Herpes Simplex Virus Co-Infection Facilitates Rolling Circle Replication of the Adeno-Associated Virus Genome

Abstract

Adeno-associated virus (AAV) genome replication only occurs in the presence of a co-infecting helper virus such as adenovirus type 5 (AdV5) or herpes simplex virus type 1 (HSV-1). AdV5-supported replication of the AAV genome has been described to occur in a strand-displacement rolling hairpin mechanism initiated at the AAV 3’ inverted terminal repeat (ITR) end. It has been assumed that the same mechanism applies to HSV-1-supported AAV genome replication. We demonstrate the formation of double-stranded head-to-tail concatemers of AAV genomes in presence of HSV-1, and thus provide evidence for an unequivocal rolling circle amplification (RCA) mechanism. This study reveals the ability of AAV to modify the canonical rolling hairpin replication mechanism and to mimic the replication strategy of a co-infecting herpesvirus. This stands in contrast to the textbook model of AAV genome replication when HSV-1 is the helper virus. Furthermore, we introduce nanopore sequencing as a novel, high-throughput approach to study viral genome replication in unprecedented detail.