Adeno-Associated Virus (AAV) Capsid Stability and Liposome Remodeling During Endo/Lysosomal pH Trafficking
"> Figure 1
<p>Adeno-associated virus 1 (AAV1) stability analysis. (<b>a</b>) Dot immunoblots of AAV1-green fluorescent protein (GFP) vectors at different pHs representative of physiological (7.4), early endosomal (6.0), late endosomal (5.5), and lysosomal (4.0) conditions. The virus was heat-shocked at the temperature indicated and blotted for intact capsids (ADK1a), denatured capsids (B1), and the accessibility of VP1u (A1). (<b>b</b>) Negative-stain electron microscopy (EM) of AAV1-GFP vectors treated as in (<b>a</b>). Scale bar: 100 nm. (<b>c</b>) T<sub>m</sub> of AAV1-VLPs determined by DSF at the pH conditions used in (<b>a</b>) and (<b>b</b>). A thermal profile for each pH condition is shown as normalized relative fluorescence units (RFUs) versus temperature (in °C). All experiments were performed in triplicate.</p> "> Figure 2
<p>AAV2 stability analysis. (<b>a</b>) Dot immunoblots of AAV2-GFP vectors at different pHs representative of physiological (7.4), early endosomal (6.0), late endosomal (5.5), and lysosomal (4.0) conditions. The virus was heat-shocked at the temperature indicated and blotted for intact capsids (A20), denatured capsids (B1), and the accessibility of VP1u (A1). (<b>b</b>) Negative-stain EM of AAV2-VLPs treated as in (<b>a</b>). Scale bar: 100 nm. (<b>c</b>) T<sub>m</sub> of AAV2-VLPs determined by DSF at the pH conditions used in (<b>a</b>) and (<b>b</b>). A thermal profile for each pH condition is shown as normalized relative fluorescence units (RFUs) versus temperature (in °C). All experiments were performed in triplicate.</p> "> Figure 3
<p>AAV5 stability analysis. (<b>a</b>) Dot immunoblots of AAV5-GFP vectors at different pHs representative of physiological (7.4), early endosomal (6.0), late endosomal (5.5), and lysosomal (4.0) conditions. The virus was heat-shocked at the temperature indicated and blotted for intact capsids (ADK5a), denatured capsids (B1), and the accessibility of VP1u (A1). (<b>b</b>) Negative-stain EM of AAV5-GFP vectors treated as in (<b>a</b>). Scale bar: 100 nm. (<b>c</b>) T<sub>m</sub> of AAV5-VLPs determined by DSF at the pH conditions used in (<b>a</b>) and (<b>b</b>). A thermal profile for each pH condition is shown as normalized relative fluorescence units (RFUs) versus temperature (in °C). All experiments were performed in triplicate.</p> "> Figure 4
<p>AAV8 stability analysis. (<b>a</b>) Dot immunoblots of AAV8-GFP vectors at different pHs representative of physiological (7.4), early endosomal (6.0), late endosomal (5.5), and lysosomal (4.0) conditions. The virus was heat-shocked at the temperature indicated and blotted for intact capsids (ADK8), denatured capsids (B1), and the accessibility of VP1u (A1). (<b>b</b>) Negative-stain EM of AAV8-GFP vectors treated as in (<b>a</b>). Scale bar: 100 nm. (<b>c</b>) T<sub>m</sub> of AAV8 VLPs determined by DSF at the pH conditions used in (<b>a</b>) and (<b>b</b>). A thermal profile for each pH condition is shown as normalized relative fluorescence units (RFUs) versus temperature (in °C). All experiments were performed in triplicate.</p> "> Figure 5
<p>POPC liposome remodeling in the presence of PLA<sub>2</sub>. Liposomes (<b>a</b>) at pH 7.4, (<b>b</b>) pH 5.5, and (<b>c</b>) in presence of bee venom PLA<sub>2</sub> (bvPLA<sub>2</sub>) at pH 7.4. Bleb formation of POPC liposomes was observed in (<b>c</b>). All experiments were performed in triplicate.</p> "> Figure 6
<p>AAV–liposome interactions. POPC liposomes in presence of (<b>a</b>) rAAV1-GFP, (<b>b</b>) AAV2-VLPs, (<b>c</b>) rAAV5-GFP, and (<b>d</b>) rAAV8-GFP at pH 7.4. POPC liposomes in presence of (<b>e</b>) rAAV1-GFP, (<b>f</b>) AAV2-VLPs, (<b>g</b>) rAAV5-GFP, and (<b>h</b>) rAAV8-GFP that were heat-shocked prior to addition to liposomes. POPC liposomes in presence of (<b>i</b>) rAAV1-GFP, (<b>j</b>) AAV2-VLPs, (<b>k</b>) rAAV5-GFP, and (<b>l</b>) rAAV8-GFP that were incubated at pH 5.5 prior to addition to liposomes. Bleb formation of POPC liposomes was observed in (<b>e</b>–<b>l</b>). All experiments were performed in triplicate.</p> "> Figure 7
<p>POPC liposome modification in the presence of AAV. Liposomes in presence of (<b>a</b>) AAV2 VP3 only VLPs at pH 7.4, (<b>b</b>) at pH 5.5, and (<b>c</b>) in presence of AAV2 VP3 only VLPs that were heat-shocked prior to addition to liposomes. POPC liposomes in presence of the AAV2 VP1u polypeptide at (<b>d</b>) pH 7.4, (<b>e</b>) at pH 5.5, and (<b>f</b>) after heat-shock to 70 °C prior to addition to liposomes. Bleb formation of POPC liposomes was observed in (<b>d</b>–<b>f</b>). All experiments were performed in triplicate.</p> "> Figure 8
<p>Effect of pH and temperature on AAV transduction. Transduction efficiency of (<b>a</b>) rAAV1, (<b>b</b>) rAAV2, (<b>c</b>) rAAV5, and (<b>d</b>) rAAV8 (all packaging the luciferase gene) in HEK293 cells infected with virus incubated for 24 h in citrate-phosphate buffer at the indicated pH and storage temperature. The transduction efficiency for each AAV serotype is shown relative to virus stored at pH 7.4 and −80 °C. The experiments were performed in triplicate and are displayed as mean+SD (n = 3). #: luciferase read-out similar to uninfected control cells (data not shown) which indicates no transduction.</p> "> Figure 9
<p>Melting temperature of AAV capsids under different pH conditions. Plot of the T<sub>m</sub> for AAV1, AAV2, AAV5, and AAV8 at physiologic (7.4), early endosome (6.0), late endosome (5.), and lysosome (4.0) conditions. The experiments were performed in triplicate and are displayed as mean+SD (n = 3).</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Culture
2.2. Cloning
2.3. Production and Purification of AAV2 VP1u Polypeptide
2.4. Production of AAV VLPs
2.5. Purification of AAV VLPs
2.6. Production and Purification of rAAV-GFP
2.7. Confirmation of purity of VLPs and rAAVs
2.8. rAAV pH and Heat Screen
2.9. Native Dot Immunoblot
2.10. Determination of the Thermal Stability of AAV Capsids
2.11. Production of Liposomes
2.12. Analysis of AAV–Liposome Interactions
2.13. Analysis of AAV Transduction Efficiency after Incubation at Different pHs and Temperatures
3. Results
3.1. AAV Capsid Stability is pH Dependent
3.2. AAVs Modify Liposomes When Heated to Externalize VP1u and at pH 5.5
3.3. AAVs Show a Temperature and pH Dependence of Transduction
4. Discussion
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Lins-Austin, B.; Patel, S.; Mietzsch, M.; Brooke, D.; Bennett, A.; Venkatakrishnan, B.; Van Vliet, K.; Smith, A.N.; Long, J.R.; McKenna, R.; et al. Adeno-Associated Virus (AAV) Capsid Stability and Liposome Remodeling During Endo/Lysosomal pH Trafficking. Viruses 2020, 12, 668. https://doi.org/10.3390/v12060668
Lins-Austin B, Patel S, Mietzsch M, Brooke D, Bennett A, Venkatakrishnan B, Van Vliet K, Smith AN, Long JR, McKenna R, et al. Adeno-Associated Virus (AAV) Capsid Stability and Liposome Remodeling During Endo/Lysosomal pH Trafficking. Viruses. 2020; 12(6):668. https://doi.org/10.3390/v12060668
Chicago/Turabian StyleLins-Austin, Bridget, Saajan Patel, Mario Mietzsch, Dewey Brooke, Antonette Bennett, Balasubramanian Venkatakrishnan, Kim Van Vliet, Adam N. Smith, Joanna R. Long, Robert McKenna, and et al. 2020. "Adeno-Associated Virus (AAV) Capsid Stability and Liposome Remodeling During Endo/Lysosomal pH Trafficking" Viruses 12, no. 6: 668. https://doi.org/10.3390/v12060668