title: Production and characterisation of Epstein-Barr virus helicase-primase complex and its accessory protein BBLF2/3.
abstract: The helicase-primase complex is part of the lytic DNA replication machinery of herpesviruses, but up to now, almost nothing is known about its structure. For Epstein-Barr virus it consists in the helicase BBLF4, the primase BSLF1 and the accessory protein BBLF2/3. The accessory protein shows only weak sequence homology within the herpesvirus family but may be related to an inactive B-family polymerase. BSLF1 belongs to the archaeo-eukaryotic primase family, whereas the helicase BBLF4 has been related either to Dda helicases of caudovirales or to Pif1 helicases. We produced the helicase-primase complex in insect cells using a baculovirus coding for all three proteins simultaneously. The soluble monomeric helicase-primase complex containing the three proteins with 1:1:1 stoichiometry showed ATPase activity, which is strongly stimulated in the presence of ssDNA oligomers. Furthermore, we expressed BBLF2/3 as soluble monomeric protein and performed small-angle X-ray scattering experiments which yielded an envelope whose shape is compatible with B-family polymerases.
authors: Univ. Grenoble Alpes, UVHCI, 38000, Grenoble, France.|CNRS, UVHCI, 38000, Grenoble, France.
authors: Eric
authors: E
authors: Thierry
authors: European Synchrotron Radiation Facility (ESRF), 38000, Grenoble, France.
authors: Martha
authors: M
authors: Brennich
authors: EMBL Grenoble Outstation, UVHCI, 38000, Grenoble, France.
authors: Adam
authors: A
authors: Round
authors: Laboratoire de Virologie, Centre Hospitalo-Universitaire de Grenoble, B.P. 217, 38043, Grenoble Cedex 9, France.
authors: Marlyse
authors: M
authors: Buisson
authors: Univ. Grenoble Alpes, UVHCI, 38000, Grenoble, France. wim.burmeister@ujf-grenoble.fr.|CNRS, UVHCI, 38000, Grenoble, France. wim.burmeister@ujf-grenoble.fr.
authors: Wim P
authors: http://orcid.org/0000-0003-0876-6118
authors: WP
authors: Burmeister
authors: Univ. Grenoble Alpes, UVHCI, 38000, Grenoble, France. hutin@uvhci.fr.|CNRS, UVHCI, 38000, Grenoble, France. hutin@uvhci.fr.
authors: Stephanie
authors: S
authors: Hutin
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors; D004854:Herpesvirus 4, Human / Q000201:enzymology* / Q000235:genetics*; D007313:Insecta; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D053838:Scattering, Small Angle; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D014764:Viral Proteins; D000255:Adenosine Triphosphate; D004265:DNA Helicases
keywords: DNA helicase; DNA replication; EBV; Epstein–Barr virus; Gammaherpesvirus; Primase