Structure-Based Mutational Analysis of the NS3 Helicase from Dengue Virus
http://www.100md.com
《病菌学杂志》
Novartis Institute for Tropical Diseases, 10 Biopolis Road, Chromos Building, Singapore 138670, Singapore
School of Biological Sciences, Nanyang Technological University, 60, Nanyang Drive, Singapore 637551, Singapore
ABSTRACT
We performed a mutational analysis of the NS3 helicase of dengue virus to test insights gleaned from its crystal structure and identified four residues in the full-length protein that severely impaired either its RTPase and ATPase (Arg-457-458, Arg-460, Arg-463) or helicase (Ile-365, Arg-376) activity. Alanine substitution of Lys-396, which is located at the surface of domain II, drastically reduced all three enzymatic activities. Our study points to a pocket at the surface of domain II that may be suitable for the design of allosteric inhibitors.
TEXT
The dengue virus NS3 protein contains protease, RNA helicase, 5'-nucleoside triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase) activities (1, 2, 5, 15, 20, 21). NS3 proteins from the four dengue virus serotypes share a minimum of 67% amino acid sequence identity. The enzymatic activities of NS3 proteins from several members of the Flaviviridae have been studied, including hepatitis C virus (HCV) (9), yellow fever virus (YFV) (19), Japanese encephalitis virus (18, 19), and West Nile virus (4). The unwinding of duplex RNA structures yielding individual RNA strands is thought to be required for an efficient viral genomic RNA synthesis by the NS5 RNA-dependent RNA polymerase. The essentiality of the helicase activity of NS3 in viral replication has been demonstrated through site-directed mutagenesis (8, 17); hence, it is an attractive target for the design of antiviral compounds. Two three-dimensional (3D) structures of active flavivirus helicase/NTPase catalytic domains from dengue virus (23) and yellow fever virus (22), respectively, were recently reported. As with the HCV NS3 helicase (12), the structure can be divided into three domains. The seven sequence motifs characteristic of superfamily 2 helicases (7) are present in domains I and II, situated at the N-terminal end of the protein. The NTPase site resides between these two domains. The C-terminal domain III differs most with its HCV counterpart (22, 23) and was suggested to bind NS5 (10). A tunnel that runs across the interface between domain III and the tip of domains I and II was proposed to accommodate a single-stranded nucleic acid tail along which the enzyme could translocate, following interdomain motions triggered by NTP hydrolysis (22, 23). Interestingly, recent studies on HCV helicase (14) suggest that the energy derived from nucleic acid binding may be used to unwind several base pairs at the fork essentially without ATP. However, the unidirectional translocation of the enzyme along a long stretch of DNA is derived from ATP hydrolysis.
Matusan et al. (17) reported mutations in the conserved motifs of dengue virus NS3 helicase. Here, based on their evolutionary conservation and structural insights (22, 23), we targeted 14 residues within the NS3 helicase (Fig. 1) using site-directed mutagenesis. The mutant proteins were tested for their involvement in the RNA-stimulated NTPase, RTPase, and double-stranded RNA (dsRNA) unwinding activity. While the truncated carboxyl-terminal two-thirds of NS3 used to determine the 3D structure displays helicase activity, we performed the mutational studies on full-length NS3 protein (NS3FL), since its dsRNA unwinding activity is approximately 30-fold greater (23). The wild-type NS3FL gene from dengue virus 2 (TSV01 strain, accession number AY037116, nucleotides 4522 to 6375) and each individual mutant was cloned and expressed in Escherichia coli as a fusion with thioredoxin reductase (Trx) with an N-terminal hexahistidine tag, as described previously (23). The TrxNS3FL fusion proteins were soluble, with yields of 4 to 6 mg of enzyme per liter of culture. The structural integrity of each mutant protein was assessed by measuring its far-UV circular dichroism spectrum and found to be similar to the wild-type enzyme (data not shown).
NTPase and RTPase activities of mutants. We tested the RNA-stimulated ATPase and RTPase activities of the mutants as previously described (1, 23). The kinetic parameters for ATP hydrolysis of wild-type TrxNS3FL and mutants are summarized in Table 1. Single alanine substitutions of the "arginine fingers" Arg-460 and Arg-463 in motif VI (corresponding to Arg-464 and Arg-467 in YFV helicase [22]) resulted in residual ATPase activities of 26% and 29%, respectively, and a comparable decrease in RTPase activity (Fig. 2A). This suggests that these two basic residues may be involved in transition-state stabilization via charge neutralization of the -phosphate of either a bound NTP or an RNA 5'-triphosphate oligonucleotide. By contrast, three strictly conserved residues, Glu-230, Asn-329, and Gly-414, which are also within a distance of 5 to 6 from the ATP substrate, showed only a slight decrease in ATPase activity, implying a lack of any direct involvement in the catalytic mechanism. Interestingly, the single mutation Lys-396-Ala completely abrogated ATPase activity (Table 1). The other mutant proteins studied retained an enzymatic activity comparable to that of the wild-type enzyme or displayed a slight increase (up to twofold) (Table 1). Overall, a good correlation exists between the ATPase and RTPase activities for the various mutants studied, and the small differences observed may be attributed to the fact that the RTPase assay uses an RNA template whose binding may influence the measured activity (e.g., residues Gly-414 and Gln-456). Alanine substitution of Lys-396, Arg-457-458, Arg-460, or Arg-463, which has impaired ATPase activity, also shows poor RTP hydrolysis, further suggesting that these two activities probably share a common catalytic site (see Table 1 and references 1 and 2). Since the Lys-396-Ala mutant did not show any ATPase activity, we determined whether it could still bind ATP. Incubation with 32P-ATP followed by short UV irradiation at various time points up to 10 min was carried out (Fig. 2B). While the wild-type enzyme showed a time-dependent binding of [32-P]ATP, no ATP binding was observed for the Lys-396-Ala mutant (Fig. 2B).
Helicase activities of mutants. Several basic or hydrophobic residues (Ile-365, Arg-342, Arg-376, Lys-381, Lys-396, Arg-398, and Lys-418) protruding from domain II of the dengue virus NS3 helicase structure (23) could make contacts with an RNA substrate. We mutated these residues to assess their role in helicase activity using a 32P-labeled dsRNA substrate containing both 5' and 3' overhangs (Fig. 2C) (23). The dsRNA unwinding activities of the mutants are shown in Fig. 2D. Two single mutations (Ile-365-Ala and Arg-376-Ala) completely abolished unwinding activity. In the Lys-396-Ala mutant, the activity is reduced by 80% compared to the wild-type enzyme. Interestingly, mutants with severely impaired helicase activity appeared to interact more tightly with the dsRNA substrate, as shown by the presence of supershifts visible in Fig. 2D. This suggested the formation of a dsRNA mutant enzyme complex more stable than the wild-type protein. To rule out the possibility that any single-stranded RNA (ssRNA) produced in the assay might comigrate with the shifted bands on the gel in Fig. 2D, we added a 100-fold excess of unlabeled competitor ssRNA to the reaction mixture and used mild denaturing conditions. As a result, these complexes could be fully (Arg-396-Ala mutant) or partially (Ile-365-Ala and Arg-376-Ala mutants) dissociated (Fig. 2E) and the dsRNA substrates released from the complex. The other mutants showed activities similar to that of the wild-type protein or an increase in dsRNA unwinding.
Structural and mechanistic interpretation. Lys-396-Ala is the only mutation identified in this study which completely abrogates the RTPase, ATPase, and helicase activity, yet the 3D structure does not suggest an obvious explanation. Lys-396 belongs to helix 2' and is located at the surface of domain II approximately midway between the ATP binding pocket and the putative single-strand binding tunnel (Fig. 3A). Its side chain is too distant to make direct contact with the phosphate groups of the polynucleotide in the tunnel but could interact with a nucleic acid segment bound at the surface of domain II. Interestingly, the segment corresponding to dengue virus NS3 helix 2' is disordered in the YFV helicase structure (22), indicating dynamic properties that might be important for NS3 enzymatic functions.
A double alanine NS3FL mutant at position Arg-457-Arg-458 (motif VI) only displays 27% residual ATPase activity. Using a truncated helicase domain, Matusan et al. (17) reported that alanine substitution of this conserved motif abolishes the helicase activity in vitro, whereas we observe a twofold increase in the helicase activity of the corresponding NS3FL mutant. The release of constraints formed by the buried Arg-457 and Arg-458 could lead to structural rearrangements in domain II, resulting in repositioning of the neighboring "arginine fingers" in a conformation less favorable for NTP hydrolysis (16). This residual ATPase activity, however, appears sufficient for duplex unwinding. Mutation of the "arginine fingers" abolished both the ATPase and helicase activities of the enzyme from HCV (11). In our study, in spite of a severe reduction in ATPase activity, only a limited reduction in helicase activity was observed for the corresponding mutants. Conversely, the substitution of either Ile-365 or Arg-376 to alanine almost completely abolished the helicase activity while fully preserving ATPase activity. This challenges the assumption that ATPase inhibitors can be used as antivirals to prevent RNA unwinding (3).
Ser-364 and Ile-365 are well-conserved residues located at the N terminus of helix 1' within a solvent-exposed pocket at the tip of domain II, which could make contact with an ssRNA tail trapped in the tunnel or with bases at the fork. Interestingly, Arg-376 and Lys-396 belong to helices 1' and 2' of domain II, respectively (Fig. 3A [23]). Whether movements in these two helices within domain II (23) are coupled with helicase activity remains to be determined. Recent single-molecule studies (6) support an inchworm-like translocation mechanism for the HCV NS3 helicase with two spatially distinct RNA binding sites. Extending their model, we propose that the pocket next to Ile-365 could act as the "helix opener" and disrupts hydrogen bonds at the fork. The basic concave face between domains II and III would act as "the translocator" by binding dsRNA ahead of the fork (Fig. 3B). The pocket next to Ile-365 appears to be an attractive region for the design of small molecules that would lock the helicase in a nonfunctional conformation.
These two authors contributed equally to this work.
REFERENCES
Bartelma, G., and R. Padmanabhan. 2002. Expression, purification, and characterization of the RNA 5'-triphosphatase activity of dengue virus type 2 nonstructural protein 3. Virology 299:122-132.
Benarroch, D., B. Selisko, G. A. Locatelli, G. Maga, J. L. Romette, and B. Canard. 2004. The RNA helicase, nucleotide 5'-triphosphatase, and RNA 5'-triphosphatase activities of Dengue virus protein NS3 are Mg2+-dependent and require a functional Walker B motif in the helicase catalytic core. Virology 328:208-218.
Borowski, P., O. Mueller, A. Niebuhr, M. Kalitzky, L. H. Hwang, H. Schmitz, M. A. Siwecka, and T. Kulikowsk. 2000. ATP-binding domain of NTPase/helicase as a target for hepatitis C antiviral therapy. Acta Biochim. Pol. 47:173-180.
Borowski, P., A. Niebuhr, O. Mueller, M. Bretner, K. Felczak, T. Kulikowski, and H. Schmitz. 2001. Purification and characterization of West Nile virus nucleoside triphosphatase (NTPase)/helicase: evidence for dissociation of the NTPase and helicase activities of the enzyme. J. Virol. 75:3220-3229.
Chambers, T. J., A. Nestorowicz, S. M. Amberg, and C. M. Rice. 1993. Mutagenesis of the yellow fever virus NS2B protein: effects on proteolytic processing, NS2B-NS3 complex formation, and viral replication. J. Virol. 67:6797-6807.
Dumont, S., W. Cheng, V. Serebov, R. K. Beran, I. Tinoco, Jr., A. M. Pyle, and C. Bustamante. 2006. RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP. Nature 439:105-108.
Gorbalenya, A. E., E. V. Koonin, A. P. Donchenko, and V. M. Blinov. 1989. Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Res. 17:4713-4730.
Grassmann, C. W., O. Isken, and S. E. Behrens. 1999. Assignment of the multifunctional NS3 protein of bovine viral diarrhea virus during RNA replication: an in vivo and in vitro study. J. Virol. 73:9196-9205.
Gwack, Y., D. W. Kim, J. H. Han, and J. Choe. 1996. Characterization of RNA binding activity and RNA helicase activity of the hepatitis C virus NS3 protein. Biochem. Biophys. Res. Commun. 225:654-659.
Johansson, M., A. J. Brooks, D. A. Jans, and S. G. Vasudevan. 2001. A small region of the dengue virus-encoded RNA-dependent RNA polymerase, NS5, confers interaction with both the nuclear transport receptor importin-beta and the viral helicase, NS3. J. Gen. Virol. 82:735-745.
Kim, D. W., J. Kim, Y. Gwack, J. H. Han, and J. Choe. 1997. Mutational analysis of the hepatitis C virus RNA helicase. J. Virol. 71:9400-9409.
Kim, J. L., K. A. Morgenstern, J. P. Griffith, M. D. Dwyer, J. A. Thomson, M. A. Murcko, C. Lin, and P. R. Caron. 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.
Levin, M. K., Y. H. Wang, and S. S. Patel. 2004. The functional interaction of the hepatitis C virus helicase molecules is responsible for unwinding processivity. J. Biol. Chem. 279:26005-26012.
Levin, M. K., M. Gurjar, and S. S. Patel. 2005. A Brownian motor mechanism of translocation and strand separation by hepatitis C virus helicase. Nat. Struct. Mol. Biol. 12:429-435.
Li, H., S. Clum, S. You, K. E. Ebner, and R. Padmanabhan. 1999. The serine protease and RNA-stimulated nucleoside triphosphatase and RNA helicase functional domains of dengue virus type 2 NS3 converge within a region of 20 amino acids. J. Virol. 73:3108-3116.
Lin, C., and J. L. Kim. 1999. Structure-based mutagenesis study of hepatitis C virus NS3 helicase. J. Virol. 73:8798-8807.
Matusan, A. E., M. J. Pryor, A. D. Davidson, and P. J. Wright. 2001. Mutagenesis of the Dengue virus type 2 NS3 protein within and outside helicase motifs: effects on enzyme activity and virus replication. J. Virol. 75:9633-9643.
Utama, A., H. Shimizu, F. Hasebe, K. Morita, A. Igarashi, I. Shoji, Y. Matsuura, M. Hatsu, K. Takamizawa, A. Hagiwara, and T. Miyamura. 2000. Role of the DExH motif of the Japanese encephalitis virus and hepatitis C virus NS3 proteins in the ATPase and RNA helicase activities. Virology 273:316-324.
Utama, A., H. Shimizu, S. Morikawa, F. Hasebe, K. Morita, A. Igarashi, M. Hatsu, K. Takamizawa, and T. Miyamura. 2000. Identification and characterization of the RNA helicase activity of Japanese encephalitis virus NS3 protein. FEBS Lett. 465:74-78.
Warrener, P., J. K. Tamura, and M. S. Collett. 1993. RNA-stimulated NTPase activity associated with yellow fever virus NS3 protein expressed in bacteria. J. Virol. 67:989-996.
Wengler, G. 1993. The NS 3 nonstructural protein of flaviviruses contains an RNA triphosphatase activity. Virology 197:265-273.
Wu, J., A. K. Bera, R. J. Kuhn, and J. L. Smith. 2005. Structure of the flavivirus helicase: implications for catalytic activity, protein interactions, and proteolytic processing. J. Virol. 79:10268-10277.
Xu, T., A. Sampath, A. Chao, D. Wen, M. Nanao, P. Chene, S. G. Vasudevan, and J. Lescar. 2005. Structure of the dengue virus helicase/nucleoside triphosphatase catalytic domain at a resolution of 2.4 . J. Virol. 79:10278-10288.(Aruna Sampath, Ting Xu, A)
School of Biological Sciences, Nanyang Technological University, 60, Nanyang Drive, Singapore 637551, Singapore
ABSTRACT
We performed a mutational analysis of the NS3 helicase of dengue virus to test insights gleaned from its crystal structure and identified four residues in the full-length protein that severely impaired either its RTPase and ATPase (Arg-457-458, Arg-460, Arg-463) or helicase (Ile-365, Arg-376) activity. Alanine substitution of Lys-396, which is located at the surface of domain II, drastically reduced all three enzymatic activities. Our study points to a pocket at the surface of domain II that may be suitable for the design of allosteric inhibitors.
TEXT
The dengue virus NS3 protein contains protease, RNA helicase, 5'-nucleoside triphosphatase (NTPase), and RNA 5'-triphosphatase (RTPase) activities (1, 2, 5, 15, 20, 21). NS3 proteins from the four dengue virus serotypes share a minimum of 67% amino acid sequence identity. The enzymatic activities of NS3 proteins from several members of the Flaviviridae have been studied, including hepatitis C virus (HCV) (9), yellow fever virus (YFV) (19), Japanese encephalitis virus (18, 19), and West Nile virus (4). The unwinding of duplex RNA structures yielding individual RNA strands is thought to be required for an efficient viral genomic RNA synthesis by the NS5 RNA-dependent RNA polymerase. The essentiality of the helicase activity of NS3 in viral replication has been demonstrated through site-directed mutagenesis (8, 17); hence, it is an attractive target for the design of antiviral compounds. Two three-dimensional (3D) structures of active flavivirus helicase/NTPase catalytic domains from dengue virus (23) and yellow fever virus (22), respectively, were recently reported. As with the HCV NS3 helicase (12), the structure can be divided into three domains. The seven sequence motifs characteristic of superfamily 2 helicases (7) are present in domains I and II, situated at the N-terminal end of the protein. The NTPase site resides between these two domains. The C-terminal domain III differs most with its HCV counterpart (22, 23) and was suggested to bind NS5 (10). A tunnel that runs across the interface between domain III and the tip of domains I and II was proposed to accommodate a single-stranded nucleic acid tail along which the enzyme could translocate, following interdomain motions triggered by NTP hydrolysis (22, 23). Interestingly, recent studies on HCV helicase (14) suggest that the energy derived from nucleic acid binding may be used to unwind several base pairs at the fork essentially without ATP. However, the unidirectional translocation of the enzyme along a long stretch of DNA is derived from ATP hydrolysis.
Matusan et al. (17) reported mutations in the conserved motifs of dengue virus NS3 helicase. Here, based on their evolutionary conservation and structural insights (22, 23), we targeted 14 residues within the NS3 helicase (Fig. 1) using site-directed mutagenesis. The mutant proteins were tested for their involvement in the RNA-stimulated NTPase, RTPase, and double-stranded RNA (dsRNA) unwinding activity. While the truncated carboxyl-terminal two-thirds of NS3 used to determine the 3D structure displays helicase activity, we performed the mutational studies on full-length NS3 protein (NS3FL), since its dsRNA unwinding activity is approximately 30-fold greater (23). The wild-type NS3FL gene from dengue virus 2 (TSV01 strain, accession number AY037116, nucleotides 4522 to 6375) and each individual mutant was cloned and expressed in Escherichia coli as a fusion with thioredoxin reductase (Trx) with an N-terminal hexahistidine tag, as described previously (23). The TrxNS3FL fusion proteins were soluble, with yields of 4 to 6 mg of enzyme per liter of culture. The structural integrity of each mutant protein was assessed by measuring its far-UV circular dichroism spectrum and found to be similar to the wild-type enzyme (data not shown).
NTPase and RTPase activities of mutants. We tested the RNA-stimulated ATPase and RTPase activities of the mutants as previously described (1, 23). The kinetic parameters for ATP hydrolysis of wild-type TrxNS3FL and mutants are summarized in Table 1. Single alanine substitutions of the "arginine fingers" Arg-460 and Arg-463 in motif VI (corresponding to Arg-464 and Arg-467 in YFV helicase [22]) resulted in residual ATPase activities of 26% and 29%, respectively, and a comparable decrease in RTPase activity (Fig. 2A). This suggests that these two basic residues may be involved in transition-state stabilization via charge neutralization of the -phosphate of either a bound NTP or an RNA 5'-triphosphate oligonucleotide. By contrast, three strictly conserved residues, Glu-230, Asn-329, and Gly-414, which are also within a distance of 5 to 6 from the ATP substrate, showed only a slight decrease in ATPase activity, implying a lack of any direct involvement in the catalytic mechanism. Interestingly, the single mutation Lys-396-Ala completely abrogated ATPase activity (Table 1). The other mutant proteins studied retained an enzymatic activity comparable to that of the wild-type enzyme or displayed a slight increase (up to twofold) (Table 1). Overall, a good correlation exists between the ATPase and RTPase activities for the various mutants studied, and the small differences observed may be attributed to the fact that the RTPase assay uses an RNA template whose binding may influence the measured activity (e.g., residues Gly-414 and Gln-456). Alanine substitution of Lys-396, Arg-457-458, Arg-460, or Arg-463, which has impaired ATPase activity, also shows poor RTP hydrolysis, further suggesting that these two activities probably share a common catalytic site (see Table 1 and references 1 and 2). Since the Lys-396-Ala mutant did not show any ATPase activity, we determined whether it could still bind ATP. Incubation with 32P-ATP followed by short UV irradiation at various time points up to 10 min was carried out (Fig. 2B). While the wild-type enzyme showed a time-dependent binding of [32-P]ATP, no ATP binding was observed for the Lys-396-Ala mutant (Fig. 2B).
Helicase activities of mutants. Several basic or hydrophobic residues (Ile-365, Arg-342, Arg-376, Lys-381, Lys-396, Arg-398, and Lys-418) protruding from domain II of the dengue virus NS3 helicase structure (23) could make contacts with an RNA substrate. We mutated these residues to assess their role in helicase activity using a 32P-labeled dsRNA substrate containing both 5' and 3' overhangs (Fig. 2C) (23). The dsRNA unwinding activities of the mutants are shown in Fig. 2D. Two single mutations (Ile-365-Ala and Arg-376-Ala) completely abolished unwinding activity. In the Lys-396-Ala mutant, the activity is reduced by 80% compared to the wild-type enzyme. Interestingly, mutants with severely impaired helicase activity appeared to interact more tightly with the dsRNA substrate, as shown by the presence of supershifts visible in Fig. 2D. This suggested the formation of a dsRNA mutant enzyme complex more stable than the wild-type protein. To rule out the possibility that any single-stranded RNA (ssRNA) produced in the assay might comigrate with the shifted bands on the gel in Fig. 2D, we added a 100-fold excess of unlabeled competitor ssRNA to the reaction mixture and used mild denaturing conditions. As a result, these complexes could be fully (Arg-396-Ala mutant) or partially (Ile-365-Ala and Arg-376-Ala mutants) dissociated (Fig. 2E) and the dsRNA substrates released from the complex. The other mutants showed activities similar to that of the wild-type protein or an increase in dsRNA unwinding.
Structural and mechanistic interpretation. Lys-396-Ala is the only mutation identified in this study which completely abrogates the RTPase, ATPase, and helicase activity, yet the 3D structure does not suggest an obvious explanation. Lys-396 belongs to helix 2' and is located at the surface of domain II approximately midway between the ATP binding pocket and the putative single-strand binding tunnel (Fig. 3A). Its side chain is too distant to make direct contact with the phosphate groups of the polynucleotide in the tunnel but could interact with a nucleic acid segment bound at the surface of domain II. Interestingly, the segment corresponding to dengue virus NS3 helix 2' is disordered in the YFV helicase structure (22), indicating dynamic properties that might be important for NS3 enzymatic functions.
A double alanine NS3FL mutant at position Arg-457-Arg-458 (motif VI) only displays 27% residual ATPase activity. Using a truncated helicase domain, Matusan et al. (17) reported that alanine substitution of this conserved motif abolishes the helicase activity in vitro, whereas we observe a twofold increase in the helicase activity of the corresponding NS3FL mutant. The release of constraints formed by the buried Arg-457 and Arg-458 could lead to structural rearrangements in domain II, resulting in repositioning of the neighboring "arginine fingers" in a conformation less favorable for NTP hydrolysis (16). This residual ATPase activity, however, appears sufficient for duplex unwinding. Mutation of the "arginine fingers" abolished both the ATPase and helicase activities of the enzyme from HCV (11). In our study, in spite of a severe reduction in ATPase activity, only a limited reduction in helicase activity was observed for the corresponding mutants. Conversely, the substitution of either Ile-365 or Arg-376 to alanine almost completely abolished the helicase activity while fully preserving ATPase activity. This challenges the assumption that ATPase inhibitors can be used as antivirals to prevent RNA unwinding (3).
Ser-364 and Ile-365 are well-conserved residues located at the N terminus of helix 1' within a solvent-exposed pocket at the tip of domain II, which could make contact with an ssRNA tail trapped in the tunnel or with bases at the fork. Interestingly, Arg-376 and Lys-396 belong to helices 1' and 2' of domain II, respectively (Fig. 3A [23]). Whether movements in these two helices within domain II (23) are coupled with helicase activity remains to be determined. Recent single-molecule studies (6) support an inchworm-like translocation mechanism for the HCV NS3 helicase with two spatially distinct RNA binding sites. Extending their model, we propose that the pocket next to Ile-365 could act as the "helix opener" and disrupts hydrogen bonds at the fork. The basic concave face between domains II and III would act as "the translocator" by binding dsRNA ahead of the fork (Fig. 3B). The pocket next to Ile-365 appears to be an attractive region for the design of small molecules that would lock the helicase in a nonfunctional conformation.
These two authors contributed equally to this work.
REFERENCES
Bartelma, G., and R. Padmanabhan. 2002. Expression, purification, and characterization of the RNA 5'-triphosphatase activity of dengue virus type 2 nonstructural protein 3. Virology 299:122-132.
Benarroch, D., B. Selisko, G. A. Locatelli, G. Maga, J. L. Romette, and B. Canard. 2004. The RNA helicase, nucleotide 5'-triphosphatase, and RNA 5'-triphosphatase activities of Dengue virus protein NS3 are Mg2+-dependent and require a functional Walker B motif in the helicase catalytic core. Virology 328:208-218.
Borowski, P., O. Mueller, A. Niebuhr, M. Kalitzky, L. H. Hwang, H. Schmitz, M. A. Siwecka, and T. Kulikowsk. 2000. ATP-binding domain of NTPase/helicase as a target for hepatitis C antiviral therapy. Acta Biochim. Pol. 47:173-180.
Borowski, P., A. Niebuhr, O. Mueller, M. Bretner, K. Felczak, T. Kulikowski, and H. Schmitz. 2001. Purification and characterization of West Nile virus nucleoside triphosphatase (NTPase)/helicase: evidence for dissociation of the NTPase and helicase activities of the enzyme. J. Virol. 75:3220-3229.
Chambers, T. J., A. Nestorowicz, S. M. Amberg, and C. M. Rice. 1993. Mutagenesis of the yellow fever virus NS2B protein: effects on proteolytic processing, NS2B-NS3 complex formation, and viral replication. J. Virol. 67:6797-6807.
Dumont, S., W. Cheng, V. Serebov, R. K. Beran, I. Tinoco, Jr., A. M. Pyle, and C. Bustamante. 2006. RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP. Nature 439:105-108.
Gorbalenya, A. E., E. V. Koonin, A. P. Donchenko, and V. M. Blinov. 1989. Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes. Nucleic Acids Res. 17:4713-4730.
Grassmann, C. W., O. Isken, and S. E. Behrens. 1999. Assignment of the multifunctional NS3 protein of bovine viral diarrhea virus during RNA replication: an in vivo and in vitro study. J. Virol. 73:9196-9205.
Gwack, Y., D. W. Kim, J. H. Han, and J. Choe. 1996. Characterization of RNA binding activity and RNA helicase activity of the hepatitis C virus NS3 protein. Biochem. Biophys. Res. Commun. 225:654-659.
Johansson, M., A. J. Brooks, D. A. Jans, and S. G. Vasudevan. 2001. A small region of the dengue virus-encoded RNA-dependent RNA polymerase, NS5, confers interaction with both the nuclear transport receptor importin-beta and the viral helicase, NS3. J. Gen. Virol. 82:735-745.
Kim, D. W., J. Kim, Y. Gwack, J. H. Han, and J. Choe. 1997. Mutational analysis of the hepatitis C virus RNA helicase. J. Virol. 71:9400-9409.
Kim, J. L., K. A. Morgenstern, J. P. Griffith, M. D. Dwyer, J. A. Thomson, M. A. Murcko, C. Lin, and P. R. Caron. 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.
Levin, M. K., Y. H. Wang, and S. S. Patel. 2004. The functional interaction of the hepatitis C virus helicase molecules is responsible for unwinding processivity. J. Biol. Chem. 279:26005-26012.
Levin, M. K., M. Gurjar, and S. S. Patel. 2005. A Brownian motor mechanism of translocation and strand separation by hepatitis C virus helicase. Nat. Struct. Mol. Biol. 12:429-435.
Li, H., S. Clum, S. You, K. E. Ebner, and R. Padmanabhan. 1999. The serine protease and RNA-stimulated nucleoside triphosphatase and RNA helicase functional domains of dengue virus type 2 NS3 converge within a region of 20 amino acids. J. Virol. 73:3108-3116.
Lin, C., and J. L. Kim. 1999. Structure-based mutagenesis study of hepatitis C virus NS3 helicase. J. Virol. 73:8798-8807.
Matusan, A. E., M. J. Pryor, A. D. Davidson, and P. J. Wright. 2001. Mutagenesis of the Dengue virus type 2 NS3 protein within and outside helicase motifs: effects on enzyme activity and virus replication. J. Virol. 75:9633-9643.
Utama, A., H. Shimizu, F. Hasebe, K. Morita, A. Igarashi, I. Shoji, Y. Matsuura, M. Hatsu, K. Takamizawa, A. Hagiwara, and T. Miyamura. 2000. Role of the DExH motif of the Japanese encephalitis virus and hepatitis C virus NS3 proteins in the ATPase and RNA helicase activities. Virology 273:316-324.
Utama, A., H. Shimizu, S. Morikawa, F. Hasebe, K. Morita, A. Igarashi, M. Hatsu, K. Takamizawa, and T. Miyamura. 2000. Identification and characterization of the RNA helicase activity of Japanese encephalitis virus NS3 protein. FEBS Lett. 465:74-78.
Warrener, P., J. K. Tamura, and M. S. Collett. 1993. RNA-stimulated NTPase activity associated with yellow fever virus NS3 protein expressed in bacteria. J. Virol. 67:989-996.
Wengler, G. 1993. The NS 3 nonstructural protein of flaviviruses contains an RNA triphosphatase activity. Virology 197:265-273.
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