The inhibition of cAMP-dependent protein kinase by full-length hepatitis C virus NS3/4A complex is due to ATP hydrolysis

Mustapha Aoubala1, John Holt1, Roger A. Clegg2, David J. Rowlands1 and Mark Harris1

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


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Hepatitis C virus (HCV) is an important cause of chronic liver disease, but the molecular mechanisms of viral pathogenesis remain to be established. The HCV non-structural protein NS3 complexes with NS4A and has three enzymatic activities: a proteinase and a helicase/NTPase. Recently, catalytically inactive NS3 fragments containing an arginine-rich motif have been reported to interact with, and inhibit, the catalytic subunit of cAMP-dependent protein kinase (PKA C-subunit). Here we demonstrate that full-length, catalytically active NS3/4A, purified from recombinant baculovirus-infected insect cells, is also able to inhibit PKA C-subunit in vitro. This inhibition was abrogated by mutation of either the arginine-rich motif or the conserved helicase motif II, both of which also abolished NTPase activity. As PKA C-subunit inhibition was also enhanced by poly(U) (an activator of NS3 NTPase activity), we hypothesized that PKA C-subunit inhibition could be due to NS3/4A-mediated ATP hydrolysis. This was confirmed by experiments in which a constant ATP concentration was maintained by addition of an ATP regeneration system – under these conditions PKA C-subunit inhibition was not observed. Interestingly, the mutations also abrogated the ability of wild-type NS3/4A to inhibit the PKA-regulated transcription factor CREB in transiently transfected hepatoma cells. Our data are thus not consistent with the previously proposed model in which the arginine-rich motif of NS3 was suggested to act as a pseudosubstrate inhibitor of PKA C-subunit. However, in vivo effects of NS3/4A suggest that ATPase activity may play a role in viral pathology in the infected liver.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Hepatitis C virus (HCV) has been identified as the causative agent of most cases of transfusion-associated and community-acquired non-A, non-B hepatitis (Booth, 1998 ; Spengler et al., 1996 ; Choo et al., 1989 ). In addition to being the major cause of post-transfusion viral hepatitis, HCV has been shown to be causally linked to hepatocellular carcinoma (Saito et al., 1990 ). HCV has been classified in a separate genus (hepaciviruses) within the family Flaviviridae, which also contains the flaviviruses and the animal pestiviruses (Booth, 1998 ). HCV is an enveloped virus with a single-stranded positive-sense RNA genome of approximately 9·5 kb that encodes a single open reading frame (ORF) of 3010 to 3033 amino acids. The ORF is flanked by 5' and 3' untranslated regions which have been shown to be involved in the initiation of translation and in virus replication respectively (Kolykhalov et al., 2000 ; Lemon & Honda, 1997 ). The primary translation product is proteolytically processed, by virally encoded and cellular proteinases, into mature structural and non-structural (NS) proteins (Clarke, 1997 ). By analogy to other flaviviruses, the NS proteins are likely to form a replication complex. The best characterized of these proteins, NS3, is a 630 amino acid protein with three enzymatic activities. A serine proteinase responsible for polyprotein processing constitutes the N-terminal 180 amino acids of NS3, whereas the C-terminal domain has both helicase and NTPase activities (Bartenschlager, 1999 ; Bartenschlager & Lohmann, 2000 ). NS4A is a short (54 amino acids) polypeptide that acts as a cofactor of the NS3 proteinase and is essential for polyprotein processing (Bartenschlager, 1999 ). The three-dimensional structure of NS3 with complexed NS4A activator peptide shows that NS4A binds tightly to the NS3 N-terminal domain and can be regarded as an integral component of the NS3 proteinase (Yan et al., 1998 ; Kim et al., 1996 ). Apart from the enzymatic activities associated with NS3, it has been suggested that this protein may have additional properties involved in interference with host cell functions such as cell metabolism, differentiation and tumour promotion (Sakamuro et al., 1995 ; Fujita et al., 1996 ; Borowski et al., 1996 , 1997 ).

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 1487–1500), 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{alpha}-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.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} DNA manipulations.
All manipulations of DNA were performed according to standard protocols (Maniatis et al., 1989 ). Pfu DNA polymerase was purchased from Stratagene. Restriction enzymes and T4 DNA ligase were purchased from New England Biolabs. Oligonucleotide primers were obtained from MWG Biotech.

{blacksquare} 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.

{blacksquare} 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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Table 1. Oligonucleotide primers for NS3/4A PCR

 
{blacksquare} Site-directed mutagenesis of the helicase/NTPase motifs in HCV NS3/4A.
Mutants of HCV NS3/4A were generated by the PCR overlap extension method (Higuchi, 1992 ) using H77 as template and overlapping internal oligonucleotides. The first pair of primers (DEforward and DEreverse – see Table 1) was designed to generate the NS3/4A(DE-AA) mutant in which residues Asp1316 and Glu1317 (within helicase motif II, DECH in the case of HCV NS3) were substituted by alanine residues. The second pair of primer sequences (4Rforward and 4Rreverse – see Table 1) were designed to generate the NS3/4A(R-A) mutant in which Arg1487, Arg1488, Arg1490 and Arg1493 (within helicase motif VI) were substituted by alanine. Recombinant baculoviruses were generated as described above for NS3/4A(wt).

{blacksquare} 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 ).

{blacksquare} Expression and purification of 6xHis–NS3/4A 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 {beta}-mercaptoethanol, 10% (v/v) glycerol, EDTA-free protease inhibitor cocktail (Boehringer)] containing 0·5% (w/v) n-dodecyl {beta}-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 Ni–NTA agarose (Qiagen) for 1 h at 4 °C. NS3/4A was eluted with 3 column volumes of buffer DL containing 0·1% n-dodecyl {beta}-D-maltoside and 250 mM imidazole. Protein concentration was estimated by Bio-Rad assay.

{blacksquare} 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{alpha} phosphorylation assays, the kinase reaction (50 µl) containing 12·5 nM PKA C-subunit, 7·5 mM magnesium acetate, 2 mM DTT, 50 µM [{gamma}-32P]ATP (1 µCi), 10 mM MOPS pH 7 and 5 µM RII{alpha} 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 SDS–PAGE and, after Coomassie blue staining, the dried gel was autoradiographed. Subsequently, the RII{alpha} 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.

{blacksquare} 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 ADP->ATP 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.

{blacksquare} 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 {beta}-galactosidase reporter construct (pcDNA3{beta}Gal) and pSG5NS3/4A constructs; 24 h after transfection, cells were harvested and assayed for luciferase activity using the Promega Luciferase Assay System. {beta}-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 {beta}-mercaptoethanol and the absorbance at 405 nm was measured. Luciferase activity was normalized to {beta}-galactosidase expression levels. Competence of the pSG5 NS3 constructs to direct protein expression was confirmed by Western blotting.

{blacksquare} 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{alpha}) 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{alpha} 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 ).


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Expression and purification of the NS3/4A complex
The NS3/4A region of the HCV genomic cDNA corresponding to amino acid residues 1027–1712 of HCV genotype 1a was PCR-amplified and cloned into the baculovirus expression plasmid pBacPAKHis-1 – this vector generates N-terminally hexahistidine-tagged proteins to facilitate purification. In parallel, the NS3/4A region of BVDV strain Oregon (Kummerer et al., 1998 ) (amino acid residue numbers 1599–2508) was also amplified and cloned. Two full-length mutated HCV NS3/4A constructs were generated by mutating two motifs shown to be critical for NTPase/helicase activity: NS3/4A(DE-AA) contained two substitutions (Asp1316–Ala and Glu1317–Ala) and NS3/4A(R-A) contained substitutions of four arginines (residues 1487, 1488, 1490 and 1493) to alanine. Following expression in Sf9 cells and purification by Ni–NTA affinity chromatography all four proteins were >90% pure as revealed by SDS–PAGE and Coomassie blue staining analysis (Fig. 1a, b). The identity of the expressed proteins was confirmed by Western blotting using either a sheep polyclonal serum raised against the protease domain of HCV NS3 (Fig. 1c) or a monoclonal antibody to BVDV NS3 (kindly provided by Gregor Meyers, Tübingen, Germany) (Fig. 1d). For HCV the presence of both NS3 and NS4A was confirmed by radioimmunoprecipitation with the polyclonal anti-NS3 antibody followed by SDS–PAGE and autoradiography (data not shown).



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Fig. 1. Expression and purification of HCV and BVDV NS3/4A. Purified NS3/4A protein preparations were generated as described in Methods and subjected to SDS–PAGE followed by Coomassie brilliant blue staining (a, b) or Western blotting (c, d). Lanes 1 and 5, HCV NS3/4A(wt); lanes 2, HCV NS3/4A(DE-AA); lanes 3, HCV NS3/4A(R-A); lanes 4, BVDV NS3/4A(wt). Western blots were probed with sheep anti-NS3 serum (c) or a monoclonal antibody to BVDV NS3 (d). Lanes M, molecular mass markers.

 
Before analysing the effect of NS3/4A on PKA C-subunit kinase activity, we first confirmed that the purified proteins were expressed in a native conformation by assessing two of the three enzymatic activities of NS3. Both HCV and BVDV NS3/4A(wt) possessed an intrinsic ATPase activity that, as previously shown, was stimulated 7-fold by the presence of poly(U). However, no ATPase activity was detected for either of the NS3/4A mutants (data not shown). These observations are in accord with previous observations (Wardell et al., 1999 ) demonstrating that both motifs II and VI are crucial for the HCV NS3 ATPase activity. Specific trans proteinase activity of HCV NS3/4A(wt) and the two mutants forms was measured by assaying cleavage of an NS5A/NS5B peptide. Both mutants showed pronounced defects in the hydrolysis of the NS5A/NS5B peptide as compared to NS3/4A(wt): residual activities were 4·4% for NS3/4A(DE-AA) and 0·74% for NS3/4A(R-A) (data not shown). However, the observation that the protein product of both mutants exhibited the same molecular mass as the wild-type, corresponding to the expected molecular mass of 6xHis–NS3 (see Fig. 1), confirms that they are able to cleave in cis between NS3–NS4A.

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{alpha} as protein substrate a similar IC50 was obtained for NS3/4A(wt) (Fig. 2bd), but again no inhibitory effect was observed with the two NS3/4A mutants (Fig. 2e).



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Fig. 2. Inhibition of PKA C-subunit-mediated phosphorylation by HCV NS3/4A is abrogated by mutations within the NTPase domain. (a) Kemptide (150 µM final concentration) was phosphorylated by 0·5 pmol PKA C-subunit. The kinase assay was performed in the presence of increasing concentrations of NS3/4A proteins. {bullet}, NS3/4A(wt); {blacktriangleup}, NS3/4A (DE-AA); {blacksquare}, NS3/4A (R-A). PKA C-subunit kinase activity is presented as the amount of phosphate transferred to Kemptide relative to a control reaction lacking NS3/4A in which the 100% value corresponded to a rate of 0·764 µmol/min/mg PKA C-subunit protein. (b) Coomassie blue-stained SDS–PAGE gel of the RII{alpha} subunit kinase reaction mixture. The kinase assay was performed in the presence of increasing amounts of NS3/4A(wt). Lanes 1–7 correspond to NS3/4A concentrations of 0·15, 0·33, 0·50, 0·75, 1·13, 1·4 and 1·7 µM, respectively. Lane 8 corresponds to the negative control in the absence of NS3/4A(wt). Positions of NS3, BSA and RII{alpha} are indicated by arrows. (c) Autoradiograph of the SDS–PAGE gel presented in (b) to detect RII{alpha} phosphorylation. (d) Phosphorylation of RII{alpha} was measured by Cerenkov counting following excision of the relevant portion of the gel. Values were normalized to the value of the control reaction (b, lane 8). (e) Autoradiograph demonstrating RII{alpha} phosphorylation in the absence (control) or the presence of 1 µM (final concentration) of NS3/4A(wt), NS3/4A(DE-AA) or NS3/4A(R-A), as indicated.

 
Poly(U) enhances the inhibitory effect of NS3/4A on PKA C-subunit kinase activity in vitro
The ability of NS3/4A(wt) to inhibit the activity of PKA C-subunit kinase activity in vitro was abrogated by mutations that abolish NS3 NTPase activity. One explanation for this observation was that ATPase activity might contribute to PKA C-subunit inhibition by hydrolysing and therefore depleting ATP within the reaction. ATP hydrolysis would additionally result in a increase in the concentration of ADP, shown to act as a competitive inhibitor of PKA C-subunit with a Ki similar to the Km of the enzyme for ATP (Whitehouse et al., 1983 ). Alternatively, the effects of these mutations might be to change the conformation of the protein such that peptide sequences required for PKA C-subunit inhibition were not exposed. To test the first hypothesis we made use of the previous observation that the addition of polyribonucleotides [e.g. poly(U)] stimulated the NTPase activity of HCV NS3 (Wardell et al., 1999 ). If NS3/4A-mediated ATP hydrolysis is important for PKA C-subunit inhibition, the addition of poly(U) should, therefore, enhance the ability of NS3/4A to inhibit PKA C-subunit. We included in this experiment purified recombinant NS3/4A from the closely related pestivirus BVDV to determine whether the ability to inhibit PKA C-subunit is restricted to HCV, or is a conserved property throughout the Flaviviridae. Fig. 3 shows that using Kemptide as substrate the addition of poly(U) resulted in a shift of the inhibition curve to the left for both HCV and BVDV NS3/4A. For HCV in this experiment the addition of poly(U) resulted in a 3-fold decrease in the IC50 value (from 90 to 30 nM). PKA C-subunit activity was unaffected by NS3/4A (DE-AA) either in the presence or absence of poly(U), thus confirming that poly(U) by itself is not inhibitory. These data clearly show that addition of poly(U) significantly enhanced the inhibitory effect of NS3/4A on PKA C-subunit kinase activity.



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Fig. 3. Poly(U) enhances HCV and BVDV NS3/4A inhibition of PKA C-subunit activity. Kemptide (150 µM final concentration) was phosphorylated by 0·5 pmol of PKA C-subunit in the presence of increasing amounts of NS3/4A(wt) ({bullet}, {circ}), NS3/4A(DE-AA) ({blacktriangleup}, {triangleup}) or BVDV NS3/4A(wt) ({diamondsuit}, {lozenge}). Assays were performed in the absence (filled symbols) or the presence (open symbols) of 3 mg/ml (~7·5 mM nucleotides) poly(U).

 
NS3/4A does not inhibit PKA kinase activity when the ATP concentration is maintained at a constant level
Borowski et al. (1996 ). suggested that inhibition of PKA by NS3 was dependent on a pseudosubstrate function of the arginine-rich motif Although the data presented above are not consistent with this, it was conceivable that any contribution of the arginine-rich motif to inhibition in our assay was masked by the inhibitory effect of the NS3/4A ATPase. The failure of the DE-AA mutant to inhibit might be due to inappropriate folding and failure to present the arginine-rich motif on the surface of the protein. In order to test for this possibility we established an assay in which the ATP concentration was maintained at a constant level by the inclusion of an ATP regeneration system, in this case phosphocreatine and creatine phosphokinase (Kholodenko et al., 1987 ). As ADP->ATP conversion would reduce the specific radioactivity of ATP during the course of the assay a non-radioactive assay system (Promega Pep-Tag) was used. In this system phosphorylation is measured by virtue of the fact that it alters the net charge of a fluorescently labelled Kemptide from +1 to -1, allowing separation of the phosphorylated and nonphosphorylated substrates by agarose gel electrophoresis. Fig. 4, lane 3 demonstrates that addition of HCV NS3/4A(wt) inhibited the production of the negatively charged phosphopeptide by purified PKA C-subunit. However, the addition of a complete ATP regeneration system (lane 5) restored the ability of PKA C-subunit to phosphorylate the substrate. As this assay was performed at a high concentration of phosphocreatine (20 mM) that might have interfered with NS3/4A function we included a control lacking the ATP regenerating enzyme creatine phosphokinase (lane 4). Under these conditions NS3/4A was still able to inhibit PKA C-subunit activity. Identical results were obtained for the BVDV NS3/4A protein (lanes 9–11) and, as previously seen (Fig. 2), the NS3/4A(DE-AA) mutant had no effect on PKA C-subunit activity (lanes 6–8). These data provide conclusive proof that inhibition of PKA C-subunit by NS3/4A(wt) is entirely due to ATP hydrolysis and the concomitant reduction of the ATP concentration in the reaction.



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Fig. 4. NS3/4A does not inhibit PKA C-subunit in the presence of a constant concentration of ATP. The Promega Pep-Tag system was used to assay PKA activity in the presence of either HCV NS3/4A(wt), NS3/4A(DE-AA) or BVDV NS3/4A (all at 0·5 µM) and an ATP regeneration system [phosphocreatine and creatine phosphokinase (Kholodenko et al., 1987 ]. The kit was used as per the manufacturer’s instructions except that an in-house preparation of PKA C-subunit (7·5 nM) was used and the ATP concentration of the assay was adjusted to 3·5 mM. Assays were assembled according to the table on the left and the products of the reaction were separated by agarose gel electrophoresis under neutral conditions – phosphorylated Kemptide migrates towards the anode whereas the non-phosphorylated form migrates towards the cathode.

 
Effect of full-length NS3/4A on PKA kinase activity in vivo
Given that ATP depletion by NS3/4A(wt) was able to inhibit PKA activity in vitro, we wished to determine whether NS3/4A had any effects on activity of PKA in vivo. To do this we made use of the pCRE-Luc (Stratagene) reporter construct in which the expression of luciferase is controlled by the cAMP response element binding protein (CREB). CREB is predominantly, though not exclusively, phosphorylated by PKA (Della Fazia et al., 1997 ). In its phosphorylated state CREB is transcriptionally active and will drive the expression of genes downstream of its cognate binding site – thus luciferase activity is an indirect measurement of PKA activity within the transfected cells. As shown in Fig. 5, NS3/4A(wt) but not NS3/4A(DE-AA) repressed CREB-driven luciferase activity in Cos7 cells. This effect was specific to CREB as expression from both SP1-responsive (Fig. 5) or AP1-responsive (data not shown) luciferase constructs was unaffected by NS3/4A(wt). Expression of NS3/4A was verified by Western blotting of parallel transfected Cos7 cells (data not shown). These data show that NS3/4A(wt) is able to specifically inhibit PKA-mediated CREB phosphorylation in vivo.



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Fig. 5. NS3/4A inhibits PKA-mediated signalling events in vivo. Cos-7 cells were transiently transfected with 1 µg of pcDNA3{beta}-gal; either pCRE-luc (Stratagene) or pSP1-Luc (a gift from Kalle Saksela, University of Tampere, Finland); and either pSG5NS3/4A(wt), pSG5NS3/4A(DE-AA) or pSG5 empty vector as indicated. Cells were harvested at 24 h post-transfection and luciferase and {beta}-galactosidase activities determined as described.

 

   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
Borowski et al. (1996 ) previously proposed on the basis of sequence analysis that HCV NS3 contained an arginine-rich motif with homology to both the inhibitory site of the heat-stable inhibitor (PKI) of PKA and the RII-subunit autophosphorylation site. Furthermore, they demonstrated experimentally that synthetic peptides corresponding to this arginine-rich motif, as well as a fragment of NS3 encompassing this sequence, could interact with and inhibit the kinase activity of PKA (Borowski et al., 1996 , 1997 ), and they proposed that the arginine-rich motif could function as a pseudosubstrate inhibitor of PKA. However, we considered that the published data merited a more rigorous analysis of the mechanism of NS3 inhibition of PKA activity in the context of the native NS3/4A complex.

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{alpha}-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. Borowski’s 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 1189–1525 of HCV. Indeed, we were also able to demonstrate that a synthetic peptide corresponding to residues 1487–1500 of HCV was able to inhibit phosphorylation of both Kemptide and RII{alpha} 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 vaccinia–T7 expression system, and is presumably therefore highly abundant. The best-characterized cellular RNA helicase, the initiation factor eIF4{alpha}, 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 ATP->cAMP 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.


   Acknowledgments
 
We would like to thank Norman Gray at GlaxoSmithKline, Stevenage for performing the specific NS3 proteinase assays. This work was supported by a project grant (24/C09593) from the Biotechnology and Biological Sciences Research Council (to M.H., R.A.C. and D.J.R.). Work in R.A.C.’s laboratory is also supported by the Scottish Executive.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 23 January 2001; accepted 13 March 2001.