Mutations in the carboxy terminus of adeno-associated virus 2 capsid proteins affect viral infectivity: lack of an RGD integrin-binding motif.

Using site-directed mutagenesis, we tested whether a potential integrin-binding site, (composed of the amino acids RGD) which is predicted in the adeno-associated virus 2 (AAV-2) capsid open reading frame (ORF), plays a role in the infectivity of AAV-2. Nucleotide sequencing of wild-type and mutant capsid protein-coding sequences, however, revealed discrepancies with the published sequence data at several positions, including a frameshift in the carboxy terminus which cancels the RGD motif and extends the capsid ORF by 27 amino acids. This sequence was confirmed by protein sequencing of proteolytic fragments of VP3. Thus, the virus mutant (pTAV-p), in which the intention was to exchange D of the putative RGD motif for E, resulted in replacing I480 by S in the newly established ORF. A second virus mutant (pTAV-d), in which the intention was to delete the RGD peptide, in fact gave a shift into the ORF of the originally published sequence. The pTAV-p mutant showed a strongly reduced infectivity compared to wild-type AAV-2, whereas pTAV-d was not infectious at all. Neither mutant accumulated viral ssDNA as detected by Hirt extraction. Analysis of virus particle formation and subcellular localization of the capsid proteins revealed a defect of the mutant capsid proteins in capsid assembly. This shows that the newly established C-terminal sequence of the AAV capsid proteins plays an important role in viral assembly.