Reprogramming the Activatable Peptide Display Function of Adeno-Associated Virus Nanoparticles

Abstract

We harnessed an intrinsic activatable peptide display behavior shared by several parvoviruses, including the adeno-associated virus (AAV), in order to design protein-based nanodevices that can carry out an exogenous functional output in response to stimulus detection. Specifically, we generated truncated viral capsid subunits that, when combined with native capsid components into mosaic capsids, can perform robust activatable peptide display. By modulating the ratio of subunits in the mosaic capsid, properties of the activatable peptide display function can be optimized. Interestingly, the truncated subunits can form homomeric capsids not observed in nature, but at the price of losing the ability to carry out activatable peptide display. Collectively, our results demonstrate the importance of capsid mosaicism when activatable peptide display is desired and help explain why the wild-type AAV capsid exists as a mosaic of different subunits. This proof-of-concept study illustrates a strategy for reprogramming a particular conformational output behavior of AAV in pursuit of the long-term vision of creating stimulus-responsive nanodevices.

An overview of functional nanoparticles as novel emerging antiviral therapeutic agents

“…Western blot assay, a method extensively used in biochemistry for qualitative detection of individual proteins and protein modifications, can identify the protein degradation of the virus treated with functional nanoparticles [34]. In a typical experiment, the sample undergoes protein denaturation, followed by gel electrophoresis characterization and creation of a primary antibody via a synthetic or animal-derived route for recognizing and binding to a specific target.…”

Adeno-associated virus (AAV) vectors: Rational design strategies for capsid engineering

“…Lastly, nonviral motifs can be inserted into the AAV capsid to bring about new functional outputs. Recently, a panel of mosaic AAV capsids with varying lengths of VP2 truncation mutant subunits with a His tag at the N-terminus have been generated [47]. By harnessing the AAV capsid’s natural mechanism of activatable peptide display, the resulting virus particles exhibit varying degrees of His tag exposure pre- and post- temperature activation.…”

Physical, chemical, and synthetic virology: Reprogramming viruses as controllable nanodevices

“…The newly externalized VP1 and VP2 domains allow for endosomal escape and nuclear localization of the virus (Bleker, Sonntag, & Kleinschmidt, 2005;Kronenberg, Böttcher, von der Lieth, Bleker, & Kleinschmidt, 2005;Sonntag, Bleker, Leuchs, Fischer, & Kleinschmidt, 2006;Thomas, Storm, Huang, & Kay, 2004). AAV's metastability has recently been harnessed to develop a platform of viruses able to externalize exogenous peptides upon activation (Thadani, Dempsey, Zhao, Vasquez, & Suh, 2017). Interestingly, mosaicism of the capsid-differing subunits assembling to form the capsid-appears to be a requirement for obtaining robust activatable peptide display with AAV.…”

“…Specifically, the N-termini of VP1 and VP2 capsid subunits are initially hidden but then are externalized onto the capsid surface upon internalization into the cellular endosome. By creating a series of truncated VP2 capsid subunits, VNPs with variable abilities to perform activatable peptide display were generated ( Figure 6) (Thadani et al, 2017). Mosaic capsids with different ratios of activatable and wt subunits display a wide range of behaviors, with some VNPs displaying peptides in a "brush-like" configuration that is always externalized and others displaying peptides only upon activation.…”

“…The wild-type capsid undergoes the structural conformational change upon incubation at~60 C. Depending on the composition of the mutant AAV capsids, some capsids display his tags prior to activation (always "ON") whereas other capsids display activatable peptide display ("activatable"). (Reprinted with permission from Thadani et al (2017). Copyright 2017 American Chemical Society)…”