Smallpox has been for centuries a major disease before it was eradicated in 1979. Still the risks of its use in bioterrorism and a reintroduction of related poxviruses from animal reservoirs persist. Poxviruses are unique in terms of their cytoplasmic replication, which relies fully on virally encoded proteins. Their genome forms a linear double-stranded DNA, which is circularized at its extremities by hairpin structures. Such genomic organization is also found in some phycodnaviruses and afsarviruses. All these viruses also share the same type of DNA polymerase, helicase-primase, and some other specificities. They have been grouped in the Nucleo-Cytoplasmic Large DNA Viruses (NCLDV) clade. In the beginning of the 80s, several models of poxvirus replication have been proposed based on nick formation, then unidirectional strand elongation followed by strand rearrangement. But the identification of a primase and the first results on the 3D structure of the replication complex obtained recently by our teams challenge these established models. We showed that the helicase-primase D5 is hexameric as other SF3 helicases. This finding suggests rather a mechanism involving a replication fork. We propose to push further the structural and functional investigations of the replication machinery involving, on one hand, the complex formed by the DNA polymerase E9, the uracil-DNA glycosylase D4, and the processivity factor A20, and on the other hand, the hexameric helicase-primase D5. Using recombinant expression of proteins from vaccinia virus, which is a safe model system 97 % identical to smallpox virus, we are able to produce milligram amounts of the four components in insect cells. Some constructs of D4 and A20 have also been expressed in bacteria. Protein crystallography and electron microscopy will be used to continue our structural work in order to determine the details of the molecular interactions within these complexes. A reconstitution of the replication machinery in vitro is also in reach and will greatly facilitate our functional studies. As we revealed recently the considerable size of the replication complex, we would like to revisit the role of D4 and the primase activity of D5 using this in vitro model. The helicase activity of D5 has still to be shown experimentally and may require the identification and inclusion of additional partner proteins. We are confident that a breakthrough in the understanding of poxvirus replication is in reach, with strong implications for other viruses within the NCLDV clade and a potential application in the design of antivirals, in particular of compounds targeting the interaction surfaces within the complex.