The N-terminal region of hepatitis C virus nonstructural protein 3 (NS3) is essential for stable complex formation with NS4A.

Hepatitis C virus proteins are produced by proteolytic processing of the viral precursor polyprotein that is encoded in the largest open reading frame of the viral genome. Processing of the nonstructural viral polyprotein requires the viral serine-type proteinase present in nonstructural protein 3 (NS3). The cleavage of the junction between NS4B and NS5A is mediated by NS3 only when NS4A is present. NS4A is thought to be a cofactor that enhances the cleavage efficiency of NS3 in hepatitis C virus protein-producing cells. Stable NS3-NS4A complex formation required the N-terminal 22 amino acid residues of NS3. This interaction contributed to stabilization of the NS3 product as well as increased the efficiency of cleavage at the NS4B/5A site. The N-terminal 22 amino acid residues fused to Escherichia coli dihydrofolate reductase also formed a stable complex with NS4A. NS3 derivatives which lacked the N-terminal 22 amino acid residues showed drastically reduced cleavage activity at the NS4B/5A site even in the presence of NS4A. These data suggested that the interaction with NS4A through the 22 amino acid residues of NS3 is primarily important for the NS4A-dependent processing of the NS4B/5A site by NS3.     

Substrate determinants for cleavage in cis and in trans by the hepatitis C virus NS3 proteinase.

Processing of the hepatitis C virus polyprotein is accomplished by a series of cotranslational and posttranslational cleavages mediated by host cell signalases and two virally encoded proteinases. Of these the NS3 proteinase is essential for processing at the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B junctions. Processing between NS3 and NS4A occurs in cis, implying an intramolecular reaction mechanism, whereas cleavage at the other sites can also be mediated in trans. Sequence analysis of the amino termini of mature cleavage products and comparisons of amino acid residues around the scissile bonds of various hepatitis C virus isolates identified amino acid residues which might contribute to substrate specificity and processing efficiency: an acidic amino acid at the P6 position, a Thr or Cys at the P1 position, and a Ser or Ala at the P1' position. To study the importance of these residues for NS3-mediated cleavage we have undertaken a mutational analysis using an NS3'-5B polyprotein expressed by recombinant vaccinia viruses in mammalian cells. For all NS3-dependent cleavage sites P1 substitutions had the most drastic effects on cleavage efficiency, showing that amino acid residues at this position are the most critical substrate determinants. Since less drastic effects were found for substitutions at the P1' position, these residues appear to be less important for proper cleavage. For all cleavage sites the P6 acidic residue was dispensable, suggesting that it is not essential for substrate recognition and subsequent cleavage. Analysis of a series of mutations at the NS3/4A site revealed great flexibility for substitutions compared with more stringent requirements at the trans cleavage sites. On the basis of these results we propose a model in which processing in cis is determined primarily by polyprotein folding, whereas cleavage in trans is governed not only by the structure of the polyprotein but also by specific interactions between the proteinase and the polyprotein substrate at or around the scissile bond.     

Hepatitis C virus polyprotein processing: kinetics and mutagenic analysis of serine proteinase-dependent cleavage.

Hepatitis C virus (HCV) serine proteinase (Cpro-2) is responsible for the processing of HCV nonstructural (NS) protein processing. To clarify the mechanism of Cpro-2-dependent processing, pulse-chase and mutation analyses were performed by using a transient protein production system in cultured cells. Pulse-chase study revealed the sequential production of HCV-NS proteins. Production of p70(NS3) and p66(NS5B) were rapid. An 89-kDa processing intermediate protein (p89) was observed during the early part of the chase. p89 seemed to be cleaved first into a 31-kDa protein (p31) and a p58/56(NS5A). p31 was further processed into p4(NS4A) and p27(NS4B). Mutation analysis of cleavage sites of NS4A/4B, NS4B/5A, and NS5A/5B revealed that cleavage at each site is essentially independent from cleavage occurring at the other site.     

Mutagenesis of folylpolyglutamate synthetase indicates that dihydropteroate and tetrahydrofolate bind to the same site

The folylpolyglutamate synthetase (FPGS) enzyme of Escherichia coli differs from that of Lactobacillus casei in having dihydrofolate synthetase activity, which catalyzes the production of dihydrofolate from dihydropteroate. The present study undertook mutagenesis to identify structural elements that are directly responsible for the functional differences between the two enzymes. The amino terminal domain (residues 1-287) of the E. coli FPGS was found to bind tetrahydrofolate and dihydropteroate with the same affinity as the intact enzyme. The domain-swap chimera proteins between the E. coli and the L. casei enzymes possess both folate or pteroate binding properties and enzymatic activities of their amino terminal portion, suggesting that the N-terminal domain determines the folate substrate specificity. Recent structural studies have identified two unique folate binding sites, the omega loop in L. casei FPGS and the dihydropteroate binding loop in the E. coli enzyme. Mutants with swapped omega loops retained the activities and folate or pteroate binding properties of the rest of the enzyme. Mutating L. casei FPGS to contain an E. coli FPGS dihydropteroate binding loop did not alter its substrate specificity to using dihydropteroate as a substrate. The mutant D154A, a residue specific for the dihydropteroate binding site in E. coli FPGS, and D151A, the corresponding mutant in the L. casei enzyme, were both defective in using tetrahydrofolate as their substrate, suggesting that the binding site corresponding to the E. coli pteroate binding site is also the tetrahydrofolate binding site for both enzymes. Tetrahydrofolate diglutamate was a slightly less effective substrate than the monoglutamate with the wild-type enzyme but was a 40-fold more effective substrate with the D151A mutant. This suggests that the 5,10-methylenetetrahydrofolate binding site identified in the L. casei ternary structure may bind diglutamate and polyglutamate folate derivatives.     

Two hepatitis C virus glycoprotein E2 products with different C termini.

Processing of the boundary region between the putative structural and nonstructural regions of the hepatitis C virus precursor polyprotein was analyzed by in vitro translation using reticulocyte lysate in the presence of canine microsomal membranes. At this boundary in the precursor polyprotein, the most carboxy-terminal of the structural proteins, gp70 (E2), is proximal to the amino terminal of the nonstructural protein p21 (NS2). The presence of a novel microsomal membrane-dependent cleavage site was observed at the region upstream of the amino-terminal end of p21 (NS2) in the precursor polyprotein. The cleavage site was assigned to amino acid residues 746/747 in the hepatitis C virus precursor polyprotein. Inefficient cleavage of this site resulted in the production of two forms of E2 products with different sizes of peptide backbones. Translation and cleavage of various C-terminal deletion constructs established the significance of the C-terminal hydrophobic amino acid sequences of E2 products in membrane anchoring.     

Investigating the structure of human RNase H1 by site-directed mutagenesis

In this study we examine for the first time the roles of the various domains of human RNase H1 by site-directed mutagenesis. The carboxyl terminus of human RNase H1 is highly conserved with Escherichia coli RNase H1 and contains the amino acid residues of the putative catalytic site and basic substrate-binding domain of the E. coli RNase enzyme. The amino terminus of human RNase H1 contains a structure consistent with a double-strand RNA (dsRNA) binding motif that is separated from the conserved E. coli RNase H1 region by a 62-amino acid sequence. These studies showed that although the conserved amino acid residues of the putative catalytic site and basic substrate-binding domain are required for RNase H activity, deletion of either the catalytic site or the basic substrate-binding domain did not ablate binding to the heteroduplex substrate. Deletion of the region between the dsRNA-binding domain and the conserved E. coli RNase H1 domain resulted in a significant loss in the RNase H activity. Furthermore, the binding affinity of this deletion mutant for the heteroduplex substrate was approximately 2-fold tighter than the wild-type enzyme suggesting that this central 62-amino acid region does not contribute to the binding affinity of the enzyme for the substrate. The dsRNA-binding domain was not required for RNase H activity, as the dsRNA-deletion mutants exhibited catalytic rates approximately 2-fold faster than the rate observed for wild-type enzyme. Comparison of the dissociation constant of human RNase H1 and the dsRNA-deletion mutant for the heteroduplex substrate indicates that the deletion of this region resulted in a 5-fold loss in binding affinity. Finally, comparison of the cleavage patterns exhibited by the mutant proteins with the cleavage pattern for the wild-type enzyme indicates that the dsRNA-binding domain is responsible for the observed strong positional preference for cleavage exhibited by human RNase H1.     

Analysis of N-terminal processing of hepatitis C virus nonstructural protein 2.

We determined the partial amino (N)-terminal amino acid sequence of hepatitis C virus p21 (nonstructural protein 2 [NS2]). Cleavage at the p21 (NS2) N terminus depended on the presence of microsomal membranes. The amino-terminal position of p21 (NS2) was assigned to amino acid 810 of the hepatitis C virus strain IIJ precursor polyprotein. Mutation of the alanine residue at position P1 of the putative cleavage site inhibited membrane-dependent processing. This alteration in processing together with the fact that hydrophobic amino acid residues are clustered upstream of the putative cleavage site suggested the involvement of a signal peptidase(s) in the cleavage. Furthermore, mutation analysis of this possible cleavage site revealed the presence of another microsome membrane-dependent cleavage site upstream of the N terminus of p21 (NS2).     

Dihydrofolate reductase from Bacillus subtilis and its artificial derivatives: expression, purification, and characterization.

The Bacillus subtilis dihydrofolate reductase (DHFR) gene was expressed in Escherichia coli. The gene product was purified to homogeneity by Butyl-Toyopearl, Toyopearl HW55, and DEAE-Toyopearl column chromatographies, and its molecular properties were compared to those of E. coli DHFR. The specific enzyme activity of the B. subtilis DHFR was 240 units/mg under the standard assay conditions, being about four times higher than that of the E. coli DHFR. Km for coenzyme NADPH was 20.7 microM, a value about three times larger than that of E. coli, whereas Km (1.5 microM) for the substrate, dihydrofolate, was similar to that of E. coli DHFR. This seems to reflect the low homology of the amino acid sequence in residues 61-88 of the two DHFRs where one of the NADPH binding sites is located [Bystrof, C. & Kraut, J. (1991) Biochemistry 30, 2227-2239]. Similar to the E. coli DHFR [Iwakura, M. et al. (1992) J. Biochem. 111, 37-45], the extension of amino acid sequences at the C-terminal end of the B. subtilis DHFR could be attained without loss of the enzyme function or decrease of the protein yield. Thus, the DHFR is useful as a carrier protein for expressing small polypeptides, such as leucine enkephalin, bradykinin, and somatostatin.     

Specificity of human cathepsin S determined by processing of peptide substrates and MHC class II-associated invariant chain

Cathepsin S (CatS) is a lysosomal cysteine protease of the papain family, the members of which possess relatively broad substrate specificities. It has distinct roles in major histocompatibility complex (MHC) class II-associated peptide loading and in antigen processing in both the MHC class I and class II pathways. It may therefore represent a target for interference with antigen presentation, which could be of value in the therapy of (auto)immune diseases. To obtain more detailed information on the specificity of CatS, we mapped its cleavage site preferences at subsites S3-S1' by in vitro processing of a peptide library. Only five amino acid residues at the substrate's P2 position allowed for cleavage by CatS under time-limited conditions. Preferences for groups of amino acid residues were also observed at positions P3, P1 and P1'. Based on these results, we developed highly CatS-sensitive peptides. After processing of MHC class II-associated invariant chain (Ii), a natural protein substrate of CatS, we identified CatS cleavage sites in Ii of which a majority matched the amino acid residue preference data obtained with peptides. These observed cleavage sites in Ii might be of relevance for its in vivo processing by CatS.     

Mutations that alter the activity of the Rous sarcoma virus protease.

Mutations designed by analysis of the Rous sarcoma virus (RSV) and human immunodeficiency virus (HIV)-1 protease (PR) crystal structures were introduced into 1) the substrate binding pocket, 2) the substrate enclosing "flaps," and 3) surface loops of RSV PR. Each mutant PR was expressed in Escherichia coli. Changes in activity were detected by following cleavage of a truncated (NC-PR) precursor polypeptide in E. coli and cleavage of synthetic peptide substrates representing RSV and HIV-1 PR cleavage sites in vitro. Mutations in the substrate binding pocket exchanged amino acid residues located close to the substrate in the HIV-1 PR for structurally equivalent residues in the RSV PR. Changing histidine 65 to glycine (H65G) gave an inactive enzyme, while a double mutant R105P,G106V, as well as the triple mutant, H65G,R105P,G106V, produced enzymes which showed significant activity toward a substrate that represented a HIV-1 cleavage site. Mutating the catalytic aspartate (D37S) or an adjacent conserved alanine to threonine (A40T), produced inactive enzymes. In contrast, the substitution A40S was active, but showed a reduced rate of catalysis. Mutations in the flaps of conserved glycines (G69L, G70L) produced inactive PRs. Two extended RSV PR surface loops were shortened to the size found in HIV-1 PR and resulted in drastically reduced activity. These results have confirmed some of the basic predictions made from structural models but have also revealed unexpected roles and interactions in the protein.     

A second virus-encoded proteinase involved in proteolytic processing of poliovirus polyprotein

The poliovirus polyprotein is cleaved at three different amino acid pairs. Viral polypeptide 3C is responsible for processing at the most common pair (glutamineglycine). We have found that a cDNA fragment encoding parts of the capsid protein region (P1) and the nonstructural protein region (P2), and including the P1-P2 processing site (tyrosine-glycine), can be expressed in E. coli. The translation product was correctly processed. Disruption of the coding sequence of 2A, a nonstructural polypeptide mapping carboxy-terminal to the tyrosine-glycine cleavage site, by linker mutagenesis or deletion, prevented processing. Deletion of the adjacent polypeptide 2B had no such effect. Antibodies against 2A specifically inhibited processing at the 3C'-3D' processing site (tyrosine-glycine) in vitro. We conclude that poliovirus encodes the second proteinase 2A, which processes the polyprotein at tyrosine-glycine cleavage sites.     

Autoproteolysis of herpes simplex virus type 1 protease releases an active catalytic domain found in intermediate capsid particles.

The UL26 gene of herpes simplex virus type 1 (HSV-1) encodes a 635-amino-acid protease that cleaves itself and the HSV-1 assembly protein ICP35cd (F. Liu and B. Roizman, J. Virol. 65:5149-5156, 1991). We previously examined the HSV protease by using an Escherichia coli expression system (I. C. Deckman, M. Hagen, and P. J. McCann III, J. Virol. 66:7362-7367, 1992) and identified two autoproteolytic cleavage sites between residues 247 and 248 and residues 610 and 611 of UL26 (C. L. DiIanni, D. A. Drier, I. C. Deckman, P. J. McCann III, F. Liu, B. Roizman, R. J. Colonno, and M. G. Cordingley, J. Biol. Chem. 268:2048-2051, 1993). In this study, a series of C-terminal truncations of the UL26 open reading frame was tested for cleavage activity in E. coli. Our results delimit the catalytic domain of the protease to the N-terminal 247 amino acids of UL26 corresponding to No, the amino-terminal product of protease autoprocessing. Autoprocessing of the full-length protease was found to be unnecessary for catalysis, since elimination of either or both cleavage sites by site-directed mutagenesis fails to prevent cleavage of ICP35cd or an unaltered protease autoprocessing site. Catalytic activity of the 247-amino-acid protease domain was confirmed in vitro by using a glutathione-S-transferase fusion protein. The fusion protease was induced to high levels of expression, affinity purified, and used to cleave purified ICP35cd in vitro, indicating that no other proteins are required. By using a set of domain-specific antisera, all of the HSV-1 protease cleavage products predicted from studies in E. coli were identified in HSV-1-infected cells. At least two protease autoprocessing products, in addition to fully processed ICP35cd (ICP35ef), were associated with intermediate B capsids in the nucleus of infected cells, suggesting a key role for proteolytic maturation of the protease and ICP35cd in HSV-1 capsid assembly.     

RNase P — a 'Scarlet Pimpernel'

RNase P is responsible for the maturation of the 5'-termini of tRNA molecules in all cells studied to date. This ribonucleoprotein has to recognize and identify its cleavage site on a large number of different precursors. This review covers what is currently known about the function of the catalytic subunit of Escherichia coli RNase P, M1 RNA, and the protein subunit, C5, in particular with respect to cleavage-site selection. Recent genetic and biochemical data show that the two C residues in the 3'-terminal CCA sequence of a precursor interact with the enzyme through Watson-Crick base-pairing. This is suggested to result in unfolding of the amino acid acceptor-stem and exposure of the cleavage site. Furthermore, other close contact points between M1 RNA and its substrate have recently been identified. These data, together with the two existing three-dimensional structure models of M1 RNA in complex with its substrate, establish a platform that will enable us to seek an understanding of the underlying mechanism of cleavage by this elusive enzyme.     

Polyprotein processing in cis and in trans by hepatitis A virus 3C protease cloned and expressed in Escherichia coli.

To determine the P3 region protein-processing sites cleaved by the hepatitis A virus 3C protease, a nested set of constructs containing a portion of 3A (3A* [the asterisk denotes an incomplete protein]), 3B and 3C and various amounts of 3D, fused in frame to Escherichia coli TrpE-coding sequences under control of the tryptophan promoter, was made. Additional plasmids that encoded a portion of 2C (2C*) and the P3 proteins, including complete or incomplete 3D sequences, were constructed. After induction, E. coli containing these recombinant plasmids produced high levels of fusion proteins as insoluble aggregates. 3C-mediated cleavage products were identified by comparison of expression with a matching set of plasmids, containing an engineered mutation in 3C. Cleavage products were detected by immunoblot analyses by using antisera against the TrpE protein, against 3D*, and against 3CD*. Scissile bonds were determined by N-terminal amino acid sequencing of the proteins formed by cleavage. The results showed that when a portion of 2C was present, the primary cleavage by the 3C protease was between 2C and 3A, and the cleavage site was QG, as predicted by J. I. Cohen, J. R. Ticehurst, R. H. Purcell, A. Buckler-White, and B. M. Baroudy, J. Virol. 61:50-59, 1987. Very little further cleavage of the released P3 protein was detected. When the fusion protein contained no 2C and included only 3A*-to-3D sequences, efficient cleavage occurred between 3B and 3C, at the QS pair, also as predicted by Cohen et al. (J. Virol. 61:50-59, 1987). The latter proteins were also cleaved between 3C and 3D, but less efficiently than between 3B and 3C. Extracts of bacteria expressing proteins from 3A* to 3D also cleaved a radiolabelled hepatitis A virus substrate containing VP1*2ABC* sequences in trans.     

Rubella virus cDNA. Sequence and expression of E1 envelope protein

A cDNA clone encoding the entire E1 envelope protein (410 amino acid residues) and a portion of the C-terminal end of the E2 envelope protein of the rubella virus has been isolated and characterized. DNA sequence analysis has revealed a region 20 nucleotides in length at the 3' end of the cloned cDNA which may be a replicase recognition site or a recognition site for encapsidation. The proteolytic cleavage site between the E1 and E2 proteins was localized based on the known amino-terminal sequence of the isolated E1 protein (Kalkkinen, N., Oker-Blom, C., and Pettersson, R. F. (1984) J. Gen. Virol. 65, 1549-1557) and the deduced amino acid sequence. The mature E1 protein is preceded by a set of 20 highly hydrophobic amino acid residues possessing characteristics of a signal peptide. This "signal peptide" is flanked on both sides by typical protease cleavage sites for trypsin-like enzyme and signal peptidase. The presence of a leader sequence in the E1 protein precursor may facilitate its translocation through the host cell membrane. The E1 protein of rubella virus shows no significant homology with alphavirus E1 envelope proteins. However, a stretch of 39 amino acids in the E1 protein of rubella virus (residues 262-300) was found to share a significant homology with the first 39 residues of bovine sperm histone. The position of 4 half-cystines and 8 arginines overlaps. The E1 protein of rubella virus has been successfully expressed in COS cells after transfecting them with rubella virus cDNA in simian virus 40-derived expression vector. This protein is antigenically similar to the one expressed by cells infected with rubella virus.     

Homology of Escherichia coli B glutathione synthetase with dihydrofolate reductase in amino acid sequence and substrate binding site

Glutathione synthetase from Escherichia coli B showed amino acid sequence homology with mammalian and bacterial dihydrofolate reductases over 40 residues, although these two enzymes are different in their reaction mechanisms and ligand requirements. The effects of ligands of dihydrofolate reductase on the reaction of E. coli B glutathione synthetase were examined to find resemblances in catalytic function to dihydrofolate reductase. The E. coli B enzyme was potently inhibited by 7,8-dihydrofolate, methotrexate, and trimethoprim. Methotrexate was studied in detail and proved to bind to an ATP binding site of the E. coli B enzyme with K1 value of 0.1 mM. The homologous portion of the amino acid sequence in dihydrofolate reductases, which corresponds to the portion coded by exon 3 of mammalian dihydrofolate reductase genes, provided a binding site of the adenosine diphosphate moiety of NADPH in the crystal structure of dihydrofolate reductase. These analyses would indicate that the homologous portion of the amino acid sequence of the E. coli B enzyme provides the ATP binding site. This report gives experimental evidence that amino acid sequences related by sequence homology conserve functional similarity even in enzymes which differ in their catalytic mechanisms.     

The C terminus of mouse ornithine decarboxylase confers rapid degradation on dihydrofolate reductase. Support for the pest hypothesis

Several years ago, we proposed that polypeptide regions rich in proline (P), glutamic acid (E), serine (S), and threonine (T) (PEST) target intracellular proteins for destruction (Rogers, S., Wells, R., and Rechsteiner, M. (1986) Science 234, 364-368). To test the PEST hypothesis, we have produced chimeric proteins in which the N or C terminus of mouse dihydrofolate reductase is extended by the PEST-containing C terminus of mouse ornithine decarboxylase. Oligonucleotides encoding the 37 C-terminal residues of mouse ornithine decarboxylase (mODC) or equivalent lengths of dissimilar amino acids were inserted at appropriate sites in a dihydrofolate reductase (DHFR) expression vector. The various fusion proteins were expressed in Escherichia coli and purified to homogeneity by enzyme affinity chromatography. All purified fusion proteins exhibited similar abilities to convert dihydrofolate to tetrahydrofolate, thereby demonstrating that the attachment of peptide extensions to either terminus did not prevent the proper folding of DHFR. Metabolic stabilities of the radioiodinated fusion proteins were assayed in rabbit reticulocyte lysate or Xenopus egg extract. Proteolysis was found to be energy-dependent with mODC-DHFR fusion proteins being degraded from 2 to almost 40-fold faster than the parental DHFR molecule or DHFR fusion proteins bearing non-PEST extensions. Deletion of most of the PEST region from the mODC extension resulted in a significantly more stable fusion protein. Rapid proteolysis of DHFR proteins containing intact mODC extensions provides support for the PEST hypothesis.     

Sequence features of substrates required for cleavage by GlpG, an Escherichia coli rhomboid protease

Rhomboids are a family of serine proteases belonging to intramembrane cleaving proteases, which are supposed to catalyse proteolysis of a substrate protein within the membrane. It remains unclear whether substrates of the rhomboid proteases have a common sequence feature that allows specific cleavage by rhomboids. We showed previously that GlpG, the Escherichia coli rhomboid, can cleave a type I model membrane protein Bla-LY2-MBP having the second transmembrane region of lactose permease (LY2) at the extramembrane region in vivo and in vitro, and that determinants for proteolysis reside within the LY2 sequence. Here we characterized sequence features in LY2 that allow efficient cleavage by GlpG and identified two elements, a hydrophilic region encompassing the cleavage site and helix-destabilizing residues in the downstream hydrophobic region. Importance of the positioning of helix-destabilizers relative to the cleavage site was suggested. These two elements appear to co-operatively promote proteolysis of substrates by GlpG. Finally, random mutagenesis of the cleavage site residues in combination with in vivo screening revealed that GlpG prefers residues with a small side chain and a negative charge at the P1 and P1' sites respectively.     

Substrate requirements of human rhinovirus 3C protease for peptide cleavage in vitro

A series of synthetic peptides representing authentic proteolytic cleavage sites of human rhinovirus type 14 were assayed as substrates for purified 3C protease. Competition cleavage assays were employed to determine the relative specificity constants (Kcat/Km) for substrates with sequences related to the viral 2C-3A cleavage site. Variable length peptides representing the 2C-3A cleavage site were cleaved with comparable efficiency. These studies defined a minimum substrate of 6 amino acids (TLFQ/GP), although retention of the residue at position P5 (ETLFQ/GP) resulted in a better substrate by an order of magnitude. Amino acid substitutions at position P5, P4, P1', or P2' indicated that the identity of the residue at position P5 was not critical, whereas substitutions at position P4, P1' or P2' resulted in substrates with Kcat/Km values varying over 2 orders of magnitude. In contrast to the 2C-3A cleavage site, small peptide derivatives representative of the 3A-3B cleavage site were relatively poor substrates, which suggested that residues flanking the minimum core sequence may influence susceptibility to cleavage. The 3C protease of rhinovirus type 14 was also capable of cleaving peptides representing comparable cleavage sites predicted for coxsackie B virus and poliovirus.     

Regulation of hepatitis C virus polyprotein processing by signal peptidase involves structural determinants at the p7 sequence junctions

The hepatitis C virus genome encodes a polyprotein precursor that is co- and post-translationally processed by cellular and viral proteases to yield 10 mature protein products (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Although most cleavages in hepatitis C virus polyprotein precursor proceed to completion during or immediately after translation, the cleavages mediated by a host cell signal peptidase are partial at the E2/p7 and p7/NS2 sites, leading to the production of an E2p7NS2 precursor. The sequences located immediately N-terminally of E2/p7 and p7/NS2 cleavage sites can function as signal peptides. When fused to a reporter protein, the signal peptides of p7 and NS2 were efficiently cleaved. However, when full-length p7 was fused to the reporter protein, partial cleavage was observed, indicating that a sequence located N-terminally of the signal peptide reduces the efficiency of p7/NS2 cleavage. Sequence analyses and mutagenesis studies have also identified structural determinants responsible for the partial cleavage at both the E2/p7 and p7/NS2 sites. Finally, the short distance between the cleavage site of E2/p7 or p7/NS2 and the predicted transmembrane alpha-helix within the P' region might impose additional structural constraints to the cleavage sites. The insertion of a linker polypeptide sequence between P-3' and P-4' of the cleavage site released these constraints and led to improved cleavage efficiency. Such constraints in the processing of a polyprotein precursor are likely essential for hepatitis C virus to post-translationally regulate the kinetics and/or the level of expression of p7 as well as NS2 and E2 mature proteins.