M. Ruffing et al., MUTATIONS IN THE CARBOXY-TERMINUS OF ADENOASSOCIATED VIRUS-2 CAPSID PROTEINS AFFECT VIRAL INFECTIVITY - LACK OF AN RGD INTEGRIN-BINDING MOTIF, Journal of General Virology, 75, 1994, pp. 3385-3392
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.