ABSTRACT: Cotesia vestalis bracoviruses (CvBVs) are domesticated endogenous viruses (DEVs) derived from ancestral nudiviruses and are integrated into the genome of the parasitoid wasp C. vestalis. The CvBV proviral genome is composed of two distinct components: one encoding genes associated with virion morphogenesis and assembly, and the other harboring virulence genes that are excised, circularized, and packaged into virions. CvBV replication and particle assembly occur exclusively in the ovaries of female wasps. While prior studies have largely focused on the function of virulence genes during parasitization, the molecular mechanisms underlying CvBV replication and assembly have remained largely unknown. In this study, we employed an integrative approach to dissect the CvBV gene set responsible for these processes. We systematically identified 71 conserved nudivirus-like genes in the C. vestalis genome and functionally characterized 21 of them. Among these, three genes involved in transcriptional regulation (p47, lef-5, lef-9) were highlighted. Notably, we discovered three previously unrecognized promoter motifs upstream of CvBV structural genes, revealing novel lineage-specific regulatory elements that may coordinate temporal gene expression via the viral RNA polymerase complex. Additionally, we characterized three CvBV replication-related genes (helicase, integrase-1/-2), eight capsid-related genes (vp39, PmV, HzNVorf9-1, HzNVorf9-2, HzNVorf106, 38k, 27b, K425_459), five genes associated with envelope formation (11k, 17a-1, 35a-1, 35a-2, K425_461), three genes required for virion assembly (vlf-1, HzNVorf140-1, HzNVorf140-2), and two viral infectivity factors (vp91, pif-0). Together, these findings provide the first comprehensive view of the key regulators controlling CvBV production, assembly, and infectivity, offering novel insights into the molecular mechanisms underlying bracovirus biogenesis and the evolutionary divergence of CvBVs from their nudiviral ancestors.
