TABLE 1.

Characterization of AAV2 pore mutants

Mutation(s)	Packaging reduction with respect to wta,b	Infectivity reduction with respect to wta,c	Heparin bindingd	Virus uptakee	Classf
E322A	UD	UD	+	ND	A
E322A/V323D/T324N	UD	UD	+	ND	A
T324Y	2.0 ± 0.8	32 ± 8	+	ND	I
Q325A	1.7 ± 0.5	4.5 ± 0.5g	+	ND	N
N326A	0.7 ± 0.1	1.2 ± 0.1	+	ND	N
N334A	3.8 ± 1.5g	1.1 ± 0.1	+	ND	P
N335A	2.0 ± 0.5g	2.1E4 ± 1.3E4g	+	+	I
L336A	1.2 ± 0.1	1.8E2 ± 0.5E2g	+	ND	I
T337A	1.1 ± 0.1	37 ± 12g	+	ND	I
S338A	8.9 ± 2.1g	1.1E3 ± 0.7E3g	+	+	P + I
D219A	4.9 ± 1.2g	1.1 ± 0.1	+	ND	P
del219/220	4.6 ± 1.5g	61 ± 14g	+	+	P + I
V221C	3.2 ± 0.3g	2.8E2 ± 0.4E2g	+	ND	P + I
V221Y	3.4 ± 0.7g	1.7E3 ± 0.8E3g	+	+	P + I
221Ins2Y	UD	UD	UD	ND	A
N223A	0.5 ± 0.1g	1.3 ± 0.1g	+	ND	N

^aValues are means plus or minus standard deviations from at least four independent experiments; UD, undetectable.

^bPackaging was measured as the ratio of capsids to viral genomes and compared to the ratio for wt AAV2. The number of assembled capsids was determined by a capsid ELISA (24); packaged DNA was quantified by a dot blot assay.

^cInfectivity was expressed as the ratio of genomes to infectious units and compared to the ratio for wt AAV2. Packaged DNA was measured by a dot blot assay; infectious AAV2 particles were quantified by a dot blot replication assay.

^dHeparin binding was measured as the percentage of viral particles which were applied to a heparin-agarose column and which were recovered by high-salt elution, as determined by a capsid ELISA; +, more than 90% binding.

^eVirus uptake (+) into cells was analyzed by indirect immunofluorescence microscopy. ND, not determined.

^fMutants were classified according to their defects: A, assembly (more than a 10-fold reduction compared to that in the wt); I, infectivity (more than a 10-fold reduction compared to that in the wt); P, packaging (more than a 2-fold reduction compared to that in the wt); N, not affected.

^gIn a comparison with the wt, the P value was <0.05.