Molecular Determinants of TRIF Proteolysis Mediated by the Hepatitis C Virus NS3/4A Protease

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. 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PubMed Scopus Google Scholar). of antiviral signaling be by inhibitors of the NS3/4A that for proteolysis or more proteins in these signaling the cellular protein that within the and which is by NS3/4A has to be we have that NS3/4A an protein within the for Toll-IL-1 receptor domain containing adaptor-inducing interferon-β or M. M. M. S.M. A. PubMed Scopus Google Scholar). TRIF is an adaptor protein that to of and H. M. T. T. Immunol. 2003; 4: PubMed Scopus Google Scholar, M. M. Kawai T. J. Immunol. 2003; PubMed Scopus Google Scholar), and its cleavage by a viral protease be to disrupt double-stranded through the with we have shown that NS3/4A the of the interferon-β by to extracellular a double-stranded RNA both in that the protease and in replication of HCV RNA M. M. M. S.M. A. PubMed Scopus Google Scholar). TRIF also signaling the of by D.R. A. J. Med. 2003; PubMed Scopus Google Scholar). It is to with the through a Toll-IL-1 receptor homology domain and to signaling through in its and J. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The signaling pathways in the of to virus and the ability of HCV to disrupt this signaling is likely to to its for infections in the of both and M. M. M. S.M. A. PubMed Scopus Google Scholar). TRIF is by the HCV protease its and M. M. M. S.M. A. PubMed Scopus Google Scholar), the domain of the protein from an interaction site required for which is a for and of as a factor M. N. J. PubMed Scopus Google Scholar). we of the of TRIF which to its ability to as a for the NS3/4A protease. that the of the protease which with TRIF to its proteolysis from with the viral substrates. The TRIF cleavage site a P6 acidic residue that has shown in to a substantial to viral and binding affinity G. M. D. M. C. PubMed Scopus Google Scholar, A. Narjes A. R. C. A. J. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). is in TRIF with an 8-residue polyproline track for which we a in with the protease. peptides representing the NS3/4A cleavage at the viral G. PubMed Scopus Google Scholar), and NS4B/5A the NS3/4A cleavage site within TRIF M. M. M. S.M. A. PubMed Scopus Google Scholar), P side cleavage and and an HCV peptide for in a fluorescence resonance energy M. Narjes M. A. C. R. A. PubMed Scopus Google from and Sos and 2003; 12: PubMed Scopus Google peptides within the and by with for by The of by at H. 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PubMed Scopus Google Scholar). with the P side that this peptide also interacts with the binding site also the and in to we for Sos peptide its binding associated with large in in and the of Sos, although and significant shift most of the to and shift that Sos binds to the 310 helix in of the Sos peptide a is of the site which is with both the binding and inhibition Because the of the Sos SH3 binding domain is present within the of a Sos interaction domain NS3 the that NS3 may with in a of the viral RNA the of the lengthy polyproline track present in TRIF, the of the TRIF peptide are likely to to the of NS3 in a to that in of viral the NMR that the upstream polyproline segment in TRIF by with the 310 may the affinity of TRIF for the NS3/4A the proteolytic we for the TRIF with the TRIF peptide the of the polyproline cleavage of a peptide substrate, we the of cleavage of the TRIF peptide with a peptide containing the upstream polyproline The of this peptide a of the cleavage at the proteolytic cleavage of the peptide at a than the TRIF peptide the P side of the cleavage of the peptide is likely to more from NS3 cleavage than the TRIF peptide P side and to significant protease as shown for the Sos peptide in this polyproline we used fluorescence spectroscopy to the difference in the NS3 binding affinity of and TRIF P side that either or include the upstream polyproline that the TRIF peptide containing the upstream polyproline segment greater affinity for the protease than a peptide the polyproline and for and TRIF have shown that there is a significant difference in the of NS3 cleavage of TRIF and the TRIF It is likely that this difference is by of the TRIF which its binding to NS3 the protease active the polyproline track upstream of the NS3 cleavage at of The NMR and increased affinity of TRIF peptides containing the polyproline track that to recognition of TRIF by is likely that other of the TRIF also to its relatively cleavage by the protease. with the affinity of the protein, the of TRIF is than that of the which has an in the N. P. P.C. 7: Full Text Full Text PDF Scopus Google with for TRIF TRIF cleavage may be than the NS4B/5A and with the the of the cleavage of TRIF in the of cellular antiviral M. M. M. S.M. A. PubMed Scopus Google Scholar). TRIF cleavage an in viral motifs are to an in cellular signaling M. J. 2000; PubMed Scopus Google Scholar, A. 2003; Scholar). these motifs may also be in viral regulatory the human immunodeficiency virus protein contains proline-rich motifs that to the Src family SH3 domain replication of the virus J. Full Text Full Text PDF PubMed Scopus Google Scholar). are by which binding may the of A. 2003; Scholar). include the of the the active site as as the of a recognition likely for the The interaction with the protease active site to be to include the 310 helix of the which with the helix of Thus, the of a P6 acidic residue in TRIF may be by the of a that extends the resulting in affinity and in TRIF also with the protease at from the a that is by NMR have shown that the nonstructural proteins the segment of the HCV polyprotein are required for RNA replication and are likely to to the of a viral V. Koch J. L. R. PubMed Scopus Google Scholar, R. D. Lohmann V. R. D. J. Virol. 2003; PubMed Scopus Google Scholar). and that NS3 interacts with M. C. J. Virol. 2003; PubMed Scopus Google and may the of P. A. R. J. Virol. PubMed Google Scholar). be moderate to for the and that are likely to be in of the HCV RNA The of the Sos peptide within with a of the affinity of Sos for NS3 the that the Sos domain of with the 310 helix of the polyproline interaction and NS3 be by the interaction of NS3 with its NS4A NS4A also interacts with M.G. PubMed Scopus Google Scholar, T. J. Virol. PubMed Google may also be interaction may have for drug discovery efforts. Because the HCV NS3 binding site is and the of inhibitors of this has and The of a polyproline binding site that is of peptides the protease active site open new for drug by both for binding drug have of for The of peptides that in at a target may in binding in the of the in the and associated with the M. J. R. R.E. 2004; PubMed Scopus Google Scholar). that the TRIF as a the polyproline as an and affinity of the for the protease. we speculate that that are to target both may protease but also of the HCV by binding mediated by the Sos in have antiviral activity. are to of the for of the and for and are also to for and and for in protein expression and

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