MUTATIONS IN THE CARBOXY-TERMINUS OF ADENOASSOCIATED VIRUS-2 CAPSID PROTEINS AFFECT VIRAL INFECTIVITY - LACK OF AN RGD INTEGRIN-BINDING MOTIF

Using site-directed mutagenesis, we tested whether a potential integri n-binding site, (composed of the amino acids RGD) which is predicted i n the adeno-associated virus 2 (AAV-2) capsid open reading frame (ORF) , plays a role in the infectivity of AAV-2. Nucleotide sequencing of w ild-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 m otif and extends the capsid ORF by 27 amino acis. This sequence was co nfirmed by protein sequencing of proteolytic fragments of VP3. Thus, t he virus mutant (pTAV-p), in which the intention was to exchange D of the putative RGD motif for E, resulted in replacing I-480 by S in the newly established ORF. A second virus mutant (pTAV-d), in which the in tention was to delete the RGD peptide, in fact gave a shift into the O RF of the originally published sequence. The pTAV-p mutant showed a st rongly reduced infectivity compared to wildtype AAV-2, whereas pTAV-d was not infectious at all. Neither mutant accumulated viral ssDNA as d etected by Hirt extraction. Analysis of virus particle formation and s ubcellular localization of the capsid proteins revealed a defect of th e mutant capsid proteins in capsid assembly. This shows that the newly established C-terminal sequence of the AAV capsid proteins plays an i mportant role in viral assembly.