Functional interaction between Epstein-Barr virus DNA polymerase catalytic subunit and its accessory subunit in vitro

Abstract

The Epstein-Barr virus (EBV) DNA polymerase catalytic subunit (BALF5 protein) and its accessory subunit (BMRF1 protein) have been independently overexpressed and purified (T. Tsurumi, A. Kobayashi, K. Tamai, T. Daikoku, R. Kurachi, and Y. Nishiyama, J. Virol. 67:4651-4658, 1993; T. Tsurumi, J. Virol. 67:1681-1687, 1993). In an investigation of the molecular basis of protein-protein interactions between the subunits of the EBV DNA polymerase holoenzyme, we compared the DNA polymerase activity catalyzed by the BALF5 protein in the presence or absence of the BMRF1 polymerase accessory subunit in vitro. The DNA polymerase activity of the BALF5 polymerase catalytic subunit alone was sensitive to high ionic strength on an activated DNA template (80% inhibition at 100 mM ammonium sulfate). Addition of the polymerase accessory subunit to the reaction greatly enhanced DNA polymerase activity in the presence of high concentrations of ammonium sulfate (10-fold stimulation at 100 mM ammonium sulfate). Optimal stimulation was obtained when the molar ratio of BMRF1 protein to BALF5 protein was 2 or more. The DNA polymerase activity of the BALF5 protein along with the BMRF1 protein was neutralized by a monoclonal antibody to the BMRF1 protein, whereas that of the BALF5 protein alone was not, suggesting a specific interaction between the BALF5 protein and the BMRF1 protein in the reaction. The processivity of nucleotide polymerization of the BALF5 polymerase catalytic subunit on singly primed M13 single-stranded DNA circles was low (approximately 50 nucleotides). Addition of the BMRF1 polymerase accessory subunit resulted in a strikingly high processive mode of deoxynucleotide polymerization (> 7,200 nucleotides). These findings strongly suggest that the BMRF1 polymerase accessory subunit stabilizes interaction between the EBV DNA polymerase and primer template and functions as a sliding clamp at the growing 3'-OH end of the primer terminus to increase the processivity of polymerization.

Immune responses to Epstein–Barr virus lytic proteins in patients with nasopharyngeal carcinoma

“…EA-D complex is an abundant EBV product during viral multiplication and has been recognized as an EBV DNA polymerase accessory protein involved in the replication of the viral genome [Tsurumi et al, 1993]. DNase and TK are also early proteins in the EBV lytic cycle.…”

Cellular transcription factors recruit viral replication proteins to activate the Epstein–Barr virus origin of lytic DNA replication, oriLyt

“…Numerous interactions between these proteins have been documented, consistent with the existence of a replication complex that is essentially viral in nature (Tsurumi et al, 1993;Zeng et al, 1997;Gao et al, 1998).…”

Bipartite DNA-binding region of the Epstein-Barr virus BMRF1 product essential for DNA polymerase accessory function

“…The EBV BMRF1 product is a DNA polymerase accessory protein which binds strongly to dsDNA (35) and is required for processive elongation activity of the EBV DNA polymerase catalytic component (36). Like several other well-characterized DNA polymerase accessory proteins, BMRF1 seems to function as a sliding clamp in that it binds to DNA and stimulates processive DNA synthesis by the catalytic component of EBVpol, but it differs from the E. coli ␤ protein, the bacteriophage T4 gp45, and eukaryotic proliferating cell nuclear antigen in that BMRF1 binds tightly to DNA while the others do not (15,18,31,35).…”

“…The in vitro activity of the Epstein-Barr virus (EBV) DNA polymerase (EBVpol) is dependent on the functional interaction between the catalytic subunit (BALF5 product) and the accessory subunit (BMRF1 product) (16,19,36,37). The genes for both components are required for in vivo replication of the viral lytic origin of replication, oriLyt, but not for oriP, which is responsible for the maintenance of latency (9).…”

“…The 110-kDa BALF5 catalytic component of EBVpol carries out nonprocessive elongation at low salt concentrations in the absence of the 50-kDa BMRF1 accessory product (37). In 100 mM ammonium sulfate, the BALF5 activity is negligible unless the BMRF1 product is present (16,36,37), and then the activity is highly processive (37). The BMRF1 product binds strongly to double-stranded DNA (dsDNA) (35); therefore, the manner by which it increases BALF5 processivity is most likely by acting as a ''sliding clamp'' to tether the catalytic subunit to the template.…”