Functional orthogonality of parvoviral phospholipase A ₂ domains in adeno-associated virus transduction

Dependoparvoviruses, which belong to the family <i>Parvoviridae,</i> are being developed as viral vectors for gene transfer. Notably, different adeno-associated viral (AAV) serotype capsids have been utilized to generate pseudotyped recombinant vectors. While capsid surface regions mediate host cell interactions, buried structural domains have been implicated in parvoviral infectivity and post-entry trafficking. In this regard, the functional diversity of highly conserved group XIII phospholipase A₂ domains (PLA₂) located within the N-terminal capsid domain of different parvoviruses is of particular interest. Here, we developed a massively parallel screen to evaluate a diverse panel of rationally engineered and naturally derived parvoviral PLA₂ domains incorporated within the human isolate, AAV9. <i>In vitro</i> infectious cycling of chimeric virions revealed a functional bias toward parvoviral PLA₂ domains of mammalian and avian origin and decreased preference for PLA₂ domains of insect, ungulate, or metagenomic origin. Notably, wild-type chimeric AAV9 virions carrying avian dependoparvovirus PLA₂ domains demonstrate increased replication over other chimeras. The best-performing recombinant avian/human origin chimera (UNY47950.1/AAV9) shows improved transduction with both single-stranded and self-complementary vector genomes. This observation is accompanied by improved cytoplasmic uptake and nuclear entry of chimeric virions compared to parental AAV9, as evidenced by subcellular fractionation and confocal microscopy. Overall, this study highlights the functional orthogonality of distinct parvoviral PLA₂ domains incorporated into AAV capsids. These chimeric virions present an opportunity to gain deeper insight into the infectious biology of parvoviruses and potentially enable new approaches to improve post-entry trafficking of AAV vectors for gene transfer applications.IMPORTANCEThis study explores the functional overlap of phospholipase domains located within the capsid lumen across the parvovirus family. The findings provide insights into parvovirus-host interactions across different genera within the context of this highly conserved capsid region and underscore its essential role in viral trafficking to the nucleus. Furthermore, incorporation of orthogonal phospholipase domains derived from diverse parvoviral family members may expand the recombinant vector toolkit of adeno-associated viruses for gene transfer applications.