Adeno-associated Virus (AAV) Assembly-Activating Protein Is Not an Essential Requirement for Capsid Assembly of AAV Serotypes 4, 5, and 11

Abstract

Adeno-associated virus (AAV) vectors have made great progress in their use for gene therapy; however, fundamental aspects of AAV's capsid assembly remain poorly characterized. In this regard, the discovery of assembly-activating protein (AAP) sheds new light on this crucial part of AAV biology and vector production. Previous studies have shown that AAP is essential for assembly; however, how its mechanistic roles in assembly might differ among AAV serotypes remains uncharacterized. Here, we show that biological properties of AAPs and capsid assembly processes are surprisingly distinct among AAV serotypes 1 to 12. In the study, we investigated subcellular localizations and assembly-promoting functions of AAP1 to -12 (i.e., AAPs derived from AAV1 to -12, respectively) and examined the AAP dependence of capsid assembly processes of these 12 serotypes using combinatorial approaches that involved immunofluorescence and transmission electron microscopy, barcode-Seq (i. e., a high-throughput quantitative method using DNA barcodes and a next-generation sequencing technology), and quantitative dot blot assays. This study revealed that AAP1 to -12 are all localized in the nucleus with serotypespecific differential patterns of nucleolar association; AAPs and assembled capsids do not necessarily colocalize; AAPs are promiscuous in promoting capsid assembly of other serotypes, with the exception of AAP4, -5, -11, and -12; assembled AAV5, -8, and -9 capsids are excluded from the nucleolus, in contrast to the nucleolar enrichment of assembled AAV2 capsids; and, surprisingly, AAV4, -5, and -11 capsids are not dependent on AAP for assembly. These observations highlight the serotypedependent heterogeneity of the capsid assembly process and challenge current notions about the role of AAP and the nucleolus in capsid assembly.IMPORTANCE Assembly-activating protein (AAP) is a recently discovered adenoassociated virus (AAV) protein that promotes capsid assembly and provides new opportunities for research in assembly. Previous studies on AAV serotype 2 (AAV2) showed that assembly takes place in the nucleolus and is dependent on AAP and that capsids colocalize with AAP in the nucleolus during the assembly process. However, through the investigation of 12 different AAV serotypes (AAV1 to -12), we find that AAP is not an essential requirement for capsid assembly of AAV4, -5, and -11, and AAP, assembled capsids, and the nucleolus do not colocalize for all the serotypes. In addition, we find that there are both serotype-restricted and serotypepromiscuous AAPs in their assembly roles. These findings challenge widely held beliefs about the importance of the nucleolus and AAP in AAV assembly and show the heterogeneous nature of the assembly process within the AAV family.

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

“…1C, compare first two data points). This suggested a critical role of AAP in AAV vector production, congruent with and extending previous data with VP3-only capsids (17)(18)(19)(20)(21).…”

“…5A). Interestingly, we noted distinct sizes of the AAP variants despite their similar lengths (Data Set S1) as well as a double band for AAP8 and AAPrh10 (plus a faint lower band for AAP5), akin to the observation by Earley and colleagues for AAP10 (21).…”

“…Previous work has almost exclusively studied the role of AAP in AAV capsid assembly in the context of virus-like particles that were composed of merely the major capsid protein VP3 (17)(18)(19)(20)(21). In contrast, the central aim of the present work was to analyze AAP biology in mammalian and insect cells during production of recombinant AAV vectors based on complete viral capsids comprising all three capsid proteins, VP1 to VP3.…”

“…In the same study, AAP2 was also used to rescue capsid assembly from serotypes 1, 2, 5, 8, and 9. More recently, the Nakai group investigated the ability of AAP from AAV1 to AAV12 to support assembly of AAV serotypes 1 to 12 (21). Importantly, in nearly all of these prior experiments, virus-like particles were produced with VP3 alone, leaving the role of the different AAP variants in formation of genuine AAV capsids and functional vectors of multiple serotypes largely unclear.…”

“…Still, it also remains possible that a key function of AAP is promotion of nucleolar VP transport to facilitate interaction with nucleolar proteins which in turn chaperone AAV capsid assembly. Arguing against this, at least as an exclusive mechanism, is that assembly of AAV serotypes other than AAV2 can occur outside the nucleolus and that AAP-independent targeting of AAV2 VP3 to the nucleolus via appropriate peptides did not foster AAV2 capsid assembly (17,21).…”