Department of Biochemistry, Oxford University, Oxford OX1 3QU, UK1
NERC Institute of Virology and Environmental Microbiology, Mansfield Road, Oxford OX1 3SR, UK2
Department of Geographic Medicine, University of Alabama at Birmingham, Birmingham, AL 35294, USA3
Author for correspondence: Polly Roy (at NERC Institute of Virology and Environmental Microbiology). Fax +44 1865 281696. e-mail por{at}wpo.nerc.ac.uk
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Abstract |
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Main text |
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NS2, the 41 kDa product of the S8 gene, binds ssRNA but does not bind dsRNA (Thomas et al., 1990 ). It is the only phosphorylated BTV protein. The effect of phosphorylation on its RNA-binding capability has been investigated: Thomas et al. (1990)
reported that there was no reduction in RNA binding when baculovirus-expressed NS2 was dephosphorylated, while Theron et al. (1994)
used E. coli-expressed NS2 and observed a reduction, but not complete elimination, of RNA binding. NS2 forms viral inclusion bodies (VIBs) in both BTV-infected mammalian cells (Brookes et al., 1993
) and insect cells when expressed from a recombinant baculovirus (Thomas et al., 1990
), suggesting that neither other viral proteins nor a full complement of viral RNAs is necessary for VIB development. Neither the amino nor carboxy terminus of NS2 is required for VIB formation (Zhao et al., 1994
). VIBs are believed to be the location at which virus assembly occurs.
In order to characterize NS2 biochemically, we obtained highly purified protein from recombinant baculovirus-infected insect cells. Spodoptera frugiperda cells, grown in suspension in TC100 medium supplemented with 5% foetal calf serum, were infected at an m.o.i. of 5 with a recombinant baculovirus (AcBTV10-NS2; Thomas et al., 1990 ) that expresses NS2 protein. Three days after infection, the cells were harvested by centrifugation at 2400 g for 10 min. The pellet was washed once with PBS and resuspended in NS2 lysis buffer [0·1 M NaH2PO4Na2HPO4 buffer (NaPi) pH 8·0, 0·1 M NaCl, 1 mM EDTA, 1 mM DTT, 0·5% Triton X-100, 1 mM Pefabloc SC (Pentapharm AG), 1 mM 4-amidinophenylmethanesulphonyl fluoride, 10 µM E-64], using 5 ml per 100 ml culture fluid. Cells were lysed by ten strokes of a Dounce homogenizer and cell debris was pelleted at 60000 r.p.m. for 45 min in a Ti70 rotor at 10 °C.
The supernatant was passed over a Q-Sepharose column (Pharmacia) equilibrated with 0·1 M NaCl, 1 mM EDTA (pH 8·0) and the protein was eluted by a gradient of 0·10·5 M NaCl plus 1 mM EDTA (pH 8·0). Fractions above 0·25 M NaCl were analysed by SDSPAGE (Fig. 1A). The peaks were pooled, diluted 1:4 with 0·1 M NaPi pH 8·0, 1 mM EDTA and passed over a heparin column (Pharmacia) equilibrated with 25 mM NaPi pH 8·0, 0·1 M NaCl, 1 mM EDTA. The column was washed with 25 mM NaPi pH 8·0, 0·1 M NaCl, 1 mM EDTA. A step gradient of 0·4, 0·5 and 1·0 M NaCl in 25 mM NaPi pH 8·0, 1 mM EDTA was applied and fractions were collected. Fractions were analysed by SDSPAGE (Fig. 1 B
). At this stage, the expressed NS2 protein was highly concentrated (approx. 1·52·0 mg/ml). A final purification step was added, exploiting the RNA-binding ability of NS2, to obtain highly purified protein. Pooled peaks, diluted 1:8 with 25 mM NaPi pH 8·0, 1 mM EDTA, were passed over a poly(U) column [prepared in-house from Sigma poly(U) agarose] equilibrated with 25 mM NaPi pH 8·0, 0·1 M NaCl, 1 mM EDTA. The column was washed with 25 mM NaPi pH 8·0, 0·1 M NaCl, 1 mM EDTA. A step gradient of 0·4, 0·5 and 1·0 M NaCl in 25 mM NaPi pH 8·0, 1 mM EDTA was applied and fractions were collected. Fractions were analysed by SDSPAGE (Fig. 1C
) and concentrated by using Centricon-10 (Ambion) centrifuge concentrators with a molecular mass cut-off of 10 kDa. Faint bands of approx. 26 kDa observed in the final preparation (Fig. 1C
, lanes 57) were identified by Western blot as NS2 degradation products (data not shown). Degradation products observed in Fig. 1(B)
lanes 5 and 6 were also identified by Western blot as NS2. Aliquots of NS2 were stored at -70 °C. Typical yields were 1 mg NS2 per 100 ml culture medium.
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Both the helicase and guanylyltransferase activities of BTV have been localized to specific proteins and therefore the significance of ATP binding by NS2 was not immediately apparent. Phosphohydrolase activity relies on the ability of a protein to bind NTPs. To test for phosphohydrolase activity associated with NS2, a TLC assay was used (Martinez-Costas et al., 1998 ). Purified NS2 (1 µg) was incubated at 37 °C for 30 min with 1 µCi [
-32P]ATP in buffers and salts as indicated in the legend of Fig. 2(A)
. The reaction was stopped by the addition of an equal volume (10 µl) of 10% TCA. The products were separated by TLC by spotting 1·5 µl of the reaction onto a PEIcellulose plate (Merck) along with known standards and the plate was developed in a TLC tank with 0·75 M KH2PO4 as the ascending phase. The results indicated that purified NS2 can hydrolyse ATP to ADP and AMP (Fig. 2A
, lanes 3 and 4). As observed for all other NTPases, the reaction requires a divalent cation, in this case Mg2+. The reducing agent DTT did not appear to influence the reaction (Fig. 2A
, lanes 1 and 2).
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To characterize further the binding of ATP and GTP by NS2, aliquots of the NTPase reactions from the previous experiments were resolved by SDSPAGE under denaturing conditions. A 10 µl aliquot of each reaction was mixed with 2 µl 5x SDSPAGE sample buffer, heated to 100 °C for 5 min and run on a 10% SDSPAGE gel. The gels were transferred to Immobilon membranes before exposure to X-ray film to reduce the excessive background radiation observed in preliminary experiments. The bound label migrated as a band identical in size to NS2. The results (Fig. 2C) suggest that NS2, and not a contaminating protein, binds ATP in the presence of Mg2+ (lanes 3 and 4). Without Mg2+, NS2 was unable to bind either nucleoside (Fig. 2C
, D
; lanes 2), suggesting that the lack of hydrolysis in the TLC assay (Fig. 2 A
, B
; lanes 2) was due to the inability of NS2 to bind ATP or GTP. These experiments indicate that NS2 binds and hydrolyses both ATP and GTP but that there appears to be a difference in the hydrolysis of the two nucleosides. The binding of UTP by NS2 was also investigated in other experiments, but the level of binding was very low compared with binding of ATP and GTP, even after long exposures to X-ray film (data not shown).
A TLC assay was used to determine any preference of NS2 for the divalent cation in the phosphohydrolase reaction. Assays were set up that differed only in their divalent cation content, with either [-32P]ATP (Fig. 3A
) or [
-32P]GTP (Fig. 3B
) as the phosphate donor. The reactions contained either MgCl2, MnCl2 or CaCl2 at a concentration of 10 mM (Fig. 3A
, B
; lanes 24) or no divalent cation (Fig. 3A
, B
; lane 1). For the ATPase activity, Mn2+ ions gave the greatest hydrolysis of ATP (Fig. 3A
, lane 3). In terms of hydrolysis to ADP, Ca2+ and Mg2+ appeared to give similar results. However, there was less hydrolysis to AMP in the presence of Ca2+ than of Mg2+ (Fig. 3B
; lanes 2 and 4). With GTP, the presence of both Mn2+ and Ca2+ gave better results than Mg2+, with little difference in the low levels of GMP produced (Fig. 3B
).
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These experiments identify enzymatic activity associated with purified NS2. NTP binding and hydrolysis by NS2 represent novel findings. The results indicate that NS2 can bind and hydrolyse ATP and GTP, with a higher affinity for the former.
NTP binding and hydrolysis are indicative of a number of enzymatic activities including the mRNA capping and dsRNA helicase activities already identified in BTV. NTP hydrolysis also plays a crucial role in the function of molecular motors, proteins or protein complexes that use chemical energy from phosphate hydrolysis to generate mechanical force. In this regard, the role of the bacteriophage 6 P4 protein and its associated NTPase activity in packaging and condensation of the positive-sense RNA genome of
6 is of interest (Gottlieb et al., 1992
; Paatero et al., 1995
).
6 is a bacteriophage with a segmented dsRNA genome. This genome consists of three segments that are packaged precisely within assembled procapsids in a specific order. Energy is required for this packaging, and the NTPase activity of P4, a component of the polymerase complex, is thought to provide this energy (Paatero et al., 1995
).
NS2 has a possible functional homology with the rotavirus non-structural protein NSP2. NS2 forms 7S multimers (Uitenweerde et al., 1995 ), while NSP2 forms 10S multimers, and both proteins bind RNA (Kattoura et al., 1992
, 1994
). Recently, NSP2 was shown to be phosphorylated and to have an NTPase activity (Taraporewala et al., 1999
). NSP2 catalyses the hydrolysis of each of the four NTPs to NDPs. Although NS2 expressed by itself can form VIBs, NSP2 assembles into viroplasms only in infected cells. Another rotavirus non-structural protein, NSP5, localizes to viroplasms in infected cells but not when transfected alone, a feature that it shares with NSP2 (Kattoura et al., 1994
; Poncet et al., 1997
). Both proteins are required for viroplasm assembly. NSP2 and NSP5 interact with each other and the detection of multiply phosphorylated forms of NSP5 has been shown to correlate with localization to viroplasms (Poncet et al., 1997
). Also, although NSP5 alone can bind poly(U) and ssRNA, binding is enhanced by the presence of NSP2 (Mattion et al., 1994
). Additionally, NSP2 is a component of the rotavirus replicase complex (Aponte et al., 1996
).
Taken together, it appears that BTV NS2 combines most of the features of rotavirus NSP2 and NSP5. Although there is no apparent sequence similarity between the three proteins (Horscroft, 1998 ), the functions performed by these proteins are clearly analogous. The NTPase activity described for both NSP2 and NS2 may play a role in providing energy for the assortment, movement or packaging of the ssRNA that they each bind, therefore making them critical in virus replication and assembly.
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Footnotes |
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References |
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Received 24 February 2000;
accepted 26 April 2000.