Low-resolution structure of vaccinia virus DNA replication machinery.

Abstract : 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.
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Article dans une revue
Journal of Virology, American Society for Microbiology, 2013, 87 (3), pp.1679-89
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Soumis le : jeudi 10 décembre 2015 - 11:18:57
Dernière modification le : lundi 11 février 2019 - 16:51:16

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Céleste Sèle, Frank Gabel, Irina Gutsche, Ivan Ivanov, Wim P Burmeister, et al.. Low-resolution structure of vaccinia virus DNA replication machinery.. Journal of Virology, American Society for Microbiology, 2013, 87 (3), pp.1679-89. 〈hal-01241253〉



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