Differential Adeno-Associated Virus Serotype-Specific Interaction Patterns with Synthetic Heparins and Other Glycans

Mietzsch, Mario; Broecker, Felix; Reinhardt, Anika; Seeberger, Peter H.; Heilbronn, Regine

doi:10.1128/jvi.03371-13

Cited by 115 publications

(95 citation statements)

References 36 publications

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“…Glycans 141/143 and 316/318 differ in that the terminal SIA in one of the branches in glycan 316/318 is linked by ␣2,6 rather than ␣2,3 to galactose. Binding of AAV5 to these sulfonated and branched glycans was also recently reported elsewhere (28).…”

Section: Resultsmentioning

confidence: 99%

Identification and Mutagenesis of the Adeno-Associated Virus 5 Sialic Acid Binding Region

Afione

DiMattia

Halder

et al. 2015

J Virol

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As a genus, the dependoviruses use a diverse group of cell surface carbohydrates for attachment and entry. Despite the fact that a majority of adeno-associated viruses (AAVs) utilize sialic acid (SIA) for binding and transduction, this virus-carbohydrate interaction is poorly understood. Utilizing X-ray crystallography, two SIA binding regions were mapped for AAV5. The first site mapped to the depression in the center of the 3-fold axis of symmetry, while the second site was located under the ␤HI loop close to the 5-fold axis. Mutagenesis of amino acids 569 and 585 or 587 within the 3-fold depression resulted in elimination or alteration in SIA-dependent transduction, respectively. This change in SIA binding was confirmed using glycan microarrays. Mutagenesis of the second site identified a role in transduction that was SIA independent. Further studies of the mutants at the 3-fold site demonstrated a change in transduction activity and cell tropism in vivo as well as resistance to neutralization by a polyclonal antibody raised against the wild-type virus. IMPORTANCEDespite the fact that a majority of AAVs utilize sialic acid for binding and transduction, this virus-carbohydrate interaction is poorly understood. Utilizing X-ray crystallography, the sialic acid binding regions of AAV5 were identified and studied using a variety of approaches. Mutagenesis of this region resulted in elimination or alteration in sialic acid-dependent transduction in cell lines. This change in sialic acid glycan binding was confirmed using glycan arrays. Further study also demonstrated a change in transduction and activity and cell tropism in vivo as well as resistance to neutralization by antibodies raised against the wild-type virus.

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Section: Resultsmentioning

confidence: 99%

Identification and Mutagenesis of the Adeno-Associated Virus 5 Sialic Acid Binding Region

Afione

DiMattia

Halder

et al. 2015

J Virol

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“…For AAV2 production, HEK 293 cells seeded at 30% confluence were transfected 24 h later with AAV plasmids and pHelper as described previously (18). AAV2 was purified from benzonase-treated, cleared freeze-thaw supernatants by one-step AVB Sepharose affinity chromatography and quantified by qPCR as described previously (19) with primers for AAV2 rep (Table 1). AAV2 infectious titers were determined by endpoint dilutions on Ad2-infected HeLa cells (20).…”

Section: Methodsmentioning

confidence: 99%

A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins

Stutika

Gogol-Döring

Botschen

et al. 2016

J Virol

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Adeno-associated virus (AAV) is recognized for its bipartite life cycle with productive replication dependent on coinfection with adenovirus (Ad) and AAV latency being established in the absence of a helper virus. The shift from latent to Ad-dependent AAV replication is mostly regulated at the transcriptional level. The current AAV transcription map displays highly expressed transcripts as found upon coinfection with Ad. So far, AAV transcripts have only been characterized on the plus strand of the AAV single-stranded DNA genome. The AAV minus strand is assumed not to be transcribed. Here, we apply Illumina-based RNA sequencing (RNA-Seq) to characterize the entire AAV2 transcriptome in the absence or presence of Ad. We find known and identify novel AAV transcripts, including additional splice variants, the most abundant of which leads to expression of a novel 18-kDa Rep/VP fusion protein. Furthermore, we identify for the first time transcription on the AAV minus strand with clustered reads upstream of the p5 promoter, confirmed by 5' rapid amplification of cDNA ends and RNase protection assays. The p5 promoter displays considerable activity in both directions, a finding indicative of divergent transcription. Upon infection with AAV alone, low-level transcription of both AAV strands is detectable and is strongly stimulated upon coinfection with Ad. A deno-associated viruses (AAV) are helper-dependent members of the parvovirus group that require coinfection with an unrelated helper virus, particularly adenovirus (Ad) for productive replication (1). Despite of several identified AAV serotypes most research has been done with prototype AAV type 2. The AAV2 genome consists of a linear, single-stranded DNA of 4.7 kb. Both ends carry identical inverted terminal repeats (ITR) of 145 bp (2), which flank the two major open reading frames (ORF), called rep and cap. The rep ORF codes for four nonstructural proteins, Rep78 and a C-terminally spliced variant, Rep68. In addition, N-terminally truncated versions thereof are expressed, called Rep52 and Rep40, respectively. The Rep proteins are required as regulators for various steps of the AAV life cycle. AAV cap harbors the ORF for the capsid proteins (VP1 to -3) and a separate ORF for the assembly-activating protein (AAP) (3).The current knowledge of AAV transcription dates back to early research in the 1980s when the three AAV2 promoters, p5, p19, or p40 and major transcripts derived thereof were characterized (4, 5). All AAV transcripts identified since then map to only one DNA plus strand (6), which led to the assumption that the complementary AAV minus strand was not transcribed. Furthermore, spliced AAV transcripts were identified displaying a single, shared intron located in the center of the AAV2 genome (4, 7). Initially, a single splice donor site at AAV nucleotide 1906 and a single splice acceptor site located at nucleotide (nt) 2228 were described. Later, an alternative splice acceptor site at nt 2201 was detected (8), allowing the expression of VP1 protein ...

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“…To date, 13 distinct human or nonhuman primate AAV serotypes have been isolated. [22][23][24] The primary receptors for 8 of these serotypes have been identified so far. Whereas the prototype AAV2, as well as AAV3 and AAV13, recognizes heparan sulfate proteoglycans, AAV1, AAV4, AAV5, and AAV6 bind Nlinked or O-linked sialic acids and AAV9 cell surface terminal galactose residues.…”

Section: Tissue Tropism and Persistence Of Aav Vectors In The Cardiovmentioning

confidence: 99%

Adeno-Associated Virus Vectors as Therapeutic and Investigational Tools in the Cardiovascular System

Zacchigna

Zentilin

Giacca

2014

Circulation Research

119

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Abstract:The use of vectors based on the small parvovirus adeno-associated virus has gained significant momentum during the past decade. Their high efficiency of transduction of postmitotic tissues in vivo, such as heart, brain, and retina, renders these vectors extremely attractive for several gene therapy applications affecting these organs. Besides functional correction of different monogenic diseases, the possibility to drive efficient and persistent transgene expression in the heart offers the possibility to develop innovative therapies for prevalent conditions, such as ischemic cardiomyopathy and heart failure. Therapeutic genes are not only restricted to protein-coding complementary DNAs but also include short hairpin RNAs and microRNA genes, thus broadening the spectrum of possible applications. In addition, several spontaneous or engineered variants in the virus capsid have recently improved vector efficiency and expanded their tropism. Apart from their therapeutic potential, adeno-associated virus vectors also represent outstanding investigational tools to explore the function of individual genes or gene combinations in vivo, thus providing information that is conceptually similar to that obtained from genetically modified animals. Finally, their single-stranded DNA genome can drive homology-directed gene repair at high efficiency. Here, we review the main molecular characteristics of adeno-associated virus vectors, with a particular view to their applications in the cardiovascular field.

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Differential Adeno-Associated Virus Serotype-Specific Interaction Patterns with Synthetic Heparins and Other Glycans

Cited by 115 publications

References 36 publications

Identification and Mutagenesis of the Adeno-Associated Virus 5 Sialic Acid Binding Region

Identification and Mutagenesis of the Adeno-Associated Virus 5 Sialic Acid Binding Region

A Comprehensive RNA Sequencing Analysis of the Adeno-Associated Virus (AAV) Type 2 Transcriptome Reveals Novel AAV Transcripts, Splice Variants, and Derived Proteins

Adeno-Associated Virus Vectors as Therapeutic and Investigational Tools in the Cardiovascular System

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