Site Specific Modification of Adeno-Associated Virus Enables Both Fluorescent Imaging of Viral Particles and Characterization of the Capsid Interactome

Jayanth S Chandran,João Miguel Da Conceição Aves-Cruzeiro,Lydia Castelli,Ian Coldicott,Evangelia Karyka,Guillaume Hautbergue,Mimoun Azzouz,Paul S Sharp,Mark O Collins

doi:10.1038/s41598-017-15255-2

Jayanth S Chandran, João Miguel Da Conceição Aves-Cruzeiro + Show 7 more

Open Access

https://doi.org/10.1038/s41598-017-15255-2

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Journal: Scientific Reports	Publication Date: Nov 7, 2017
Citations: 24	License type: open-access

Affiliation: University of Sheffield

Abstract

Adeno-associated viruses (AAVs) are attractive gene therapy vectors due to their low toxicity, high stability, and rare integration into the host genome. Expressing ligands on the viral capsid can re-target AAVs to new cell types, but limited sites have been identified on the capsid that tolerate a peptide insertion. Here, we incorporated a site-specific tetracysteine sequence into the AAV serotype 9 (AAV9) capsid, to permit labelling of viral particles with either a fluorescent dye or biotin. We demonstrate that fluorescently labelled particles are detectable in vitro, and explore the utility of the method in vivo in mice with time-lapse imaging. We exploit the biotinylated viral particles to generate two distinct AAV interactomes, and identify several functional classes of proteins that are highly represented: actin/cytoskeletal protein binding, RNA binding, RNA splicing/processing, chromatin modifying, intracellular trafficking and RNA transport proteins. To examine the biological relevance of the capsid interactome, we modulated the expression of two proteins from the interactomes prior to AAV transduction. Blocking integrin αVβ6 receptor function reduced AAV9 transduction, while reducing histone deacetylase 4 (HDAC4) expression enhanced AAV transduction. Our method demonstrates a strategy for inserting motifs into the AAV capsid without compromising viral titer or infectivity.

Highlights

Several reports have demonstrated that the loop region of associated viruses (AAVs) exposed to the surface and defined by amino acids 584–590 in VP1 can tolerate peptide insertions without a significant loss of titer[7,10,11]
Toward validation of these interactomes, we assessed the biological relevance of two novel AAV serotype 9 (AAV9) interactors in vivo. αV and β6 subunits were identified as AAV9 interactors in HEK cells and we found that blocking the function of αVβ6 integrin led to a decrease in AAV9 transduction
The three viral proteins (VP1, VP2, and VP3) that form the AAV capsid lattice differ at the N-terminus so that the entire VP3 sequence is contained within VP2, which is in turn contained within VP1 (Fig. 1C)

Summary

Introduction

Several reports have demonstrated that the loop region of AAVs exposed to the surface and defined by amino acids 584–590 in VP1 can tolerate peptide insertions without a significant loss of titer[7,10,11]. We demonstrate the potential of this technology as a fluorescently labelled viral particle to examine viral particle dynamics, and use a biotinylated viral particle to characterize the capsid interactome using two different paradigms (1) whole cell analysis in HEK cells through direct labeling of newly formed AAV9 and (2) probing interactors in tissue by incubating biotinylated AAV9 capsids with mouse brain lysates. Toward validation of these interactomes, we assessed the biological relevance of two novel AAV9 interactors in vivo. Our successful insertion of a site-specific sequence onto AAV9 provides a template for designing new tools to facilitate viral gene transfer and understanding AAV function

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