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• W4296008462 abstract "Hepatitis C virus (HCV) is a major cause of liver-related diseases and hepatocellular carcinoma. The helicase domain of one of the nonstructural proteins of HCV, NS3 (nonstructural protein 3), is essential for viral replication; however, its specific biological role is still under investigation. Here, we set out to determine the interaction between a purified recombinant full length NS3 and synthetic guanine-rich substrates that represent the conserved G-quadruplex (G4)-forming sequences in the HCV-positive and HCV-negative strands. We performed fluorescence anisotropy binding, G4 reporter duplex unwinding, and G4RNA trapping assays to determine the binding and G4 unfolding activity of NS3. Our data suggest that NS3 can unfold the conserved G4 structures present within the genome and the negative strand of HCV. Additionally, we found the activity of NS3 on a G4RNA was reduced significantly in the presence of a G4 ligand. The ability of NS3 to unfold HCV G4RNA could imply a novel biological role of the viral helicase in replication. Hepatitis C virus (HCV) is a major cause of liver-related diseases and hepatocellular carcinoma. The helicase domain of one of the nonstructural proteins of HCV, NS3 (nonstructural protein 3), is essential for viral replication; however, its specific biological role is still under investigation. Here, we set out to determine the interaction between a purified recombinant full length NS3 and synthetic guanine-rich substrates that represent the conserved G-quadruplex (G4)-forming sequences in the HCV-positive and HCV-negative strands. We performed fluorescence anisotropy binding, G4 reporter duplex unwinding, and G4RNA trapping assays to determine the binding and G4 unfolding activity of NS3. Our data suggest that NS3 can unfold the conserved G4 structures present within the genome and the negative strand of HCV. Additionally, we found the activity of NS3 on a G4RNA was reduced significantly in the presence of a G4 ligand. The ability of NS3 to unfold HCV G4RNA could imply a novel biological role of the viral helicase in replication. Infection with Hepatitis C Virus (HCV) causes several liver-related health problems including hepatocellular carcinoma (1Manns M.P. Buti M. Gane E. Pawlotsky J.M. Razavi H. Terrault N. et al.Hepatitis C virus infection.Nat. Rev. Dis. Prim. 2017; 3: 1-19Google Scholar). It was estimated in 2015 that around 71.1 million people in the world were infected with the virus (2Polaris Observatory HCV CollaboratorsGlobal prevalence and genotype distribution of hepatitis C virus infection in 2015: a modelling study.Lancet Gastroenterol. Hepatol. 2017; 2: 161-176Google Scholar). In addition, a report from World Health Organization suggests that close to 290,000 people died in 2019 of liver issues linked to HCV infection https://www.who.int/news-room/fact-sheets/detail/hepatitis-c (Accessed November 8, 2021). HCV is a positive-sense, ssRNA virus with a single ORF that encodes for a single polyprotein, which then is cleaved into structural and nonstructural proteins (1Manns M.P. Buti M. Gane E. Pawlotsky J.M. Razavi H. Terrault N. et al.Hepatitis C virus infection.Nat. Rev. Dis. Prim. 2017; 3: 1-19Google Scholar). The positive strand of HCV inside a host cell is copied to a negative strand RNA from which a large number of positive strands are made (1Manns M.P. Buti M. Gane E. Pawlotsky J.M. Razavi H. Terrault N. et al.Hepatitis C virus infection.Nat. Rev. Dis. Prim. 2017; 3: 1-19Google Scholar). The nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B make up the complex that is necessary for viral replication (1Manns M.P. Buti M. Gane E. Pawlotsky J.M. Razavi H. Terrault N. et al.Hepatitis C virus infection.Nat. Rev. Dis. Prim. 2017; 3: 1-19Google Scholar, 3Paul D. Madan V. Bartenschlager R. Hepatitis C virus RNA replication and assembly: living on the fat of the land.Cell Host Microbe. 2014; 16: 569-579Google Scholar). Nonstructural protein 3 (NS3) is a dual-function protein with a protease domain at the N-terminus and a helicase domain at the C-terminus (1Manns M.P. Buti M. Gane E. Pawlotsky J.M. Razavi H. Terrault N. et al.Hepatitis C virus infection.Nat. Rev. Dis. Prim. 2017; 3: 1-19Google Scholar, 3Paul D. Madan V. Bartenschlager R. Hepatitis C virus RNA replication and assembly: living on the fat of the land.Cell Host Microbe. 2014; 16: 569-579Google Scholar). Studies suggest that the helicase activity of NS3 is essential for viral replication; however, its exact biological role in the life cycle of HCV is still under investigation (4Kolykhalov A.A. Mihalik K. Feinstone S.M. Rice C.M. Hepatitis C virus-encoded enzymatic activities and conserved RNA elements in the 3’ nontranslated region are essential for virus replication in vivo.J. Virol. 2000; 74: 2046-2051Google Scholar, 5Scheel T.K.H. Rice C.M. Understanding the hepatitis C virus life cycle paves the way for highly effective therapies.Nat. Med. 2013; 19: 837-849Google Scholar). The possible roles of NS3 helicase include the following: (1) unwinding the duplex formed by the positive and negative strand during replication, (2) unfolding secondary structures within the HCV genome, and (3) displacing proteins that could potentially deter the viral replication process (5Scheel T.K.H. Rice C.M. Understanding the hepatitis C virus life cycle paves the way for highly effective therapies.Nat. Med. 2013; 19: 837-849Google Scholar, 6Raney K.D. Sharma S.D. Moustafa I.M. Cameron C.E. Hepatitis C virus non-structural protein 3 (HCV NS3): a multifunctional antiviral target.J. Biol. Chem. 2010; 285: 22725-22731Google Scholar). NS3 is a member of the DExH helicase family, which is under superfamily 2 (7Byrd A.K. Raney K.D. Superfamily 2 helicases.Front. Biosci. 2012; 17: 2070-2088Google Scholar). It has unwinding activity on RNA or DNA substrates by translocating in the 3′ to 5′ direction (8Frick D.N. The hepatitis C virus NS3 protein: a model RNA helicase and potential drug target.Curr. Issues Mol. Biol. 2007; 9: 1-20Google Scholar). In addition to unwinding dsRNA and DNA molecules, some helicases can resolve noncanonical DNA or/and RNA secondary structures known as G-quadruplexes (G4s) (9Mendoza O. Bourdoncle A. Bouí J.-B. Brosh R.M. Mergny J.-L. Survey and summary G-quadruplexes and helicases.Nucl. Acids Res. 2016; 44: 1989-2006Google Scholar). G4 structures are formed when tandem repeats of guanine bases of an RNA or a DNA molecule interact with one another through Hoogsteen hydrogen bonding to form a planer structure known as a quartet or a tetrad (10Rhodes D. Lipps H.J. G-quadruplexes and their regulatory roles in Biology.Nucl. Acids Res. 2015; 43: 8627-8637Google Scholar). When two or more tetrads stack on top of each other, they form a stable G4 (9Mendoza O. Bourdoncle A. Bouí J.-B. Brosh R.M. Mergny J.-L. Survey and summary G-quadruplexes and helicases.Nucl. Acids Res. 2016; 44: 1989-2006Google Scholar, 10Rhodes D. Lipps H.J. G-quadruplexes and their regulatory roles in Biology.Nucl. Acids Res. 2015; 43: 8627-8637Google Scholar, 11Kharel P. Balaratnam S. Beals N. Basu S. The role of RNA G-quadruplexes in human diseases and therapeutic strategies.Wiley Inter. Rev. RNA. 2020; 11: e1568Google Scholar, 12Song J. Perreault J.-P. Topisirovic I. Ephane Richard S. RNA G-quadruplexes and their potential regulatory roles in translation.Translation. 2016; 41244031Google Scholar, 13Varshney D. Spiegel J. Zyner K. Tannahill D. Balasubramanian S. The regulation and functions of DNA and RNA G-quadruplexes.Nat. Rev. Mol. Cell Biol. 2020; 21: 459-474Google Scholar). G-quadruplex RNA (G4RNA) is more stable than its counterpart G4DNA (11Kharel P. Balaratnam S. Beals N. Basu S. The role of RNA G-quadruplexes in human diseases and therapeutic strategies.Wiley Inter. Rev. RNA. 2020; 11: e1568Google Scholar). G4 stability is determined by the number of tetrads, loop length, and the number of strands involved in forming the tetrad (10Rhodes D. Lipps H.J. G-quadruplexes and their regulatory roles in Biology.Nucl. Acids Res. 2015; 43: 8627-8637Google Scholar, 11Kharel P. Balaratnam S. Beals N. Basu S. The role of RNA G-quadruplexes in human diseases and therapeutic strategies.Wiley Inter. Rev. RNA. 2020; 11: e1568Google Scholar). G4 is also stabilized by monovalent cations such as K+ and Na+ that coordinate in the central channel of the four-stranded structure (11Kharel P. Balaratnam S. Beals N. Basu S. The role of RNA G-quadruplexes in human diseases and therapeutic strategies.Wiley Inter. Rev. RNA. 2020; 11: e1568Google Scholar). Quadruplexes can further be stabilized by ligands such as Phen-DC3, NMM, TMPyP4, Pyridostatin (PDS) (9Mendoza O. Bourdoncle A. Bouí J.-B. Brosh R.M. Mergny J.-L. Survey and summary G-quadruplexes and helicases.Nucl. Acids Res. 2016; 44: 1989-2006Google Scholar, 11Kharel P. Balaratnam S. Beals N. Basu S. The role of RNA G-quadruplexes in human diseases and therapeutic strategies.Wiley Inter. Rev. RNA. 2020; 11: e1568Google Scholar). In addition to the human genome, viral genomes could also harbor G4 structures (14Lavezzo E. Berselli M. Frasson I. Perrone R. Palù G. Brazzale A.R. et al.G-Quadruplex forming sequences in the genome of all known human viruses: a comprehensive guide.PLoS Comput. Biol. 2018; 14e1006675Google Scholar). Recently, Lavezzo et al. explored the presence of potential G4-forming sequences in the genome of all human viruses discovered so far (14Lavezzo E. Berselli M. Frasson I. Perrone R. Palù G. Brazzale A.R. et al.G-Quadruplex forming sequences in the genome of all known human viruses: a comprehensive guide.PLoS Comput. Biol. 2018; 14e1006675Google Scholar). The G4s in the genome of viruses could regulate translation, replication, and capsid formation (11Kharel P. Balaratnam S. Beals N. Basu S. The role of RNA G-quadruplexes in human diseases and therapeutic strategies.Wiley Inter. Rev. RNA. 2020; 11: e1568Google Scholar). Bioinformatics analysis on a number of HCV genotypes and subtypes revealed that both the positive and negative strands of the HCV genome contained conserved G4-forming sequences (15Wang S.R. Min Y.Q. Wang J.Q. Liu C.X. Fu B.S. Wu F. et al.A highly conserved G-rich consensus sequence in hepatitis C virus core gene represents a new anti–hepatitis C target.Sci. Adv. 2016; 2e1501535Google Scholar, 16Jaubert C. Bedrat A. Bartolucci L. Di Primo C. Ventura M. Mergny J.L. et al.RNA synthesis is modulated by G-quadruplex formation in Hepatitis C virus negative RNA strand.Sci. Rep. 2018; 8: 8120Google Scholar). Folded G4s within viral genomes, particularly those stabilized further with ligands, could deter successful viral replication (15Wang S.R. Min Y.Q. Wang J.Q. Liu C.X. Fu B.S. Wu F. et al.A highly conserved G-rich consensus sequence in hepatitis C virus core gene represents a new anti–hepatitis C target.Sci. Adv. 2016; 2e1501535Google Scholar, 17Artusi S. Nadai M. Perrone R. Biasolo M.A. Palù G. Flamand L. et al.The Herpes Simplex Virus-1 genome contains multiple clusters of repeated G-quadruplex: implications for the antiviral activity of a G-quadruplex ligand.Antivir. Res. 2015; 118: 123-131Google Scholar, 18Madireddy A. Purushothaman P. Loosbroock C.P. Robertson E.S. Schildkraut C.L. Verma S.C. G-quadruplex-interacting compounds alter latent DNA replication and episomal persistence of KSHV.Nucl. Acids Res. 2016; 44: 3675-3694Google Scholar, 19Perrone R. A dynamic G-quadruplex region regulates the HIV-1 long terminal repeat promoter.J. Med. Chem. 2013; 56: 6521-6530Google Scholar). As a result, viruses need to control the folding and unfolding of these nucleic acid structures in a regulated manner. There are several helicases reported to have the ability to unfold RNA or/and DNA G4s (20Sauer M. Paeschke K. G-quadruplex unwinding helicases and their function in vivo.Biochem. Soc. Trans. 2017; 45: 1173-1182Google Scholar, 21Caterino M. Paeschke K. Action and function of helicases on RNA G-quadruplexes.Methods. 2022; 204: 110-125Google Scholar). For instance, helicases of the same family as NS3, such as DHX36 and DHX9, unfold G4RNAs (21Caterino M. Paeschke K. Action and function of helicases on RNA G-quadruplexes.Methods. 2022; 204: 110-125Google Scholar, 22Booy E.P. Meier M. Okun N. Novakowski S.K. Xiong S. Stetefeld J. et al.The RNA helicase RHAU (DHX36) unwinds a G4-quadruplex in human telomerase RNA and promotes the formation of the P1 helix template boundary.Nucl. Acids Res. 2012; 40: 4110-4124Google Scholar, 23Chakraborty P. Grosse F. Human DHX9 helicase preferentially unwinds RNA-containing displacement loops (R-loops) and G-quadruplexes.DNA Repair (Amst). 2011; 10: 654-665Google Scholar, 24Creacy S.D. Routh E.D. Iwamoto F. Nagamine Y. Akman S.A. Vaughn J.P. G4 resolvase 1 binds both DNA and RNA tetramolecular quadruplex with high affinity and is the major source of tetramolecular quadruplex G4-DNA and G4-RNA resolving activity in HeLa cell lysates.J. Biol. Chem. 2008; 283: 34626-34634Google Scholar, 25Tippana R. Chen M.C. Demeshkina N.A. Ferré-D’Amaré A.R. Myong S. RNA G-quadruplex is resolved by repetitive and ATP-dependent mechanism of DHX36.Nat. Commun. 2019; 10: 1855Google Scholar). By unfolding these stable secondary structures, helicases remove the roadblocks to translation machineries (26Liu H. Lu Y.N. Paul T. Periz G. Banco M.T. Ferré-D’Amaré A.R. et al.A helicase unwinds hexanucleotide repeat RNA G-quadruplexes and facilitates repeat-associated non-AUG translation.J. Am. Chem. Soc. 2021; 143: 7368-7379Google Scholar, 27Huang W. Smaldino P.J. Zhang Q. Miller L.D. Cao P. Stadelman K. et al.Yin Yang 1 contains G-quadruplex structures in its promoter and 5′-UTR and its expression is modulated by G4 resolvase 1.Nucl. Acids Res. 2012; 40: 1033-1049Google Scholar). HCV replication takes places at a vesicle termed the “membranous web (28Gosert R. Egger D. Lohmann V. Bartenschlager R. Blum H.E. Bienz K. et al.Identification of the hepatitis C virus RNA replication complex in Huh-7 cells harboring subgenomic replicons.J. Virol. 2003; 77: 5487-5492Google Scholar)”. It is likely that NS3 is the only helicase inside the vesical because, according to the model proposed by Quinkert et al., only molecules of a specific size (not bigger than 16 kDa) can enter through the “neck’ of the membranous web into the vesicle where replication takes place (29Quinkert D. Bartenschlager R. Lohmann V. Quantitative analysis of the hepatitis C virus replication complex.J. Virol. 2005; 79: 13594-13605Google Scholar). Therefore, it is likely that HCV uses its own helicase (NS3) to unfold the conserved G4 structures within its genome to allow effective replication. In our study, we investigated the activity of NS3 on G4RNA-forming sequences derived from the positive or the negative strand of HCV. Our data suggests that NS3 unfolds G4RNA. However, its G4RNA-unfolding activity is significantly reduced when the G4 is inherently stable or stabilized with a G4 ligand. To determine whether and how NS3 interacts with G4RNA, we first design three different G-rich sequences: HCVG4, NEGG4, and NONHCVG4 (see Table S1 for sequences). HCVG4 contains the G-rich sequences from the positive strand of genotype 3a (15Wang S.R. Min Y.Q. Wang J.Q. Liu C.X. Fu B.S. Wu F. et al.A highly conserved G-rich consensus sequence in hepatitis C virus core gene represents a new anti–hepatitis C target.Sci. Adv. 2016; 2e1501535Google Scholar) that can theoretically form a three-tetrad G4 structure. NEGG4 substrate contains the conserved G-rich sequences from the negative strand of various strains of HCV (16Jaubert C. Bedrat A. Bartolucci L. Di Primo C. Ventura M. Mergny J.L. et al.RNA synthesis is modulated by G-quadruplex formation in Hepatitis C virus negative RNA strand.Sci. Rep. 2018; 8: 8120Google Scholar) and has the potential to form a two-tetrad G4 structure. Unlike HCVG4 and NEGG4, NONHCVG4 does not represent a sequence from HCV; however, it is a useful model substrate because it can potentially fold into a highly stable three-tetrad G4 since it has only one nucleotide loop length. For G4RNA structures containing relatively shorter loops, loop length has an inverse relationship with G4 stability (30Pandey S. Agarwala P. Maiti S. Effect of loops and G-quartets on the stability of RNA G-quadruplexes.J. Phys. Chem. B. 2013; 117: 6896-6905Google Scholar). Therefore, the shorter loop length results in more stable G4. NONHCVG4 was included in this study to understand how NS3 interacts with an RNA substrate that is likely to fold into an inherently stable G4. The helicase domain of NS3 interacts with oligonucleotides in an orientation-specific manner (31Gwack Y. Kim D.W. Han J.H. Choe J. Characterization of RNA binding activity and RNA helicase activity of the hepatitis C virus NS3 protein.Biochem. Biophys. Res. Commun. 1996; 225: 654-659Google Scholar, 32Tai C.L. Chi W.K. Chen D.S. Hwang L.H. The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3).J. Virol. 1996; 70: 8477-8484Google Scholar, 33Levin M.K. Gurjar M. Patel S.S. A Brownian motor mechanism of translocation and strand separation by hepatitis C virus helicase.Nat. Struct. Mol. Biol. 2005; 12: 429-435Google Scholar). For instance, Levin and Patel reported that the HCV helicase bound to a duplex DNA substrate containing a 3′ overhang around 50 times tighter than a similar substrate with a 5′ overhang (33Levin M.K. Gurjar M. Patel S.S. A Brownian motor mechanism of translocation and strand separation by hepatitis C virus helicase.Nat. Struct. Mol. Biol. 2005; 12: 429-435Google Scholar). In addition, Tai et al. and Gwack et al. independently demonstrated that effective duplex substrate–unwinding activity of NS3 helicase was dependent on the presence of a 3′ overhang (31Gwack Y. Kim D.W. Han J.H. Choe J. Characterization of RNA binding activity and RNA helicase activity of the hepatitis C virus NS3 protein.Biochem. Biophys. Res. Commun. 1996; 225: 654-659Google Scholar, 32Tai C.L. Chi W.K. Chen D.S. Hwang L.H. The helicase activity associated with hepatitis C virus nonstructural protein 3 (NS3).J. Virol. 1996; 70: 8477-8484Google Scholar). Therefore, a 3′ overhang has a significant effect on the affinity and activity of NS3 on a partial duplex. With the aim of understanding the effect of a 3′ overhang on the interaction between NS3 and G4RNA, 20 nucleotide (nt) adenosine monophosphates (A20) were added to each of the above three substrates to form HCVG4-A20, NEGG4-A20, and NONHCVG4-A20. CD spectra of all of the G-rich substrates mentioned above indicate that the sequences fold into a parallel G4 structures in 100 mM KCl (Fig. 1). A parallel G4 possesses a distinct CD spectrum with a peak around 265 nm and a valley around 240 nm (34Kypr J. Kejnovská I. Renčiuk D. Vorlíčková M. Circular dichroism and conformational polymorphism of DNA.Nucl. Acids Res. 2009; 37: 1713-1725Google Scholar, 35Oliver A.W. Kneale G.G. Structural characterization of DNA and RNA sequences recognized by the gene 5 protein of bacteriophage fd.Biochem. J. 1999; 339: 525-531Google Scholar, 36Del Villar-Guerra R. Trent J.O. Chaires J.B. G-quadruplex secondary structure obtained from circular dichroism spectroscopy.Angew. Chem. Int. Ed. Engl. 2018; 57: 7171-7175Google Scholar). We conclude that the presence of 20-nt adenosine monophosphates at the 3′ end of HCVG4, NEGG4, or NONHCVG4 does not hinder the formation of a parallel G4. To test whether NS3 binds to the G4RNA structures, a fluorescence anisotropy–binding assay was performed in 100 mM KCl binding buffer containing fluorescently labeled HCVG4, NEGG4, or NONHCVG4 and increasing concentration of NS3. NS3 bound tightly to the ssRNA, A20-3′FAM, with similar affinity (Kd = 6 ± 1 nM) to what was reported in the past for NS3 binding to a ssRNA (37Beran R.K.F. Serebrov V. Pyle A.M. The serine protease domain of hepatitis C viral NS3 activates RNA helicase activity by promoting the binding of RNA substrate.J. Biol. Chem. 2007; 282: 34913-34920Google Scholar) (Fig. 2). Because A20 has the highest amplitude and two NS3 molecules could potentially bind to the same oligonucleotide (based on eight nucleotides-binding site size for NS3 (38Raney V.M. Reynolds K.A. Harrison M.K. Harrison D.K. Cameron C.E. Raney K.D. Binding by the hepatitis C virus NS3 helicase partially melts duplex DNA.Biochemistry. 2012; 51: 7596-7607Google Scholar, 39Levin M.K. Patel S.S. Helicase from hepatitis C virus, energetics of DNA binding.J. Biol. Chem. 2002; 277: 29377-29385Google Scholar)), we also fit the data points to a two-sites binding model and determined the Kd1 and Kd2 to be 4.9 nM and 860 nM, respectively (Fig. S1). NS3 bound to HCVG4, NEGG4, or NONHCVG4 tightly with Kd values in the nano-molar range (Kd = 17 ± 4 nM, 19 ± 5 nM, and 23 ± 7 nM, respectively) (Fig. 2, A, B, and D). Similarly, NS3 binds tightly to the 3′ overhang–containing substrates, HCVG4-A20, NEGG4-A20, or NONHCVG4-A20 (Kd= 11 ± 3 nM, 6 ± 2 nM, and 12 ± 3 nM, respectively) (Fig. 2, A, C, and D). The binding affinity of NS3 to the 3′ overhang containing substrates is within two-fold of the single-stranded substrate. However, the substrates that lack a 3′ overhang bind to NS3 with a dissociation constant as high as four-fold than the ssRNA substrate. Nonetheless, the absence of a single-strand protein-loading zone does not prevent the NS3 from binding to the G4-forming substrates. Unfolding of a G4 structure requires a reaction buffer containing enough cations to maintain the folding of the G4 during the span of the reaction. Therefore, it is necessary to first verify the activity of the helicase in a buffer in which the G-rich oligonucleotides fold to form a stable G4. Several previous studies that examined the interaction of a helicase with a G4-forming oligonucleotides reported of using a buffer containing 100 mM KCl (26Liu H. Lu Y.N. Paul T. Periz G. Banco M.T. Ferré-D’Amaré A.R. et al.A helicase unwinds hexanucleotide repeat RNA G-quadruplexes and facilitates repeat-associated non-AUG translation.J. Am. Chem. Soc. 2021; 143: 7368-7379Google Scholar, 40Griffin W.C. Gao J. Byrd A.K. Chib S. Raney K.D. A biochemical and biophysical model of G-quadruplex DNA recognition by positive coactivator of transcription 4.J. Biol. Chem. 2017; 292: 9567-9582Google Scholar, 41Yangyuoru P.M. Bradburn D.A. Liu Z. Xiao T.S. Russell R. The G-quadruplex (G4) resolvase DHX36 efficiently and specifically disrupts DNA G4s via a translocation-based helicase mechanism.J. Biol. Chem. 2018; 293: 1924-1932Google Scholar). Therefore, we first determined the ATPase activity of a recombinant NS3 protein in 100 mM KCl buffer by conducting enzyme-coupled spectrophotometric ATPase assay that links ATP hydrolysis with the oxidation of NADH (Fig. 3A). In saturating conditions, which is the concentration of Poly (U) at which the specific activity is maximum, NS3 has ATPase activity of 52.5 ± 1.8 s−1 in low salt (50 mM KCl) buffer and 49.8 ± 5.0 s−1 in high salt (100 mM KCl) buffer, which is not significantly different (Fig. 3B). Therefore, the purified NS3 is as active at high salt concentration as it is at low salt concentration. To test whether NS3 can unfold G4-forming substrates to which it binds, we conducted a G4 reporter duplex unwinding assay. A radiolabeled–G4 reporter duplex substrate that contains the G-rich sequences from HCVG4, NEGG4, or NONHCVG4 oligonucleotides flanked by a 25 base pair duplex region and A20 3′ overhang was made as described in the Experimental procedures and illustrated in Fig. S2. Three nucleotides were added as a linker between the duplex region and the G4-forming sequences to allow proper folding of the G4 structure. The reporter substrate was incubated with the enzyme and the reaction was initiated with the addition of ATP (Fig. 4, A and B). In the reporter assay, the unfolding of the G4 structure by the enzyme is reported by the unwinding of the duplex region (Fig. 4B). Initially, we evaluated the amount of NS3 needed for the G4 reporter assay to saturate the substrate. We carried out the G4 reporter assay with varying concentrations of NS3 (400 nM, 600 nM, 800 nM, or 1000 nM) and 2 nM HCVG4-A20 reporter duplex substrate. The product formed from 400 nM NS3 was significantly lower than the product formed from the other NS3 concentrations; however, the amount of products obtained from 600 nM, 800 nM, and 1000 nM NS3 are not significantly different (Figs. 4, C, D and S3), suggesting 600 nM NS3 is enough to saturate the substrate. Consequently, 600 nM NS3 is the concentration used for subsequent dsRNA or G4 reporter duplex unwinding reactions. We used these reaction conditions as our standard condition: 600 nM NS3 incubated with 2 nM-radiolabeled HCVG4-A20 reporter duplex substrate in a buffer containing 100 mM KCl (Fig. 5A). Under standard conditions, NS3 unwinds HCVG4-A20 reporter duplex with an amplitude of product formation of 50 ± 1% at 20 min. The product formation is drastically reduced to 11 ± 1% when 0.25 μM PDS, a known G4-stabilizing ligand, is added to the reaction (Fig. 5, B and C). The same concentration of PDS has less effect on the activity of NS3 on a partial duplex substrate with the same length and sequence as the HCVG4-A20 reporter duplex, except that the guanines within the G-rich sequence are mutated to adenine (MUTHCVG4-A20; Table S1 and Fig. S4). For the HCVG4-A20 reporter duplex unwinding, there is a small (<5%) but measurable amount of product observed in the absence of ATP during the course of the reaction. In contrast, virtually no product is measured in the absence of NS3, suggesting that the product observed under the standard reaction conditions is driven by the enzyme. Interestingly, some product is measured from the zero second-time point (Fig. 5B). It is possible that this product is formed during preincubation in an ATP-independent manner. Reynolds et al. reported NS3 unwinding of 3′- or 5′-tailed short duplexes in an ATP-independent manner and proposed a noncanonical duplex melting mechanism by NS3, which involved local strand separation utilizing binding energy (42Reynolds K.A. Cameron C.E. Raney K.D. Melting of duplex DNA in the absence of ATP by NS3 helicase domain through specific interaction with a single-strand/double-strand junction.Biochemistry. 2015; 54: 4248-4258Google Scholar). Therefore, the product we observe from the G4 reporter duplex unwinding reaction in the absence of ATP could be due to NS3 binding and unwinding of the duplex region adjacent to the G4 structure in an ATP-independent manner. A single-turnover reaction involves product formation by the actions of NS3 molecules that are bound to the substrate at the initiation of the reaction. To create single-turnover reaction conditions, a protein trap is added in the reaction buffer to keep the dissociated NS3 molecules from rebinding to the substrate. The amount of product formed from a single-turnover reaction indicates the level of processivity of NS3 with the substrate studied. Under single-turnover conditions, 11 ± 1% product is formed from HCVG4-A20 reporter duplex, which is a 78% reduction from the product measured under a multi-turnover reaction condition in which NS3 molecules are allowed to bind, unwind, dissociate, and rebind for another cycle of enzymatic action (Fig. 6, A and B). The low product formation from the single-turnover reaction indicates NS3 might need to undergo a series of association and dissociation events from the substrate in order to unfold the G4 and then unwind the adjacent duplex region. Unlike KCl and NaCl, LiCl does not support the formation of a stable G4 (43Bhattacharyya D. Arachchilage G.M. Basu S. Metal cations in G-quadruplex folding and stability.Front. Chem. 2016; 4: 38Google Scholar). To test the effect of not having a stable G4 on the unfolding activity of NS3, we allowed the enzyme to unfold the same substrate, HCVG4-A20 reporter duplex, in 100 mM LiCl reaction buffer, which resulted in 91 ± 2% product formation (Fig. 6, C and D). The product obtained in 100 mM KCl is significantly lower than in 100 mM LiCl, implying that a stable G4 could be a formidable barrier, keeping the NS3 from unwinding the adjoining duplex region of the G4 reporter duplex substrate. In addition to the external factors discussed above, the length of the 3′ overhang could also regulate the G4-unfolding activity of NS3. Efficient partial duplex unwinding by NS3 is highly dependent on the length of the 3′ overhang; the longer the tail, the higher the unwinding amplitude (44Zhou T. Ren X. Adams R.L. Pyle A.M. NS3 from hepatitis C virus strain JFH-1 is an unusually robust helicase that is primed to bind and unwind viral RNA.J. Virol. 2018; 92e01253-17Google Scholar). In order to investigate the effect of the length of a 3′ overhang on G4-unfolding activity of NS3, a G4 reporter duplex unwinding reaction was conducted with HCVG4 reporter substrate containing 20-nt, 9-nt, or 2-nt 3′ overhangs. We observed 50 ± 1%, 40 ± 3%, and 20 ± 2% product at 20 min for substrates with 20-nt, 9-nt, and 2-nt of 3′ overhangs, respectively, in a multi-turnover reaction (Fig. 7). The result strongly implies that the length of the single-strand extension is important for efficient unfolding of a G4 by NS3. Under multi-turnover conditions, 82 ± 4% of the MUTHCVG4-A20 control duplex is unwound, which is significantly higher than the product obtained from HCVG4-A20 reporter duplex substrate (50 ± 1%) (Fig. 8, A and B). In addition, the fraction of the duplex substrate unwound under single-turnover conditions was considerably higher than the amount of G4 containing reporter duplex unwound (Fig. 8, A and B). This higher activity of NS3 with the control duplex substrate than with the G4 reporter duplex implies that the helicase is more processive on the former than on the later. It also indicates that G4 is a strong deterrent for helicase progression. Unlike for HCVG4-A20 reporter duplex unwi" @default.
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• W4296008462 date "2022-11-01" @default.
• W4296008462 modified "2023-10-18" @default.
• W4296008462 title "Hepatitis C virus nonstructural protein NS3 unfolds viral G-quadruplex RNA structures" @default.
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• W4296008462 doi "https://doi.org/10.1016/j.jbc.2022.102486" @default.
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