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.

Targeted Integration of siRNA against Porcine Cytomegalovirus (PCMV) Enhances the Resistance of Porcine Cells to PCMV

“…The main results indicate that siRNAs targeting three sites-U77 (a component of the helicase-primase complex HP1), U51 (a G protein-coupled receptor), and U12 (a G protein-coupled receptor-like protein)-significantly reduce the copy number of PCMV. This finding suggested that these three sites are important and conserved targets for PCMV replication, with the G protein-coupled receptor being of interest in murine cytomegalovirus and the component of the helicase-primase complex being associated with the lytic DNA replication machinery of herpesviruses [21,22]. Interfering with their expression or function can effectively inhibit virus replication, providing new insights and methods for the prevention and treatment of PCMV infection.…”

Targeted Integration of siRNA against Porcine Cytomegalovirus (PCMV) Enhances the Resistance of Porcine Cells to PCMV

“…The main results indicate that siRNAs targeting three sites-U77 (a component of the helicase-primase complex HP1), U51 (a G protein-coupled receptor), and U12 (a G protein-coupled receptor-like protein)-significantly reduce the copy number of PCMV. These findings suggested that these three sites are important and conserved targets for PCMV replication, with the G protein-coupled receptor being of interest in murine cytomegalovirus and the component of the helicase-primase complex being associated with the lytic DNA replication machinery of herpesviruses [39,40]. Interfering with their expression or function can effectively inhibit virus replication, providing new insights and methods for the prevention and treatment of PCMV infection.…”

Interplay between Epstein-Barr virus infection and environmental xenobiotic exposure in cancer

“…In any case, the expression of both Zta and Rta proteins is always required for subsequent expression of lytic early proteins [ 41 ] such as BMRF1, SM, BHLF1, and BHRF1. Additionally, viral DNA polymerase (BALF5) [ 42 ], the DNA polymerase processivity factor (BMRF1) [ 43 ], the viral helicase (BBLF4) [ 44 ], and viral primase (BSLF1) [ 44 ], among others are currently expressed at this stage. BMRF1 and BRRF1 are transcription factors that activate the oriLyt (Lytic replication origin).…”