Functional expression and characterization of the Epstein-Barr virus DNA polymerase catalytic subunit

The Epstein-Barr virus (EBV) BMRF1 gene product is necessary for DNA polymerase catalytic subunit (BALF5) activity in 100 mM ammonium sulfate. To map regions of BMRF1 necessary for polymerase accessory function, linker insertion and deletion mutant BMRF1 polypeptides were expressed by in vitro transcriptiontranslation and assayed for DNA polymerase elongation activity and binding to double-stranded DNA (dsDNA)-cellulose. Amino-terminal deletions up to residue 303 were defective for stimulation of elongation. Deletions between residues 44 and 194 and residues 238 and 303 abolished binding to dsDNA-cellulose. The region from residues 194 to 238, therefore, is necessary for stimulation of BALF5 elongation but dispensable for dsDNA-cellulose binding. Deletion analysis also localized reactive epitopes of two neutralizing monoclonal antibodies to BMRF1 to a carboxy-terminal region which is dispensable for activity. These data suggest that a bipartite DNA-binding region is an essential component of the DNA polymerase accessory function and that the two noncontiguous regions are separated by a region (residues 194 to 217) which is essential for stimulation; therefore, it may interact with the BALF5 catalytic subunit of EBV DNA polymerase. Both EBV BMRF1 and herpes simplex virus UL42 gene products are DNA polymerase accessory proteins which bind dsDNA and increase the processivity of their corresponding catalytic components. Outstanding similarities between their primary amino acid sequences are not evident. However, it appears that their structural organizations are similar.

Section: Discussionsupporting

confidence: 70%

mentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

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

Kiehl

Dorsky

1995

“…EA-D is a component of the EBV DNA polymerase complex (9,35,72) and is essential for EBV lytic replication (25,27). The protein functions as a DNA processivity factor (8,71).…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Epstein-Barr Virus Replication by a Benzimidazole l -Riboside: Novel Antiviral Mechanism of 5,6-Dichloro-2-(Isopropylamino)-1-β- l -Ribofuranosyl-1H-Benzimidazole

Zacny

Gershburg

Davis

et al. 1999

Although a number of antiviral drugs inhibit replication of Epstein-Barr virus (EBV) in cell culture, and acyclovir (ACV) suppresses replication in vivo, currently available drugs have not proven effective for treatment of EBV-associated diseases other than oral hairy leukoplakia. Benzimidazole riboside compounds represent a new class of antiviral compounds that are potent inhibitors of human cytomegalovirus (HCMV) replication but not of other herpesviruses. Here we characterize the effects of two compounds in this class against lytic replication of EBV induced in a Burkitt lymphoma cell line latently infected with EBV. We analyzed linear forms of EBV genomes, indicative of lytic replication, and episomal forms present in latently infected cells by terminal probe analysis followed by Southern blot hybridization as well as the high-molecular-weight unprocessed viral DNA by pulsed-field gel electrophoresis. d-Ribofuranosyl benzimidazole compounds that act as inhibitors of HCMV DNA maturation, including BDCRB (5,6-dichloro-2-bromo-1-β-d-ribofuranosyl-1H-benzimidazole), did not affect the accumulation of high-molecular-weight or monomeric forms of EBV DNA in the induced cells. In contrast, the generation of linear EBV DNA as well as precursor viral DNA was sensitive to thel-riboside 1263W94 [5,6-dichloro-2-(isopropylamino)-1-β-l-ribofuranosyl-1H-benzimidazole]. The 50% inhibitory concentration range for 1263W94 was 0.15 to 1.1 μM, compared with 10 μM for ACV. Thus, 1263W94 is a potent inhibitor of EBV. In addition, 1263W94 inhibited the phosphorylation and the accumulation of the essential EBV replicative cofactor, early antigen D.

“…In investigations of protein-protein interactions between the subunits of the EBV DNA Pol holoenzyme, the overexpression and purification systems of the individual components have been developed by using a baculovirus expression system (19,22). The BMRF1 gene products exist as phosphorylated forms of 52 and 50 kDa and an unphosphorylated form of 48 kDa in recombinant baculovirus-infected insect cells, although the functional relationship of the phosphorylation is not yet known.…”

mentioning

confidence: 99%

Further characterization of the interaction between the Epstein-Barr virus DNA polymerase catalytic subunit and its accessory subunit with regard to the 3'-to-5' exonucleolytic activity and stability of initiation complex at primer terminus

Tsurumi

Daikoku

Nishiyama

1994

Self Cite

The Epstein-Barr virus (EBV) DNA polymerase catalytic subunit, BALF5 gene product, possesses an intrinsic 3'-to 5' proofreading exonuclease activity in addition to 5'-to-3' DNA polymerase activity (T. Tsurumi, A. Kobayashi, K. Tamai, T. Daikoku, R. Kurachi, and Y. Nishiyama, J. Virol. 67:4651-4658, 1993). The exonuclease hydrolyzed both double-and single-stranded DNA substrates with 3'-to-5' directionality, releasing deoxyribonucleoside 5'-monophosphates. The double-strand exonucleolytic activity catalyzed by the BALF5 polymerase catalytic subunit was very sensitive to high ionic strength, whereas the single-strand exonucleolytic activity was moderately resistant. The addition of the BMRF1 polymerase accessory subunit to the reaction enhanced the double-strand exonucleolytic activity in the presence of high concentrations of ammonium sulfate (fourfold stimulation at 75 mM ammonium sulfate). Optimal stimulation was obtained when the molar ratio of BMRF1 protein to BALF5 protein was 2 and higher, identical to the values required for reconstituting the optimum DNA polymerizing activity (T. Tsurumi, T. Daikoku, R. Kurachi, and Y. Nishiyama, J. Virol. 67:7648-7653, 1993). Furthermore, product size analyses revealed that the polymerase catalytic subunit alone excised a few nucleotides from the 3' termini of the primer hybridized to template DNA and that the addition of the BMFR1 polymerase accessory subunit stimulated the nucleotide excision several times. In contrast, the hydrolysis of single-stranded DNA by the BALF5 protein was not affected by the addition of the BMRF1 polymerase accessory subunit at all. These observations suggest that the BMRF1 polymerase accessory subunit forms a complex with the BALF5 polymerase catalytic subunit to stabilize the interaction of the holoenzyme complex with the 3'-OH end of the primer on the template DNA during exonucleolysis. On the other hand, challenger DNA experiments revealed that the BALF5 polymerase catalytic subunit alone stably binds to the primer terminus in a stationary state, whereas the reconstituted polymerase holoenzyme is unstable. The instability of the initiation complex of the EBV DNA polymerase would allow the rapid removal of the EBV DNA polymerase holoenzyme from the lagging strand after it has replicated up to the previous Okazaki fragment. This feature of the EBV DNA polymerase holoenzyme in a stationary state is in marked contrast to the moving holoenzyme complex tightly bound to the primer end during polymerization and exonucleolysis.