Journal: Scientific Reports

Article Title: Functional roles of the membrane-associated AAV protein MAAP

doi: 10.1038/s41598-021-01220-7

Figure Lengend Snippet: MAAP sequence. MAAP is encoded in the wt-AAV2 genome (GenBank Sequence ID: AF043303.1) at nucleotides 2282-2641. The ORF is located on the same DNA region encoding the VP1/2 unique domains. VP1/2 are encoded on reading frame + 1 and MAAP on reading frame + 2. MAAP translation initiates at a CTG codon (start 1), encoding a leucine which corresponds in position to VP1 proline in position 27. The next potential start codons are MAAP-T13 (ACG) and MAAP-R14 (AGG) (start 2-3). In red are the three basic-amino-acid-rich clusters MAAP2BR1, MAAP2BR2 and MAAP2BR3. Underlined is the sequence of the peptide used to generate MAAP polyclonal antiserum. All studied amino acid substitutions are shown below the sequence.

Article Snippet: To determine the ratios of capsids containing AAV2 genomes from total AAV2 capsids, A20 capsid ELISA was first performed on 500–10,000 serial dilutions of the virus preparation using the AAV2 titration ELISA kit (Progen, ref # PRATV) according to the manufacturer’s instructions.

Techniques: Sequencing

Journal: Scientific Reports

Article Title: Functional roles of the membrane-associated AAV protein MAAP

doi: 10.1038/s41598-021-01220-7

Figure Lengend Snippet: MAAP start codon identification and MAAP inactivation. ( A ) MAAP detected by immunoblot from 293 T cell lysates 24 hpt with wt- or MAAP mutants and Ad helper plasmids. Immunoblots of MAAP (top panel) and of α-Tubulin (lower panel) for equal sample loading. Left to right: (1) MW. (2) wt-AAV2. (3) AAV2 with MAAP-L1 (CTG) start codon modified to R1 (CGG). (4) MAAP-Q9. (5) MAAP-S39. (6) MAAP-S33-S39-S47. (7) non-transfected 293 T cells. ( B ) Start codon usage for MAAP translation. MAAP expressed from the first or second potential start codon and detected by immunoblot. Left to right: (1) MW. (2) wt-AAV2. (3) MAAP expressed from L1 (CTG) mutated to M (ATG). (4) MAAP expressed from R13 (AGG) mutated to M (ATG). (5) wt-AAV2. (6) v/v mix of MAAP expressed from L1 (CTG) mutated to M (ATG) and MAAP expressed from R13 (AGG) mutated to M (ATG). (7) MW. (8) non-transfected 293 T cells.

Article Snippet: To determine the ratios of capsids containing AAV2 genomes from total AAV2 capsids, A20 capsid ELISA was first performed on 500–10,000 serial dilutions of the virus preparation using the AAV2 titration ELISA kit (Progen, ref # PRATV) according to the manufacturer’s instructions.

Techniques: Western Blot, Modification, Transfection

Journal: Scientific Reports

Article Title: Functional roles of the membrane-associated AAV protein MAAP

doi: 10.1038/s41598-021-01220-7

Figure Lengend Snippet: Kinetics of AAV2 protein production. Translation of AAV2 MAAP, VP, Rep, AAP and α-Tubulin studied by immunoblot from wt-AAV2 and Ad helper plasmid-transfected 293 T cells. From left to right: MW, non-transfected 293 T, wt-AAV2 cell lysates harvested from 6.5 to 48 hpt. Degradation products of VP proteins are annotated, the 32 kDa fragment (−), 18 kDa (*) and 12 kDa (#).

Article Snippet: To determine the ratios of capsids containing AAV2 genomes from total AAV2 capsids, A20 capsid ELISA was first performed on 500–10,000 serial dilutions of the virus preparation using the AAV2 titration ELISA kit (Progen, ref # PRATV) according to the manufacturer’s instructions.

Techniques: Western Blot, Plasmid Preparation, Transfection

Journal: Scientific Reports

Article Title: Functional roles of the membrane-associated AAV protein MAAP

doi: 10.1038/s41598-021-01220-7

Figure Lengend Snippet: 3D visualization of MAAP cellular distribution in transfected cells. 3D confocal analysis of representative cytoplasmic distribution of MAAP at 24 hpt in cells co-transfected with wt-AAV2 and Ad helper plasmids. ( A ) xy, ( B ) yz and ( C ) xz slices of confocal z-stack of cytoplasmic MAAP (green), and DAPI-stained cellular DNA (blue). Inset shows higher magnification image of the cytoplasmic MAAP (boxed area), scale bars, 3 μm. ( D ) 3D reconstruction of the nucleus and distribution of MAAP obtained by confocal microscopy. The MAAP appears in green, scale bar, 3 μm. See also S2 movie . ( E ) Quantitative analysis of MAAP distribution as function of distance from the nuclear envelope towards the nuclear centre, where nuclear envelope is located in 0.0. The 3D nuclear envelope boundary reconstruction was based on distribution of DAPI-labelled chromatin. The shaded areas around the data points represent the standard error of the mean (SEM, n = 6).

Article Snippet: To determine the ratios of capsids containing AAV2 genomes from total AAV2 capsids, A20 capsid ELISA was first performed on 500–10,000 serial dilutions of the virus preparation using the AAV2 titration ELISA kit (Progen, ref # PRATV) according to the manufacturer’s instructions.

Techniques: Transfection, Staining, Confocal Microscopy

Journal: Scientific Reports

Article Title: Functional roles of the membrane-associated AAV protein MAAP

doi: 10.1038/s41598-021-01220-7

Figure Lengend Snippet: Localization and intensity of MAAP and AAV-2 capsid proteins in transfected cells. ( A ) Representative confocal images of intracellular localisation of MAAP and capsid proteins at 24 hpt. Cells were co-transfected with wt-AAV2 and MAAP-S33-S39-S47 variant together with Ad helper plasmid, or with recombinant MAAP expression plasmid without Ad helper plasmid. The cells were immunolabelled with polyclonal antibody against MAAP (green) and monoclonal antibody against viral capsid proteins VP1, VP2 and VP3 (red). Blue corresponds to DAPI staining. Scale bars, 3 µm. For analyses, the cells were either mock transfected (n = 19), co-transfected with wt-AAV2 (n = 11), MAAP-S33-S39-S47 (n = 19), MAAP-W105 (n = 20), MAAP-L106 (n = 17), MAAP-L110 (n = 20) together with Ad helper plasmid, or transfected with the recombinant MAAP plasmid (n = 18). ( B ) Quantitative analyses of the total intensities of MAAP (light grey) and capsid proteins (dark grey) at 24 hpt. ( C ) Quantitative analyses of the relative intensity of nuclear (dark grey) in comparison to cytoplasmic capsid proteins (light grey) at 24 hpt. For analysis, the cells were immunolabelled with antibodies against viral capsid protein VP1, VP2 and VP3 (red) and the nuclear boundary segmentation was based on distribution of DAPI-labelled chromatin (blue). The error bars show the SEM.

Article Snippet: To determine the ratios of capsids containing AAV2 genomes from total AAV2 capsids, A20 capsid ELISA was first performed on 500–10,000 serial dilutions of the virus preparation using the AAV2 titration ELISA kit (Progen, ref # PRATV) according to the manufacturer’s instructions.

Techniques: Transfection, Variant Assay, Plasmid Preparation, Recombinant, Expressing, Staining, Comparison

Journal: Scientific Reports

Article Title: Functional roles of the membrane-associated AAV protein MAAP

doi: 10.1038/s41598-021-01220-7

Figure Lengend Snippet: Intracellular distribution and intensity of AAV2 capsids in MAAP transfected cells. ( A ) Representative confocal images of intracellular localisation of capsids (red) in wt-AAV2 or MAAP-S33-S39-S47-transfected cells at 24 hpt . Cells were co-transfected with Ad helper plasmid. The cells were immunolabelled with an antibody against intact viral capsids (red). Blue corresponds to NucBlue chromatin staining. Nuclear border is indicated by dashed white line, scale bars, 3 µm. ( B ) Quantitative analyses of the total intensity of capsids in cells either mock transfected (n = 15) or co-transfected with wt-AAV2 (n = 12), MAAP-S33-S39-S47 (n = 12), MAAP-W105 (n = 11), MAAP-L106 (n = 13), MAAP-L110 (n = 7) together with Ad helper plasmid. ( C ) Analyses of the relative intensity of nuclear capsids (dark grey) in comparison to cytoplasmic capsids (light grey). For analyses, the cells were immunolabelled with antibodies against viral capsids (red) and the nuclear boundary segmentation was based on distribution of NucBlue-labelled chromatin (blue). Nuclear border is indicated by dashed white line. The error bars show the SEM.

Article Snippet: To determine the ratios of capsids containing AAV2 genomes from total AAV2 capsids, A20 capsid ELISA was first performed on 500–10,000 serial dilutions of the virus preparation using the AAV2 titration ELISA kit (Progen, ref # PRATV) according to the manufacturer’s instructions.

Techniques: Transfection, Plasmid Preparation, Staining, Comparison

Journal: Scientific Reports

Article Title: Functional roles of the membrane-associated AAV protein MAAP

doi: 10.1038/s41598-021-01220-7

Figure Lengend Snippet: MAAP variants and their effect on AAV proteins. To study the effects of MAAP variants on capsid proteins, 293 T cells were co-transfected with wt-AAV2 or MAAP variant plasmids and Ad helper plasmid. From cell extracts harvested at 24 hpt, we detected α-Tubulin, MAAP, VPs, Rep and AAP by immunoblotting.

Article Snippet: To determine the ratios of capsids containing AAV2 genomes from total AAV2 capsids, A20 capsid ELISA was first performed on 500–10,000 serial dilutions of the virus preparation using the AAV2 titration ELISA kit (Progen, ref # PRATV) according to the manufacturer’s instructions.

Techniques: Transfection, Variant Assay, Plasmid Preparation, Western Blot

Journal: Scientific Reports

Article Title: Functional roles of the membrane-associated AAV protein MAAP

doi: 10.1038/s41598-021-01220-7

Figure Lengend Snippet: MAAP effects on capsid levels. Plasmids with wt-AAV2 or AAV2 encoding MAAP variants were co-transfected with Ad helper plasmid. From cell extracts harvested 72 hpt, we performed AAV2 capsids ELISA ( A ), expressed as AAV capsids per mL with mean and the standard deviation (SD). Statistical significance between wt-AAV2 and AAV2 encoding MAAP mutants was evaluated using ANOVA followed by Dunnett’s multiple comparison test. Table ( B ) indicates the average AAV per mL capsid titer measured for each virus, along with the fold difference compared to wt-AAV2.

Article Snippet: To determine the ratios of capsids containing AAV2 genomes from total AAV2 capsids, A20 capsid ELISA was first performed on 500–10,000 serial dilutions of the virus preparation using the AAV2 titration ELISA kit (Progen, ref # PRATV) according to the manufacturer’s instructions.

Techniques: Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Standard Deviation, Comparison, Virus

Journal: Scientific Reports

Article Title: Functional roles of the membrane-associated AAV protein MAAP

doi: 10.1038/s41598-021-01220-7

Figure Lengend Snippet: Effect of MAAP inactivation on AAV2 vg titers. wt-AAV2 or AAV2 MAAP variants and Ad helper plasmids were co-transfected in 293 T cells. At 24 hpt ( A ) and 72 hpt ( B ) AAV vg titers were quantified, with results expressed as vg mL −1 with mean and SD. Statistical significance between wt-AAV2 and AAV2 MAAP variants was evaluated using ANOVA followed by Dunnett’s multiple comparison test. The table represents the average vg mL −1 titer measured for each virus, along with the fold difference compared to wt-AAV2 at 24 hpt ( C ) and 72 hpt ( D ). Experiments were performed independently between 3 and 7 times (indicated as N), each time with two replicate samples.

Article Snippet: To determine the ratios of capsids containing AAV2 genomes from total AAV2 capsids, A20 capsid ELISA was first performed on 500–10,000 serial dilutions of the virus preparation using the AAV2 titration ELISA kit (Progen, ref # PRATV) according to the manufacturer’s instructions.

Techniques: Transfection, Comparison, Virus

Journal: Scientific Reports

Article Title: Functional roles of the membrane-associated AAV protein MAAP

doi: 10.1038/s41598-021-01220-7

Figure Lengend Snippet: MAAP trans-complementation. We performed wt-AAV2 and MAAP-S33-S39-S47 variant production with or without the addition of recombinant MAAP. Vg titers (vg mL −1 ) were measured from cell extract samples harvested 24 hpt ( A ) and 72 hpt ( B ). Graphics show individual samples with mean and SD. Statistical significance was evaluated for recombinant MAAP addition using ANOVA followed by Dunnett’s multiple comparison test. Tables ( C ) and ( D ) represent the average titer (vg mL −1 ) at 24 hpt and 72 hpt, with fold difference to wt-AAV2. The samples from left to right are: (1) wt-AAV2. (2) MAAP-S33-S39-S47. (3) wt-AAV2 trans-complemented with MAAP expressing plasmid. (4) MAAP-S33-S39-S47 trans-complemented with MAAP expressing plasmid. (5) wt-AAV2 trans-complemented with a GFP plasmid of similar size to the recombinant MAAP plasmid. (6) MAAP-S33-S39-S47 complemented with a GFP plasmid of similar size to the recombinant MAAP plasmid.

Article Snippet: To determine the ratios of capsids containing AAV2 genomes from total AAV2 capsids, A20 capsid ELISA was first performed on 500–10,000 serial dilutions of the virus preparation using the AAV2 titration ELISA kit (Progen, ref # PRATV) according to the manufacturer’s instructions.

Techniques: Variant Assay, Recombinant, Comparison, Expressing, Plasmid Preparation

Journal: Scientific Reports

Article Title: Functional roles of the membrane-associated AAV protein MAAP

doi: 10.1038/s41598-021-01220-7

Figure Lengend Snippet: Effect of MAAP inactivation on kanamycin resistance gene packaging. wt-AAV2 or AAV2 MAAP variants and Ad helper plasmids were co-transfected in 293 T cells. At 24 hpt ( A ) and 72 hpt ( B ), AAV vg titers were quantified. In parallel, the kanamycin resistance gene carried by the AAV2 genome and the Ad helper plasmids was quantified. We present the ratio of kanamycin resistance gene packaging relative to the AAV2 genome packaging, expressed as percentage. Individual samples are represented, along with the mean and the standard deviation. Statistical significance between the wt-AAV2 and AAV encoding MAAP mutants was evaluated using ANOVA followed by Dunnett’s multiple comparison test. Tables ( C ) and ( D ) show the average percentage of kanamycin resistance gene packaging relative to AAV2 genome packaging, measured for each virus at 24 hpt and 72 hpt, and the fold difference compared to wt-AAV2. Experiments were performed independently between 3 and 6 times (indicated as N), each time with two samples.

Article Snippet: To determine the ratios of capsids containing AAV2 genomes from total AAV2 capsids, A20 capsid ELISA was first performed on 500–10,000 serial dilutions of the virus preparation using the AAV2 titration ELISA kit (Progen, ref # PRATV) according to the manufacturer’s instructions.

Techniques: Transfection, Standard Deviation, Comparison, Virus

Journal: Scientific Reports

Article Title: Functional roles of the membrane-associated AAV protein MAAP

doi: 10.1038/s41598-021-01220-7

Figure Lengend Snippet: Effect of MAAP inactivation on genome packaging. wt-AAV2 or AAV2 MAAP variants and Ad helper plasmids were co-transfected in 293 T cells. At 72 hpt ( A ) AAV vg titers were quantified. In parallel, we quantified the total number of AAV capsids from the same samples by ELISA. We present the ratio of capsid containing AAV2 genome versus total capsids, expressed as percentage. Samples are represented, with mean and SD. Statistical significance between wt-AAV2 and AAV encoding MAAP variants was evaluated using ANOVA followed by Dunnett’s multiple comparison test. Table ( B ) shows the average percentage of capsid containing AAV2 genomes and the fold difference compared to wt-AAV2.

Article Snippet: To determine the ratios of capsids containing AAV2 genomes from total AAV2 capsids, A20 capsid ELISA was first performed on 500–10,000 serial dilutions of the virus preparation using the AAV2 titration ELISA kit (Progen, ref # PRATV) according to the manufacturer’s instructions.

Techniques: Transfection, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Scientific Reports

Article Title: Functional roles of the membrane-associated AAV protein MAAP

doi: 10.1038/s41598-021-01220-7

Figure Lengend Snippet: MAAP inactivation and its effect, in the context of AAV2 and Adenovirus 5 co-infection. We generated wt-AAV2, AAV2-MAAP-S33-S39-S47 and AAV2-MAAP-L100 viruses using plasmid transfection of 293 T cells. The harvested viruses were used to infect 293 T cells at an MOI of 500 with or without Adenovirus 5 added at an MOI of 50. Samples were harvest at 12, 18, 24, 48, 72 hpi and AAV vg titers ( A ), adenovirus vg titers ( B ), and kanamycin resistance gene titers ( C ) were quantified. Medians of the titers are represented. Statistical significance between samples was evaluated using Kruskal–Wallis test followed by Dunn’s multiple comparison test.

Article Snippet: To determine the ratios of capsids containing AAV2 genomes from total AAV2 capsids, A20 capsid ELISA was first performed on 500–10,000 serial dilutions of the virus preparation using the AAV2 titration ELISA kit (Progen, ref # PRATV) according to the manufacturer’s instructions.

Techniques: Infection, Generated, Plasmid Preparation, Transfection, Comparison