JVI Accepts, published online ahead of print on 26 March 2008

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J. Virol. doi:10.1128/JVI.02721-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Surface Loop Dynamics in AAV Capsid Assembly

Nina DiPrimio, Aravind Asokan, Lakshmanan Govindasamy, Mavis Agbandje-McKenna, and R. Jude Samulski^*

Gene Therapy Center, Department of Pharmacology, School of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Department of Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL 32610, USA

^* To whom correspondence should be addressed. Email: rjs{at}med.unc.edu.

The HI loop is a prominent domain on the AAV capsid surface that extends from each viral protein (VP) subunit overlapping the neighboring five-fold VP. Despite the highly conserved nature of the residues at the five-fold pore, the HI loops surrounding this critical region vary significantly in amino acid sequence between the AAV serotypes. In order to understand the role of this unique capsid domain, we ablated side chain interactions between the HI loop and the underlying EF loop in the neighboring VP subunit by generating a collection of deletion, insertion and substitution mutants. A mutant lacking the HI loop was unable to assemble particles while a substitution mutant (ten glycine residues) assembled particles but was unable to package viral genomes. Substitution mutants carrying corresponding regions from AAV1, AAV4, AAV5 and AAV8 yielded; a) particles with titers and infectivity identical to AAV2 (AAV2 HI1 & HI8), b) particles with decreased virus titer (one log), but normal infectivity (HI4), and c) particles that synthesized VPs but were unable to assemble into intact capsids (HI5). AAV5 HI is shorter than all other HI loops by one amino acid. Replacing the missing residue (threonine) in AAV2 HI5 resulted in a moderate particle assembly rescue. In addition, we substituted the HI loop with peptides varying in length and amino acid sequence. This region tolerated seven-amino acid peptide substitutions, unless spanning a conserved phenylalanine at amino acid position 661. Mutation of this highly conserved phenylalanine to a glycine resulted in a modest decrease in virus titer, but a substantial decrease (one log order) in infectivity. Subsequently, confocal studies revealed that AAV2 F661G is incapable of efficiently completing a key step in the infectious pathway, nuclear entry, hinting at a possible perturbation of VP1 phospholipase activity. Molecular modeling studies with the F661G mutant suggest that disruption of interactions between F661 and an underlying P373 residue in the EF loop of the neighboring subunit might adversely affect incorporation of the VP1 subunit at the five-fold axis. Western blot analysis confirmed inefficient incorporation of VP1 as well as a proteolytically processed VP1 subunit that could account for the markedly reduced infectivity. In summary, our studies show that the HI loop, while flexible in amino acid sequence, is critical for AAV capsid assembly, proper VP1 subunit incorporation, and viral genome packaging all of which implicate a potential role for this unique surface domain in viral infectivity.

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