Istituto di Ricerche di Biologia Molecolare P. Angeletti (IRBM), Via Pontina Km 30.600, 00040 Pomezia (Rome), Italy1
Author for correspondence: Paola Gallinari. Fax +39 06 91093225. e-mail Gallinari{at}IRBM.it
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Abstract |
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Introduction |
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Different three-dimensional structures of the isolated NS3 helicase domain have been determined (Cho et al., 1998 ; Kim et al., 1998
; Yao et al., 1997
). The enzyme comprises three domains termed 1, 2 and 3, with domains 1 and 2 being structurally similar. The ATP-binding site is situated in a cleft between domains 1 and 2 that is lined with conserved sequence motifs that are the hallmarks of helicases (Gorbalenya & Koonin, 1993
). NS3 is a member of a large class of helicases that have a 3' to 5' directionality and share a number of structural features (Bird et al., 1998
). Other members of this class include the Rep, PcrA and UvrD bacterial DNA helicases for which much structural data became available recently (Korolev et al., 1997
; Subramanya et al., 1996
). NS3 helicase domains 1 and 2 fold similar to domains 1A and 2A of the above-mentioned enzymes, but with a slightly different connectivity (Bird et al., 1998
). The third domain has no structural similarity with Rep or PcrA but sits in a position approximately equivalent to that occupied by domain 1B in the Rep helicase (Korolev et al., 1998
). The structure of a complex of the NS3 helicase domain with a (dU)8 oligonucleotide (Kim et al., 1998
) demonstrated the nucleic acid in a channel that separates domain 3 from domains 1 and 2, in a position equivalent to the binding site for ssDNA in the Rep (Korolev et al., 1997
) and PcrA (Velankar et al., 1999
) enzymes. Three different models for the mechanism of NS3 helicase have been proposed, one to accompany each of the three published crystal structures. To get more insight on the overall mechanism of unwinding, we have analysed the effect that binding of ATP (or non-hydrolysable ATP analogues) to the full-length (FL) enzyme exerts on its relative affinity for ssRNA and dsRNA.
In order for a helicase to unwind duplex nucleic acids in a processive manner, the enzyme should destabilize the hydrogen bonds between the base pairs, translocate to the next base-paired region, and repeat the cycle without dissociating from the RNA substrate. Here we have analysed the FL-NS3 processive unwinding under single cycle conditions using heparin as a trapping molecule both in helicase and ATPase assays.
The helicase activity of NS3 was previously shown to require substrates with a free 3' single-stranded tail (Gwack et al., 1996 ; Tai et al., 1996
). In this study, we have measured NS3 unwinding activity on double-hybrid substrates designed to mimic stemloop structures, containing internal ssRNA regions of different lengths. The ability of NS3 to interact preferentially with HCV genomic or antigenomic RNA has not been addressed so far. We and others have previously shown that in the absence of ATP, NS3 binds ssRNA tightly with no particular sequence specificity and with efficiency depending only on the length of ssRNA (Gallinari et al., 1998
; Gwack et al., 1996
; Kanai et al., 1995
; Tai et al., 1996
). Therefore, it was of interest to assess whether NS3 shows a selective interaction with the 3' ends of the viral genome and antigenome, the presumed initiation sites for negative- and positive-strand RNA synthesis, respectively. We have studied the interaction and the helicase function of FL-NS3 with a series of RNA oligonucleotides derived from the 46 nt stemloop structure (SL I) at the 3' terminus of HCV genomic positive-strand RNA (Blight & Rice, 1997
; Kolykhalov et al., 1996
; Tanaka et al., 1996
). The stability and structural conservation of SL I suggested that it represents a recognition site for viral and/or cellular proteins involved in virus replication. While some of the proteinRNA interactions at the 3'-end of the genome have been recently elucidated (Cheng et al., 1999
; Ito & Lai, 1997
; Tsuchihara et al., 1997
), our data represent the first evidence of the ability of HCV NS3 to recognize and resolve RNA secondary structures within the viral 3'-NTR.
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Methods |
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ATPase activity assays.
ATPase activity was directly determined by monitoring [-32P]ATP hydrolysis by thin-layer chromatography, as described by Gallinari et al. (1998)
. RNA titration assays were carried out by incubating 20 nM enzyme in the presence of increasing concentrations of ssRNA or partial dsRNA (0 to 10 µM) for 30 min at 37 °C in helicase activity buffer [25 mM MOPSNaOH (pH 7), 2·5 mM DTT, 2·5 U RNasin (Promega), 100 µg/ml BSA, 5% glycerol, 3 mM MgCl2] containing 1 mM ATP and 2 µCi [
-32P]ATP (6000 Ci/mmol, 10 mCi/ml; Dupont NEN) in a volume of 10 µl. For analysis under single processive cycle conditions, increasing amounts of heparin were added to the samples as described in the legend to Fig. 6
. After termination with 5 mM EDTA, 0·5 µl aliquots were spotted onto polyethyleneimine cellulose sheets and developed by ascending chromatography in 150 mM LiCl, 150 mM formic acid (pH 3·0). The cellulose sheets were dried and released [32P]phosphoric acid was quantified with a PhosphorImager using ImageQuant software.
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Results |
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Effect of nucleotides on the affinity of NS3 for ssRNA and dsRNA
Although we have shown that NS3 is able to form stable complexes with double-hybrid substrates containing single-stranded regions down to 3 nt in length, no direct binding was observed on ssRNA molecules shorter than 18 nt in the same experimental conditions (unpublished results). Since NS3 bound ssRNA molecules with lower affinity than partial dsRNA molecules containing ssRNA regions of equal size, a contribution to the overall binding affinity of NS3 for these latter substrates could be given by additional interactions with the dsRNA region. We were interested in evaluating the effect that binding of ATP to NS3 might exert on its relative affinity for ssRNA and dsRNA. Previously we demonstrated that formation of a stable complex of NS3 with a ssRNA oligonucleotide was severely impaired by the addition of 5 mM ATP (Gallinari et al., 1998 ). Here we compared the binding affinity of the enzyme for the helicase double-hybrid substrate in the presence or absence of 5 mM ATP (or its non-hydrolysable analogue AMP-PCP) (Fig. 3
). In the experiment shown in Fig. 3 (A
, B
) addition of ATP to the reaction mixture prior to addition of the enzyme determined a significant decrease of the binding efficiency, which probably reflected a diminished affinity for the ssRNA region of the probe. Moreover, in these conditions, strand release was also inhibited (not shown), presumably as a consequence of a reduction in the number of pre-formed enzymesubstrate complexes. Substituting AMP-PCP for ATP produced a similar decrease in the binding efficiency (not shown), thus suggesting that binding to the nucleotide and not hydrolysis was responsible for the weakened enzymeRNA interaction. When ATP was added after pre-incubation of the enzyme with the labelled substrate, no retarded band was visible and release of the labelled strand was observed, as expected (not shown). However, addition of AMP-PCP, which does not support NS3 unwinding activity (Gallinari et al., 1998
), after pre-incubating the enzyme with the substrate caused a marked increase in the binding efficiency (Fig. 3C
, D
), which probably reflected an increased affinity for the dsRNA region of the probe. In conclusion, the effect of the order of addition of ATP or AMP-PCP on the level of binding activity to the partial dsRNA substrate suggests that the enzymenucleotide complex has a lower affinity for ssRNA and a higher affinity for duplex RNA than does the non-complexed protein.
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NS3 can resolve an SL I-containing stemloop RNA structure
Next we addressed whether the helicase activity of FL-NS3 was able to resolve the SL I RNA structure by unwinding the stem region upon binding to the 6 nt loop. First attempts to devise an unwinding assay using optimized NS3 helicase conditions and the 46-mer SL I RNA oligonucleotide as a substrate failed to reveal any reaction product co-migrating with the corresponding linearized form in a native gel (not shown). A possible explanation resides in the extreme thermodynamic stability of SL I [G=-26·5 kcal/mol with a melting temperature of 85 °C; Blight & Rice, 1997
], which probably favours re-annealing of the base-paired region, thus preventing the isolation of enzymatically melted RNA molecules. Therefore, by annealing oligonucleotides 1 and 2 schematized in Fig. 5(A)
, we created a new stemloop substrate in which we extended both the 5'- and 3'-ends of SL I stem with complementary tails of 10 nt and introduced a nick in the middle of the original base-paired region. This new stemloop substrate (SL I-l, Fig. 5A
) allowed us to monitor strand release rather than a melting product, with the assumption that the two unwinding reactions were equivalent in all other respects. To verify that the presence of the nick in the stem structure would not destabilize the base-paired region by creating an artificial single-stranded tail which could trigger unwinding, we designed two negative control substrates. By annealing oligonucleotide 1 either with oligonucleotides 3 and 4 or 3a and 4a, blunt-ended linear dsDNA molecules were constructed which no longer included the single-stranded loop but retained the nick-containing extended stem with (C1) or without (C2) bulges, respectively (Fig. 5A
). As shown in Fig. 5(B
, C
), the labelled oligonucleotide 1 was efficiently released from the SL I-derived stemloop structure by the NS3-associated helicase activity and the enzyme showed a significant preference for this substrate compared with the negative controls. This difference was reflected in a higher binding affinity for the stemloop than for the blunt-ended substrates (Fig. 5D
), thus confirming that NS3 was able to interact with the 6 nt single-stranded loop and to unwind the double-stranded SL I stem starting from it. The presence of the two bulges did not cause any significant additional destabilization of the base-paired region as judged by the two control curves in Fig. 5(C)
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Effect of heparin on ATP hydrolysis and helicase activities
NS3 helicase has a large intrinsic ATPase activity (kcat=3 s-1) which is stimulated up to 30-fold by ssRNA or DNA (Preugschat et al., 1996 ). We have compared the stimulation of NS3 ATPase activity in the presence of increasing concentrations of either a 20 nt ssRNA or a dsRNA substrate containing a 10 nt 3'-tail (Gallinari et al., 1998
). The two titration curves shown in Fig. 6(A)
were very similar, indicating that the degree of stimulation of the ATPase activity observed with the ssRNA and the tailed dsRNA was identical (approximately 10-fold). The dissociation constant values for the two activator RNAs were also very similar (2 and 2·1 µM, respectively). This result suggests that the presence of a duplex region does not influence the efficiency of the RNA-mediated activation of ATP hydrolysis. Although it is known that NS3 unwinding activity is ATP-dependent (Tai et al., 1996
), the mechanism of coupling ATP hydrolysis to unwinding of the duplex is not completely understood. We reasoned that if ATPase activity in the presence of a 3'-tailed dsRNA was effectively coupled with the unwinding reaction, we should be able to determine the enzyme processivity by measuring the hydrolysis of ATP under single processive cycle conditions. To this aim, we added increasing concentrations of heparin in a standard ATPase reaction stimulated by the addition of a saturating amount of 3'-tailed dsRNA (Fig. 6B
). In parallel, we performed the same heparin titration experiment adding the trapping molecule in a standard helicase assay (Fig. 6C
). In both experiments heparin was added either before the enzyme or together with ATP after pre-incubation of the protein with the helicase substrate. Heparin completely inhibited both NS3 enzymatic activities when present in the reaction before the enzyme was added (Fig. 6B
, C
). In the helicase experiment (Fig. 6C
), residual activity was observed when heparin was added together with ATP. In this latter case, the strand release measured (about 0·02 nM product/nM enzyme) was the result of a single processive cycle of unwinding and represents therefore an index of helicase processivity. In contrast, the RNA-stimulated ATPase activity was completely inhibited by heparin in these conditions (Fig. 6B
), suggesting that most of the observed RNA-stimulated ATP hydrolysis is not directly coupled with the translocation of the enzyme on the dsRNA substrate. On the contrary, binding of the protein to ssRNA appears the only event important for the stimulation of ATPase activity. Interestingly, the residual RNA-stimulated activity following heparin addition was equal to the intrinsic ATPase, confirming that the observed effect depended on the competition of the trapping molecule for binding to RNA rather than on a non-specific inhibitory interaction with the enzyme.
Time-courses of duplex unwinding (Fig. 6D) revealed that NS3 reached a plateau in the reaction after only 10 min, both in the absence and in the presence of heparin. Under single processive cycle conditions, the maximal concentration of strand released was 0·3 nM, about 40% of that measured in the absence of the trapping molecule. In our time-course experiments, we were not able to measure with accuracy the amplitude of the rapid phase of the reaction. This value depends also on the length of the duplex region that is unwound by the enzyme in a single cycle, 15 base pairs in our substrate, and further experiments on much longer duplex substrates are needed to more precisely define the intrinsic rate of unwinding by NS3.
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Discussion |
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It is reasonable to assume that following HCV infection, the initiation of negative-strand RNA synthesis depends on an initial recognition and specific binding of the replicative complex to the 3'-end of the viral genomic RNA. NS5B polymerase has been recently shown to specifically interact with conserved stemloop structures in the 3' coding region of the HCV genomic RNA (Cheng et al., 1999 ). Since the 98 nt X region at the 3' terminus of the HCV genome is highly conserved in sequence and has a stable secondary structure (Blight & Rice, 1997
; Ito & Lai, 1997
; Kolykhalov et al., 1996
; Tanaka et al., 1996
), it has been proposed to be involved in viral RNA synthesis and in multiple proteinRNA interactions. It has been recently suggested that NS5B polymerase can use this region as a cis-acting sequence to initiate HCV RNA synthesis in vitro (Oh et al., 1999
), although previous studies failed to demonstrate a specific interaction between NS5B and a viral RNA containing the 98 nt sequence (Cheng et al., 1999
; Lohmann et al., 1997
). Furthermore, cellular proteins including polypyrimidine tract-binding protein have been demonstrated to bind specifically to the SL 2 and SL 3 stemloop structures of the conserved X region (Ito & Lai, 1997
; Tsuchihara et al., 1997
). For efficient transcription initiation at the 3'-end of the HCV positive- and negative-strands, NS3-associated unwinding activity might be required to remove the secondary structure on the template RNA. Our analysis of NS3 unwinding activity on double-hybrid substrates indicates that the minimal internal non-base-paired region for optimal strand displacement lies between 9 and 6 nt, while shortening this region down to 3 nt causes a decrease to 25% of maximal activity. This would suggest that NS3 might require three or more non-base-paired ribonucleotides to trigger its unwinding activity and resolve the secondary structure elements on the template RNA. NS3 protein from HCV-related dengue virus has been recently demonstrated to interact with stemloop structures in the 3' non-coding region of the genomic RNA that plays an important role in the initiation of the negative-strand RNA synthesis (Chen et al., 1997
; Cui et al., 1998
). Our data indicate that HCV NS3 protein is able to bind tightly and with some specificity to the stable stemloop structure SL I formed by the 3'-terminal 46 bases of HCV positive-strand RNA (Blight & Rice, 1997
). Furthermore, NS3-associated helicase activity is able to resolve this kind of structure in a standard unwinding assay, presumably through the initial binding to the 6 nt loop followed by the ATP-dependent translocation of the enzyme along the base-paired stem. The specificity of the interaction might depend only on the presence of RNA secondary structure and not on the primary sequence. Indeed all base changes identified within SL I in different HCV genotypes occur either in the single-stranded loop or, when they arise in the double-stranded stem, compensatory mutations are always present which preserve the secondary structure (Blight & Rice, 1997
). It would be of interest to assess whether NS3 could interact with other stemloop structures within the ends of the genomic and anti-genomic RNA, and the degree of binding selectivity in the absence or presence of other replication factors.
Processivity of the NS3 helicase was inferred by its capacity to unwind a tailed RNA substrate containing a 15 nt duplex region under single processive cycle conditions. We included heparin to trap the enzyme not bound to RNA. Heparin has been used as a nucleic acid analogue in studies of a number of enzymes including DNA helicases (Korangy & Julin, 1992 , 1993
) and affinity chromatography on a heparin column was used in the purification protocol of native FL NS3 (Gallinari et al., 1998
). The effect exhibited by the order of addition of the trapping molecule on the amount of strand released by NS3 helicase activity is consistent with a processive action of the enzyme. This implies that heparin does not bind to the enzyme while unwinding its substrate. A similar inhibitory effect on the helicase activity of NS3 was observed by including excess amounts of oligo(U)18 in the assay (not shown). Assuming that the enzyme progresses on the duplex RNA by two base pairs for every molecule of ATP hydrolysed (Porter et al., 1998
), theoretically 200 nM ATP (half of the amount of base pairs contained in 20 nM of 20 nt dsRNA) should be hydrolysed in the presence of heparin by 20 nM enzyme during a complete single cycle of unwinding (assuming that all the enzyme is catalytically active). On the contrary, the ATPase activity stimulated by partial dsRNA was completely inhibited by heparin, independent of the order of addition. This would suggest that the extent of ATP hydrolysis measured in this assay is not coupled, for the most part, with the translocation of the enzyme on duplex RNA but is only reflecting NS3 binding to the ssRNA tail. This is consistent with the unanticipated features of a K1235E mutant in motif I of NS3 helicase (Kim et al., 1997
). This mutation almost completely abolished both the intrinsic ATPase and helicase activities of the isolated helicase domain, although the RNA-stimulated ATPase activity was only partially reduced. Similar results were also reported for a different mutation at the same K residue in FL-NS3 (Wardell et al., 1999
). It has been reported that, under single cycle conditions, the processivity of the NS3 helicase domain is low (Porter et al., 1998
). The evidence presented here with FL-NS3 does not support or disagree with this observation and additional experiments are required to establish whether the isolated helicase domain and the FL enzyme are equally processive in vitro. Furthermore, the association to other cellular and/or viral proteins might increase the processivity of NS3 in vivo as has been shown for several viral and cellular replication helicases (Boehmer, 1998
; Dong et al., 1996
; Phillips et al., 1997
).
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Acknowledgments |
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References |
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Blight, K. J. & Rice, C. M. (1997). Secondary structure determination of the conserved 98-base sequence at the 3' terminus of hepatitis C virus genome RNA.Journal of Virology 71, 7345-7352.[Abstract]
Boehmer, P. E. (1998). The herpes simplex virus type-1 single-strand DNA-binding protein, ICP8, increases the processivity of the UL9 protein DNA helicase.Journal of Biological Chemistry 273, 2676-2683.
Chen, C.-J., Kuo, M.-D., Chien, L.-J., Hsu, S.-L., Wang, Y.-M. & Lin, J.-H. (1997). RNAprotein interactions: involvement of NS3, NS5, and 3' noncoding regions of Japanese encephalitis virus genomic RNA.Journal of Virology 71, 3466-3473.[Abstract]
Cheng, J.-C., Chang, M.-F. & Chang, S. C. (1999). Specific interaction between the hepatitis C virus NS5B RNA polymerase and the 3' end of the viral RNA.Journal of Virology 73, 7044-7049.
Cho, H.-S., Ha, N.-C., Kang, L.-W., Chung, K. M., Back, S. H., Jang, S. K. & Oh, B.-H. (1998). Crystal structure of RNA helicase from genotype 1b hepatitis C virus.Journal of Biological Chemistry 273, 15045-15052.
Clarke, B. (1997). Molecular virology of hepatitis C virus.Journal of General Virology 78, 2397-2410.
Cui, T., Sugrue, R. J., Xu, Q., Lee, A. K. W., Chan, Y.-C. & Fu, J. (1998). Recombinant dengue virus type 1 NS3 protein exhibits specific viral RNA binding and NTPase activity regulated by the NS5 protein.Virology 246, 409-417.[Medline]
De Francesco, R., Pessi, A. & Steinkühler, C. (1998). The hepatitis C virus NS3 proteinase: structure and function of a zinc-containing serine proteinase. In Therapies for Viral Hepatitis, pp. 235-245. Edited by R. F. Schinazi, J.-P. Sommadossi & H. C. Thomas. London: International Medical Press.
Dong, F., Weitzel, S. E. & von Hippel, P. H. (1996). A coupled complex of T4 DNA replication helicase (gp41) and polymerase (gp43) can perform rapid and processive DNA strand-displacement synthesis.Proceedings of the National Academy of Sciences, USA 93, 14456-14461.
Gallinari, P., Brennan, D., Nardi, C., Brunetti, M., Tomei, L., Steinkühler, C. & De Francesco, R. (1998). Multiple enzymatic activities associated with recombinant NS3 protein of hepatitis C virus.Journal of Virology 72, 6758-6769.
Gallinari, P., Paolini, C., Brennan, D., Nardi, C., Steinkühler, C. & De Francesco, R. (1999). Modulation of hepatitis C virus NS3 protease and helicase activities through the interaction with NS4A.Biochemistry 38, 5620-5632.[Medline]
Gorbalenya, A. E. & Koonin, E. V. (1993). Helicases: amino acid sequence comparison and structurefunction relationship.Current Opinion in Structural Biology 3, 419-429.
Gwack, Y., Wook, D., Han, J. H. & Choe, J. (1995). NTPase activity of hepatitis C virus NS3 protein expressed in insect cells.Molecular Cell 5, 171-175.
Gwack, Y., Kim, D. W., Han, J. H. & Choe, J. (1996). Characterization of RNA binding activity and RNA helicase activity of the hepatitis C virus NS3 protein.Biochemical and Biophysical Research Communications 225, 654-659.[Medline]
Houghton, M. (1996). Hepatitis C viruses. In Fields Virology, pp. 1035-1058. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. New York: Raven Press.
Ito, T. & Lai, M. C. (1997). Determination of the secondary structure of and cellular protein binding to the 3'-untranslated region of the hepatitis C virus RNA genome.Journal of Virology 71, 8698-8706.[Abstract]
Jin, L. & Peterson, D. L. (1995). Expression, isolation, and characterization of the hepatitis C virus ATPase/RNA helicase.Archives of Biochemistry & Biophysics 323, 47-53.[Medline]
Kanai, A., Tanabe, K. & Kohara, M. (1995). Poly(U) binding activity of hepatitis C virus NS3 protein, a putative RNA helicase.FEBS Letters 376, 221-224.[Medline]
Kim, D. W., Gwack, Y., Han, J. H. & Choe, J. (1995). C-terminal domain of the hepatitis C virus NS3 protein contains an RNA helicase activity.Biochemical and Biophysical Research Communications 215, 160-166.[Medline]
Kim, D. W., Kim, J., Gwack, Y., Han, J. H. & Choe, J. (1997). Mutational analysis of the hepatitis C virus RNA helicase.Journal of Virology 71, 9400-9409.[Abstract]
Kim, J. R., Morgernstern, K. A., Griffith, J. P., Dwyer, M. D., Thomson, J. A., Murcko, M. A., Lin, C. & Caron, P. R. (1998). Hepatitis C virus NS3 RNA helicase domain with a bound oligonucleotide: the crystal structure provides insights into the mode of unwinding.Structure 6, 89-100.[Medline]
Kolykhalov, A. A., Feinstone, S. M. & Rice, C. M. (1996). Identification of a highly conserved sequence element at the 3' terminus of hepatitis C virus genome RNA.Journal of Virology 70, 3363-3371.[Abstract]
Korangy, F. & Julin, D. A. (1992). A mutation in the consensus ATP-binding sequence of the RecD subunit reduces the processivity of the RecBCD enzyme from Escherichia coli.Journal of Biological Chemistry 267, 3088-3095.
Korangy, F. & Julin, D. A. (1993). Kinetics and processivity of ATP hydrolysis and DNA unwinding by the RecBC enzyme from Escherichia coli.Biochemistry 32, 4873-4880.[Medline]
Korolev, S., Hsieh, J., Gauss, G. H., Lohman, T. M. & Waksman, G. (1997). Major domain swiveling revealed by the crystal structures of complexes of E. coli Rep helicase bound to single-stranded DNA and ADP.Cell 90, 635-647.[Medline]
Korolev, S., Yao, N., Lohman, T. M., Weber, P. C. & Waksman, G. (1998). Comparisons between the structures of HCV and Rep helicases reveal structural similarities between SF1 and SF2 super-families of helicases.Protein Science 7, 605-610.
Kwong, A. D., Kim, J. L., Rao, G., Lipovsek, D. & Raybuck, S. A. (1998). Hepatitis C virus NS3/4A protease.Antiviral Research 40, 1-18.[Medline]
Lohman, T. M. & Bjornson, K. P. (1996). Mechanisms of helicase-catalyzed DNA unwinding.Annual Review of Biochemistry 65, 169-214.[Medline]
Lohmann, V., Koch, J. O. & Bartenschlager, R. (1996). Processing pathways of the hepatitis C virus proteins.Journal of Hepatology 24, 11-19.[Medline]
Lohmann, V., Körner, F., Herian, U. & Bartenschlager, R. (1997). Biochemical properties of hepatitis C virus NS5B RNA-dependent RNA polymerase and identification of amino acid sequence motifs essential for enzymatic activity.Journal of Virology 71, 8416-8428.[Abstract]
Oh, J.-W., Ito, T. & Lai, M. M. C. (1999). A recombinant hepatitis C virus RNA-dependent RNA polymerase capable of copying the full-length viral RNA.Journal of Virology 73, 7694-7702.
Phillips, R. J., Hickleton, D. C., Boehmer, P. E. & Emmerson, P. T. (1997). The RecB protein of Escherichia coli translocates along single-stranded DNA in the 3' to 5' direction: a proposed ratchet mechanism.Molecular and General Genetics 254, 319-329.[Medline]
Porter, D. J. T. (1998). A kinetic analysis of the oligonucleotide-modulated ATPase activity of the helicase domain of the NS3 protein from hepatitis C virus.Journal of Biological Chemistry 273, 14247-14253.
Porter, D. J. T., Short, S. A., Hanlon, M. H., Preugschat, F., Wilson, J. E., Willard, D. H.Jr & Consler, T. G. (1998). Product release is the major contributor to kcat for the hepatitis C virus helicase-catalyzed strand separation of short duplex DNA.Journal of Biological Chemistry 273, 18906-18914.
Preugschat, F., Averett, D. R., Clarke, B. E. & Porter, D. J. T. (1996). A steady-state and pre-steady state kinetic analysis of the NTPase activity associated with the hepatitis C virus NS3 helicase domain.Journal of Biological Chemistry 271, 24449-24457.
Rice, C. M. (1996). Flaviviridae: the viruses and their replication. In Fields Virology, pp. 931-960. Edited by B. N. Fields, D. M. Knipe & P. M. Howley. New York: Raven Press.
Studier, F. W., Rosenberg, A. H., Dunn, J. J. & Dubendorff, J. W. (1998). Use of the T7 RNA polymerase to direct expression of cloned genes.Methods in Enzymology 185, 60-89.
Subramanya, H. S., Bird, L. E., Brannigan, J. A. & Wigley, D. B. (1996). Crystal structure of a DExx box helicase.Nature 384, 379-383.[Medline]
Tai, C.-L., Chi, W.-K., Chen, D.-S. & Hwang, L.-H. (1996). The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3).Journal of Virology 70, 8477-8484.[Abstract]
Tanaka, T., Kato, N., Cho, M. J. & Shimotohno, K. (1996). Structure of the 3' terminus of the hepatitis C virus.Journal of Virology 70, 3307-3312.[Abstract]
Tsuchihara, K., Tanaka, T., Hijikata, M., Kuge, S., Toyoda, H., Nomoto, A., Yamamoto, N. & Shimotohno, K. (1997). Specific interaction of polypyrimidine tract-binding protein with the extreme 3'-terminal structure of the hepatitis C virus genome, the 3'X.Journal of Virology 71, 6720-6726.[Abstract]
Velankar, S. S., Soultanas, P., Dillingham, M. S., Subramanya, H. S. & Wigley, D. B. (1999). Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism.Cell 97, 75-84.[Medline]
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]
Wong, I. & Lohman, T. M. (1992). Allosteric effects of nucleotide cofactors on Escherichia coli Rep helicase-DNA binding.Science 256, 350-355.[Medline]
Yao, N., Hesson, T., Cable, M., Hong, Z., Kwong, A. D., Le, H. V. & Weber, P. C. (1997). Structure of the hepatitis C virus RNA helicase domain.Nature Structural Biology 4, 463-467.[Medline]
Received 12 October 1999;
accepted 5 January 2000.