The aim of this project is two-fold: To establish clearly the poxvirus replication mechanism and to provide high-resolution structures of the key players in this process: the DNA polymerase holoenzyme and the helicase-primase. This work will give insight into the poxvirus DNA replication, which shows a different organisation than previously described ones. On the long run the structural information will provide a breakthrough in the structure-based design of new drugs against orthopoxviruses.
Variola virus (family: Poxviridae, genus: Orthopoxvirus), was responsible of smallpox, the most lethal and devastating viral infection in history. After an intense campaign of vaccination led by the World Health Organization, smallpox was declared eradicated in 1979. Shortly after the vaccination program was stopped which results currently in a high proportion of non-immune people worldwide. Therefore, if used as a biological weapon, variola virus could represent a serious threat to the civilian population since no specific antiviral treatment is yet available against orthopoxvirus infections. Furthermore there is a considerable risk of zoonoses or the reintroduction from an animal reservoir. <br />Since the terrorist attacks of September 2001 the French Armed Biomedical Research Institute (IRBA) has made important efforts to develop new countermeasures against variola virus. One line of research done in a collaborative way between the IRBA and the “Integrated biology of persistent viruses” group at the UVHCI is focusing on the structural study of essential proteins involved in poxvirus DNA synthesis. Ultimately this work will facilitate the design of new drugs targeting the DNA replication machinery.
Working on the safe model system vaccinia virus which was used in vaccination, we study the heart of DNA replication formed by the polymerase holoenzyme consisting of the polymerase E9, the processivity factor composed of the uracil-DNA glycosylase D4 and a bridging protein A20 on one hand and the hexameric helicase-primase D5 on the other hand. These proteins are more than 95 % identical to their smallpox virus counterparts. We have set up the expression of D5, D4, A20 and E9 in the insect cell - baculovirus system whereas fragments of these proteins are expressed in bacteria. Combining biophysical methods (such as crystallography, electron microscopy, small-angle x-ray scattering, multi-angle laser light scattering) in a multi-scale approach we progress towards the structure. Other techniques are used in order to understand interactions and functions of the machinery such as surface plasmon resonance and fluorescence anisotropy.
Main advances concerned the structure of the polymerase holoenzyme and the organization of the helicase-primase D5. We expressed and purified the complex formed by D4 and the first 50 amino acids of A20 (D4/A20(1-50)). Whereas D4 forms homodimers in solution when expressed alone, D4/A20(1-50) clearly behaves as a heterodimer. Interestingly, small molecule docking with anti-poxvirus inhibitors selected to interfere with D4/A20 binding could reproduce several key features of the D4/A20(1-50) interaction. Overall, our data give new insights into the assembly of the poxvirus DNA polymerase processivity factor and may be useful for the design and rational improvement of antivirals targeting the D4/A20 interface. We also obtained the structure of uracil-DNA glycosylase D4/A20(1-50) bound to a 10-mer DNA duplex containing an abasic site resulting from the cleavage of a uracil base. Comparison with human uracil-DNA glycosylase revealed major divergences in the contacts between protein and DNA and in the enzyme orientation on the DNA. However, the conformation of the dsDNA within both structures is very similar. In contrast to human UNG, D4 is rigid and does not adopt an open to close conformational change upon DNA binding. This second DNA complex structure of a family I UNG gives new insight into the role of D4 as a co-factor of vaccinia virus DNA polymerase and allows a better understanding of the structural determinants required for UNG action. We obtained first insights into the overall structure and function of the essential vaccinia virus helicase-primase D5 and showed that the active helicase domain of D5 builds a hexameric ring structure. This hexamer has nucleoside hydrolase activity, independent of the nature of the nucleoside and binds strongly to single- and double stranded DNA. The organization of helicase and primase domains differs from known helicase-primase proteins.
Future work will concentrate on the high-resolution structure of the polymerase subunit E9, its interfaces with A20. This will create a wider base of structural knowledge for the design of anti-poxvirus drugs. Another aim is to push the structure of the helicase-primase D5 to higher resolution, helped by the understanding of its domain organization which we obtained. Further studies will be carried out in order to understand loading and activation of the helicase.
Contesto-Richefeu, C., Tarbouriech, N., Brazzolotto, X., Betzi, S., Morelli, X., Burmeister, W. P., & Iseni, F. Crystal structure of the Vaccinia virus DNA polymerase holoenzyme subunit D4 in complex with the A20 N-terminus domain. PLoS Pathogens 10, e1003978 (2014).
Burmeister, W.P., Tarbouriech, N., Fender, P., Contesto-Richefeu, C., Peyrefitte, C.N., Iseni & F. Crystal structure of the vaccinia virus uracil DNA-glycosylase in complex with DNA. J Biol. Chem. pii: jbc.M115.648352. (2015).
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.
Monsieur Wim Burmeister (Unit of Virus Host Cell Interactions)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
UMI 3265 UJF-EMBL-CNRS Unit of Virus Host Cell Interactions
CEA/DSV/IBS Commissariat a l'energie atomique et aux energies alternatives
IRBA Institut de Recherche Biomédical des Armées
Help of the ANR 350,000 euros
Beginning and duration of the scientific project: December 2012 - 42 Months