Graduate Program in Genetics1 and Department of Entomology2, University of California, Riverside, CA 92521, USA
Author for correspondence: Brian Federici (at Department of Entomology). Fax +1 909 787 3086 or 3681. e-mail brian.federici{at}ucr.edu
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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 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, IIVI) of these proteins and those of enzymes that unwind duplex-DNA (Lu & Carstens, 1991 ; Gorbalenya et al., 1988
, 1989a
, 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 non-specifically (Laufs et al., 1997
). However, the two most important characteristics of the DNA helicases, the ability to hydrolyse ATP and unwind duplex DNA (Matson & Kaiser-Rogers, 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 non-denaturing imidazole-based buffers and nickelnitrilotriacetic acid (NiNTA) 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 °C in 500 ml of TED buffer (10 mM TrisHCl, pH 7·0, 1·0 mM EDTA, 1 mM DTT) and stored at -70 °C in storage buffer (10 mM TrisHCl, pH 7·0, 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 °C 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).
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We do not know why the ATPase activity of rP137 decreased substantially in the presence of TnGV and DNAs (Fig. 2B
). One possibility is that the decreased activity was due to the sequestration of Mg2+ 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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References |
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Received 12 January 2000;
accepted 3 March 2000.