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 mpox caused by the monkeypox virus (MPXV), 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 mpox virus towards more efficient human transmission cannot be excluded once it has been introduced into the human population where an increased mutation rate has been observed. 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.
The replication of the poxvirus genome is fully cytoplasmic and can occur even in absence of the cell nucleus. Furthermore, the poxvirus replication machinery is unique due to the structure of the genome best described as linear double-stranded DNA circularized at the extremities by hairpin loops. These telomers have a peculiar structure as the terminal loops are preceded by a stretch of incompletely paired dsDNA, a conserved telomere resolution sequence required to form viral genomes from concatemeric intermediates and terminal repeat sequences (Figure 1).
A better knowledge of the unique DNA replication machinery and its interplay with virus uncoating and assembly will make it possible to develop new compounds (reviewed in [1]).
Figure 1 : A) Organization of the VACV genome with the flip telomere at the left and the flop telomere at the right side, with a zoom view indicating the repeat sequences. B) The extremity of the flip telomere hairpin with a proposed base pairing obtained with the RNAstructure webserver. The position of the proposed origin of replication (red arrow) and part of the site of concatemer resolution (green line) are indicated. C) 3-D model of the disturbed DNA obtained by the 3dRNA/DNA Web Server, not taking into account the effect of bound proteins.
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 an uracil N-glycosylase D4.
Recently, we and other groups determined the structure of the polymerase holoenzyme ( [2], Figure 2) and of the helicase domain of D5 ( [3], Figure 3). This progress could build on the three-dimensional structure of these proteins and their interfaces determined in our group at increasing resolution culminating in the X-ray structure of the E9 polymerase [4]. More recently in a structure of the A20-E9 interface obtained by structural nuclear magnetic resonance (NMR) [5].
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 2 : 3.9 Å Cryo-EM density and model of The E9-A20-D4 polymerase holoenzyme. E9 polymerase : orange ; A20 processivity factor : violet ; D4 uracil-DNA glycosylase : green.
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 resolution information on the structure and domain organisation of D5 with a N-terminal primase domain and a C-terminal helicase domain [6]. The entrance to the helicase domain is formed by a collar domain, which forms a hexameric closed ring. 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 with Alphafold2, we obtained the 4.1 Å cryo-EM structure of the hexameric helicase fragment in complex with DNA [3]. It revealed the architecture of a whole class of hexameric helicases present in bacteriophages and autonomously replicating DNA elements. Still, the strand separation by the D5 helicase has not been demonstrated clearly and as the structures show always a collar domain forming a closed hexameric ring (Figure 3). A key for the enigmatic mechanism of the helicase entry into the replication fork may lay in the peculiar telomere structure of the poxvirus genome (Figure 1) containing the proposed origins of replication. In order to investigate the role of the telomere structure further, we initiated a study of its structure and the one of the associated proteins.
For the polymerase holoenzyme, we obtained the structure of the apo-form (Figure 2) and could show that more open forms must be present in solution [3]. Despite several DNA-bound structures, which have been published recently, several functions of the holoenzyme such as proofreading or the synchronisation with the primase domain of D5 in the context of the replication fork are not understood. A recent publication of a role of the H5 hub protein as additional processivity factor showed the requirement to revisit the role of the processivity factors.
Figure 3 : 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.
The recent explosion of structural information provides us with snapshots of the replication machinery, which still have to be put into context urging for more biochemical work on the polymerase holoenzyme, the helicase-primase and their interplay. In order to advance in the understanding of poxvirus DNA replication, we will use the techniques our team is spezialised in : cryo-electron microscopy (cryo-EM), X-ray crystallography and small-angle X-ray scattering (SAXS) combined with other biophysical techniques such as Multi-Angle Light Scattering (MALS), BioLayer Interferometry (BLI), polyacrylamide gel electrophoresis (PAGE), Electrophoretic Mobility Shift Assays (EMSA) etc.
Our team collaborates closely with Frédéric Iseni who heads the Virology Laboratory at IRBA, Bretigny-sur-Orge, in the Paris region on the validation of structural results by the generation of mutant viruses.
Keywords
poxvirus, DNA replication, DNA polymerase, DNA helicase, primase, processivity factor, telomere
Techniques
- Production of recombinant proteins in the baculovirus-insect cell system and in E. coli
- X-ray crystallography
- Cryo-EM
- Biochemical and biophysical characterisation (fluorescence anisotropy, surface plasmon resonance, SAXS, circular dichroism, MALS, BLI)