ORIGINAL ARTICLE

Expression, purification, and enzymatic characterizationof Bombyx mori nucleopolyhedrovirus DNA polymerase

Liu Liu • Huifang Song • Lei Zhang •

Xiaoting Fan • Qian Zhang • Keping Chen •

Huiqing Chen • Yajing Zhou

Received: 20 March 2013 / Accepted: 2 May 2013

� Springer-Verlag Wien 2013

Abstract Bombyx mori nucleopolyhedrovirus (BmNPV)

is a major viral agent that causes deadly grasserie disease in

silkworms. BmNPV DNA polymerase (Bm-DNAPOL),

encoded by the ORF53 gene, plays a central role in viral

DNA replication. In this work, a His-tagged Bm-DNAPOL

fusion protein, constructed using a novel MultiBac

expression system, was overexpressed in Sf-9 insect cells,

purified to near homogeneity on Ni-NTA agarose beads

and further purified by ion-exchange chromatography.

About 0.4 mg of enzyme was obtained from about 1 9 109

infected Sf-9 cells in suspension culture. Characterization

of the highly purified enzyme indicated that Bm-DNAPOL

is a monomer with an apparent molecular mass of

approximately 110,000 Da. It possessed a specific activity

of 15,126.3 U/mg under optimal in vitro reaction condi-

tions and behaved in the manner of a proliferating cell

nuclear antigen (PCNA)-independent DNA polymerase on

both poly(dA)/oligo(dT) primer/template and singly pre-

miered M13 DNA. BmNPV viral replication may be

independent of replication factor C and a PCNA complex,

while single-stranded DNA binding protein might play an

important role in BmNPV DNA replication. These findings

will be significant in studies on BmNPV-based disease in

silkworms and for using silkworms as a bioreactor for the

production of biomolecules of commercial importance.

Introduction

The silkworm (Bombyx mori) is not only an economically

important insect but also a model animal for studies in life

sciences. China has a history of over 5,000 years of raising

silkworms and is still a primary silk-producing country

today. Viral silkworm diseases cause great losses in cocoon

production, among which grasserie disease caused by

Bombyx mori nucleopolyhedrovirus (BmNPV) is one of the

most disastrous. Among the 700 silkworm strains at the

National Center for Silkworm Genetic Resources Preser-

vation of the Chinese Academy of Agricultural Sciences,

most Bombyx strains are highly susceptible to BmNPV

infection, while only a few strains are completely resistant

to BmNPV even after being fed with a high concentration

of virus inoculum.

BmNPV is the type member of the Baculoviridae, a

large family of viruses containing circular, supercoiled,

double-stranded DNA genomes [3, 18]. Although knowl-

edge about silkworm defense at the molecular level has

been extended rapidly in recent years, its antiviral immune

mechanism remains unclear. Previous studies revealed that

BmNPV could invade the midguts of both susceptible and

resistant strains, but viral proliferation was greatly slowed

in the resistant strain by unknown mechanisms [5].

Viral DNA synthesis is a crucial step in virus life cycles,

and virus-encoded DNA polymerase is essential for ensuring

the faithful and efficient reproduction of the progeny virus.

All nucleopolyhedrovirus (NPVs) encode their own DNA

polymerases [2, 7, 19, 20, 28]. Functional analysis of

Autographa californica multiple nucleopolyhedrovirus

L. Liu and H. Song are contributed equally to this work

L. Liu � H. Song � L. Zhang � X. Fan � Q. Zhang � K. Chen �H. Chen (&) � Y. Zhou (&)

Institute of Life Sciences, Jiangsu University, Zhenjiang 212013,

Jiangsu, People’s Republic of China

e-mail: chq2008@ujs.edu.cn

Y. Zhou

e-mail: yajingzhou@ujs.edu.cn

123

Arch Virol

DOI 10.1007/s00705-013-1758-8

(AcMNPV) DNA polymerase revealed that it harbors both

DNA polymerase and exonuclease activities as well as

processivity and moderate strand-displacement activity [19,

30]. Despite the high degree of homology between the

genomes of AcMNPV and BmNPV [18], little is known

concerning the function of genes involved in BmNPV DNA

synthesis, and this hinders understanding of the defense

mechanism of Bombyx mori against BmNPV. Bioinformat-

ics analysis has indicated that BmNPV polymerase (Bm-

DNAPOL) encoded by ORF53 shares significant amino acid

sequence similarity with other NPV DNA polymerases.

Alignment of NPV DNAPOL amino acid sequences

revealed that the conserved amino acid motifs are similar to

those found in mammalian DNA polymerases [21], which

have been demonstrated to interact with a processivity fac-

tor, proliferating cell nuclear antigen (PCNA) [46]. In

eukaryotic cells, DNA replication is carried out by coordi-

nated actions of many proteins, including DNA polymer-

ases, replication factor C (RFC), PCNA, and replication

protein A (RPA) which functions as a single-stranded DNA

binding protein (SSB) [22, 25, 44]. The polymerases are

required for chromosomal DNA synthesis on both the

leading and lagging strands. RFC has DNA-dependent

ATPase activity, loading sliding clamp PCNA onto DNA

[26, 41]. The likely role of PCNA is in stabilizing the

polymerase-DNA interaction to maintain the processivity of

the polymerase [8, 14]. SSB is required for the initiation and

elongation phases of DNA replication [4, 45]. For baculo-

viruses, LEF-3 is characterized as an SSB whose function is

well studied. In AcMNPV, Ac 49, a homolog of PCNA, is

not an essential gene [23] and did not appear to elevate DNA

replication in transient replication assays, but in later studies

it was found that Sf-9 cellular PCNA is involved in

AcMNPV DNA replication. Comparison of genome

sequences showed that BmNPV lacks the homologue of

AcMNPV PCNA. Hence, many questions arise regarding

viral DNA replication: What are the features of Bm-DNA-

POL? Does Bm-DNAPOL behave as a PCNA-dependent

DNA polymerase? If so, does it require host PCNA? Could

the AcMNPV PCNA substitute for the host PCNA?

To further address the role of Bm-DNAPOL in baculo-

virus DNA replication, in this work, we overexpressed and

purified Bm-DNAPOL as a His-tagged fusion protein in

infected Sf-9 cells using the novel MultiBac expression

system [6]. The highly purified enzyme was characterized

by determining its molecular weight by size-exclusion

chromatography on a Superose 6 column, measuring its

activity on both poly(dA)/oligo(dT) primer/template and

singly primed M13 DNA, and investigating its relationship

to PCNAs from different sources, RFC, and SSB in a

reconstitution system. Our data provide significant infor-

mation on the enzymology of Bm-DNAPOL that could lead

to a better understanding of BmNPV viral DNA replication.

Materials and methods

Reagents and chemicals

All reagents and chemicals used in this study were pur-

chased from Sigma-Aldrich, Invitrogen, and Gibco-BRL

except as otherwise indicated.

Cells and virus

BmNPV (Zhenjiang strain) [11] viruses were propagated in

BmN cells maintained at 27 �C in TC-100 insect medium

supplemented with 10 % (v/v) fetal bovine serum (Gibco-

BRL). Spodoptera frugiperda Sf-9 cells were cultured at

27 �C in SFX-insect (Hyclone) medium supplemented with

2.5 % fetal bovine serum.

Proteins used in Bm-DNAPOL activity assays

Recombinant human PCNA, AcMNPV PCNA (AcPCNA)

and host PCNA (BmPCNA) were overexpressed in E. coli

DH5a cells and purified to near-homogeneity as described

previously [47]. S. cerevisiae RFC was a generous gift

kindly provided by Dr. Gregory Bowman. E. coli single-

strand binding protein (SSB) was purchased from Sigma-

Aldrich. Recombinant human DNA Pol d (hPol d) four-

subunit complex was purified to near-homogeneity from

the hemolymph of infected silkworm larvae as described

previously [47]. The recombinant BmNPV DNA poly-

merase was constructed, expressed and purified to near-

homogeneity as described below.

Construction and expression of a recombinant

baculovirus harboring Bm-DNAPOL

The construction of recombinant BmNPV DNA polymer-

ase was performed with a novel MultiBac expression sys-

tem as described [47], according to manufacturer’s

instructions (kindly provided by Dr. Timothy J. Richmond,

Institute for Molecular Biology and Biophysics, ETH

Zurich, Switzerland) [6]. PCR amplification was carried

out with a forward primer, 50- CGCGGATCCCCAT

GAAAATATATTCTTACAATG-30, and a reverse primer,

50-CCCAAGCTTTTATTAGTGATGGTGATGGTGATG

TTTTTTTATTTTATACAAAC-30 (the BamHI and Hin-

dIII sides are underlined, and a synthetic 69His tag in the

reverse primer is double underlined), using viral DNA

isolated from BmNPV as the template. The PCR products

that were generated were digested with BamHI and

HindIII, subcloned in-frame into the BamHI-HindIII sites

of MCS1 of transfer vector pFBDM (under the control of

the polyhedrin promoter), and sequenced. Thus, the

recombinant plasmid pFBDM-Bm-DNAPOL, harboring a

L. Liu et al.

123

full-length sequence of BmNPV DNA polymerase with a

69His tag at its C-terminus, was obtained.

The resulting recombinant plasmid pFBDM-Bm-DNA-

POL was then introduced by transformation into competent

E. coli DH10MultiBac cells, which contain a modified

baculovirus genome in which two baculoviral genes, v-cath

and chiA, are disrupted, leading to improved maintenance

of cellular compartments during infection and protein

production [6]. Recombinant bacmids were then con-

structed by transposing a mini-Tn7 element from a pFBDM

derivative to the mini-attTn7 attachment site on the bac-

mid, with the Tn7 transposition functions provided in trans

by a helper plasmid. High-molecular-weight miniprep

DNA was prepared from selected E. coli with a white

phenotype and used to transfect Sf-9 cells for the genera-

tion of recombinant baculovirus particles according to the

manufacturer’s instructions (Invitrogen).

For the expression of Bm-DNAPOL, 500 ml of Sf-9

cells at 2 9 106 cells/ml in suspension culture was infected

with the generated recombinant baculoviruses at a multi-

plicity of infection (MOI) of 2. The cells were collected 72

hours postinfection, and the cell pellets were either treated

directly with lysis buffer or stored at -80 �C.

Purification of Bm-DNAPOL in Sf-9 cells

His-tagged Bm-DNAPOL expressed in Sf-9 cells was

purified using Ni-NTA agarose beads (QIAGEN) and fur-

ther purified by ion-exchange chromatography on an FPLC

Mono Q 5/50 GL column (GE Healthcare) as described

previously [35]. Eluted fractions were analyzed on 10 %

SDS-PAGE, followed by Coomassie blue staining.

Western blot analysis

Electrophoresis was performed by loading samples onto a

10 % SDS-PAGE gel. The separated proteins were trans-

ferred onto a nitrocellulose membrane. The membrane was

blocked with 5 % w/v nonfat dry milk in TBST buffer (20

mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05 % Tween-20)

for 30 min at room temperature and then incubated with

anti-His monoclonal antibody (Santa Cruz Biotechnology)

for 1 hour at room temperature. After three 15-min washes

in TBST, the membrane was incubated with AP-conjugated

goat anti-mouse IgG (Pierce) for 1 hour and washed with

TBST 3 times for 10 min. A Perfect Protein Western Blot

Kit (Novagen) was used for signal generation.

FPLC gel filtration chromatography

A 250-ll sample from the peak fraction after FPLC Mono

Q ion-exchange chromatography was loaded onto a Supe-

rose 6 10/300 GL column (GE Healthcare) equilibrated

with 150 mM NaCl in TGEED buffer (40 mM Tris-HCl, pH

7.8, 10 % glycerol, 0.5 mM EDTA, 0.1 mM EGTA, 1 mM

DDT). The column was eluted at a flow rate of 0.25 ml/min,

and a total of 120 fractions of 0.2 ml each were collected.

Bm-DNAPOL activity assays

Assays for DNA polymerase activity were performed by

determining the amount of [3H]dTMP incorporated into

poly(dA)/oligo(dT) template/primer as described previ-

ously [32, 47]. To determine the optimum reaction tem-

perature, the enzymatic reactions were performed at

different temperatures, ranging from 20 �C to 37 �C in a

reaction mixture containing 0.25 OD units/ml of sparsely

primed poly(dA)4000/oligo(dT)50 (Supertechs, Bethesda,

MD) in 50 mM Tris-HCl, pH8.0, 5 % glycerol, 0.1 mg/ml

BSA, 5 mM MgCl2, 5 lM dTTP, 0.5 lM [3H]dTTP, and

*0.2 units of Bm-DNAPOL in a total volume of 30 ll.

Reaction mixtures were incubated for 30 min and termi-

nated by spotting onto DE81 papers that were then washed

four times with 0.3 M ammonium formate, pH 7.8, and

once with 95 % ethanol and then dried and counted using a

liquid scintillation counter. The optimum reaction pH and

salt concentration were determined similarly at the opti-

mum temperature of 27 �C and pH values ranging from 6.5

to 10, and different concentrations of KCl ranging from 0

to 200 mM. The effects of DMSO and aphidicolin on the

Bm-DNAPOL activity were examined by the addition of

these inhibitors to the reaction mixture at various concen-

trations. All reactions were repeated in triplicate. Statistical

analysis (ANOVA) was done using Microsoft Excel 2007.

The specific activity of Bm-DNAPOL was determined

under the optimum reaction conditions. One unit of Bm-

DNAPOL activity corresponds to the incorporation of 1

nmol of dTMP in cpm (counts per minute; preset time, 1

min) per hour at 27 �C.

Assays using singly primed M13 DNA as the template

were performed as described previously with some modifi-

cations [47]. Single-stranded M13mp18 DNA (7250 bp,

New England Biolabs) was primed with a 20-mer oligonu-

cleotide (50-CTAGAGGATCCCCGGGTACC-30) comple-

mentary to nucleotides 6262-6243 of the M13 genome. The

standard 30-ll reaction mixtures contained 40 mM Tris-HCl

(pH 7.8), 10 mM MgCl2, 50 mM NaCl, 0.2 mg/ml bovine

serum albumin, 1 mM dithiothreitol, 5 mM each dATP,

dCTP, and dGTP, 0.5 mM [3H] dTTP, 0.5 mM ATP, 100 ng

of singly primed M13mp18 DNA, and *0.2 units of Bm-

DNAPOL. The complete reaction mixtures were incubated

at 27 �C for 30 min, terminated by the addition of 20 mM

EDTA, and then spotted onto DE81 paper, which was

washed three times with 0.3 M ammonium formate, pH 7.8,

and once with 95 % ethanol and then dried and counted in a

liquid scintillation counter. To examine the effects of

Bombyx mori nucleopolyhedrovirus DNA polymerase

123

AcPCNA, BmPCNA, RFC, and SSB on the elongation

activity of Bm-DNAPOL on singly primed M13 template,

the reaction mixtures were supplemented with the recom-

mended optimal amounts of S. cerevisiae RFC (80 ng), SSB

(200 ng), AcPCNA, or BmPCNA [35, 47].

Results

Construction, expression, and purification of Bm-

DNAPOL

To obtain soluble active enzyme for the examination of the

enzymology of Bm-DNAPOL in well-defined preparations,

we used a novel MultiBac system for Bm-DNAPOL

expression. Restriction analysis showed that a *3000-bp

fragment was successfully cloned into MCS1 of pFBDM

(Fig. 1A). DNA sequencing confirmed that the full-length

cDNA of wild-type Bm-dnapol was correctly fused to the

His-tag at its C-terminal end with the correct DNA

sequence. The resulting recombinant plasmid, pFBDM-

Bm-dnapol, was then transformed into MultiBac bacul-

oviral DNA in competent DH10MultiBacCre E. coli cells.

White clones were selected and recombinant bacmid DNAs

were isolated. Eight correct phenotypes verified by PCR

analysis were selected for transfection of Sf-9 cells. At 5

days post-transfection, recombinant baculoviruses con-

taining Bm-dnapol were successfully obtained, as shown

by western blot analysis with anti-His monoclonal

antibody.

To facilitate the purification of Bm-DNAPOL expressed

in Sf-9 cells, we developed a procedure for purification on

Ni-NTA agarose beads in combination with ion-exchange

chromatography (Materials and methods). Lysates of

transfected Sf-9 cells were cleared by high-speed centri-

fugation, and 500 ml of the supernatant was applied to a

1-ml Ni-NTA column and washed with lysis buffer con-

taining 30 mM imidazole. Although other concentrations of

imidazole in the wash buffer may be used for the wash, we

found that 30 mM imidazole is optimal for removing most

contaminants while retaining the majority of the DNA

polymerase proteins. The bound proteins were eluted in

1-ml fractions with elution buffer containing 300 mM

imidazole. The eluted fractions were analyzed by 10 %

SDS-PAGE (Fig. 1B) and confirmed by western blot

analysis with anti-His antibody (Fig. 1C). The amino acid

sequence of the protein that was obtained was verified by

mass spectrometry on a 4800 Plus MALDI TOF/TOFTM

Fig. 1 Cloning, expression and purification of recombinant Bm-

DNAPOL. Panel A: Agarose gel electrophoresis. Lane M, DNA

marker in bp; lanes 1-2, pFBDM-Bm-dnapol and empty vector

pFBDM digested with BamHI and HindIII, respectively. Panel B:

Coomassie-blue-stained 10 % SDS-PAGE analysis for monitoring the

purification of His-Bm-DNAPOL on an Ni-NTA agarose column. The

lanes from left to right are as follows: M, protein maker in kDa; Bc,

lysates; Ft, flow-through; W1 and W7, washing fractions; E1-E5,

eluted fractions. The position of His-Bm-DNAPOL is indicated by an

arrow. Panel C: western blot analysis for monitoring the purification

of His-Bm-DNAPOL on an Ni-NTA agarose column, with the lanes

exactly the same as in panel B. Panel D: Coomassie-blue-stained

10 % SDS-PAGE analysis for the purification of His-Bm-DNAPOL

on a Mono Q column. The lanes from left to right are as follows: M,

protein maker in kDa; Bc, the dialyzed combination of eluted

fractions (E1-E5) from Ni-NTA agarose column; 5-20, eluted

fractions. The position of His-Bm-DNAPOL is indicated by an arrow

L. Liu et al.

123

Analyzer (AB SCIEX, USA). Four peptide fragments

(further analysis after a first custom MALDI-TOF Bio-

Numerics experiment type) were identified, with a high

MOWSE score of 401, and matched against the deduced

amino acid sequence of Bm-DNAPOL (Fig. 2).

Although Bm-DNAPOL was eluted as a major compo-

nent, a number of contaminants were observed (Fig. 1B).

In order to eliminate these contaminants from the prepa-

ration on Ni-NTA agarose beads, a further purification was

performed using a Mono Q column. The eluted peak

fractions from the Ni-NTA column were combined, dia-

lyzed with TGEED buffer, and loaded onto a 1-ml Mono Q

5/5 column. The fractions, eluted with 10 bed volumes of a

linear gradient of NaCl from 0.1 to1 M in TGEED buffer,

were run on a 10 % SDS-PAGE gel and stained with

Coomassie blue as shown in Fig. 1D. The peak activities of

Bm-DNAPOL were eluted between 250 and 350 mM NaCl

in TGEED buffer. Corresponding to the peak activities, the

peak fractions of the Bm-DNAPOL enzyme were eluted

between fractions 7 and 10 as the only bands, while all

contaminants were separated after fraction 11 (Fig. 1D).

Thus, a highly pure Bm-DNAPOL with His-tagged

fusion protein was prepared by this procedure. From about

500 ml of infected Sf-9 cells (about 1 9 109 cells), as much

as 0.4 mg of Bm-DNAPOL protein was obtained.

Molecular weight determination by Superose 6 gel

filtration shows that Bm-DNAPOL is a monomer

Normally, DNA polymerases of yeast or mammalians exist

as a complex consisting of multiple subunits. To determine

the oligomeric state of Bm-DNAPOL in infected Sf-9 cells,

the molecular weight of purified Bm-DNAPOL was esti-

mated by comparison to protein standards by Superose 6

gel filtration using a logarithm plot of its molecular weight

versus elution volume.

Fig. 2 MALDI spectra of

tryptic digests of Bm-DNAPOL.

The identified protein, score,

amino acid sequence coverage,

and number of identified

peptides are shown. The

sequences of identified peptides

(further analysis after a first

custom MALDI-TOF BioNu-

merics experiment type) shown

in bold red covered 6 % of the

deduced amino acid sequence of

Bm-DNAPOL

Bombyx mori nucleopolyhedrovirus DNA polymerase

123

A 250-ll sample from Mono Q peak fraction 9 (Fig. 1D)

was passed through a Superose 6 column. Analysis by 10 %

SDS-PAGE with Coomassie blue staining (Fig. 3B), western

blot analysis (Fig. 3C), and activity assays of the collected

fractions (Fig. 3A) showed that Bm-DNAPOL was eluted

around peak fraction 78. The estimated molecular weight

was about 110,000 based on a calibration curve as shown in

Fig. 3D, indicating that the Bm-DNAPOL expressed and

assembled in infected Sf-9 cells is a monomer.

Enzymatic characterization of Bm-DNAPOL

Optimization of reaction conditions for Bm-DNAPOL

activity on poly(dA)/oligo(dT) template/primer

To determine the biochemical properties of the purified

recombinant BmNPV DNA polymerase, enzymatic assays

were carried out under different reaction conditions. First,

the optimal conditions for the DNA polymerase activity

were tested using a poly(dA)/oligo(dT) template/primer.

The activity of Bm-DNAPOL was examined at various

reaction temperatures ranging from 20 �C to 37 �C. As

shown in Fig. 4A, Bm-DNAPOL was most active at 27 �C

and decreased by about 50 % and 30 % at 20 �C and 37 �C,

respectively, compared with the highest activity at 27 �C.

The effect of pH on Bm-DNAPOL activity was mea-

sured in the range of pH 6.5 and pH 10.0. As shown in

Fig. 4B, Bm-DNAPOL was most active at pH 8.0. The pH

optimum for the enzyme activity was found to be in the

range of pH 8.0-pH 10.0, and only about 30 % of the

maximal activity was observed at pH 6.5.

The effect of salt concentration on Bm-DNAPOL

activity was investigated by increasing the concentration of

KCl in the reaction buffer. As shown in Fig. 4C, no sig-

nificant effect of KCl concentration on Bm-DNAPOL

activity was observed within the range of 0-50 mM.

However, increasing the KCl concentration to 100 mM

resulted in a decrease in activity of about 40 %, and 200

mM KCl or more led to an almost total loss of activity.

The dose dependence and time course of enzyme

activity were also examined under optimized conditions.

About 50 fmol of enzyme resulted in a saturation level of

the activity, and a 30-min reaction time brought the activity

to 92 % of the saturation level. Defining of one unit as the

incorporation of 1 nmol of dTMP per hour at 27 �C, the

specific activity of isolated recombinant Bm-DNAPOL was

calculated to be 15,126 units/mg.

Effect of DNA polymerase inhibitors on Bm-DNAPOL

activity

The influence of selected inhibitors on the DNA

polymerase activity of Bm-DNAPOL was investigated.

The concentration dependence of the inhibition of

Fig. 3 Determination of molecular weight by size-exclusion chro-

matography. A 250-ll sample from Mono Q peek fraction 9 was

passed through a Superose 6 column pre-calibrated with molecular

weight standards. Panel A: The polymerase activity of collected

fractions across the peak was measured by poly(dA)/oligo(dT) assay.

The vertical axis indicates the activities as incorporated dTMP in

cpm, and the horizontal axis shows the Bc (starting material) and

fraction numbers. Panels B and C: Coomassie-blue-stained 10 %

SDS-PAGE and western blot for collected fractions as shown in Panel

A. Lane M, protein maker in kDa; the position of Bm-DNAPOL is

indicated by an arrow. Panel D: The diagram shows the calibration of

the column that was used to estimate the molecular weight. The

standards used here were as follows: a, thyroglobulin (667,000); b,

ferritin (445,000); c, aldolase (158,000); d, bovine serum albumin

(67,000); e, ribonuclease A (13,700). The position of eluted

recombinant Bm-DNAPOL is indicated by a vertical arrow. A

calibration curve was plotted by the logarithm of molecular weights

versus calculated Kav

L. Liu et al.

123

Bm-DNAPOL activity by DMSO is shown in Fig. 4D.

When the concentration of DMSO was increased to 20 %

(v/v), Bm-DNAPOL activity gradually decreased to about

one-third.

The Bm-DNAPOL was sensitive to the tetracyclic dit-

erpenoid aphidicolin, which competes with each dNTP for

binding to DNA polymerase. This compound strongly

inhibits DNA replication of eukaryotic family B DNA

polymerases. As shown in Fig. 4E, treatment with 0.25 lg

aphidicolin per ml resulted in a loss of 45 % of the activity,

while the Bm-DNAPOL activity decreased to about 20 %

at an aphidicolin concentration of 1 lg/ml.

Bm-DNAPOL behaves in the manner of PCNA-independent

DNA polymerase

PCNA was originally identified as a processivity factor for

DNA polymerase d (Pol d) that functions as a molecular

sliding clamp and stimulates Pol d activity, playing a

crucial and versatile role in many DNA transactions [40].

Fig. 4 Enzymatic characterization of highly purified recombinant

Bm-DNAPOL. Effects of different components and conditions were

assayed using poly(dA)/oligo(dT) as a primer/template. The standard

assays, as described in ‘Materials and methods’, were carried out with

about 0.2 units of purified enzyme at different (A) temperatures and

(B) pH values or in the presence of various concentrations of (C) KCl,

(D) DMSO and (E) aphidicolin. Panel F indicates that PCNA

stimulation was tested with both host PCNA (BmPCNA) and

AcMNPV PCNA (AcPCNA). Samples in which PCNA was omitted

were included as negative controls. Stimulation of human Pol d by

human PCNA was included as a positive control

Bombyx mori nucleopolyhedrovirus DNA polymerase

123

To examine whether Bm-DNAPOL activity requires

PCNA, we tested the effect of PCNA from different

sources on Bm-DNAPOL activity. As shown in Fig. 4F,

neither PCNA from the host (Bombyx mori) nor PCNA

from AcMNPV stimulated Bm-DNAPOL activity on

poly(dA)/oligo(dT) template/primer, even when the con-

centration of PCNA was increased to 1600 ng in the

reaction. Human PCNA, as a positive control in the assays,

stimulated the activity of human DNA polymerase delta

(hPol d) near 20 times, and as little as 100 ng of human

PCNA led to a saturating level of activity.

Next, we tested the effect of PCNA treatment on Bm-

DNAPOL elongation activity on a single-stranded M13

DNA template. This assay is a standard one that is widely

used to assess the ability of DNA polymerases to carry out

processive DNA synthesis. It requires the loading of PCNA

by RFC in the presence of SSB. According to the optimal

conditions for this holoenzyme assay [35, 47], the reaction

mixture was supplemented with 80 ng of S. cerevisiae

RFC, 200 ng of SSB and increasing amounts of PCNA

(AcPCNA or BmPCNA) ranging from 0 to 800 ng per

reaction. Surprisingly, neither host PCNA nor AcMNPV

PCNA appeared to stimulate Bm-DNAPOL synthesis

activity. As shown in Fig. 5A, there was no stimulation of

Bm-DNAPOL elongation activity observed when AcPCNA

(column 2) or BmPCNA (column 3) was included, even

when the amount of PCNA in the reaction was increased to

800 ng, while only 100 ng of human PCNA in the reaction

could lead to a saturating level of human Pol d activity

(column 1), which is consistent with the observations in

poly(dA)/oligo(dT) assays.

The responses of Bm-DNAPOL to SSB, RFC, and

PCNA were examined in the same assay as shown in

Fig. 5B. RFC also appeared to have no effect on the syn-

thesis activity of Bm-DNAPOL, because it was not nec-

essary for PCNA to be loaded onto replication fork in this

situation. A twofold stimulation of activity was observed

with SSB alone or in concert with PCNA and RFC,

implying that it is necessary for SSB to be coated onto

ssDNA in this assay.

Thus, Bm-DNAPOL behaves as a PCNA-independent

DNA polymerase, both on poly(dA)/oligo(dT) template/

primer and on singly primed M13 DNA.

Discussion

BmNPV is a viral agent that causes deadly grasserie disease

in silkworms and is therefore harmful to sericulture. The

BmNPV genome was sequenced by Gomi et al [18]. and

found to share a high degree of homology with those of

other NPVs. The DNA polymerases encoded by viruses

play a central role in the invasion of their hosts, ensuring

the faithful and efficient reproduction of the progeny virus.

Although the basic characterizations of virus-encoded

DNA polymerases, for example, of AcMNPV [19, 30] or

SpliNPV [20], have been extensively studied, little is

known about features of BmNPV DNA polymerase, with

Fig. 5 Measurement of Bm-DNAPOL synthetic activities by M13

assay. Analysis of DNA activity by recombinant Bm-DNAPOL

holoenzyme on primed M13 DNA is described in ‘Materials and

methods’. Panel A: A direct side-by-side comparison of highly

purified recombinant Bm-DNAPOL with the human Pol d enzyme

purified from the hemolymph of infected silkworm larvae in response

to PCNA. The lanes in column 1 show the stimulation of human Pol dby human PCNA. The lanes, from left to right, show 0, 25, 50, 100,

400, and 800 ng per reaction. The lanes in columns 2 and 3 show the

responses of Bm-DNAPOL to the same amounts of AcPCNA and

BmPCNA as those in column 1. The vertical axis indicates the

synthetic activity as incorporated dTMP in cpm. Panel B: The

responses of Bm-DNAPOL to SSB, RFC, and PCNA in the M13

assay. The vertical axis indicates the relative activity (in %), and the

horizontal axis shows the reaction in the presence of Bm-DNAPOL

together with, from left to right, SSB, BmPCNA, AcPCNA, RFC only,

holoenzyme without SSB, and holoenzyme. The activity in the

reaction containing Bm-DNAPOL alone was taken as 100 %

L. Liu et al.

123

only an early description of the nucleotide sequence and

transcriptional analysis for this gene [9]. The AcMNPV

polymerase has been shown to possess processivity and

moderate strand-displacement activity [30]. The SpliNPV

DNAPOL has both DNA polymerase and 30-50 exonuclease

activity, and the deletion of the first 80 amino acid residues

does not affect these enzymatic activities [20]. In order to

understand the relationship between BmNPV and Bombyx

mori for the selection of completely BmNPV-resistant

strains, there is an urgent need to study the functions of this

enzyme, which is encoded by the BmNPV ORF53 gene.

Our initial efforts to study the enzymology of BmNPV

DNA polymerase were unsuccessful because of the

unavailability of soluble active enzyme. We attempted to

overexpress soluble GST- or His-tagged Bm-DNAPOL

protein in E. coli using several bacterial expression vectors,

such as pET-28c(?) (Novagen) and pGEX-5X-3 (GE

Healthcare), and in insect cells using the Bac-to-Bac Bac-

oluvirus Expression System (Invitrogen), but none of these

resulted in successful high-level expression of protein in a

soluble form. Alternatively, we chose the novel MultiBac

expression system [6, 47] to generate recombinant Bm-

DNAPOL. This system includes two important elements:

transfer vectors (pFBDM and pUCDM) and an engineered

baculovirus genome (MultiBac). The transfer vector

pFBDM contains two expression cassettes in a head-to-head

arrangement with multiple cloning sites (MCS1 and MCS2)

flanked by polh or p10 promoters and SV40 or HSVtk polyA

signal sequences, respectively. It is particularly suited for

generating multigene expression cassettes. In the engineered

baculovirus genome, two baculoviral genes, v-cath which

encodes the viral protease V-CATH, which is activated upon

cell death by a process dependent on a juxtaposed gene on

the viral DNA, and chiA, which encodes a chitinase, were

disrupted. Therefore, the quality of proteins produced by this

system is significantly improved through a reduction in

virus-dependent proteolytic activity and reduced cell lysis.

We successfully produced a four-subunit complex of human

polymerase delta using this system [47]. Here, we have

constructed and expressed Bm-DNAPOL in Sf-9 insect cells

using this novel system. The infection time was extended

from the normal 48 hours to 72 hours or even more, which is

beneficial for the expression and assembly of Bm-DNAPOL

in infected Sf-9 cells with no significant cell lysis. Rigorous

isolation of the enzyme led to near homogeneity, using

purification on an Ni-NTA agarose column and further

purification on an FPLC Mono Q column (Fig. 1D, eluted

fractions 7 to 10). About 0.4 mg of soluble enzyme was

obtained from 500 ml of infected Sf-9 cells (about 1 9 109

cells).

The highly purified enzyme possessed a specific activity

of 15,126.3 units/mg, which is higher than that of AcMNPV

DNAPOL (5,332 units/mg) [19] or SpliNPV DNAPOL

(6,890 units/mg) [20]. Gel filtration analysis indicated that

our highly purified Bm-DNAPOL is a monomer with an

apparent molecular mass of 110,000 Da by comparison to

protein standards on a Superose 6 column using a logarithm

plot of molecular weight versus elution volume (Fig. 3D),

and the identity of the enzyme in the peak fraction that eluted

in fraction 78 was confirmed by DNA polymerase activity

assays (Fig. 3A), Coomassie-blue-stained SDS-PAGE gel

(Fig. 3B), and western blot analysis (Fig. 3C).

The conditions for DNA polymerase activity assays

were optimized on a poly(dA)/oligo(dT) template-primer.

The optimal pH for viral DNA replication by Bm-DNA-

POL was within the alkaline range, i.e., pH 8.0-pH 10

(Fig. 4B). The optimal reaction temperature was close to

that for larval growth (Fig. 4A). The optimal salt concen-

tration was found to be 0-50 mM KCl (Fig. 4C). For further

characterization of highly purified BmNPV viral DNA

polymerase, the effects of different chemical compounds

on its activity were examined using a poly(dA)/oligo(dT)

template-primer. DMSO is frequently used as a solvent for

antifungal drugs for the determination of their minimal

inhibitory concentration [15, 16, 24, 43]. It has also been

used in the formulation of idoxouridine, adenine arabino-

side, acyclovir and cidofovir [1, 17, 38]. There are differ-

ential effects of DMSO on the activities of mammalian

DNA polymerase a (Pol a) and d (Pol d). The activity of

DNA polymerase a is inhibited by DMSO, whereas DNA

polymerase d is significantly activated. In our assays, Bm-

DNAPOL was apparently inhibited by DMSO (Fig. 4D).

Another compound, aphidicolin, was tested in this study.

Aphidicolin is a tetracyclic diterpenoid antibiotic that has

been shown to be a potent inhibitor of eukaryotic DNA

polymerases a and d as well as DNA polymerases encoded

by several large DNA viruses such as herpes virus and

vaccinia virus [34, 37]. We showed that aphidicolin is an

efficient inhibitor of Bm-DNAPOL (Fig. 4E), in agreement

with a previous report [19], demonstrating the inhibition of

AcMNPV DNA polymerase by aphidicolin.

The nucleotide sequence of an AcMNPV gene (pcna)

encoding a PCNA-like protein had been reported [12, 33],

and this protein has 42 % amino acid sequence identify to

mammalian PCNAs. Homologues of this gene code for

DNA polymerase processivity factors and have been found

to be essential in many DNA replication systems [39].

Although eukaryotic PCNA lacks an enzymatic function, it

plays a crucial and versatile role in DNA synthesis, DNA

repair, and cell cycle progression [39]. In AcMNPV, it is

not an essential gene and did not appear to elevate DNA

replication in transient replication assays [12, 23]. To test

whether our highly purified Bm-DNAPOL exhibits PCNA-

dependent DNA activity, both AcMNPV PCNA and host

Bombyx mori PCNA were used to examine its stimulation

of Bm-DNAPOL activity on both poly(dA)/oligo(dT)

Bombyx mori nucleopolyhedrovirus DNA polymerase

123

template-primer and singly primed M13 ssDNA (M13

assay). The M13 assay is a standard one that uses singly

primed M13 DNA to assess the ability of polymerase to

carry out processive DNA synthesis. This assay requires

the loading of PCNA by RFC onto the SSB-coated DNA

and allows the assessment of the ability of polymerase to

synthesize full-length M13 DNA. However, neither assay

detected any stimulation by PCNA from either source

(Figs. 4F, 5A). These results, together with the fact that a

gene encoding a PCNA-like protein is absent from the

genomes of BmNPV and Orgyia pseudotsugata MNPV,

suggest that these PCNA-like proteins, AcMNPV PCNA

and host Bombyx mori PCNA, may not be directly involved

in baculovirus DNA replication.

Based on the general model of DNA replication, bac-

uloviruses encode most of the corresponding genes that are

involved in this process. However, there are some factors

that have not been identified – for example, RFC. In all

replication systems studied thus far, DNA is synthesized by

a complex apparatus consisting of many protein compo-

nents. One of the key proteins involved in loading the

replicative polymerases to create the replication fork is

RFC, a complex of five subunits that is conserved in all

eukaryotes [10]. Functional homologues exist in bacteria,

some bacteriophages, and Archea [13]. The main role for

RFC is to load the trimeric, ring-like structure of PCNA

onto DNA at a primer-template junction or to load it onto a

nicked site in duplex DNA [27, 41, 42]. RFC-catalyzed

PCNA loading is a prerequisite for assembly of Pol d onto

the template DNA to form a processive holoenzyme [41,

42]. In this study, we demonstrated that BmNPV DNA

polymerase was not stimulated by RFC (Fig. 5B), sug-

gesting that the BmNPV replication model is not dependent

on an RFC-PCNA complex.

A component present in many DNA replication systems

is single-stranded-DNA (ssDNA) binding protein (SSB),

which coats ssDNA to prevent the formation of secondary

structure, thereby allowing DNA polymerase to access its

substrate[29, 31, 36]. In addition to its intrinsic ability to

bind ssDNA, SSB has an important role in recruiting

genome maintenance proteins to their target ssDNA

through physical interactions [36]. In this study, addition of

SSB to Bm-DNAPOL reactions significantly increased the

polymerase activity (Fig. 5B). Human RPA, replication

factor A, a kind of SSB, or SSB from other sources was

able to substitute for viral SSB (LEF-3) even though they

do not share any sequence homology to LEF-3 and are very

different in structure and size. This finding suggests that

SSB (LEF-3) might play an important role in BmNPV

DNA polymerase activity in viral infection.

In summary, we have made a major advance in the

expression of Bm-DNAPOL using a MultiBac expression

system. Rigorous isolation of the enzyme led to near

homogeneity by purification on Ni-NTA agarose and ion-

exchange chromatography. About 0.4 mg of enzyme was

obtained from 500 ml of infected Sf-9 cells. Character-

ization of the highly purified enzyme indicated that

BmNPV polymerase is a monomer. It behaves as a PCNA-

independent DNA polymerase on both poly(dA)/oligo(dT)

primer/template and singly primed M13 DNA. BmNPV

replication may be not dependent on an RFC-PCNA-like

complex, and SSB might play an important role in BmNPV

DNA replication. These findings will be significant for

studies of disease in silkworms caused by BmNPV and

using silkworms as a bioreactor for the production of

biomolecules of commercial importance.

Acknowledgments We wish to thank Dr. Timothy J. Richmond for

providing the vector pFBDM, and Dr. Gregory Bowman for the

generous gift of S. cerevisiae RFC. This research was supported by

the National Natural Science Foundation of China (31100118,

30970612), Natural Science Foundation of Jiangsu Province

(BK2011495), China Postdoctoral Science Foundation

(20110491361), Natural Science Foundation of the Higher Education

Institutions of Jiangsu Province (09KJB180001) and Startup Scientific

Research Fund from Jiangsu University for Advanced Professionals

(09JDG002, 09JDG006).

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