We found a match
Your institution may have access to this item. Find your institution then sign in to continue.
- Title
104. Role of the HI Loop in the Adeno-Associated Virus (AAV) Life Cycle.
- Authors
DiPrimio, Nina; Asokan, Aravind; Samulski, Richard J.
- Abstract
The fivefold axis of symmetry associated with pore region in icosahedral AAV capsids has been extensively studied in regard to its role in deploying the phospholipase A2 domain and packaging of the 4.7kb AAV genome. However, the role of the HI loops surrounding the fivefold pore, which appear to vary significantly in amino acid sequence between AAV serotypes has not been defined thus far. The HI loop is a surface-displayed peptide motif of 10-15 amino acid residues connecting the H and I beta-strands of the VP3 subunit of AAV and extends to interact with a fivefold-related neighboring subunit. In order to understand the role of this capsid region on the AAV life cycle, we substituted the HI loop in the VP3 subunit of the AAV2 capsid with corresponding regions from the VP3 subunits of AAV1, AAV4, AAV5 or AAV8. In addition, we created a deletion mutant lacking the HI loop and an AAV2 mutant substituted with a loop containing ten glycine residues. Determination of the mutant virus titers by dot blot analysis revealed that vector genome titers of AAV2HI1 (AAV1 HI loop substituted in AAV2) and AAV2HI8 displayed a two-fold decrease in titer, while AAV2HI4 displayed a ten-fold decrease in titer when compared to parental AAV2. On the other end of this spectrum, AAV2HI5, AAV2HIdel (deletion mutant), and AAV2HIpolyG (poly-glycine loop) were unable to package vector genomes. Further western and dot blot western analysis revealed that AAV2HI5 and AAV2HIdel mutants were able to synthesize capsid subunits, but unable to assemble into capsids. Interestingly, the AAV2HIpolyG mutant assembled into empty capsids highlighting the plasticity of this loop region. Further characterization of AAV HI loop mutants using transduction assays, heparin binding assays and electron microscopy are currently in progress. Our studies implicate a potential role for the HI loop in AAV genome packaging and capsid assembly.Molecular Therapy (2006) 13, S43–S43; doi: 10.1016/j.ymthe.2006.08.124
- Subjects
ADENOVIRUSES; AMINO acids; GENETICS; GENOMES; ACETIC acid
- Publication
Molecular Therapy, 2006, Vol 13, pS43
- ISSN
1525-0016
- Publication type
Article
- DOI
10.1016/j.ymthe.2006.08.124