Browsing by Author "Brooke, Dewey"

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Adeno-Associated Virus (AAV) Capsid Stability and Liposome Remodeling During Endo/Lysosomal pH Trafficking
(2020-06) Lins-Austin, Bridget; Patel, Saajan; Mietzsch, Mario; Brooke, Dewey; Bennett, Antonette; Venkatakrishnan, Balasubramanian; Van Vliet, Kim; Smith, Adam N.; Long, Joanna R.; McKenna, Robert; Potter, Mark; Byrne, Barry; Boye, Sanford L.; Bothner, Brian; Heilbronn, Regine; Agbandje-McKenna, Mavis
Adeno-associated viruses (AAVs) are small, non-pathogenic ssDNA viruses being used as therapeutic gene delivery vectors for the treatment of a variety of monogenic diseases. An obstacle to successful gene delivery is inefficient capsid trafficking through the endo/lysosomal pathway. This study aimed to characterize the AAV capsid stability and dynamics associated with this process for a select number of AAV serotypes, AAV1, AAV2, AAV5, and AAV8, at pHs representative of the early and late endosome, and the lysosome (6.0, 5.5, and 4.0, respectively). All AAV serotypes displayed thermal melt temperatures that varied with pH. The stability of AAV1, AAV2, and AAV8 increased in response to acidic conditions and then decreased at pH 4.0. In contrast, AAV5 demonstrated a consistent decrease in thermostability in response to acidification. Negative-stain EM visualization of liposomes in the presence of capsids at pH 5.5 or when heat shocked showed induced remodeling consistent with the externalization of the PLA2 domain of VP1u. These observations provide clues to the AAV capsid dynamics that facilitate successful infection. Finally, transduction assays revealed a pH and temperature dependence with low acidity and temperatures > 4 °C as detrimental factors.
Analysis of phospholipase activity in adeno-associated virus particles by liquid-chromatography/mass-spectrometry
(2013-03) Brooke, Dewey; Bothner, Brian; Agbandje-McKenna, Mavis
Adeno-associated virus (AAV) belongs to the Parvovridae, a family of small, non-enveloped isosahedral viruses. The viral capsid has T=1 symmetry and is composed of 60 subunits, made up from three proteins (VP1, VP2, VP3) in a ratio of 1:1:10. The minor proteins are the same as VP3 in their C-termini region, but they have additional domains on their N-termini that play essential roles in cellular entry and trafficking. Structural studies of AAV have shown that the N-termini of VP1 and VP2 are initially internalized in the capsid and become externalized, most likely during endocytosis. Based on sequence and structural similarity, VP1 contains a phospholipase A2 domain (PLA2) which, when mutated, dramatically reduces infectivity. Currently, little is known about the mechanism of VP1 externalization or the role of the lipase in escape of the virus particle from the endosome. Also, due to low sequence similarity, there is even concern over whether this is a true PLA2 type domain. To address these questions, we have developed a liquid-chromatography mass-spectrometry based assay for lipase activity. To date, we have tested factors such as receptor binding, heat, and pH on the externalization of the PLA2 and are addressing the question of substrate specificity.