The long-term goal of this proposal is to understand the molecular mechanisms of the replication cycle ofnucleocytoplasmic large DNA viruses that attracted attention after the discovery of the giant Mimivirus adecade ago. Here, we focus on ranaviruses from the family Iridoviridae that have caused large economiclosses in Europe and Asia and threaten wildlife biodiversity worldwide. The ranavirus disease has thereforebeen listed by the World Organization for Animal Health. Ranaviruses have a unique replication cycle thatleads to two coexisting forms of infectious particles, naked capsids and enveloped virions, each withdifferent mechanisms of cell egress and cell entry. Additionally, the proteinaceous capsid contains aninternal membrane that presumably assists to assembly of a large DNA-free icosahedral shell. This shellis subsequently filled by the viral genome and transforms into the mature capsid by a headful packaging mechanism that is also used by some dsDNA bacteriophages. We aim to characterize the structural and mechanistic features of ranavirusassembly, maturation and cell entry by combination of cryo-electron microscopy and tomography with othercomplementary techniques. The novelty in our plan is application of cryo-FIB micromachining and cryo-electrontomography to visualize the intracellular steps of the ranavirus replication cycle in 3D at nativeconditions. The three objectives of this research project are to: (i) identify the structural basis of subunitrecognition that guides assembly of large icosahedral capsids of type ranaviruses and elucidate the functionof the internal membrane in this process, (ii) determine the conformational changes that accompanymaturation of ranavirus capsids and regulate the headful packaging of the dsDNA genome, and (iii) ascertainthe mechanisms of cell entry by the naked capsids and enveloped ranavirus virions.