Evidence for pH-dependent protease activity in the adeno-associated virus capsid.
Incubation of highly purified adeno-associated virus (AAV) capsids in vitro at pH 5.5 induced significant autocleavage of capsid proteins at several amino acid positions. No autocleavage was seen at pH 7.5. Examination of other AAV serotypes showed at least two different pH-induced cleavage patterns, suggesting that different serotypes have evolved alternative protease cleavage sites. In contrast, incubation of AAV serotypes with an external protease substrate showed that purified AAV capsid preparations have robust protease activity at neutral pH but not at pH 5.5, opposite to what is seen with capsid protein autocleavage. Several lines of evidence suggested that protease activity is inherent in AAV capsids and is not due to contaminating proteins. Control virus preparations showed no protease activity on external substrates, and filtrates of AAV virus preparations also showed no protease activity contaminating the capsids. Further, N-terminal Edman sequencing identified unique autocleavage sites in AAV1 and AAV9, and mutagenesis of amino acids adjacent to these sites eliminated cleavage. Finally, mutation of an amino acid in AAV2 (E563A) that is in a conserved pH-sensitive structural region eliminated protease activity on an external substrate but did not seem to affect autocleavage. Taken together, our data suggested that AAV capsids have one or more protease active sites that are sensitive to pH induction. Further, it appears that acidic pHs comparable to those seen in late endosomes induce a structural change in the capsid that induces autolytic protease activity. The pH-dependent protease activity may have a role in viral infection.
Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, Florida, USA.
Maxim
M
Salganik
Balasubramanian
B
Venkatakrishnan
Antonette
A
Bennett
Bridget
B
Lins
Joseph
J
Yarbrough
Nicholas
N
Muzyczka
Mavis
M
Agbandje-McKenna
Robert
R
McKenna
D002213:Capsid / Q000187:drug effects* / Q000201:enzymology*; D036022:Capsid Proteins / Q000378:metabolism*; D000229:Dependovirus / Q000187:drug effects* / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D010447:Peptide Hydrolases / Q000378:metabolism*; D011487:Protein Conformation / Q000187:drug effects; D013379:Substrate Specificity
D036022:Capsid Proteins; D050505:Mutant Proteins; D010447:Peptide Hydrolases