Relevance of Assembly-Activating Protein for Adeno-associated Virus Vector Production and Capsid Protein Stability in Mammalian and Insect Cells

Abstract

The discovery that adeno-associated virus 2 (AAV2) encodes an eighth protein, called assembly-activating protein (AAP), transformed our understanding of wild-type AAV biology. Concurrently, it raised questions about the role of AAP during production of recombinant vectors based on natural or molecularly engineered AAV capsids. Here, we show that AAP is indeed essential for generation of functional recombinant AAV2 vectors in both mammalian and insect cell-based vector production systems. Surprisingly, we observed that AAV2 capsid proteins VP1 to -3 are unstable in the absence of AAP2, likely due to rapid proteasomal degradation. Inhibition of the proteasome led to an increase of intracellular VP1 to -3 but neither triggered assembly of functional capsids nor promoted nuclear localization of the capsid proteins. Together, this underscores the crucial and unique role of AAP in the AAV life cycle, where it rapidly chaperones capsid assembly, thus preventing degradation of free capsid proteins. An expanded analysis comprising nine alternative AAV serotypes (1, 3 to 9, and rh10) showed that vector production always depends on the presence of AAP, with the exceptions of AAV4 and AAV5, which exhibited AAP-independent, albeit low-level, particle assembly. Interestingly, AAPs from all 10 serotypes could cross-complement AAP-depleted helper plasmids during vector production, despite there being distinct intracellular AAP localization patterns. These were most pronounced for AAP4 and AAP5, congruent with their inability to rescue an AAV2/AAP2 knockout. We conclude that AAP is key for assembly of genuine capsids from at least 10 different AAV serotypes, which has implications for vectors derived from wild-type or synthetic AAV capsids.IMPORTANCE Assembly of adeno-associated virus 2 (AAV2) is regulated by the assembly-activating protein (AAP), whose open reading frame overlaps with that of the viral capsid proteins. As the majority of evidence was obtained using virus-like particles composed solely of the major capsid protein VP3, AAP's role in and relevance for assembly of genuine AAV capsids have remained largely unclear. Thus, we established a trans-complementation assay permitting assessment of AAP functionality during production of recombinant vectors based on complete AAV capsids and derived from any serotype. We find that AAP is indeed a critical factor not only for AAV2, but also for generation of vectors derived from nine other AAV serotypes. Moreover, we identify a new role of AAP in maintaining capsid protein stability in mammalian and insect cells. Thereby, our study expands our current understanding

The Assembly-Activating Protein Promotes Stability and Interactions between AAV’s Viral Proteins to Nucleate Capsid Assembly

“…This is likely because capsids are assembled rapidly in the presence of AAP, and because assembled VP proteins are not susceptible to degradation, a VP band persists. Grosse et al (2017) performed a similar experiment and found that unassembled AAV2 VP proteins have a half-life of 2–3 hr in the absence of AAP and that VP proteins are rapidly assembled into capsids when AAP is present. Importantly, when AAP is present in these experiments, CHX treatment also results in loss of AAP protein, so it is difficult to determine whether AAP directly affects monomer stability or whether stability is solely a result of assembly into the stable icosahedron.…”

“…It is difficult to dissect the effect of AAP on the stability of monomeric VP proteins, considering how rapidly VPs are assembled into the stable icosahedron in its presence. Experiments assaying AAP’s effect on monomer turnover rates necessitates blocking translation of new VP proteins (Grosse et al, 2017), which, unfortunately, also blocks translation of AAP protein, resulting in AAP loss over time and an inability to draw conclusions regarding the effect of AAP on monomer stability. It is unclear whether AAP interacts directly or indirectly with VP proteins; VP proteins purified from Sf9 cells by chemical extraction methods showed increased oligomerization when HeLa cell extract was added, suggesting that host factors are required or at least increase the efficiency of oligomerization (Steinbach et al, 1997).…”

“…AAP of AAV2 (referred to here as AAP2) has been shown to shuttle VP3 proteins to the nucleolus (Earley et al, 2015), but recent findings demonstrate that AAV2 is unique in this respect, with no other AAV serotype demonstrating strict nucleolar assembly (Earley et al, 2017). To date, most studies of AAP’s requirement for particle assembly employ VP3-only particles, and studies of the full VP1, VP2, and VP3 capsid were only very recently published (Grosse et al, 2017). …”

Using a barcoded AAV capsid library to select for novel clinically relevant gene therapy vectors

“…A recent report by the Grimm group laid to rest the concern that functionality of AAV capsid libraries generated for directed evolution studies might be severely compromised by inactivation of the assembly activating protein (AAP) in a large proportion of the chimeric variant pool (Herrmann et al, 2018b). Although this small protein had previously been described to play an important role in virus assembly (Grosse et al, 2017;Naumer et al, 2012;Sonntag et al, 2011;Sonntag et al, 2010), it appears that AAV is strikingly tolerant towards recombination within the AAP coding sequence.…”

Residues on Adeno-associated Virus Capsid Lumen Dictate Interactions and Compatibility with the Assembly-Activating Protein

“…Universally, AAP is required to achieve maximal production titers, but recent studies have shown that the absolute requirement for AAP across AAV variants ranges broadly. Some serotypes, such as AAV4 and AAV5, can assemble appreciable capsid quantities in the absence of any AAP, and many serotypes, such as AAV2 and AAV8, require the near-full-length AAP protein, but several serotypes only need the N-terminal 60 amino acids of AAP (AAPN) to assemble capsids, including AAV3 and AAV9 (15)(16)(17). A set of 9 putative ancestral capsid variants (AncAAVs), inferred by ancestral sequence reconstruction (18), show a similar range in requirement for AAP, as well as a range in functionality of their cognate AAP, although preserving the AAP reading frame was not taken into account in the design of these variants.…”