaction with the capsid depends on a defined capsid conformation. Both mutant types together support the conclusion that the pores at the fivefold symmetry axes are involved in genome packaging and that capsid conformation-dependent Rep-capsid interactions play an essential role in the packaging process.

SUBMITTER: Bleker S

PROVIDER: S-EPMC1346863 | biostudies-literature | 2006 Jan

REPOSITORIES: biostudies-literature

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Impact of capsid conformation and Rep-capsid interactions on adeno-associated virus type 2 genome packaging.

Bleker Svenja S Pawlita Michael M Kleinschmidt Jürgen A JA

Journal of virology 20060101 2

Single-stranded genomes of adeno-associated virus (AAV) are packaged into preformed capsids. It has been proposed that packaging is initiated by interaction of genome-bound Rep proteins to the capsid, thereby targeting the genome to the portal of encapsidation. Here we describe a panel of mutants with amino acid exchanges in the pores at the fivefold axes of symmetry on AAV2 capsids with reduced packaging and reduced Rep-capsid interaction. Mutation of two threonines at the rim of the fivefold p ...[more]

PMID: 16378983

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Project description:Recombinant adeno-associated viruses (rAAVs) are one of the most commonly used vectors for a variety of gene therapy applications. In the last 2 decades, research focused primarily on the characterization and isolation of new cap, genes resulting in hundreds of natural and engineered AAV capsid variants, while the rep gene, the other major AAV open reading frame, has been less studied. This is due to the fact that the rep gene from AAV serotype 2 (AAV2) enables the single-stranded DNA packaging of recombinant genomes into most AAV serotype and engineered capsids. However, a major by-product of all vector productions is empty AAV capsids, lacking the encapsidated vector genome, especially for non-AAV2 vectors. Despite the packaging process being considered the rate-limiting step for rAAV production, none of the rep genes from the other AAV serotypes have been characterized for their packaging efficiency. Thus, in this study AAV2 rep was replaced with the rep gene of a select number of AAV serotypes. However, this led to a lowering of capsid protein expression, relative to the standard AAV2-rep system. In further experiments the 3' end of the AAV2 rep gene was reintroduced to promote increased capsid expression and a series of chimeras between the different AAV Rep proteins were generated and characterized for their vector genome packaging ability. The utilization of these novel Rep hybrids increased the percentage of genome containing (full) capsids approximately 2- to -4-fold for all of the non-AAV2 serotypes tested. Thus, these Rep chimeras could revolutionize rAAV production. IMPORTANCE A major by-product of all adeno-associated virus (AAV) vector production systems are "empty" capsids, void of the desired therapeutic gene, and thus do not provide any curative benefit for the treatment of the targeted disease. In fact, empty capsids can potentially elicit additional immune responses in vivo gene therapies if not removed by additional purification steps. Thus, there is a need to increase the genome packaging efficiency and reduce the number of empty capsids from AAV biologics. The novel Rep hybrids from different AAV serotypes described in this study are capable of reducing the percentage of empty capsids in all tested AAV serotypes and improve overall yields of genome-containing AAV capsids at the same time. They can likely be integrated easily into existing AAV manufacturing protocols to optimize the production of the generated AAV gene therapy products.

Project description:The Rep proteins of adeno-associated virus type 2 (AAV) are known to bind to Rep recognition sequences (RRSs) in the AAV inverted terminal repeats (ITRs), the AAV p5 promoter, and the preferred AAV integration site in human chromosome 19, called AAVS1. Integration of the AAV genome into AAVS1 appears to be mediated by an interaction between the Rep proteins of AAV and Rep binding sites within the viral genome and the integration locus. In an attempt to identify potential alternate integration sites, we looked for recognition sites for AAV Rep proteins in the human genome by performing a BLASTN computerized homology search. We used the 16-mer core sequences of the RRSs in the AAV ITRs and AAVS1 separately as query sequences and identified 18 new RRSs in or flanking the genes coding for the following: tyrosine kinase activator protein 1 (TKA-1); colony stimulating factor-1; insulin-like growth factor binding protein 2 (IGFBP-2); histone H2B.1; basement membrane heparan sulfate proteoglycan, also known as perlecan; the AF-9 gene product, which is involved in the chromosomal translocation t (9:11)(p22:q23); the betaB subunit of the hormone known as inhibin; interleukin-2 enhancer binding factor; an endoplasmic reticulum-Golgi intermediate compartment resident protein called p63; a global transcription activator (hSNF2L); the beta-actin repair domain; a retinoic acid-inducible factor, also known as midkine; a breast tumor autoantigen; a growth-arrest- and DNA-damage-inducible protein called gadd45; the cyclin-dependent kinase inhibitor called KIP2, which inhibits several G1 cyclin-cyclin-dependent kinase complexes; and the hereditary breast and ovarian cancer gene (BRCA1). RRSs were also identified in a newly discovered open reading frame on chromosome 10 and in the ERCC1 locus on human chromosome 19. The ability of a maltose binding protein-Rep68 fusion protein to bind to these sequences was confirmed by electrophoretic mobility shift assays. These sites may serve as alternate integration sites for AAV or play a role in Rep-mediated effects on human cells.

Project description:The adeno-associated viruses (AAV) are promising therapeutic gene delivery vectors and better understanding of their capsid assembly and genome packaging mechanism is needed for improved vector production. Empty AAV capsids assemble in the nucleus prior to genome packaging by virally encoded Rep proteins. To elucidate the capsid determinants of this process, structural differences between wild-type (wt) AAV2 and a packaging deficient variant, AAV2-R432A, were examined using cryo-electron microscopy and three-dimensional image reconstruction both at an ?5.0-Å resolution (medium) and also at 3.8- and 3.7-Å resolutions (high), respectively. The high resolution structures showed that removal of the arginine side chain in AAV2-R432A eliminated hydrogen bonding interactions, resulting in altered intramolecular and intermolecular interactions propagated from under the 3-fold axis toward the 5-fold channel. Consistent with these observations, differential scanning calorimetry showed an ?10°C decrease in thermal stability for AAV2-R432A compared to wt-AAV2. In addition, the medium resolution structures revealed differences in the juxtaposition of the less ordered, N-terminal region of their capsid proteins, VP1/2/3. A structural rearrangement in AAV2-R432A repositioned the ?A strand region under the icosahedral 2-fold axis rather than antiparallel to the ?B strand, eliminating many intramolecular interactions. Thus, a single amino acid substitution can significantly alter the AAV capsid integrity to the extent of reducing its stability and possibly rendering it unable to tolerate the stress of genome packaging. Furthermore, the data show that the 2-, 3-, and 5-fold regions of the capsid contributed to producing the packaging defect and highlight a tight connection between the entire capsid in maintaining packaging efficiency.The mechanism of AAV genome packaging is still poorly understood, particularly with respect to the capsid determinants of the required capsid-Rep interaction. Understanding this mechanism may aid in the improvement of AAV packaging efficiency, which is currently ?1:10 (10%) genome packaged to empty capsid in vector preparations. This report identifies regions of the AAV capsid that play roles in genome packaging and that may be important for Rep recognition. It also demonstrates the need to maintain capsid stability for the success of this process. This information is important for efforts to improve AAV genome packaging and will also inform the engineering of AAV capsid variants for improved tropism, specific tissue targeting, and host antibody escape by defining amino acids that cannot be altered without detriment to infectious vector production.

Project description:Adeno-associated viruses (AAVs) are being developed as clinical gene therapy vectors. One issue undermining their broad use in the clinical setting is the high prevalence of circulating antibodies in the general population capable of neutralizing AAV vectors. Hence, there is a need for AAV vectors that can evade the preexisting immune response. One possible source of human naive vectors are AAVs that do not disseminate in the primate population, and one such example is serpentine AAV (SAAV). This study characterizes the structural and biophysical properties of the SAAV capsid and its receptor interactions and antigenicity. Single particle cryo-electron microscopy (cryo-EM) and thermal stability studies were conducted to characterize the SAAV capsid structure at pH 7.4, 6.0, 5.5, and 4.0, conditions experienced during cellular trafficking. Cell binding assays using Chinese hamster ovary (CHO) cell lines identified terminal sialic acid as the primary attachment receptor for SAAV similar to AAV1, 4, 5, and 6. The binding site of sialic acid to the SAAV capsid was mapped near the 2-fold axis toward the 2/5-fold wall, in a different location than AAV1, 4, 5, and 6. Towards determining the SAAV capsid antigenicity native immunodot blots showed that SAAV evades AAV serotype-specific mouse monoclonal antibodies. However, despite its reptilian origin, it was recognized by ~25% of 50 human sera tested, likely due to the presence of cross-reactive antibodies. These findings will inform future gene delivery applications using SAAV-based vectors and further aid the structural characterization and annotation of the repertoire of available AAV capsids. IMPORTANCE AAVs are widely studied therapeutic gene delivery vectors. However, preexisting antibodies and their detrimental effect on therapeutic efficacy are a primary challenge encountered during clinical trials. In order to circumvent preexisting neutralizing antibodies targeting mammalian AAV capsids, serpentine AAV (SAAV) was evaluated as a potential alternative to existing mammalian therapeutic vectors. The SAAV capsid was found to be thermostable at a wide range of environmental pH conditions, and its structure showed conservation of the core capsid topology but displays high structural variability on the surface. At the same time, it binds to a common receptor, sialic acid, that is also utilized by other AAVs already being utilized in gene therapy trials. Contrary to the initial hypothesis, SAAV capsids were recognized by one in four human sera tested, pointing to conserved amino acids around the 5-fold region as epitopes for cross-reacting antibodies.

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Impact of capsid conformation and Rep-capsid interactions on adeno-associated virus type 2 genome packaging.

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Impact of capsid conformation and Rep-capsid interactions on adeno-associated virus type 2 genome packaging.

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