Division of Microbiology, School of Biochemistry and Molecular Biology, University of Leeds, Leeds LS2 9JT, UK1
Hannah Research Institute, Ayr KA6 5HL, UK2
Author for correspondence: Mark Harris. Fax +44 113 233 5638. e-mail mharris{at}bmb.leeds.ac.uk
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Abstract |
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Introduction |
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Cyclic AMP (cAMP)-dependent protein kinase (PKA) is a family of isozymes involved in signal transduction in all eukaryotic cells (Walsh & Van Patten, 1994 ; Francis & Corbin, 1994
). The catalytically inactive PKA holoenzyme is a heterotetrameric molecule; in most cells its localization is predominantly cytosolic, although a proportion of cellular PKA is anchored to intracellular membranes and cytoskeletal elements (Edwards & Scott, 2000
). It is composed of two catalytic (C-) subunits and two regulatory (R-) subunits. Two types of R-subunit (RI and RII) define the two major sub-classes (type I and type II) of the PKA holoenzyme. Each R-subunit has two high-affinity binding sites for cAMP, binding of which results in the dissociation of the dimeric R-subunit from the catalytically active C-subunit monomers. Thus the activation status of PKA is primarily a function of the cAMP concentration this is generally elevated by the activity of G-protein-coupled adenylate cyclase. Once dissociated from the holoenzyme, free PKA C-subunit can diffuse through the cytosol or into the nucleus catalysing the phosphorylation of cognate substrates. In either of these locations, PKA C-subunit may encounter a member of a family of heat-stable inhibitor proteins PKI (Olsen & Uhler, 1991
). These bind to PKA C-subunit with high affinity (Kd=2·3 nM), promote its export from the nucleus and inhibit catalytic activity through occupancy of the catalytic site with a pseudo-substrate motif. Whereas RI-subunits employ a similar inhibitory mechanism, the motif in RII that blocks the catalytic site is a phospho-acceptor substrate, such that RII-subunits are phosphorylated during association with PKA C-subunits. PKA activation affects both the regulation of key metabolic enzymes and the expression of numerous target genes.
Within NS3 a short arginine-rich motif (HCV polyprotein residues 14871500), described by Borowski et al. (1996) , shows similarities to both the inhibitory site of PKI and the RII phosphorylation site. Synthetic peptides corresponding to this NS3 sequence, as well as a catalytically inert fragment of NS3 fused to glutathione S-transferase, were shown to inhibit the phosphorylation of different protein substrates by PKA C-subunit in vitro (Borowski et al., 1996
). Moreover, it was shown that this NS3 fragment could inhibit PKA C-subunit-mediated histone phosphorylation in vivo (Borowski et al., 1997
). Although these data appear to show a pseudo-substrate inhibitor-like action of NS3 on PKA C-subunit, the detailed mechanism of these effects is not well-understood and the possible role of ATP sequestration or hydrolytic depletion by NS3 has not been fully explored. In this regard, NS3 belongs to the family of DExH RNA helicases. As such, it contains four conserved regions common to these helicases: motifs I (GxGKS), II (DExH) and III (TAT box) are required for ATP binding and hydrolysis, and motif VI (QRXGRXGR) is involved in ATP hydrolysis and RNA unwinding (Kadare & Haenni, 1997
). Interestingly, motif VI coincides with the arginine-rich motif discussed above. Together, these observations highlight the importance of investigating the effect of native full-length NS3 to fully elucidate its role in PKA modulation. Here, we report that native full-length NS3/4A complex from both HCV and the related pestivirus bovine viral diarrhoea virus (BVDV) inhibit phosphotransferase activity of PKA C-subunit in vitro as assayed using two substrates, synthetic peptide (Kemptide) and the RII
-subunit. Furthermore, we report that mutations in the conserved helicase motifs (motifs II and VI) of HCV NS3 that eliminate NTPase activity also abolish the ability of NS3/4A to inhibit PKA C-subunit. Our data are consistent with the conclusion that inhibition of PKA C-subunit by NS3/4A is entirely due to ATP hydrolysis.
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Methods |
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Cell culture.
Cos-7 cells were cultured in DMEM supplemented with 10% foetal calf serum, 2 mM glutamine, penicillin (200 units/ml) and streptomycin (200 units/ml). Sf9 cells were cultured in TC100 medium supplemented with 10% foetal calf serum.
Construction of recombinant baculoviruses expressing the NS3/4A complex.
To generate wild-type full-length NS3/4A [NS3/4A(wt)] construct, the complete NS3/4A coding region from the H77 infectious clone of HCV genotype 1a (Yanagi et al., 1997 ) (kindly provided by Jens Bukh, NIH, Bethesda, MD, USA) was amplified by PCR using the oligonucleotide primers NS3forward and NS4Areverse (see Table 1
). To generate full-length BVDV NS3/4A, the complete NS3/4A coding region from the Oregon strain (Kummerer et al., 1998
) (kindly provided by Gregor Meyers, Tübingen, Germany) was amplified by PCR using the oligonucleotide primers BVDVforward and BVDVreverse (see Table 1
). PCR products were cloned into pCR-Blunt (Invitrogen) and, after sequence confirmation, the NS3/4A coding fragment was excised and cloned into pBacPAKHis1 (Clontech). The resulting transfer vectors were co-transfected into Sf9 cells with linearized BacPAK6 DNA (Kitts et al., 1990
) by lipofection; recombinant baculoviruses expressing NS3/4A were harvested 3 days post-transfection and were plaque purified. Expression of NS3 protein in infected Sf9 cells was verified by Western blotting.
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Construction of recombinant mammalian vectors expressing NS3/4A.
NS3/4A(wt) and NS3/4A(DE-AA) were amplified by PCR from H77 cDNA using the oligonucleotide primers NS3SG5forward and NS4ASG5reverse (see Table 1), cloned into pCR-Blunt, and sub-cloned into the expression vector pSG5 (Green et al., 1988
).
Expression and purification of 6xHisNS3/4A proteins.
NS3/4A and NS3/4A mutants were expressed and purified as previously described (Sali et al., 1998 ), with minor modifications. Briefly, Sf9 cells were infected (5 p.f.u. per cell) and harvested 60 h later by scraping and centrifugation at 1500 g for 15 min at 4 °C. Cell pellets were washed once with cold PBS and Dounce homogenized in buffer DL [50 mM sodium phosphate, pH 8, 0·3 M NaCl, 10 mM
-mercaptoethanol, 10% (v/v) glycerol, EDTA-free protease inhibitor cocktail (Boehringer)] containing 0·5% (w/v) n-dodecyl
-D-maltoside. Insoluble material was pelleted by centrifugation at 100000 g for 1 h at 4 °C. Clarified supernatant, containing about 80% of the recombinant NS3/4A, was diluted 5-fold with buffer DL lacking detergent and batch-adsorbed onto NiNTA agarose (Qiagen) for 1 h at 4 °C. NS3/4A was eluted with 3 column volumes of buffer DL containing 0·1% n-dodecyl
-D-maltoside and 250 mM imidazole. Protein concentration was estimated by Bio-Rad assay.
Radiometric assays of PKA C-subunit activity in vitro.
In vitro kinase assays were performed using two different substrates. To measure phosphorylation of the synthetic substrate, Kemptide, PKA C-subunit was used at a final concentration of 7·5 nM and the assay was performed at 30 °C as previously described (Roskoski et al., 1983 ). For RII
phosphorylation assays, the kinase reaction (50 µl) containing 12·5 nM PKA C-subunit, 7·5 mM magnesium acetate, 2 mM DTT, 50 µM [
-32P]ATP (1 µCi), 10 mM MOPS pH 7 and 5 µM RII
subunit was incubated for 3 min at 30 °C. The reaction was terminated by adding 10 µl 6x denaturing sample buffer and incubated for 10 min at 100 °C. The protein samples were separated by SDSPAGE and, after Coomassie blue staining, the dried gel was autoradiographed. Subsequently, the RII
bands were excised and the radioactivity was measured. To study the effect of NS3/4A on the kinase activity of PKA C-subunit, purified PKA C-subunit was preincubated with increasing concentrations of purified NS3/4A proteins prior to addition of substrate.
Non-radiometric assay of PKA C-subunit activity in vitro.
For the analysis of PKA C-subunit activity in the presence of an ATP regeneration system it was not possible to use the standard radiometric assay as ADPATP conversion would reduce the specific radioactivity of ATP during the assay. To overcome this a commercial assay (Pep-Tag; Promega) utilizing a fluorescently labelled Kemptide substrate was used. PKA C-subunit (7·5 nM final concentration) and HCV or BVDV NS3/4A (0·5 µM final concentration) were incubated in 25 µl of kinase assay buffer containing the Pep-Tag substrate, 3·5 mM ATP, 20 mM phosphocreatine (Sigma) and 1 Unit of creatine phosphokinase (Sigma). After incubation at 30 °C for 30 min, the reaction was terminated by boiling and the products were separated by 0·8% agarose gel electrophoresis at neutral pH. The unphosphorylated peptide has a net charge of +1 whereas phosphorylated peptide has a net charge of -1, allowing rapid separation of the two species.
Effect of NS3/4A on PKA function in vivo.
To study the effects of NS3/4A(wt) and NS3/4A(DE-AA) on PKA activity in vivo, the luciferase-based reporter construct pCRE-Luc (Stratagene) was used. Cos-7 cells, seeded at 6x105 cells per T25 flask, were co-transfected using lipofectamine (Life Technologies) with 1 µg each of pCRE-Luc, a -galactosidase reporter construct (pcDNA3
Gal) and pSG5NS3/4A constructs; 24 h after transfection, cells were harvested and assayed for luciferase activity using the Promega Luciferase Assay System.
-Galactosidase activity was measured by ONPG assay: lysate was incubated in 25 mM ONPG, 160 mM phosphate buffer, pH 7·3, 2 mM MgCl2 and 10 mM
-mercaptoethanol and the absorbance at 405 nm was measured. Luciferase activity was normalized to
-galactosidase expression levels. Competence of the pSG5 NS3 constructs to direct protein expression was confirmed by Western blotting.
Other protocols.
A plasmid driving expression of the His-tagged NS3 protease domain (kindly provided by Hans-Georg Krausslich, Heidelberg, Germany) was used to generate recombinant protein in E. coli BL21(DE3). Purified protein was used to raise a polyclonal sheep serum for immunodetection of full-length NS3. Recombinant fusion protein comprising the catalytic (C) subunit of murine PKA with a C-terminal hexahistidine tag was expressed in E. coli BL21(DE3) and purified by metal chelate affinity chromatography. Detailed descriptions of plasmid construction, protein expression and purification will be published elsewhere. The plasmid driving expression of murine PKA RII
subunit in E. coli was a generous gift from John Scott (Portland, OR, USA). RII expression and purification were achieved as previously described (Scott et al., 1990
).
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Results |
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Effect of full-length NS3/4A on PKA C-subunit kinase activity in vitro
HCV NS3 contains an arginine-rich motif (corresponding to the conserved helicase motif VI) which shows homology to both the pseudosubstrate sequence in the regulatory subunit of PKA and recognition sequences within the protein substrates of this enzyme (Borowski et al., 1996 ). Moreover, different synthetic peptides corresponding to this arginine-rich region, as well as a truncated form of HCV NS3, had been shown to inhibit the kinase activity of PKA both in vitro and in vivo (Borowski et al., 1996
, 1997
). The mechanism of this inhibition is still not well understood and pertinently the inhibitory effect of HCV NS3 protein has not been investigated with full-length enzymatically active NS3 protein. Therefore, using purified full-length NS3/4A(wt) complex, we attempted to characterize and mutagenize the potential NS3 regions that may be involved in the PKA inhibitory effect of HCV NS3. The effect of NS3/4A(wt) and the two mutants on the kinase activity of PKA C-subunit were studied using two different substrates. Experiments performed with a synthetic peptide substrate (Kemptide) revealed a concentration-dependent inhibition of PKA C-subunit kinase activity by NS3/4A(wt) (Fig. 2a
). An NS3/4A concentration of 150 nM was found to give 50% inhibition of substrate phosphorylation (IC50). However neither of the two NS3/4A mutants exhibited any inhibitory effect on PKA C-subunit kinase activity (Fig. 2a
). By using recombinant RII
as protein substrate a similar IC50 was obtained for NS3/4A(wt) (Fig. 2b
d
), but again no inhibitory effect was observed with the two NS3/4A mutants (Fig. 2e
).
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Discussion |
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To this end we have investigated the effect of full-length NS3/4A complex on PKA C-subunit kinase activity in vitro and in vivo. Specifically, we addressed the questions: (1) is the putative arginine-rich pseudosubstrate motif of NS3 necessary and sufficient for PKA C-subunit inhibition by native NS3/4A and (2) does the ATPase activity of NS3/4A play a role in PKA C-subunit inhibition? NS3/4A(wt) and two mutants were expressed as His-tagged proteins in the Sf9/recombinant baculovirus system and purified to >90% homogeneity (Fig. 1). The correct folding of purified NS3/4A(wt) was confirmed by analysing its specific proteinase and ATPase activities (data not shown). NS3/4A(wt) was found to possess both activities. As expected neither of the NS3/4A mutants hydrolysed ATP either in the presence or absence of poly(U). This observation is consistent with previously reported results (Wardell et al., 1999
), showing that mutagenesis within motifs II and VI of NS3 totally abrogated the ATPase activity of the protein. The two mutants also exhibited dramatically reduced specific proteinase activity suggesting an interdependence between the proteinase and the helicase domains in NS3 protein as shown by the three-dimensional structure of NS3/4A (Yao et al., 1999
).
The production of the three NS3/4A variants allowed us to analyse whether amino acid substitutions within the Walker motifs II and VI modulated the inhibitory effect of NS3 on PKA kinase activity. The data clearly demonstrate that whereas NS3/4A(wt) could inhibit both Kemptide and RII-subunit phosphorylation mediated by PKA C-subunit, neither of the two mutant forms of NS3/4A were able to inhibit PKA C-subunit kinase activity (Fig. 2
). The inability of the NS3/4A(DE-AA) mutant to inhibit PKA C-subunit is not consistent with the notion that the arginine-rich motif, acting only as a pseudo-substrate inhibitor, is necessary and sufficient to account for the inhibition of PKA C-subunit by NS3/4A. Alternatively, the inability of the NS3/4A(DE-AA) mutant to inhibit PKA C-subunit may be due to a conformational re-arrangement that renders the arginine-rich motif inaccessible for interaction with the catalytic cleft of the kinase. However, this argument is not supported by the ability of both NS3/4A(wt) and NS3/4A(DE-AA) to bind to poly(U)Sepharose with equal affinity (data not shown) or, as discussed below, the observation that poly(U) enhances NS3/4A-mediated inhibition of PKA C-subunit. Since the interaction with poly(U) is likely mediated by the arginine-rich motif (Pause et al., 1993
), this motif evidently remains accessible in the DE-AA mutant.
The presence (and presumably binding of) polyribonucleotides has been shown to stimulate the NTPase activity of NS3 7-fold (Wardell et al., 1999 ). We decided to exploit this phenomenon to obtain more information about the mechanism of inhibition. We reasoned that there would be two alternative outcomes of adding poly(U) to the assay system. Firstly, if NS3/4A ATPase activity contributed to PKA C-subunit inhibition, the addition of poly(U) to the assay should result in more efficient inhibition. Secondly, if the arginine-rich motif, acting as a pseudo-substrate inhibitor, was necessary and sufficient to account for the inhibition of PKA C-subunit then the addition of poly(U) would most likely abrogate this inhibition. This would be because the arginine-rich motif is involved in RNA binding, and thus in the presence of RNA would be unavailable for binding to the catalytic cleft of PKA.
Fig. 3 clearly demonstrates that the ability of both HCV and BVDV NS3/4A to inhibit PKA C-subunit is stimulated by addition of poly(U). The IC50 for both proteins is reduced
3-fold by the presence of the polyribonucleotide. As predicted, poly(U) had no effect on PKA C-subunit activity in the absence of NS3/4A(wt) (data not shown) or in the presence of the ATPase defective NS3/4A(DE-AA) mutant. This observation provides more evidence for the contribution of NS3/4A ATPase activity to the inhibition of PKA C-subunit. However, given that the addition of poly(U) stimulates the ATPase activity of the protein, it may result in a conformational change that could expose an (as yet uncharacterized motif) capable of PKA C-subunit inhibition. To distinguish between these possibilities and conclusively address the question as to whether ATPase activity might mask any specific inhibition of PKA C-subunit by NS3/4A we performed assays in the presence of the phosphocreatine/creatine phosphokinase ATP regenerating system. Phosphocreatine is a high-energy phosphate store from muscle that acts as a phosphate donor for the conversion of ADP to ATP by creatine phosphokinase (Kholodenko et al., 1987
). For the reasons discussed above (Results) this was performed with a non-radioactive PKA assay system (Promega Pep-Tag) which measures phosphorylation by virtue of the fact that it alters the net charge of a fluorescently labelled Kemptide from +1 to -1. Fig. 4
conclusively demonstrates that in the presence of a functional ATP regeneration system the activity of PKA C-subunit is unaffected by NS3/4A.
Our data are therefore not consistent with the previously proposed model in which the arginine-rich motif in NS3 functions as a pseudo-substrate inhibitor of PKA. On the contrary, we believe that full-length, native NS3/4A inhibits PKA C-subunit simply through depletion of the substrate for the enzyme by ATP hydrolysis since, under conditions in which a high ATP concentration is maintained, NS3/4A has no effect on the activity of PKA C-subunit. Fig. 4 shows that this mechanism of inhibition is conserved in both hepaci- and pestiviruses. Although we did not test any non-viral ATPases our data suggest that any active ATPase would have similar effects; however, the magnitude of the effect would presumably be dependent on the activity of the enzyme.
How can our data can be reconciled with the results of Borowski et al. (1996 , 1997
)? We believe that the two sets of data are in fact addressing different parameters. Borowskis data show that the arginine-rich motif can inhibit substrate phosphorylation by PKA by a pseudo-substrate mechanism when presented either as a synthetic peptide or as part of a polypeptide spanning residues 11891525 of HCV. Indeed, we were also able to demonstrate that a synthetic peptide corresponding to residues 14871500 of HCV was able to inhibit phosphorylation of both Kemptide and RII
in vitro (data not shown). However, the data presented here show that, in the context of a physiologically relevant protein (full-length catalytically active NS3/4A), the arginine-rich motif is unable to interact with and inhibit PKA C-subunit. In the context of the HCV-infected hepatocyte we believe that NS3/4A would therefore not inhibit PKA by acting as a pseudo-substrate for the enzyme.
Our data show that both HCV and BVDV NS3/4A are highly active ATPases. What might be the physiological consequence of expression of this ATPase in virus-infected cells? HCV NS3 has been detected by immunostaining in a small proportion (4%) of hepatocytes from patients with chronic HCV infection (Errington et al., 1999 ), although these authors do not discuss absolute expression levels. In BVDV-infected cells (Tautz et al., 1996
) NS3 can be readily detected by RIPA, at levels similar to those obtained using the vacciniaT7 expression system, and is presumably therefore highly abundant. The best-characterized cellular RNA helicase, the initiation factor eIF4
, will clearly also be abundant in all cells; however, the activity of such cellular RNA helicases is tightly regulated. such that they only exhibit ATPase activity when bound to RNA (Pause et al., 1993
). The activity of other intrinsic ATPases (e.g. those associated with ion pumps), and other enzymes consuming cytosolic ATP, are also generally subject to restraint by coupled regulatory events.
In contrast, NS3/4A can hydrolyse ATP in the absence of RNA (Wardell et al., 1999 ; data not shown). The presence of this unregulated enzymatic activity in infected cells might contribute to virus pathology by increasing the rate of ATP turnover within the cell. As the product of ATPase (ADP) is recycled via the reaction 2xADP
ATP+AMP, this would increase the AMP concentration with the potential to activate AMP-activated kinase, a master switch involved in cellular responses to metabolic stress (Winder & Hardie, 1999
). Furthermore, an increase in the cytosolic AMP/ATP ratio and a decrease in the concentration of cAMP (see below) would, in the context of HCV-infected hepatocytes, favour acceleration in the rate of glycolysis. This effect, particularly in the post-prandial state, would cause substantial disruption of normal hepatocyte metabolism.
Our observation that NS3/4A specifically inhibits the transcription factor CREB is consistent with NS3/4A-mediated ATP depletion. CREB is predominantly activated by PKA, which is itself activated by adenylate cyclase mediated ATPcAMP conversion. Cytoplasmic ATP depletion by NS3/4A could, therefore, indirectly inhibit CREB by reducing the pool of ATP available for conversion to cAMP by adenylate cyclase. CREB is involved in the transcription of many cellular genes: thus inhibition of CREB phosphorylation would result in global changes to hepatocyte gene expression patterns. Our current work is focussing on the functional consequences of NS3/4A expression in vivo to elucidate whether this multi-functional enzyme might contribute to virus pathology by dysregulating cellular metabolism.
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Acknowledgments |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Bartenschlager, R. & Lohmann, V. (2000). Replication of hepatitis C virus. Journal of General Virology 81, 1631-1648.
Booth, J. C. L. (1998). Chronic hepatitis C: the virus, its discovery and the natural history of the disease. Journal of Viral Hepatitis 5, 213-222.[Medline]
Borowski, P., Heiland, M., Oehlmann, K., Becker, B., Kornetzky, L., Feucht, H. & Laufs, R. (1996). Nonstructural protein-3 of hepatitis-C virus inhibits phosphorylation mediated by cAMP-dependent protein-kinase. European Journal of Biochemistry 237, 611-618.[Abstract]
Borowski, P., Oehlmann, K., Heiland, M. & Laufs, R. (1997). Nonstructural protein 3 of hepatitis C virus blocks the distribution of the free catalytic subunit of cyclic AMP-dependent protein kinase. Journal of Virology 71, 2838-2843.[Abstract]
Choo, Q. L., Kuo, G., Weiner, A. J., Overby, L. R., Bradley, D. W. & Houghton, M. (1989). Isolation of a cDNA clone derived from a blood-borne non-A, non-B viral-hepatitis genome. Science 244, 359-362.[Medline]
Clarke, B. (1997). Molecular biology of hepatitis C virus. Journal of General Virology 78, 2397-2410.
Della Fazia, M. A., Servillo, G. & Sassone Corsi, P. (1997). Cyclic AMP signalling and cellular proliferation: regulation of CREB and CREM. FEBS Letters 410, 22-24.[Medline]
Edwards, A. S. & Scott, J. D. (2000). A-kinase anchoring proteins: protein kinase A and beyond. Current Opinion in Cell Biology 12, 217-221.[Medline]
Errington, W., Wardell, A. D., McDonald, S., Goldin, R. D. & McGarvey, M. J. (1999). Subcellular localisation of NS3 in HCV-infected hepatocytes. Journal of Medical Virology 59, 456-462.[Medline]
Francis, S. H. & Corbin, J. D. (1994). Structure and function of cyclic nucleotide-dependent protein-kinases. Annual Review of Physiology 56, 237-272.[Medline]
Fujita, T., Ishido, S., Muramatsu, S., Itoh, M. & Hotta, H. (1996). Suppression of actinomycin D-induced apoptosis by the NS3 protein of hepatitis C virus. Biochemical and Biophysical Research Communications 229, 825-831.[Medline]
Green, S., Issemann, I. & Sheer, E. (1988). A versatile in vivo and in vitro eukaryotic expression vector for protein engineering. Nucleic Acids Research 16, 369.[Medline]
Higuchi, R. (1992). Using PCR to engineer DNA. In PCR Technology. Principles andApplications , pp. 61-70. Edited by H. A. Erlich. New York:W. H. Freeman.
Kadare, G. & Haenni, A. L. (1997). Virus-encoded RNA helicases. Journal of Virology 71, 2583-2590.
Kholodenko, B., Zilinskiene, V., Borutaite, V., Ivanoviene, L., Toleikis, A. & Praskevicius, A. (1987). The role of adenine-nucleotide translocators in regulation of oxidative-phosphorylation in heart-mitochondria. FEBS Letters 223, 247-250.[Medline]
Kim, J. L., Morgenstern, K. A., Lin, C., Fox, T., Dwyer, M. D., Landro, J. A., Chambers, S. P., Markland, W., Lepre, C. A., Omalley, E. T., Harbeson, S. L., Rice, C. M., Murcko, M. A., Caron, P. R. & Thomson, J. A. (1996). Crystal-structure of the hepatitis-C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide. Cell 87, 343-355.[Medline]
Kitts, P. A., Ayers, M. D. & Possee, R. D. (1990). Linearization of baculovirus DNA enhances recovery of recombinant virus expression vectors. Nucleic Acids Research 18, 5667-5672.[Abstract]
Kolykhalov, A. A., Mihalik, K., Feinstone, S. M. & Rice, C. M. (2000). Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3' nontranslated region are essential for virus replication in vivo. Journal of Virology 74, 2046-2051.
Kummerer, B. M., Stoll, D. & Meyers, G. (1998). Bovine viral diarrhea virus strain Oregon: a novel mechanism for processing of NS2-3 based on point mutations. Journal of Virology 72, 4127-4138.
Lemon, S. M. & Honda, M. (1997). Internal ribosome entry sites within the RNA genomes of hepatitis C virus and other flaviviruses. Seminars in Virology 8, 274-288.
Maniatis, T., Fritsch, E. F. & Sambrook, J. (1989). Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Olsen, S. R. & Uhler, M. D. (1991). Isolation and characterization of cDNA clones for an inhibitor protein of cAMP-dependent protein kinase. Journal of Biological Chemistry 266, 11158-11162.
Pause, A., Methot, N. & Sonenberg, N. (1993). The HRIGRXXR region of the DEAD box RNA helicase eukaryotic translation initiation factor-4a is required for RNA-binding and ATP hydrolysis. Molecular and Cellular Biology 13, 6789-6798.[Abstract]
Roskoski, R. J. (1983). Assays of protein kinase. Methods in Enzymology 99, 3-6.[Medline]
Saito, I., Miyamura, T., Ohbayashi, A., Harada, H., Katayama, T., Kikuchi, S., Watanabe, Y., Koi, S., Onji, M., Ohta, Y., Choo, Q. L., Houghton, M. & Kuo, G. (1990). Hepatitis-C virus-infection is associated with the development of hepatocellular-carcinoma. Proceedings of the National Academy of Sciences, USA 87, 6547-6549.[Abstract]
Sakamuro, D., Furukawa, T. & Takegami, T. (1995). Hepatitis C virus nonstructural protein NS3 transforms NIH 3T3 cells. Journal of Virology 69, 3893-3896.[Abstract]
Sali, D. L., Ingram, R., Wendel, M., Gupta, D., McNemar, C., Tsarbopoulos, A., Chen, J. W., Hong, Z., Chase, R., Risano, C., Zhang, R. M., Yao, N. H., Kwong, A. D., Ramanathan, L., Le, H. V. & Weber, P. C. (1998). Serine protease of hepatitis C virus expressed in insect cells as the NS3/4A complex. Biochemistry 37, 3392-3401.[Medline]
Scott, J. D., Stofko, R. E., McDonald, J. R., Comer, J. D., Vitalis, E. A. & Mangili, J. A. (1990). Type-II regulatory subunit dimerization determines the subcellular-localization of the cAMP-dependent protein-kinase. Journal of Biological Chemistry 265, 21561-21566.
Spengler, U., Lechmann, M., Irrgang, B., Dumoulin, F. L. & Sauerbruch, T. (1996). Immune responses in hepatitis C virus infection. Journal of Hepatology 24, 20-25.[Medline]
Tautz, N., Meyers, G., Stark, R., Dubovi, E. J. & Thiel, H. J. (1996). Cytopathogenicity of a pestivirus correlates with a 27-nucleotide insertion. Journal of Virology 70, 7851-7858.[Abstract]
Walsh, D. A. & Van Patten, S. M. (1994). Multiple pathway signal-transduction by the cAMP-dependent protein-kinase. FASEB Journal 8, 1227-1236.
Wardell, A. D., Errington, W., Ciaramella, G., Merson, J. & McGarvey, M. J. (1999). Characterization and mutational analysis of the helicase and NTPase activities of hepatitis C virus full-length NS3 protein. Journal of General Virology 80, 701-709.[Abstract]
Whitehouse, S., Feramisco, J. R., Casnellie, J. E., Krebs, E. G. & Walsh, D. A. (1983). Studies on the kinetic mechanism of the catalytic subunit of the cAMP-dependent protein-kinase. Journal of Biological Chemistry 258, 3693-3701.
Winder, W. W. & Hardie, D. G. (1999). AMP-activated protein kinase, a metabolic master switch: possible roles in Type 2 diabetes. American Journal of PhysiologyEndocrinology And Metabolism 40, E1-E10.
Yan, Y. W., Li, Y., Munshi, S., Sardana, V., Cole, J. L., Sardana, M., Steinkuehler, C., Tomei, L., Defrancesco, R., Kuo, L. C. & Chen, Z. G. (1998). Complex of NS3 protease and NS4A peptide of BK strain hepatitis C virus: a 2·2 angstrom resolution structure in a hexagonal crystal form. Protein Science 7, 837-847.
Yanagi, M., Purcell, R. H., Emerson, S. U. & Bukh, J. (1997). Transcripts from a single full-length cDNA clone of hepatitis C virus are infectious when directly transfected into the liver of a chimpanzee. Proceedings of the National Academy of Sciences, USA 94, 8738-8743.
Yao, N. H., Reichert, P., Taremi, S. S., Prosise, W. W. & Weber, P. C. (1999). Molecular views of viral polyprotein processing revealed by the crystal structure of the hepatitis C virus bifunctional proteasehelicase. Structure With Folding & Design 7, 1353-1363.[Medline]
Received 23 January 2001;
accepted 13 March 2001.