Enzymatic activities of overexpressed herpes simplex virus DNA polymerase purified from recombinant baculovirus-infected insect cells

Abstract

Biochemical characterization of the herpes simplex virus (HSV) DNA polymerase, a model DNA polymerase and an important target for antiviral drugs, has been limited by a lack of pure enzyme in sufficient quantity. To overcome this limitation, the HSV DNA polymerase gene was introduced into the baculovirus, Autographa californica nuclear polyhedrosis virus, under the control of the polyhedrin promoter to give rise to a recombinant baculovirus, BP58. BP58-infected Spodoptera frugiperda insect cells expressed a polypeptide that was indistinguishable from authentic polymerase by several immunological and biochemical properties, at levels approximately ten-fold higher per infected cell than found in HSV-infected Vero cells. The DNA polymerase was purified to apparent homogeneity from BP58-infected insect cells. Using activated DNA as primer-template, the purified enzyme exhibited specific activity similar to that of enzyme isolated from HSV-infected Vero cells, indicating that additional polymerase-associated proteins from HSV-infected cells are not critical for activity with this primer-template. 3'-5' exonuclease activity co-purified with the BP58-expressed HSV DNA polymerase, demonstrating that this activity is intrinsic to the polymerase polypeptide. The purified enzyme also exhibited RNAse H activity. The recombinant baculovirus should permit detailed biochemical and biophysical studies of this enzyme.

The effect of the UL42 protein on the DNA polymerase activity of the catalytic subunit of the DNA polymerase encoded by herpes simplex virus type 1

“…It has been reported [10,11] that the specific activity of the purified Pol is very similar to that of the DNA polymerase (PoI-UI.A2 protein heterodimer) isolated from HSV-l-infected mammalian cells. The conclusion drawn from this was that any stimulatory activity by the UIA2 protein on the Pol activity was slight, in contrast to the results of Gallo and co-workers [fl], although the UIA2 protein was found to increase the processivity of the polymerisation ([I0], A. Owsianka and H.S.…”

“…The reported activation [8] was observed under high salt conditions (100 mM Tris buffer, pH 8.0, 200 mM KCi), while lack of any effect [10,11] was found under conditions (20 mM Tris-HCl, pH 7.5, 150 mM (NH~)2SO~) that seem similar to those (75 mM Tris-HCl, pH 7.5, 70 mM (NH4)2SO4; Fig. 1) under which the UL42 protein has little or no effect on the polymerasc activity of Pol.…”

“…By using appropriate recombinant baculoviruscs both subunits of the HSV-1 DNA polymerase have been obtained free of the other subunit [9][10][11].…”

Herpes simplex virus type 1 origin-dependent DNA replication in insect cells using recombinant baculoviruses

“…Indeed, six of the replication proteins have already been expressed in a functional form in Spodoptera frugiperda (SO insect cells infected with appropriate (AcNPV) recombinants (Olivo et al, 1988;Dodson et al, 1989;Gottlieb et al, 1990;Marcy et al, 1990;Calder & Stow, 1990). Because many future efforts are aimed at utilizing these heterologously expressed proteins in cell-free systems, it is important that they should be able to perform all the replicative functions normally carried out by their counterparts during a lytic infection of permissive cells, and not merely those activities for which convenient assays are presently available.…”

Contribution of the 3′- to 5′-Exonuclease Activity of Herpes Simplex Virus Type 1 DNA Polymerase to the Fidelity of DNA Synthesis

“…Viruses in this family encode most of the proteins essential for and directly involved in DNA replication (2)(3)(4), including a well conserved DNA polymerase catalytic subunit (pol), which is a member of the polymerase B family (5,6). HSV-1 pol possesses 5Ј-to 3Ј-polymerizing and 3Ј-to 5Ј-exonuclease (exo) activities (7,8), the latter of which is involved in the removal of incorrectly incorporated deoxyribonucleoside triphosphates (9 -12). The importance of this proofreading activity for maintaining fidelity of DNA replication was suggested by studies from our laboratory that demonstrated the relatively poor ability of HSV-1 pol to discriminate between the correct and incorrect nucleotide for incorporation in single turnover experiments (13).…”