Green Fluorescent Protein-Tagged Adeno-Associated Virus Particles Allow the Study of Cytosolic and Nuclear Trafficking

Abstract

To allow the direct visualization of viral trafficking, we genetically incorporated enhanced green fluorescent protein (GFP) into the adeno-associated virus (AAV) capsid by replacement of wild-type VP2 by GFP-VP2 fusion proteins. High-titer virus progeny was obtained and used to elucidate the process of nuclear entry. In the absence of adenovirus 5 (Ad5), nuclear translocation of AAV capsids was a slow and inefficient process: at 2 h and 4 h postinfection (p.i.), GFP-VP2-AAV particles were found in the perinuclear area and in nuclear invaginations but not within the nucleus. In Ad5-coinfected cells, isolated GFP-VP2-AAV particles were already detectable in the nucleus at 2 h p.i., suggesting that Ad5 enhanced the nuclear translocation of AAV capsids. The number of cells displaying viral capsids within the nucleus increased slightly over time, independently of helper virus levels, but the majority of the AAV capsids remained in the perinuclear area under all conditions analyzed. In contrast, independently of helper virus and with 10 times less virions per cell already observed at 2 h p.i., viral genomes were visible within the nucleus. Under these conditions and even with prolonged incubation times (up to 11 h p.i.), no intact viral capsids were detectable within the nucleus. In summary, the results show that GFP-tagged AAV particles can be used to study the cellular trafficking and nuclear entry of AAV. Moreover, our findings argue against an efficient nuclear entry mechanism of intact AAV capsids and favor the occurrence of viral uncoating before or during nuclear entry.

Off-target-free gene delivery by affinity-purified receptor-targeted viral vectors

“…This is in line with our initial finding demonstrating that insertion of DARPins at the N terminus of VP2 does not interfere with capsid assembly or packaging efficiency 11 . DARPins are thus a further example proving the suitability of this insertion site for the display of functionally active polypeptides while keeping basic viral vector functions active 21,22 . After cell entry, Her2-AAV showed an overall similar intracellular distribution as the unmodified vector, AAV-2, with a small shift from nuclear to membrane-bound localization ( Supplementary Fig.…”

“…20) were amplified by PCR using the primer pair DARPin-for/DARPin-rev or DARPin-for/DARPin EC1-rev respectively, (Supplementary Table 1) and were inserted into pGFP (green fluorescent protein)-VP2 (ref. 21) by sticky-end ligation using AgeI and BsrGI restriction sites resulting in plasmids pHer2-VP2, pCD4-VP2 and pEpCAM-VP2. Plasmids encoding VP2 fused to His-tagged or Myc-tagged DARPins were generated analogously using the primer pairs His-forw/DARPin-rev or His-forw/DARPin EC1-rev and Myc-forw/DARPin-rev or Myc-forw/DARPin Ec1-rev, respectively.…”

Heart-targeted adeno-associated viral vectors selected by in vivo biopanning of a random viral display peptide library

“…Genetic modification of the AAV2 capsid by oligonucleotide insertions, random mutagenesis or DNA shuffling is able to circumvent some of these limitations. [8][9][10][17][18][19] Whereas larger peptides up to the size of GFP (238 amino acids) could be accommodated at the VP2 N-terminus 15,[20][21][22][23][24][25] the addition of small peptides up to 34 amino acids is tolerated at several positions of the AAV2 capsid. 1 Among the tolerated insertion sites for small peptides, insertions in the AAV2 heparin binding domain 26,27 adjacent to amino acids 587 or 588 was most successful.…”

Directed evolution of novel adeno-associated viruses for therapeutic gene delivery

“…2 The virus's capsid governs its ability to transduce cells, from initial cell surface receptor binding to gaining entry into the nucleus. Briefly, AAV2-the variant most broadly studied to date-is internalized via receptor-mediated endocytosis, with evidence supporting a role for both the clathrin-coated pit pathway 4,5 and the clathrinindependent carriers/glycosylphosphatidylinositol-anchored-proteinenriched endosomal compartment pathway. 6 Following cellular entry, the virion escapes from early endosomes and traffics to the perinuclear area.…”