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

Abstract

The adeno-associated virus (AAV) serves as a broadly used vector system for in vivo gene delivery. The process of AAV capsid assembly remains poorly understood. The viral cofactor assembly-activating protein (AAP) is required for maximum AAV production and has multiple roles in capsid assembly, namely, trafficking of the structural proteins (VP) to the nuclear site of assembly, promoting the stability of VP against multiple degradation pathways, and facilitating stable interactions between VP monomers. The N-terminal 60 amino acids of AAP (AAPN) are essential for these functions. Presumably, AAP must physically interact with VP to execute its multiple functions, but the molecular nature of the AAP-VP interaction is not well understood. Here, we query how structurally related AAVs functionally engage AAP from AAV serotype 2 (AAP2) toward virion assembly. These studies led to the identification of key residues on the lumenal capsid surface that are important for AAP-VP and for VP-VP interactions. Replacing a cluster of glutamic acid residues with a glutamine-rich motif on the conserved VP beta-barrel structure of variants incompatible with AAP2 creates a gain-of-function mutant compatible with AAP2. Conversely, mutating positively charged residues within the hydrophobic region of AAP2 and conserved core domains within AAPN creates a gain-of-function AAP2 mutant that rescues assembly of the incompatible variant. Our results suggest a model for capsid assembly where surface charge/neutrality dictates an interaction between AAPN and the lumenal VP surface to nucleate capsid assembly. IMPORTANCE Efforts to engineer the AAV capsid to gain desirable properties for gene therapy (e.g., tropism, reduced immunogenicity, and higher potency) require that capsid modifications do not affect particle assembly. The relationship between VP and the cofactor that facilitates its assembly, AAP, is central to both assembly preservation and vector production. Understanding the requirements for this compatibility can inform manufacturing strategies to maximize production and reduce costs. Additionally, library-based approaches that simultaneously examine a large number of capsid variants would benefit from a universally functional AAP, which could hedge against overlooking variants with potentially valuable phenotypes that were lost during vector library production due to incompatibility with the cognate AAP. Studying interactions between the structural and nonstructural components of AAV enhances our fundamental knowledge of capsid assembly mechanisms and the protein-protein interactions required for productive assembly of the icosahedral capsid.

Completion of the AAV Structural Atlas: Serotype Capsid Structures Reveals Clade-Specific Features

“…An analysis of residues shared between AAV4 and AAV11 but not with AAV12 revealed a total of 23 aa. Previous studies suggested that interior residues are involved in the AAP function [ 74 , 75 ]. Only four of 23 aa differences are located in the interior of the capsid, A301S, A338T, I619V, and R688H (first aa type = AAV4/11, second aa type = AAV12).…”

The Carboxyl Terminus of the Porcine Circovirus Type 2 Capsid Protein Is Critical to Virus-Like Particle Assembly, Cell Entry, and Propagation

“…Two charged residues ( 227 KD 228 ) were also conserved among all the strains of PCV1 and PCV2. In general, strictly conserved residues in viral structure proteins, particularly when exposed on the surface of the virus, may play important roles in the life cycle of the virus, such as virus-host interactions, virion assembly, cell entry, and/or egress from host cells (45,46). Second, the 3D structure from X-ray crystallography (4) of PCV2 VLPs demonstrated that the CT (red) is located adjacent to the edge of the 5-fold axes of the capsid, which was decorated by loop BC (blue) (Fig.…”

Establishment of a Recombinant AAV2/HBoV1 Vector Production System in Insect Cells

“…Infection of BEV-Rep2Cap2, which was made following the Kotin strategy of Bac-AAV2Rep-Cap [56], expressed AAP in Sf9 cells, and knockout of the AAP decreased rAAV2 yield by 10 times [55]. This suggested that the AAP plays an important role in rAAV vector production in Sf9, which is likely through facilitation of the assembly of AAV capsids [22][23][24]. We currently do not know whether HBoV1 cap also expresses an AAP-like protein that may facilitate the assembly of the HBoV1 capsid, which warrants further investigation.…”

“…Three AAV Cap proteins, VP1, VP2 and VP3, are expressed from the mRNA transcribed by the P40 promoter [2,19]. In addition, a small NS protein, assembly-associated protein (AAP), is alternatively translated from the P40-transcribed Cap-coding mRNA [20], which plays a role in capsid assembly [21][22][23][24]. Recently, another small NS protein, membrane-associated accessory protein (MAAP), has been identified, which is also expressed from the P40-transcribed mRNA through alternative translation [25].…”