Evolution of dependoparvoviruses across geological timescales—implications for design of AAV-based gene therapy vectors

Abstract

Endogenous viral elements (EVEs) are genetic remnants of viruses that have integrated into host genomes millions of years ago and retained as heritable elements passed on to offspring until present-day. As a result, EVEs provide an opportunity to analyze the genomes of extinct viruses utilizing these genomic viral fossils to study evolution of viruses over large timescales. Analysis of sequences from near full-length EVEs of dependoparvoviral origin identified within three mammalian taxa, Whippomorpha (whales and hippos), Vespertillionade (smooth-nosed bats), and Lagomorpha (rabbits, hares, and pikas), indicates that distinct ancestral dependoparvovirus species integrated into these host genomes approximately 77-23 million years ago (MYA). These ancestral viruses are unique relative to modern adeno-associated viruses (AAVs), and distinct from extant species of genus Dependoparvovirus. These EVE sequences show characteristics previously unseen in modern, mammalian AAVs, but instead appear more similar to the more primitive, autonomously-replicating and pathogenic waterfowl dependoparvoviruses. Phylogeny reconstruction suggests that the whippomorph EVE orthologue derives from exogenous ancestors of autonomous and highly pathogenic dependoparvovirus lineages, believed to have uniquely co-evolved with waterfowl birds to present date. In contrast, ancestors of the two other mammalian orthologues (Lagomorpha and Vespertilionidae) likely shared the same lineage as all other known mammalian exogenous AAVs. Comparative in silico analysis of the EVE genomes revealed remarkable overall conservation of AAV rep and cap genes, despite millions of years of integration within the host germline. Modeling these proteins identified unexpected variety, even between orthologues, in previously defined capsid viral protein (VP) variable regions (VRs), especially in those related to the threefold and fivefold symmetry axes of the capsid. Moreover, the normally well-conserved phospholipase A2 (PLA2) domain of the predicted minor VP1 also exhibited a high degree of sequence variance. These findings may indicate unique biological properties for these virus “fossils” relative to extant dependoparvoviruses and suggest key regions to explore within capsid sequences that may confer novel properties for engineered gene therapy vectors based upon paleovirology data.

Optimization of AAV vectors to target persistent viral reservoirs

“…Reconstructed ancestral AAV capsids generated via this approach appear to be more thermostable and are highly potent for several different cell and tissue types [ 93 , 94 ]. In a similar approach, germline endogenous viral elements (EVEs) from the Dependoparvovirus lineage have also been identified in a number of mammals, birds and marsupials [ 95 – 99 ]. These EVEs are now being used to guide the rational design of novel AAV vectors based on their distinct structural elements that could provide unique vector properties or tropisms [ 99 ].…”

“…In a similar approach, germline endogenous viral elements (EVEs) from the Dependoparvovirus lineage have also been identified in a number of mammals, birds and marsupials [ 95 – 99 ]. These EVEs are now being used to guide the rational design of novel AAV vectors based on their distinct structural elements that could provide unique vector properties or tropisms [ 99 ]. As we continue to learn more about AAV and how it interacts with cells and tissues, new rational design strategies will be developed that enable us to discover improved vectors for different therapeutic applications.…”

Adeno-Associated Virus (AAV) Gene Delivery: Dissecting Molecular Interactions upon Cell Entry

“…Viral genome integration occasionally occurs in animal germ cells, and these ancient insertion events are ubiquitous in extant animal genomes. The insertion events are referred to as “EVEs” (EVEs: Endogenous Viral Elements), and ancient AAV insertions are AAV-EVEs [ 78 ]. Comparisons of AAV-EVEs from sequenced mammalian host genomes provide estimates of divergence times based on the last common ancestors of EVE hosts.…”

“…Comparisons of AAV-EVEs from sequenced mammalian host genomes provide estimates of divergence times based on the last common ancestors of EVE hosts. Current estimates suggest AAV integrated into mammalian genomes 23–77 millions of years ago (MYA) [ 78 ].…”

“…Capsid differences are observed between extant primate AAVs and ancient mammalian AAV-EVEs [ 78 ]. Some of the differences are found in the mammalian AAV-EVE VP1u domain.…”