Despite tight genetic compression, viral genomes are often organized into functional gene clusters, a modular structure that might favor their evolvability. This has greatly facilitated biotechnological developments such as the recombinant adeno-associated virus (AAV) systems for gene therapy. Following this lead, we endeavored to engineer the related insect parvovirus Junonia coenia densovirus (JcDV) to create addressable vectors for insect pest biocontrol. To enable safer manipulation of capsid mutants, we translocated the nonstructural (ns) gene cluster outside the viral genome. To our dismay, this yielded a virtually nonreplicable clone. We linked the replication defect to an unexpected modularity breach, as ns translocation truncated the overlapping 3' untranslated region (UTR) of the capsid transcript (vp). We found that the native vp 3' UTR is necessary for high-level VP production but that decreased expression does not adversely impact the expression of NS proteins, which are known replication effectors. As nonsense vp mutations recapitulate the replication defect, VP proteins appear to be directly implicated in the replication process. Our findings suggest intricate replication-encapsidation couplings that favor the maintenance of genetic integrity. We discuss possible connections with an intriguing cis-packaging phenomenon previously observed in parvoviruses whereby capsids preferentially package the genome from which they were expressed. IMPORTANCE Densoviruses could be used as biological control agents to manage insect pests. Such applications require an in-depth biological understanding and associated molecular tools. However, the genomes of these viruses remain difficult to manipulate due to poorly tractable secondary structures at their extremities. We devised a construction strategy that enables precise and efficient molecular modifications. Using this approach, we endeavored to create a split clone of Junonia coenia densovirus (JcDV) that can be used to safely study the impact of capsid mutations on host specificity. Our original construct proved to be nonfunctional. Fixing this defect led us to uncover that capsid proteins and their correct expression are essential for continued rolling-hairpin replication. This points to an intriguing link between replication and packaging, which might be shared with related viruses. This serendipitous discovery illustrates the power of synthetic biology approaches to advance our knowledge of biological systems.
Keywords: capsid; densovirus; replication; synthetic biology.