Download DNA-independent ATPase activity of the Trichoplusia ni

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of General Virology (2000), 81, 1601–1604. Printed in Great Britain
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SHORT COMMUNICATION
DNA-independent ATPase activity of the Trichoplusia ni
granulovirus DNA helicase
D. K. Bideshi1 and B. A. Federici1, 2
Graduate Program in Genetics1 and Department of Entomology2, University of California, Riverside, CA 92521, USA
DNA helicases of baculoviruses are essential for
virus replication and have been implicated as
molecular determinants of host range. Although
these proteins contain seven motifs (I, Ia, II–VI)
characteristic of DNA helicases, the two most important characteristics of helicases – duplex-DNA
unwinding and ATPase activity – have not been
demonstrated. In the present study, a recombinant
putative DNA helicase (rP137) of Trichoplusia ni
granulovirus (TnGV) was purified from insect cells
infected with a recombinant Autographa californica
multicapsid nucleopolyhedrovirus that overproduced rP137. The rP137 protein exhibited an
intrinsic DNA-independent ATPase activity that
required Mg2M as a co-factor, an activity that was
reduced in the presence of TnGV and phage λ DNAs.
These results provide further evidence that baculovirus helicase genes encode proteins with biochemical properties similar to those of classical
DNA helicases.
The family Baculoviridae is composed of a large and diverse
group of circular dsDNA occluded viruses isolated primarily
from lepidopteran insects (Martignoni & Iwai, 1986 ; Volkman
et al., 1995). Two groups are recognized based on occlusion
body morphology : the nucleopolyhedroviruses (NPVs) and
granuloviruses (GVs). Though the NPVs and GVs are related phylogenetically (Zanotto et al., 1993 ; Cowan et al.,
1994 ; Bulach et al., 1999), members within each group vary in
their host range, tissue tropism and pathobiology (Federici,
1997). The genetic determinants and molecular mechanisms
responsible for these differences are largely unknown.
In this regard, studies on baculovirus putative DNA
helicases are of considerable interest because these enzymes
are essential for virus replication and potentially play a
significant role in baculovirus host range. For example, in the
Author for correspondence : Brian Federici (at Department of
Entomology). Fax j1 909 787 3086 or 3681.
e-mail brian.federici!ucr.edu
0001-6920 # 2000 SGM
accompanying paper (Bideshi & Federici, 2000) we report that
Trichoplusia ni (Tn)GV helicase does not support replication of
Autographa californica (Ac)MNPV in cells and larvae of T. ni.
However, changes in only one or a few amino acids in the
helicase (P143) of AcMNPV enable this virus to replicate in
cells and larvae of Bombyx mori, where its replication is
normally restricted (Maeda et al., 1993 ; Croizier et al.,
1994 ; Kamita & Maeda, 1997 ; Argaud et al., 1998). In addition,
P143 is required for replication of reporter plasmids in transient
assays (Lu & Miller, 1995 ; Kool et al., 1995 ; Ahrens &
Rohrmann, 1996 ; Heldens et al., 1997), results which support
earlier studies suggesting that P143 is an essential component
of the AcMNPV replisome (Lu & Carstens, 1991).
The classification of baculovirus helicases as DNA helicases
is based on similarities between several conserved amino acid
motifs (I, Ia, II–VI) of these proteins and those of enzymes that
unwind duplex-DNA (Lu & Carstens, 1991 ; Gorbalenya et al.,
1988, 1989 a, b ; Hodgeman, 1988 ; Matson & Kaiser-Rogers,
1990 ; Linder et al., 1989 ; Saraste et al., 1990 ; Walker et al.,
1982). AcMNPV P143 has been shown to bind DNA nonspecifically (Laufs et al., 1997). However, the two most
important characteristics of the DNA helicases, the ability to
hydrolyse ATP and unwind duplex DNA (Matson & KaiserRogers, 1990), have not been demonstrated for any baculovirus
helicase.
The objective of the present study was to determine
whether the putative DNA helicase (P137 ; Bideshi et al., 1998)
encoded by TnGV had ATPase or DNA binding and
unwinding activities. In the present study we show that a
recombinant P137 (rP137) has an intrinsic DNA-independent
ATPase activity, an enzymatic function associated with
helicase motifs I and II (Hodgeman, 1988 ; Linder et al.,
1989 ; Matson & Kaiser-Rogers, 1990 ; Gorbalenya et al., 1988).
However, we were unable to demonstrate either DNA binding
or unwinding activity for TnGV P137.
To study TnGV P137, the gene ( p137) encoding this
putative helicase was hyper-expressed in BTI-TN-5B1-4 cells
(Invitrogen) using the Bac-to-Bac HT AcMNPV Baculovirus
Expression (Bacmid) system (Gibco BRL). To facilitate purification, codons for a histidine tag were added to the 5’ end of
the gene. The histidine-tagged recombinant P137 protein
(rP137) was purified by column chromatography using nondenaturing imidazole-based buffers and nickel–nitrilotriacetic
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BGAB
D. K. Bideshi and B. A. Federici
Fig. 1. Purified recombinant helicase (rP137) of TnGV. The rTEv proteasetreated (j) and untreated (k) samples were subjected to
electrophoresis to demonstrate purity in a 12 % SDS–polyacrylamide gel
stained with silver.
acid (Ni–NTA) resin (Gibco BRL). After purification, two
proteins (Fig. 1), a 140 kDa protein (rP137 including the
histidine tag) and a 137 kDa protein (rP137 protein from which
the histidine tag had been removed by cleavage with rTEv
protease from Gibco BRL), were dialysed at 4 mC in 500 ml of
TED buffer (10 mM Tris–HCl, pH 7n0, 1n0 mM EDTA, 1 mM
DTT) and stored at k70 mC in storage buffer (10 mM
Tris–HCl, pH 7n0, 2 mM EDTA, 2 mM DTT, 5 mM NaCl,
5 mM KCl, 20 % glycerol). A 100 ng sample of each
preparation was used in ATPase and helicase assays (Pause et
al., 1993 ; Matson & Kaiser-Rodgers, 1990). Removal of the
histidine tag with the rTEv protease, and incubation of rP137
at 37 mC for at least 30 min before performing the ATPase
assay, significantly lowered enzymatic activity (data not
shown). Therefore, the results reported here are based on
assays performed with the histidine-tagged P137 (rP137).
Like all DNA helicases, the ATPase activity of rP137
required Mg#+ as a co-factor, with optimal enzymatic activity
occurring in the presence of 5 mM Mg#+ (Fig. 2 B). However,
unlike many DNA helicases (Matson & Kaiser-Rogers, 1990),
rP137 activity did not require the presence of DNA in the
assay (Fig. 2). Furthermore, ATPase activity was inhibited
substantially in the presence of 10 ng of TnGV DNA. The
maximal ATPase activity of rP137, for example, was 8-fold
greater in the absence of TnGV DNA (1n17i10& versus
1n48i10% c.p.m. ; Fig. 2 B). A similar result was observed when
the assay was performed with 10 ng of phage λ DNA
(Fig. 2 B).
BGAC
Fig. 2. ATPase activity of the recombinant TnGV helicase, rP137.
(A) Results of assays performed at 37 mC with 2n5 mM magnesium acetate
in the presence (j) or absence (k) of TnGV rP137. (B) Results of
assays with different concentrations of magnesium acetate and 10 ng of
TnGV or λ DNAs.
The DNA-independent ATPase activity of rP137 has
several possible explanations. The conformation of this enzyme
may have been altered by either the presence of the histidine
tag or the method of purification, thereby exposing the
catalytic site for ATP hydrolysis. Alternatively, the activity
could be an intrinsic property of rP137. There is precedence for
this in that at least two DNA helicases are known which
exhibit DNA-independent ATPase activity : bovine papillomavirus type 1 (BPV-1) E1 and simian virus (SV) 40 large T
antigen. The BPV-1 E1 protein requires ATP to unwind DNA
(Seo et al., 1993 a, b), but exhibits ATPase activity in the
absence of DNA (Santucci et al., 1995 ; Bream et al.,
1993 ; Hughes & Romanos, 1993). The multifunctional SV40
large T antigen hydrolyses ATP in the absence of DNA, and
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TnGV p137 encodes an ATPase
though this activity is enhanced 5-fold in the presence of
poly(dT), other nucleic acids have a minimal effect (Giachero &
Hagar, 1979 ; Clark et al., 1981).
We do not know why the ATPase activity of rP137
decreased substantially in the presence of TnGV and λ DNAs
(Fig. 2 B). One possibility is that the decreased activity was due
to the sequestration of Mg#+ by viral DNA. Another possible
explanation is that rP137 binds DNA, but upon binding,
additional host- or TnGV-encoded protein(s) not present in
our assay were required for efficient enzymatic activity.
Although we demonstrated ATPase activity as a property of
TnGV rP137, we were unable to show that this enzyme bound
DNA or unwound partial duplex-DNA substrates. Therefore,
as suggested for P143 (Laufs et al., 1997), it is possible that
additional proteins encoded by TnGV or T. ni are required for
rP137 full helicase activity.
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