FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2017462823> ?p ?o ?g. }

• W2017462823 endingPage "20492" @default.
• W2017462823 startingPage "20483" @default.
• W2017462823 abstract "Persistent infections with hepatitis C virus (HCV) are a major cause of liver disease and reflect its ability to disrupt virus-induced signaling pathways activating cellular antiviral defenses. HCV evasion of double-stranded RNA signaling through Toll-like receptor 3 is mediated by the viral protease NS3/4A, which directs proteolysis of its proline-rich adaptor protein, Toll-IL-1 receptor domain containing adaptor-inducing interferon-β (TRIF). The TRIF cleavage site has remarkable homology with the viral NS4B/5A substrate, although an 8-residue polyproline track extends upstream from the P6 position in lieu of the acidic residue present in viral substrates. Circular dichroism (CD) spectroscopy confirmed that a substantial fraction of TRIF exists as polyproline II helices, and inclusion of the polyproline track increased affinity of P side TRIF peptides for the HCV-BK protease. A polyproline II peptide representing an SH3 binding motif (PPPVPPRRR, Sos) bound NS3 with moderate affinity, resulting in inhibition of proteolytic activity. Chemical shift perturbations in NMR spectra indicated that Sos binds a 310 helix close to the protease active site. Thus, a polyproline II interaction with the 310 helix likely facilitates NS3/4A recognition of TRIF, indicating a significant difference from NS3/4A recognition of viral substrates. Because SH3 binding motifs are also present in NS5A, a viral protein that interacts with NS3, we speculate that the NS3 310 helix may be a site of interaction with other viral proteins. Persistent infections with hepatitis C virus (HCV) are a major cause of liver disease and reflect its ability to disrupt virus-induced signaling pathways activating cellular antiviral defenses. HCV evasion of double-stranded RNA signaling through Toll-like receptor 3 is mediated by the viral protease NS3/4A, which directs proteolysis of its proline-rich adaptor protein, Toll-IL-1 receptor domain containing adaptor-inducing interferon-β (TRIF). The TRIF cleavage site has remarkable homology with the viral NS4B/5A substrate, although an 8-residue polyproline track extends upstream from the P6 position in lieu of the acidic residue present in viral substrates. Circular dichroism (CD) spectroscopy confirmed that a substantial fraction of TRIF exists as polyproline II helices, and inclusion of the polyproline track increased affinity of P side TRIF peptides for the HCV-BK protease. A polyproline II peptide representing an SH3 binding motif (PPPVPPRRR, Sos) bound NS3 with moderate affinity, resulting in inhibition of proteolytic activity. Chemical shift perturbations in NMR spectra indicated that Sos binds a 310 helix close to the protease active site. Thus, a polyproline II interaction with the 310 helix likely facilitates NS3/4A recognition of TRIF, indicating a significant difference from NS3/4A recognition of viral substrates. Because SH3 binding motifs are also present in NS5A, a viral protein that interacts with NS3, we speculate that the NS3 310 helix may be a site of interaction with other viral proteins. Hepatitis C virus (HCV) 1The abbreviations used are: HCV, hepatitis C virus; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DTT, dithiothreitol; ERK, extracellular signal-regulated kinase; FL, full-length; FRET, fluorescence resonance energy transfer; HPLC, high performance liquid chromatography; HSQC, heteronuclear single quantum correlation; IRF-3, interferon regulatory factor 3; NS, nonstructural; PPII, polyproline II; sc, single-chain; SH, Src homology; Sos, Son of Sevenless; TIR, Toll-IL-1 receptor; TLR, Toll-like receptor; TRIF, Toll-IL-1 receptor domain containing adaptor-inducing interferon-β. 1The abbreviations used are: HCV, hepatitis C virus; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DTT, dithiothreitol; ERK, extracellular signal-regulated kinase; FL, full-length; FRET, fluorescence resonance energy transfer; HPLC, high performance liquid chromatography; HSQC, heteronuclear single quantum correlation; IRF-3, interferon regulatory factor 3; NS, nonstructural; PPII, polyproline II; sc, single-chain; SH, Src homology; Sos, Son of Sevenless; TIR, Toll-IL-1 receptor; TLR, Toll-like receptor; TRIF, Toll-IL-1 receptor domain containing adaptor-inducing interferon-β. is the causative agent of chronic hepatitis C, a globally distributed infection that affects more than 170 million persons worldwide and results in 8,000–10,000 deaths from liver disease annually in the United States alone (1Wong J.B. McQuillan G.M. McHutchison J.G. Poynard T. Am. J. Public Health. 2000; 90: 1562-1569Crossref PubMed Scopus (525) Google Scholar, 2Alter M.J. Mast E.E. Moyer L.A. Margolis H.S. Infect. Dis. Clin. North Am. 1998; 12: 13-26Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Currently available therapeutic regimens include combination therapy with interferon and ribavirin, but these are limited in efficacy, frequently associated with adverse reactions, and costly (3McHutchison J.G. Fried M.W. Clin. Liver Dis. 2003; 7: 149-161Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Thus, there is a compelling need for new antiviral drugs possessing greater efficacy against this virus. HCV is a member of the family Flaviviridae, classified within the genus Hepacivirus. It has a relatively small, 9.7-kb positive-strand RNA genome, which contains a large open reading frame that spans most of the genomic RNA (4Reed K.E.F. Rice C.M. Curr. Top. Microbiol. Immunol. 2000; 242: 55-84Crossref PubMed Scopus (473) Google Scholar). Translation of genomic RNA results in the expression of a lengthy polyprotein that is co- and post-translationally processed into at least 10 functional proteins by both host and viral protease activities. The processing events that liberate the nonstructural HCV proteins required for viral RNA replication (NS3, NS4A, NS4B, NS5A, and NS5B) are directed either in cis or in trans by a serine protease formed by the noncovalent association of NS3 with a segment of NS4A (5Grakoui A. McCourt D.W. Wychowski C. Feinstone S.M. Rice C.M. J. Virol. 1993; 67: 2832-2843Crossref PubMed Google Scholar, 6Bartenschlager R. Lohmann V. Wilkinson T. Koch J.O. J. Virol. 1995; 69: 7519-7528Crossref PubMed Google Scholar). Given the clinical success of inhibitors of the human immunodeficiency virus protease (7Randolph J.T. DeGoey D.A. Curr. Top. Med. Chem. 2004; 4: 1079-1095Crossref PubMed Scopus (85) Google Scholar), the HCV NS3/4A protease has become a leading target for drug discovery efforts. Candidate NS3/4A protease inhibitors have entered clinical trials and have shown substantial promise (8Lamarre D. Anderson P.C. Bailey M. Beaulieu P. Bolger G. Bonneau P. Bos M. Cameron D.R. Cartier M. Cordingley M.G. Faucher A.M. Goudreau N. Kawai S.H. Kukolj G. Lagace L. LaPlante S.R. Narjes H. Poupart M.A. Rancourt J. Sentjens R.E. St. George R. Simoneau B. Steinmann G. Thibeault D. Tsantrizos Y.S. Weldon S.M. Yong C.L. Llinas-Brunet M. Nature. 2003; 426: 186-189Crossref PubMed Scopus (842) Google Scholar). However, highly active NS3/4A inhibitors have proved to be quite challenging to develop because the active site of the viral protease is unusually featureless (9Love R.A. Parge H.E. Wickersham J.A. Hostomsky Z. Habuka N. Moomraw E.W. Adachi T. Hostomska Z. Cell. 1996; 87: 331-342Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 10Kim J.L. Morgenstern K.A. Lin C. Fox T. Dwyer M.D. Landro J.A. Chambers S.P. Markland W. Lepre C.A. O'Malley E.T. Harbeson S.L. Rice C.M. Murcko M.A. Caron P.R. Thomson J.A. Cell. 1996; 87: 343-355Abstract Full Text Full Text PDF PubMed Scopus (668) Google Scholar, 11De Francesco R. Steinkuhler C. Curr. Top. Microbiol. Immunol. 2000; 242: 149-169PubMed Google Scholar). The shallow contour and solvent-exposed nature of the NS3/4A substrate binding site lacks the pockets or crevices that would facilitate design of small molecule inhibitors with high affinity and binding specificity. In addition to its critical role in processing the viral proteins that comprise the viral RNA replicase, the NS3/4A protease disrupts innate intracellular antiviral defenses by blocking virus activation of interferon regulatory factor 3 (IRF-3) and NF-κB (12Foy E. Li K. Wang C. Sumter R. Ikeda M. Lemon S.M. Gale Jr., M. Science. 2003; 300: 1145-1148Crossref PubMed Scopus (697) Google Scholar, 13Li K. Foy E. Ferreon J.C. Nakamura M. Ferreon A.C.M. Ikeda M. Ray S.C. Gale Jr., M. Lemon S.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2992-2997Crossref PubMed Scopus (903) Google Scholar). These are cellular transcription factors that induce the expression of a large number of cellular antiviral defense genes, including the type 1 α/β interferons and interferon-stimulated genes, as well as chemokines and proinflammatory cytokines (14Hiscott J. Pitha P. Genin P. Nguyen H. Heylbroeck C. Mamane Y. Algarte M. Lin R. J. Interferon Cytokine Res. 1999; 19: 1-13Crossref PubMed Scopus (192) Google Scholar, 15Santoro M.G. Rossi A. Amici C. EMBO J. 2003; 22: 2552-2560Crossref PubMed Scopus (294) Google Scholar). Recent work indicates that the products of viral replication may lead to IRF-3 and NF-κB activation through two distinct and independent pathways, one involving engagement of Toll-like receptor 3 (TLR3) within endocytic vesicles by viral double-stranded RNA, and the other involving recognition of intracellular structured viral RNAs by the cellular DExH/D RNA helicase, retinoic acid-inducible gene I (16Yoneyama M. Kikuchi M. Natsukawa T. Shinobu N. Imaizumi T. Miyagishi M. Taira K. Akira S. Fujita T. Nat. Immunol. 2004; 5: 730-737Crossref PubMed Scopus (3023) Google Scholar, 17Alexopoulou L. Holt A.C. Medzhitov R. Flavell R.A. Nature. 2001; 413: 732-738Crossref PubMed Scopus (4796) Google Scholar). Both pathways are specifically disrupted by the protease activity of the NS3/4A complex (13Li K. Foy E. Ferreon J.C. Nakamura M. Ferreon A.C.M. Ikeda M. Ray S.C. Gale Jr., M. Lemon S.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2992-2997Crossref PubMed Scopus (903) Google Scholar, 18Foy E. Li K. Sumpter Jr., R. Loo M.Y. Johnson C. Wang C. Fish P. Yoneyama M. Fujita T. Lemon S.M. Gale Jr., M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2986-2991Crossref PubMed Scopus (470) Google Scholar). This disruption of antiviral signaling can be reversed by specific peptidomimetic inhibitors of the NS3/4A protease, suggesting that it targets for proteolysis one or more proteins involved in these signaling pathways. Although the cellular protein that resides within the retinoic acid-inducible gene I pathway and which is putatively cleaved by NS3/4A has yet to be identified, we have recently demonstrated that NS3/4A targets an essential protein within the TLR3 pathway for proteolysis: Toll-IL-1 receptor domain containing adaptor-inducing interferon-β (TRIF or TICAM-1) (13Li K. Foy E. Ferreon J.C. Nakamura M. Ferreon A.C.M. Ikeda M. Ray S.C. Gale Jr., M. Lemon S.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2992-2997Crossref PubMed Scopus (903) Google Scholar). TRIF is an essential adaptor protein that links TLR3 to downstream activation of IRF-3 and NF-κB (19Oshiumi H. Matsumoto M. Funami K. Akazawa T. Seya T. Nat. Immunol. 2003; 4: 161-167Crossref PubMed Scopus (988) Google Scholar, 20Sato S. Sugiyama M. Yamamoto M. Watanabe Y. Kawai T. Takeda K. Akira S. J. Immunol. 2003; 171: 4304-4310Crossref PubMed Scopus (567) Google Scholar), and its cleavage by a viral protease would be expected to disrupt double-stranded RNA-induced signal transduction through the TLR3 pathway. Consistent with this, we have shown that NS3/4A blocks the activation of the interferon-β promoter normally induced by exposure to extracellular poly(I·C, a synthetic double-stranded RNA analog, both in osteosarcoma cells that conditionally express the protease and in HeLa cells supporting ongoing replication of subgenomic HCV RNA (13Li K. Foy E. Ferreon J.C. Nakamura M. Ferreon A.C.M. Ikeda M. Ray S.C. Gale Jr., M. Lemon S.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2992-2997Crossref PubMed Scopus (903) Google Scholar). TRIF also supports MyD88-independent signaling after the engagement of TLR4 by pathogen-specific ligands (21Fitzgerald K.A. Rowe D.C. Barnes B.J. Caffrey D.R. Visintin A. Latz E. Monks B. Pitha P.M. Golenbock D.T. J. Exp. Med. 2003; 198: 1043-1055Crossref PubMed Scopus (901) Google Scholar). It is thought to interact with the TLRs through a Toll-IL-1 receptor (TIR) homology domain and to recruit multiple molecular signaling partners through specific domains in its N-terminal and C-terminal sequences (22Akira S. J. Biol. Chem. 2003; 278: 38105-38108Abstract Full Text Full Text PDF PubMed Scopus (610) Google Scholar). The signaling pathways it activates play critical roles in the response of cells to virus infection, and the ability of HCV to disrupt this signaling is likely to contribute substantially to its capacity for sustaining persistent infections in the face of both innate and adaptive immune responses (13Li K. Foy E. Ferreon J.C. Nakamura M. Ferreon A.C.M. Ikeda M. Ray S.C. Gale Jr., M. Lemon S.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2992-2997Crossref PubMed Scopus (903) Google Scholar). TRIF is cleaved proteolytically by the HCV protease between its Cys-372 and Ser-373 residues (13Li K. Foy E. Ferreon J.C. Nakamura M. Ferreon A.C.M. Ikeda M. Ray S.C. Gale Jr., M. Lemon S.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2992-2997Crossref PubMed Scopus (903) Google Scholar), effectively separating the TIR domain of the protein from an N-terminal TANK-binding kinase 1 interaction site required for IRF-3 phosphorylation, which is a prerequisite for dimerization, nuclear translocation, and activation of IRF-3 as a transcriptional factor (23Servant M. Grandvaux N. Hiscott J. Biochem. Pharmacol. 2002; 64: 985-992Crossref PubMed Scopus (132) Google Scholar). Here, we describe studies of the molecular properties of TRIF which contribute to its ability to function as a substrate for the NS3/4A protease. We demonstrate that the amino acid residues of the protease which interact with TRIF to facilitate its proteolysis differ significantly from those interacting with the canonical viral substrates. The TRIF cleavage site lacks a conserved P6 acidic residue that has been shown in several studies to make a substantial contribution to viral substrate specificity and binding affinity (24Koch U. Biasiol G. Brunetti M. Fattori D. Pallaoro M. Steinkuhler C. Biochemistry. 2001; 40: 631-640Crossref PubMed Scopus (37) Google Scholar, 25Urbani A. Bianchi E. Narjes F. Tramontano A. De Francesco R. Steinkuhler C. Pessi A. J. Biol. Chem. 1997; 272: 9204-9209Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). This is replaced in TRIF with an 8-residue polyproline track for which we demonstrate a unique role in interactions with the protease. Peptides—Synthetic peptides representing sequences spanning the NS3/4A cleavage sites at the viral NS5A/5B (H-EDVVαC/SMSY-OH) (26Landro J.A. Raybuck S.A. Luong Y.P. O'Malley E.T. Harbeson S.L. Morgenstern K.A. Rao G. Livingston D.J. Biochemistry. 1997; 36: 9340-9348Crossref PubMed Scopus (104) Google Scholar), NS4A/4B (H-EFDEMEEC/ASHLPYI-OH), and NS4B/5A (H-ECT-TPC/SGSWLRD-OH) junctions, the NS3/4A cleavage site within TRIF (Ac-PSSTPC/SAHL-amide, Ac-PPPPPPPPSSTPC/SAHL-amide) (13Li K. Foy E. Ferreon J.C. Nakamura M. Ferreon A.C.M. Ikeda M. Ray S.C. Gale Jr., M. Lemon S.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 2992-2997Crossref PubMed Scopus (903) Google Scholar), their P side cleavage products (Ac-PSSTPC-OH, and Ac-PPPPPPPPSSTPC-OH), and an HCV peptide suitable for use in a fluorescence resonance energy transfer (FRET) assay (Ac-DED(EDANS)EEAbuψ [COO]ASK-(DABCYL)-am) (27Taliani M. Bianchi E. Narjes F. Fossatelli M. Urbani A. Steinkuhler C. De Francesco R. Pessi A. Anal. Biochem. 1996; 240: 60-67Crossref PubMed Scopus (105) Google Scholar) were purchased from Anaspec and were >95% pure. Sos (Ac-PPPVPPRRR-amide) and SosY (Ac-VPPPVPPRRRY-amide) (28Ferreon J.C. Hilser V.J. Protein Sci. 2003; 12: 447-457Crossref PubMed Scopus (72) Google Scholar) peptides were synthesized within the UTMB Peptide Synthesis Core Laboratory and kindly provided by Dr. Vincent Hilser with >95% purity. All peptides, except for SosY, were quantified by amino acid analysis. The concentration of SosY was measured by absorbance at 280 nm using Edelhoch reagent (29Edelhoch H. Biochemistry. 1967; 6: 1948-1954Crossref PubMed Scopus (2987) Google Scholar). Protein Overexpression and Purification—TRIF was cloned as a His tag fusion protein into pET21d vector (Novagen) and recovered from Escherichia coli grown in a 20-liter fermenter as follows. A single colony of E. coli, carrying the expression plasmid, was inoculated into a small flask (37 °C, overnight) and then transferred into 20 liters of LB broth supplemented with 100 μg/ml ampicillin. Cells were grown at 37 °C until the A600 reached 0.6–0.8, then induced by addition of 1 mm isopropyl 1-thio-β-d-galactopyranoside followed by further incubation at 37 °C for 4–5 h. Cells were harvested and stored at –80 °C prior to purification. Immunoblots suggested that almost all of the expressed TRIF was present in inclusion bodies; therefore purification was carried under denaturing conditions. The cell pellet was dissolved in extraction buffer (6 m guanidine HCl, 100 mm sodium phosphate, 10 mm Tris, 2 mm β-mercaptoethanol, pH 8, 5 ml of buffer/g of cell pellet) for 24 h at 4 °C, with stirring. Cell debris was removed by ultracentrifugation, and the supernatant passed through a Ni2+ affinity fast protein liquid chromatography column. The column was rinsed with 10–15 volumes of wash buffer (8 m urea, 100 mm sodium phosphate, 10 mm Tris, 2 mm β-mercaptoethanol, pH 7.5) containing 50 mm imidazole. Bound proteins were eluted with wash buffer containing 500 mm imidazole. The initial fractions contained TRIF and other low molecular mass contaminants, whereas the final fractions contained mostly TRIF (>90% purity). Fractions with impurities were subjected to size exclusion chromatography (Superdex 200). TRIF was refolded by dialysis against 20 mm Tris, 2 mm DTT, pH 7.5, with the signature of the folded protein monitored by fluorescence and CD spectroscopy. From a 100-liter large scale purification, ∼50 mg was obtained with purity of >90%. The majority of the contaminants appeared to be TRIF fragments or degradation products. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (UTMB Protein Facility) confirmed that the molecular mass was ∼77.2 kDa. The HCV NS3 single-chain protease (scNS3) and a full-length single-chain NS3 (FL scNS3) containing the helicase domain were kindly provided by Bruce Malcolm (Schering-Plough Research Institute); both were derived from the BK strain of HCV (30Howe A.Y. Chase R. Taremi S.S. Risano C. Beyer B. Malcolm B. Lau J.Y. Protein Sci. 1999; 8: 1332-1341Crossref PubMed Scopus (51) Google Scholar). CD Spectroscopy—Far-ultraviolet CD spectra of TRIF were recorded from 200 to 260 nm using an AVIV™ CD spectrometer with the temperature maintained at 25 °C and the bandwidth set at 1 nm with a 0.5-s averaging time. The protein concentration was ∼0.4 mg/ml in 20 mm Tris, 2 mm DTT, pH 7.5. Buffer spectra were collected and subtracted from the protein spectra. Comparative Kinetic Analyses of NS3 Protease Activity with Different Viral and TRIF Substrates—In vitro cleavage of the different peptide substrates (5A/5B, 4A/4B, 4B/5A, TRIF p372, and HCV-FRET) was monitored using an HPLC Shimadzu™ chromatograph, equipped with UV-visible detection. For the 5A/5B, 4A/4B, 4B/5A substrates, the peptide fragments were monitored at a 280 nm wavelength, versus 220 nm for TRIF p372 and 512 nm for the FRET peptide. The reaction mix consisted of varying concentrations of the peptide substrate and enzyme (scNS3, BK strain) in 25 mm Tris, 300 mm NaCl, 5 μm EDTA, 10% glycerol, 0.05% n-dodecyl-β-d-maltoside, 10 mm DTT, pH 7.5. Enzyme concentrations were 4.2 nm for the 5A/5B substrate, 85.7 nm for 4A/4B, 1 μm for 4B/5A, and 3.3 μm for TRIF p372 substrate, with substrate concentrations ranging from 15 to 70 μm for 5A/5B, 70 to 700 μm for 4A/4B, 100 to 1,800 μm for 4B/5A, and 100 to 3500 μm for the TRIF p372 substrate. For most of the peptide substrates, such as 4B/5A and TRIF p372, the final peptide concentrations did not exceed the Km because of poor solubility. Nonetheless, enzyme concentrations generally were at least 100-fold less than substrate concentrations. Preliminary experiments ensured linearity in the initial velocity under these conditions and demonstrated that the enzyme concentration was directly proportional to the initial velocity. Reactions were incubated at 30 °C for a period of time sufficient to achieve ∼20% cleavage, then quenched with 1% trifluoroacetic acid. The products were analyzed by HPLC, with the fragments separated using a 10–50% acetonitrile gradient at 2.5 ml/min. Areas of product peaks were integrated and quantitated with calibration standards. Kinetic parameters (kcat and Km) were obtained by nonlinear fitting of the data (initial rates versus substrate concentration) with the Michaelis-Menten equation, V0=kcat[Et][S]/([S]+Km)(Eq. 1) where V0 is the initial velocity, Et is the total enzyme concentration, S is the substrate concentration, kcat is the catalytic turnover, and Km is the Michaelis constant. Full-length TRIF Cleavage by Full-length NS3 and scNS3—For proteolytic cleavage of TRIF by the full-length NS3 (both protease and helicase domains), reactions contained 4 μm TRIF (20 mm Tris, 30 mm DTT, pH 7.5) and 2 μm NS3 or scNS3 (BK strain), with or without 10 μm SCH6, a ketoamide, peptidomimetic NS3/4A protease inhibitor (12Foy E. Li K. Wang C. Sumter R. Ikeda M. Lemon S.M. Gale Jr., M. Science. 2003; 300: 1145-1148Crossref PubMed Scopus (697) Google Scholar). Reactions were incubated at 28 °C for 2 h. Estimated EC50 for scNS3 Cleavage of Full-length TRIF—To determine the enzyme concentration required to hydrolyze 50% of the full-length TRIF protein (EC50), different reaction tubes were prepared with 4 μm TRIF and varying scNS3 concentrations (0.25, 0.5, 1, 2, 3, 5 μm) in 20 mm Tris, 30 mm DTT, pH 7.5. Reactions were incubated at 30 °C for 30 min, quenched with gel loading buffer, and frozen at –20 °C prior to SDS-PAGE analysis. The quantity of the remaining full-length TRIF species present in each reaction mix was estimated by densitometric analysis. The data (TRIF intensity versus enzyme concentration) was fitted in a nonlinear fashion to the following simplified four-parameter logistic function (27Taliani M. Bianchi E. Narjes F. Fossatelli M. Urbani A. Steinkuhler C. De Francesco R. Pessi A. Anal. Biochem. 1996; 240: 60-67Crossref PubMed Scopus (105) Google Scholar) derived from the Hill equation, y=100/(1+([E]/EC50)n(Eq. 2) where y is the substrate intensity (TRIF), normalized to 100% for no cleavage, E is the enzyme concentration, and n is the Hill slope. Estimated EC50 for scNS3 Cleavage of the TRIF p372 Peptide— Reaction mixtures contained 4 μm TRIF p372 and varying scNS3 concentrations: 0, 1, 2, 5, 8.3, and 16.7 μm. The buffer conditions were identical to those for the full-length TRIF cleavage assay. The reactions were incubated at 30 °C for 30 min and quenched with an equal volume of 1% trifluoroacetic acid, prior to HPLC analysis. The decrease in the quantity of p372 was plotted against the enzyme concentration and fitted to Equation 2. Fluorescence Spectroscopy—Binding of the scNS3 protease to the Sos peptide, and a short and long TRIF peptide (Ac-PSSTPC-OH and Ac-PPPPPPPPSSTPC-OH, respectively) were followed by fluorescence spectroscopy (SPEX FluoroMax™ spectrofluorometer). The excitation and emission wavelengths were fixed at 275 and 340 nm, respectively, to monitor tyrosine and tryptophan fluorescence changes. The observed decreases in the fluorescence intensity signal that occurred upon ligand binding were fitted to a single-site binding model equation, y=A∗x/(Kd+x)(Eq. 3) where y is the relative fluorescence intensity, x is the ligand concentration, A is the maximum fluorescence intensity, and Kd is the dissociation constant. The experiments were performed at 25 °C and in 75 mm potassium phosphate buffer, 10 mm DTT, pH 6.5. IC50 Determination of Sos Peptide Inhibition of NS3/4A Protease Activity—Protease activity was assayed using the FRET-based HCV peptide as substrate, monitored by HPLC at 512 nm. The concentration of the SosY peptide necessary to inhibit 50% of the reaction (IC50) was determined from the decrease in the initial velocity of the reaction observed with increasing concentrations of the SosY peptide, fitted to the equation y=A/(1+([I]/IC50)n)(Eq. 4) where y is the initial velocity, A is the maximum initial velocity, I is the inhibitor concentration, and n is the Hill slope. The buffer conditions were 50 mm Tris, 50% glycerol, 2% CHAPS, 30 mm DTT, pH 7.5. NMR Spectroscopy—NMR spectra were collected at 25 °C using a Varian UnityPlus 750 MHz instrument equipped with a triple resonance probe and a pulsed field gradient. Sensitivity-enhanced 1H-15N HSQC spectra were recorded with identical acquisition parameters and 0.5 mm 15N-labeled scNS3 protein (75 mm potassium phosphate, 5% glycerol, 25 mm DTT, 0.015% NaN3, 10% D2O, pH 6.5) with different Sos peptide concentrations (0, 0.3, 1.3, 2.2 mm final). Data were processed and visualized on an SGI work station using Felix 98.0 software. To increase spectral resolution, time domain data were zero-filled twice, and 90° phase-shifted sine bell apodization functions were applied in both dimensions. The protein and chemical shift assignments (31McCoy M.A. Senior M.M. Gesell J.J. Ramanathan L. Wyss D.F. J. Mol. Biol. 2001; 305: 1099-1110Crossref PubMed Scopus (50) Google Scholar) were kindly provided by Bruce Malcolm (Schering Plough Research Institute). Visualizations—The Molmol program (32Koradi R. Billeter M. Wuthrich K. J. Mol. Graph. 1996; 14 (51–55): 29-32Crossref Scopus (6454) Google Scholar) was used to generate ribbon models of the NS3 protease (PDB entry 1NS3) (see Fig. 7) and the ball-and-stick model of the Sos peptide (see Fig. 4B), adopted from the co-crystal structure of the SEM-5 C-terminal SH3 domain with the Sos peptide (PDB entry 1SEM, chain C) (33Lim W.A. Richards F.M. Fox R.O. Nature. 1994; 372: 375-379Crossref PubMed Scopus (446) Google Scholar).Fig. 4The HCV NS5A protein contains a highly conserved Sos SH3 binding domain sequence (PPVPPRR). A, at the top is shown an alignment of the murine Sos homolog 1 sequence, NS5A sequence from the genotype 1b BK virus (the source of NS3/4A protease used in this study), and genotype 1a H77c virus, and TRIF (Gen-Bank accession numbers are shown) in the regions surrounding the putative SH3 binding domains. The SH3 binding domain motif is boxed and consists of ΦPXΦPX+ where Φ is a hydrophobic residue, X is variable, and + is generally basic (46Tan S.L. Nakao H. He Y. Vijaysri S. Neddermann P. Jacobs B.L. Mayer B.J. Katze M.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5533-5538Crossref PubMed Scopus (207) Google Scholar). Below is shown a schematic representation of the viral NS5A domain structure highlighting the NS4A interaction site (56Asabe S.I. Tanji Y. Satoh S. Kaneko T. Kimura K. Shimotohno K. J. Virol. 1997; 71: 790-796Crossref PubMed Google Scholar) as well as the Sos homology sequence that has been shown to interact with the cellular Grb2 SH3 domain in vivo. B, stereo view showing the characteristic PPII conformation of the Sos peptide (PPPVPPR; PDB entry 1SEM, chain C).View Large Image Figure ViewerDownload Hi-res image Download (PPT) TRIF Is a Novel Host Substrate for the HCV NS3/4A Protease—NS3 is a bifunctional protein, with a serine protease domain located within its N-terminal third, and ATPase/RNA helicase activity located within its C-terminal two-thirds (34Yao N. Reichert P. Taremi S.S. Prosise W.W. Weber P.C. Struct. Fold. Des. 1999; 7: 1353-1363Abstract Full Text Full Text PDF Scopus (365) Google Scholar). The NS3/4A protease is responsible for directing cleavage within the HCV polyprotein at the NS3/4A, 4A/4B, 4B/5A, and 5A/5B junctions. NS4A remains noncovalently associated with NS3 after scission at the NS3–4A junction, and it is a necessary cofactor for the full expression of NS3 protease activity (6Bartenschlager R. Lohmann V. Wilkinson T. Koch J.O. J. Virol. 1995; 69: 7519-7528Crossref PubMed Google Scholar, 35Gallinari P. Paolini C. Brennan D. Nardi C. Steinkuhler C. De Francesco R. Biochemistry. 1999; 38: 5620-5632Crossref PubMed Scopus (65) Google Scholar, 36Lin C. Thomson J.A. Rice C.M. J. Virol. 1995; 69: 4373-4380Crossref PubMed Google Scholar). In the studies presented here, we used a single-chain protease, scNS3, in which residues 21–32 of NS4A are fused to the N terminus of the protease domain of NS3 (30Howe A.Y. Chase R. Taremi S.S. Risano C. Beyer B. Malcolm B. Lau J.Y. Protein Sci. 1999; 8: 1332-1341Crossref PubMed Scopus (51) Google Scholar, 37Taremi S.S. Beyer B. Maher M. Yao N. Prosise W. Weber P.C. Malcolm B.A. Protein Sci. 1998; 7: 2143-2149Crossref PubMed Scopus (99) Google Scholar). This construction allows the NS4A cofactor peptide sequence to fold properly to form a mature NS3/4A protease possessing activity that is very similar if not identical to that of the noncovalent NS3/4A complex. In previous demonstrations of TRIF cleavage by NS3/4A, we used both an scNS3 protein and a bacterially expressed product representing the NS3 protease domain complexed with an NS4A peptide cofactor, both derived from the HCV-N strain of H" @default.
• W2017462823 created "2016-06-24" @default.
• W2017462823 creator A5026233059 @default.
• W2017462823 creator A5029720502 @default.
• W2017462823 creator A5049293174 @default.
• W2017462823 creator A5076060154 @default.
• W2017462823 date "2005-05-01" @default.
• W2017462823 modified "2023-10-16" @default.
• W2017462823 title "Molecular Determinants of TRIF Proteolysis Mediated by the Hepatitis C Virus NS3/4A Protease" @default.
• W2017462823 cites W1587896012 @default.
• W2017462823 cites W1612448259 @default.
• W2017462823 cites W1622437624 @default.
• W2017462823 cites W1913862483 @default.
• W2017462823 cites W1975451946 @default.
• W2017462823 cites W1978273560 @default.
• W2017462823 cites W1986178002 @default.
• W2017462823 cites W1989142184 @default.
• W2017462823 cites W1991243909 @default.
• W2017462823 cites W1993056035 @default.
• W2017462823 cites W1997307715 @default.
• W2017462823 cites W1998074525 @default.
• W2017462823 cites W1999824494 @default.
• W2017462823 cites W2001804120 @default.
• W2017462823 cites W2002195659 @default.
• W2017462823 cites W2009484032 @default.
• W2017462823 cites W2012172587 @default.
• W2017462823 cites W2013601771 @default.
• W2017462823 cites W2019080843 @default.
• W2017462823 cites W2022201177 @default.
• W2017462823 cites W2037368492 @default.
• W2017462823 cites W2042579519 @default.
• W2017462823 cites W2044278888 @default.
• W2017462823 cites W2046714103 @default.
• W2017462823 cites W2049115723 @default.
• W2017462823 cites W2056189051 @default.
• W2017462823 cites W2056931443 @default.
• W2017462823 cites W2057065474 @default.
• W2017462823 cites W2059280966 @default.
• W2017462823 cites W2062345648 @default.
• W2017462823 cites W2064779031 @default.
• W2017462823 cites W2076361525 @default.
• W2017462823 cites W2082049978 @default.
• W2017462823 cites W2082890018 @default.
• W2017462823 cites W2083785179 @default.
• W2017462823 cites W2088461289 @default.
• W2017462823 cites W2091937293 @default.
• W2017462823 cites W2096215839 @default.
• W2017462823 cites W2098552577 @default.
• W2017462823 cites W2098676417 @default.
• W2017462823 cites W2110671968 @default.
• W2017462823 cites W2115726565 @default.
• W2017462823 cites W2119586900 @default.
• W2017462823 cites W2119867428 @default.
• W2017462823 cites W2120245356 @default.
• W2017462823 cites W2123893936 @default.
• W2017462823 cites W2141137217 @default.
• W2017462823 cites W2142684817 @default.
• W2017462823 cites W2145130602 @default.
• W2017462823 cites W2150639096 @default.
• W2017462823 cites W2151898297 @default.
• W2017462823 cites W2155706758 @default.
• W2017462823 cites W2156638064 @default.
• W2017462823 cites W2161945006 @default.
• W2017462823 cites W4235571857 @default.
• W2017462823 doi "https://doi.org/10.1074/jbc.m500422200" @default.
• W2017462823 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15767257" @default.
• W2017462823 hasPublicationYear "2005" @default.
• W2017462823 type Work @default.
• W2017462823 sameAs 2017462823 @default.
• W2017462823 citedByCount "116" @default.
• W2017462823 countsByYear W20174628232012 @default.
• W2017462823 countsByYear W20174628232013 @default.
• W2017462823 countsByYear W20174628232014 @default.
• W2017462823 countsByYear W20174628232015 @default.
• W2017462823 countsByYear W20174628232016 @default.
• W2017462823 countsByYear W20174628232017 @default.
• W2017462823 countsByYear W20174628232018 @default.
• W2017462823 countsByYear W20174628232019 @default.
• W2017462823 countsByYear W20174628232020 @default.
• W2017462823 countsByYear W20174628232021 @default.
• W2017462823 countsByYear W20174628232022 @default.
• W2017462823 countsByYear W20174628232023 @default.
• W2017462823 crossrefType "journal-article" @default.
• W2017462823 hasAuthorship W2017462823A5026233059 @default.
• W2017462823 hasAuthorship W2017462823A5029720502 @default.
• W2017462823 hasAuthorship W2017462823A5049293174 @default.
• W2017462823 hasAuthorship W2017462823A5076060154 @default.
• W2017462823 hasBestOaLocation W20174628231 @default.
• W2017462823 hasConcept C132379496 @default.
• W2017462823 hasConcept C136449434 @default.
• W2017462823 hasConcept C159047783 @default.
• W2017462823 hasConcept C170493617 @default.
• W2017462823 hasConcept C181199279 @default.
• W2017462823 hasConcept C185592680 @default.
• W2017462823 hasConcept C2522874641 @default.
• W2017462823 hasConcept C2776408679 @default.
• W2017462823 hasConcept C2776709828 @default.
• W2017462823 hasConcept C2776714187 @default.

• next