Cryo-electron microscopy structure of the poxvirus DNA replication machinery – PoxRep

We propose to elucidate structure and function of the poxvirus DNA replication machinery by single particle cryo-electron microscopy (cryo-EM). The poxvirus family comprises members with a strong potential of spillover from the animal kingdom, such as cowpox and monkeypox, which leads to local epidemic outbreaks in Africa with a fatality rate of about 2 %. Furthermore poxviruses may be employed in the context of bioterrorism. They replicate without the requirement of host cell protein synthesis in cytoplasmic viral factories. The structure of the large DNA genome is only shared with asfarviruses and consists of a double-stranded DNA with circularized ends. There is no consensus model for poxvirus DNA replication. The DNA replication of vaccinia virus, a safe model system, involves the DNA polymerase holoenzyme complex, built from the polymerase E9, a structural protein A20 and an uracil-DNA glycosylase D4, which form the processivity factor. Furthermore it requires the helicase-primase D5, the ssDNA-binding protein I3 and the DNA ligase A50. We propose to determine structures of the E9-A20-D4 DNA polymerase holoenzyme in complex with different DNA substrates mimicking different steps in polymerase action and to approach in the same way the helicase-primase D5 in order to reconstitute components of the replication fork. In a second stage, the analysis may include the direct interaction between E9-A20-D4 and D5 or further proteins involved in DNA replication. The dynamic structure of the complexes with DNA precluded so far structure determination by crystallography, but with the classification techniques of cryo-EM these structures are now within reach. We can build on our X-ray structure determination and biophysical characterisations of several of the central partners, existing expression systems and our preliminary results on the cryo-EM of DNA-free forms of the complexes.