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). Large quantities of DNA-containing IV virions are produced in ovary calyx cells during the pupal and adult stages of female wasps. Females parasitize host insects by injecting eggs and virions into the body cavity. After injection, virions rapidly infect host cells which is followed by expression of IV genes that promote the successful development of wasp offspring. IV genomes consist of two components: proviral segment loci that serve as templates for circular dsDNAs that are packaged into capsids, and genes from an ancestral virus that produce virions. In this study, we generated a chromosome-scale genome assembly for Hyposotor didymator that harbors H. didymator ichnovirus (HdIV). We identified a total of 67 HdIV loci that are amplified in calyx cells during the wasp pupal stage. We then focused on an HdIV gene, U16, which is transcribed in calyx cells during the initial stages of replication. Sequence analysis indicated that U16 contains a conserved domain in primases from select other viruses. Knockdown of U16 by RNA interference inhibited virion morphogenesis in calyx cells. Genome-wide analysis indicated U16 knockdown also inhibited amplification of HdIV loci in calyx cells. Altogether, our results identified several previously unknown HdIV loci, demonstrated that all HdIV loci are amplified in calyx cells during the pupal stage, and showed that U16 is required for amplification and virion morphogenesis. Author summary: Parasitoid "domesticated endogenous viruses" (DEVs) provide a fascinating example of eukaryotes acquiring new functions through integration of a viral genome. DEVs consist of multiple loci in the genomes of wasps. Upon activation, these elements collectively orchestrate the production of virions or virus-like particles that are crucial for successful parasitism of host insects. Despite the significance of DEVs for parasitoid biology, the mechanisms regulating key steps in virion morphogenesis are largely unknown. In this study, we focused on the ichneumonid parasitoid Hyposoter didymator, which harbors an ichnovirus consisting of 67 proviral loci. Our findings reveal that all proviral loci are simultaneously amplified in ovary calyx cells of female wasps during the pupal stage suggesting the hijacking of cellular replication complexes by viral proteins. We functionally studied the ichnovirus gene U16, which encodes a protein with a weakly conserved primase C-terminal domain. Silencing U16 inhibited viral DNA amplification and virion production, underscoring the key role of this gene for ichnovirus replication. Our study provides evidence that many genes involved in viral DNA replication have been conserved during the domestication of ichnoviruses. [ABSTRACT FROM AUTHOR]