Low-Resolution Structure of Vaccinia Virus DNA Replication Machinery

Abstract

c Smallpox caused by the poxvirus variola virus is a highly lethal disease that marked human history and was eradicated in 1979 thanks to a worldwide mass vaccination campaign. This virus remains a significant threat for public health due to its potential use as a bioterrorism agent and requires further development of antiviral drugs. The viral genome replication machinery appears to be an ideal target, although very little is known about its structure. Vaccinia virus is the prototypic virus of the Orthopoxvirus genus and shares more than 97% amino acid sequence identity with variola virus. Here we studied four essential viral proteins of the replication machinery: the DNA polymerase E9, the processivity factor A20, the uracil-DNA glycosylase D4, and the helicase-primase D5. We present the recombinant expression and biochemical and biophysical characterizations of these proteins and the complexes they form. We show that the A20D4 polymerase cofactor binds to E9 with high affinity, leading to the formation of the A20D4E9 holoenzyme. Small-angle X-ray scattering yielded envelopes for E9, A20D4, and A20D4E9. They showed the elongated shape of the A20D4 cofactor, leading to a 150-Å separation between the polymerase active site of E9 and the DNA-binding site of D4. Electron microscopy showed a 6-fold rotational symmetry of the helicase-primase D5, as observed for other SF3 helicases. These results favor a rolling-circle mechanism of vaccinia virus genome replication similar to the one suggested for tailed bacteriophages.

Crystal Structure of the Vaccinia Virus Uracil-DNA Glycosylase in Complex with DNA

“…The current model of D4 tethering the polymerase holoenzyme to the DNA may be too simplistic because of its weak interaction with DNA at physiological ionic strength, although mutants affecting polymerase processivity point to a role of the DNA-binding site of D4 (40). The simultaneous presence of two DNA interacting domains, the polymerase active site in one hand and the D4 DNA binding site on the other hand, may still lead to an increased avidity, but this effect is hard to predict because of the long distance (ϳ150 Å) between the two DNA interacting domains (47). It is likely that the bridging A20 protein contributes also to the interaction with DNA.…”

“…D4 appears to interact preferentially with uracil in the context of dsDNA narrowing down potential models of position and orientation of the polymerase holoenzyme within the replication fork (47). The current model of D4 tethering the polymerase holoenzyme to the DNA may be too simplistic because of its weak interaction with DNA at physiological ionic strength, although mutants affecting polymerase processivity point to a role of the DNA-binding site of D4 (40).…”

Genetic Confirmation that the H5 Protein Is Required for Vaccinia Virus DNA Replication

“…6B) or for the assembly or activity of the DNA Pol or the holoenzyme (data not shown). Purified preparations of DNA polymerase, as visualized by silver stain analysis, appear to be free of other proteins, and the processive holoenzyme can be assembled from separate baculovirus infections expressing Pol and A20/UDG (66). Moreover, active holoenzyme can be assembled using in vitro-transcribed/translated proteins (67).…”

Domain Organization of Vaccinia Virus Helicase-Primase D5

“…The full-length D5 construct was cloned as a His-tagged tobacco etch virus (TEV) protease-cleavable construct and expressed in SF21 cells (9).…”

“…1A), which participate in a modular architecture in a number of proteins with a helicase and/or primase function within the NCLDV clade, mimiviruses (8), as well as the P4 bacteriophages (3,5), the only known high-resolution structures concern the primase domain (reviewed in reference 5). For the SF3 helicase domains, a ring-like structure, often with a 6-fold symmetry like that for D5, can be inferred (6,9). Information on the arrangement of the individual domains is still lacking.…”