Introduction
One of the great successes of modern medicine was the eradication of the smallpox virus declared in 1979 after a long vaccination campaign with the prototype poxvirus, vaccinia virus (VACV), which is also a safe model system. Yet the poxvirus family includes members with a high potential for spread from the animal kingdom, where monkeypox and cowpox viruses present the main risk. In the early summer of 2022, this fear became reality with a worldwide outbreak of monkeypox, transmitted mainly through sex between men. Previously, monkeypox has resulted in local epidemics in the Democratic Republic of Congo and West Africa with about 5,000 cases per year and a mortality rate of about 2%. Even with the current decline in the number of cases, a future evolution of the virus towards more efficient human transmission cannot be excluded once the virus has been introduced into the human population.
It is therefore essential to prepare for poxvirus infections by having a range of antivirals available, but to date there are only two molecules, brincidofovir (which proved too toxic during the current epidemic) and tecovirimat. A better knowledge of the structure of poxvirus proteins in general and of the DNA polymerase in particular will make it possible to develop new compounds and to better understand the mechanisms of resistance to current molecules.
Our project
We are working on vaccinia virus, which is 98% identical to smallpox virus and monkeypox virus at the amino acid level of the essential DNA replication proteins : the helicase-primase D5, the DNA polymerase holoenzyme built from of the catalytic subunit E9 and the processivity factor composed of the accessory protein A20 and uracil N-glycosylase D4 [1]. In recent years, we have obtained information on the three-dimensional structure of these proteins and their interfaces at increasing resolution culminating in the X-ray structure of the E9 polymerase [2] and more recently in a structure of the A20-E9 interface obtained by structural nuclear magnetic resonance (NMR) [3].
The detailed knowledge of the interfaces of the different subunits of the polymerase can be used for the design of inhibitors that interfere with the assembly of the protein complex.
Figure 1 : Model of the E9-A20-D4 polymerase holoenzyme. The maximal dimension has been obtained by SAXS.. The high-resolution structures obtained by our team have been positioned. The DNA bound to the polymerase has been modeled whereas the DNA bound to A20(rés. 1-50)-D4 [4] is part of the crystal structure of a A20(rés. 1-50)-D4-dsDNA complex [5].
Due to its dynamics and flexibility, the high-resolution structure of the D5 helicase-primase remained inaccessible for a long time. We have obtained low information on the structure and domain organisation in an N-terminal primase domain and a C-terminal helicase domain [6]. D5 has been extensively studied by small angle X-ray scattering (SAXS), which has also led to new methodological developments in the combination of SAXS and column chromatography. With the rapid evolution of cryo-electron microscopy and aided by structure prediction by Alphafold2, we obtained the 4.1 Å cryo-EM structure of the helicase fragment in complex with DNA [7]. It revealed the architecture of a whole class of hexameric viral helicases.
Figure 2 : Reconstruction of the hexameric D5 helicase domain with bound double-stranded DNA shown in green. The structural domains are the collar domain in yellow, the AAA+ helicase domain in orange and the C-terminal domain in purple.
Based on these results, we continue to unravel the replication mechanism that replicates the unique DNA structure of the poxvirus genome consisting of a linear double-stranded DNA that is circularised at the ends. With this aim, the study of the E9-A20-D4 holoenzyme and D5 in complex with different DNAs will be conducted.
The project of our team is based on a collaboration with Frédéric Iseni who heads the Virology Laboratory at IRBA, Bretigny-sur-Orge in the Paris region.
Keywords
poxvirus, DNA replication, DNA polymerase, helicase, primase, processivity factor
Techniques
Production of recombinant proteins in insect cells and E. coli
X-ray crystallography
Cryo-electron microscopy
Biochemical and biophysical characterisation (fluorescence anisotropy, surface plasmon resonance, SAXS, circular dichroism, MALLS, BLI)
Electron microscopy in collaboration with the Electron Microscopy and Methods (MEM) group at IBS.