The assembly of replication factors into functional complexes is crucial for the initiation of viral genome replication and processing of nascent viral DNA. Binding to viral DNA and interaction of protein domains presumably guide compartmentalization of replication factors. The phase separation due to hydrophilicity and hydrophobicity of components may also contribute to the assembling process. However, phase separation effects are poorly investigated in the infection cycle of baculoviruses, large DNA viruses infecting Diptera, Hymenoptera, and Lepidoptera insects. Herein, we describe an investigation on a possible role of phase separation in the assembly of nuclear replication factories in Spodoptera frugiperda Sf9 cells infected with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The inhibitory effect of 1,6-Hexanediol on the translocation of a viral DNA binding protein (DBP) to the replicative centers has revealed the involvement of liquid phases separation in the assembly of these centers. DBP is a structural component of the virogenic stroma, a sub-nuclear membrane-less compartment involved in viral DNA replication and the production of nucleocapsids. This sub-nuclear structure is presumably assembled via a biomolecular condensation mechanism.
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