Identification of a viral gene essential for the genome replication of a domesticated endogenous virus in ichneumonid parasitoid wasps

Thousands of endoparasitoid wasp species in the families Braconidae and Ichneumonidae harbor "domesticated endogenous viruses" (DEVs) in their genomes. This study focuses on ichneumonid DEVs, named ichnoviruses (IVs), which derive from an unknown virus and produce virions in ovary calyx cells during the pupal and adult stages of female wasps. Females inject IV virions into host insects when laying eggs. Virions infect cells which express IV genes with functions required for wasp progeny development. IVs have a dispersed genome consisting of two genetic components: proviral segment loci that serve as templates for circular dsDNAs that are packaged into capsids, and genes from an ancestral virus controlling virion production. Because of the lack of homology with known viral genes, the molecular control mechanisms of IV genome are largely uncharacterized. We generated a chromosome-scale genome assembly for Hyposoter didymator and identified a total of 67 H. didymator ichnovirus (HdIV) loci distributed across the 12 wasp chromosomes. By analyzing genomic DNA levels, we found that all HdIV loci were locally amplified in calyx cells during the wasp pupal stage, suggesting the implication of viral proteins in DNA replication. We tested a candidate HdIV gene, U16, encoding a protein with a conserved domain found in primases and which is transcribed in calyx cells during the initial stages of replication. Knockdown of U16 by RNA interference inhibited amplification of all HdIV loci, as well as HdIV gene transcription, circular molecule production and virion morphogenesis in calyx cells. Altogether, our results showed that viral DNA amplification is an early step of IV replication essential for virions production, and demonstrated the implication of the viral gene U16 in this process.