Single-cell resolution imaging of membrane-anchored hepatitis C virus NS3/4A protease activity

The study of host and viral membrane-associated proteases has been hampered due to a lack of in vivo assays. We report here the development of a cell-based fluorescence assay for detecting hepatitis C virus (HCV) NS3/4A juxtamembrane protease activity. Intracellular membrane-anchored protein substrates were engineered comprising: (1) an endoplasmic reticulum targeting domain, the HCV NS5A N-terminal amphipathic alpha-helix; (2) a NS3/4A-specific cleavage site; and (3) a red fluorescent reporter group, DsRed. The results of our immunofluorescence and Western blotting studies demonstrate that our membrane-bound fluorescent probe was cleaved specifically and efficiently by NS3/4A expressed in human cells.     

Control of innate immune signaling and membrane targeting by the Hepatitis C virus NS3/4A protease are governed by the NS3 helix α0

Hepatitis C virus (HCV) infection is sensed in the host cell by the cytosolic pathogen recognition receptor RIG-I. RIG-I signaling is propagated through its signaling adaptor protein MAVS to drive activation of innate immunity. However, HCV blocks RIG-I signaling through viral NS3/4A protease cleavage of MAVS on the mitochondrion-associated endoplasmic reticulum (ER) membrane (MAM). The multifunctional HCV NS3/4A serine protease is associated with intracellular membranes, including the MAM, through membrane-targeting domains within NS4A and also at the amphipathic helix α(0) of NS3. The serine protease domain of NS3 is required for both cleavage of MAVS, a tail-anchored membrane protein, and processing the HCV polyprotein. Here, we show that hydrophobic amino acids in the NS3 helix α(0) are required for selective cleavage of membrane-anchored portions of the HCV polyprotein and for cleavage of MAVS for control of RIG-I pathway signaling of innate immunity. Further, we found that the hydrophobic composition of NS3 helix α(0) is essential to establish HCV replication and infection. Alanine substitution of individual hydrophobic amino acids in the NS3 helix α(0) impaired HCV RNA replication in cells with a functional RIG-I pathway, but viral RNA replication was rescued in cells lacking RIG-I signaling. Therefore, the hydrophobic amphipathic helix α(0) of NS3 is required for NS3/4A control of RIG-I signaling and HCV replication by directing the membrane targeting of both viral and cellular substrates.     

Hepatitis C virus NS3 serine proteinase: trans-cleavage requirements and processing kinetics.

The hepatitis C virus H strain (HCV-H) polyprotein is cleaved to produce at least 10 distinct products, in the order of NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B -COOH. An HCV-encoded serine proteinase activity in NS3 is required for cleavage at four sites in the nonstructural region (3/4A, 4A/4B, 4B/5A, and 5A/5B). In this report, the HCV-H serine proteinase domain (the N-terminal 181 residues of NS3) was tested for its ability to mediate trans-processing at these four sites. By using an NS3-5B substrate with an inactivated serine proteinase domain, trans-cleavage was observed at all sites except for the 3/4A site. Deletion of the inactive proteinase domain led to efficient trans-processing at the 3/4A site. Smaller NS4A-4B and NS5A-5B substrates were processed efficiently in trans; however, cleavage of an NS4B-5A substrate occurred only when the serine proteinase domain was coexpressed with NS4A. Only the N-terminal 35 amino acids of NS4A were required for this activity. Thus, while NS4A appears to be absolutely required for trans-cleavage at the 4B/5A site, it is not an essential cofactor for serine proteinase activity. To begin to examine the conservation (or divergence) of serine proteinase-substrate interactions during HCV evolution, we demonstrated that similar trans-processing occurred when the proteinase domains and substrates were derived from two different HCV subtypes. These results are encouraging for the development of broadly effective HCV serine proteinase inhibitors as antiviral agents. Finally, the kinetics of processing in the nonstructural region was examined by pulse-chase analysis. NS3-containing precursors were absent, indicating that the 2/3 and 3/4A cleavages occur rapidly. In contrast, processing of the NS4A-5B region appeared to involve multiple pathways, and significant quantities of various polyprotein intermediates were observed. NS5B, the putative RNA polymerase, was found to be significantly less stable than the other mature cleavage products. This instability appeared to be an inherent property of NS5B and did not depend on expression of other viral polypeptides, including the HCV-encoded proteinases.     

Molecular mechanisms of viral and host cell substrate recognition by hepatitis C virus NS3/4A protease

Hepatitis C NS3/4A protease is a prime therapeutic target that is responsible for cleaving the viral polyprotein at junctions 3-4A, 4A4B, 4B5A, and 5A5B and two host cell adaptor proteins of the innate immune response, TRIF and MAVS. In this study, NS3/4A crystal structures of both host cell cleavage sites were determined and compared to the crystal structures of viral substrates. Two distinct protease conformations were observed and correlated with substrate specificity: (i) 3-4A, 4A4B, 5A5B, and MAVS, which are processed more efficiently by the protease, form extensive electrostatic networks when in complex with the protease, and (ii) TRIF and 4B5A, which contain polyproline motifs in their full-length sequences, do not form electrostatic networks in their crystal complexes. These findings provide mechanistic insights into NS3/4A substrate recognition, which may assist in a more rational approach to inhibitor design in the face of the rapid acquisition of resistance.     

丙型肝炎病毒感染过程中其NS3/4A蛋白酶切割线粒体抗病毒信号蛋白的动态过程

已知丙型肝炎病毒(hepatitis C virus,HCV)可通过其蛋白酶NS3/4A切割线粒体抗病毒信号蛋白(mitochondrial antiviral signaling protein,MAVS)来逃逸天然免疫识别,但尚不清楚其切割动力学及切割在抑制干扰素中的作用。本研究旨在细胞模型中探讨HCV感染过程中病毒复制建立及病毒NS3/4A切割MAVS的动态过程,探究NS3/4A切割MAVS对病毒逃逸宿主天然免疫建立感染的贡献。首先构建基于绿色荧光蛋白(green fluorescent protein,GFP)的MAVS切割报告系统(GFP-NLS-MAVS-TM462),用 HCV Jc1-Gluc 感染Huh7.5/GFP-NLS-MAVS-TM462细胞。结果显示,病毒复制早期MAVS切割效率较低;NS3/4A高效切割MAVS发生于HCV复制晚期,且其切割效率与NS3蛋白水平相关。利用带有GFP ypet的HCV报告病毒Jc1-378-1感染Huh7.5/RFP-NLS-MAVS-TM462细胞,在单细胞水平观察HCV感染阳性细胞中MAVS被切割情况,发现HCV复制细胞中仅部分细胞MAVS被切割。进一步研究发现,不同基因型NS3/4A切割MAVS的效率仅与NS3表达水平相关。以上结果提示,HCV蛋白酶NS3/4A切割MAVS依赖NS3/4A蛋白在病毒复制过程中的累积,对在病毒复制早期逃逸宿主天然免疫建立感染可能无显著贡献。     

丙型肝炎病毒NS3蛋白酶在酵母系统中的可溶性表达

利用毕赤酵母系统表达具有催化活性的丙型肝炎病毒 (HCV)NS3蛋白酶 .将PCR直接扩增的病毒NS3丝氨酸蛋白酶基因和重组的带有辅酶的单链NS3 NS4A蛋白酶基因 ,分别插入表达载体pPICZαA的EcoRⅠ和XbaⅠ克隆位点 ,转化毕赤酵母GS115 ,可溶性表达NS3蛋白酶和单链NS3 4A蛋白酶 ;ELISA法测定表达蛋白酶的抗原性 ;原核高效表达载体pBVIL1表达酶切底物NS5A B片段 ,体外与蛋白酶共同温育 ,SDS PAGE鉴定蛋白酶催化活性 .载体测序结果表明 ,重组载体pPICZαA NS3和pPICZαA NS3 4A中的目的基因序列插入正确 ;SDS PAGE结果显示 ,培养物上清中存在分泌型目的蛋白带 ;ELISA结果证实 ,表达蛋白与HCV阳性血清有结合活性 ;蛋白酶与底物NS5A B片段不同作用时间的SDS PAGE ,看到约 2 4kD处底物条带的分解 .说明用毕赤酵母表达系统成功地表达了可溶性HCVNS3和单链NS3 4A蛋白酶 ;两种结构形式的蛋白酶在体外系统中都有催化活性 ,同时也都具有抗原性 .该研究为大量和方便地获得有催化活性的HCVNS3蛋白酶提供了有效途径 .     

Charged residues in hepatitis C virus NS4B are critical for multiple NS4B functions in RNA replication

The nonstructural 4B (NS4B) protein of hepatitis C virus (HCV) plays a central role in the formation of the HCV replication complex. To gain insight into the role of charged residues for NS4B function in HCV RNA replication, alanine substitutions were engineered in place of 28 charged residues residing in the N- and C-terminal cytoplasmic domains of the NS4B protein of the HCV genotype 1b strain Con1. Eleven single charged-to-alanine mutants were not viable, while the remaining mutants were replication competent, albeit to differing degrees. By selecting revertants, second-site mutations were identified for one of the lethal NS4B mutations. Second-site mutations mapped to NS4B and partially suppressed the lethal replication phenotype. Further analyses showed that three NS4B mutations disrupted the formation of putative replication complexes, one mutation altered the stability of the NS4B protein, and cleavage at the NS4B/5A junction was significantly delayed by another mutation. Individual charged-to-alanine mutations did not affect interactions between the NS4B and NS3-4A proteins. A triple charged-to-alanine mutation produced a temperature-sensitive replication phenotype with no detectable RNA replication at 39°C, demonstrating that conditional mutations can be obtained by altering the charge characteristics of NS4B. Finally, NS4B mutations dispensable for efficient Con1 RNA replication were tested in the context of the chimeric genotype 2a virus, but significant defects in infectious-virus production were not detected. Taken together, these findings highlight the importance of charged residues for multiple NS4B functions in HCV RNA replication, including the formation of a functional replication complex.     

The C-terminal transmembrane domain of hepatitis C virus (HCV) RNA polymerase is essential for HCV replication in vivo

Hepatitis C virus (HCV) RNA replication is dependent on the enzymatic activities of the viral RNA-dependent RNA polymerase NS5B, which is a membrane-anchored protein. Recombinant NS5B lacking the C-terminal transmembrane domain (21 amino acids) is enzymatically active. To address the role of this domain in HCV replication in vivo, we introduced a series of mutations into the NS5B of an HCV subgenomic replicon and examined the replication capabilities of the resultant mutants by a colony formation assay. Replicons lacking the transmembrane domain did not yield any colonies. Furthermore, when Huh-7 cells harboring the HCV subgenomic replicon were treated with a synthetic peptide consisting of the NS5B transmembrane domain fused to the antennapedia peptide, the membrane association of NS5B was completely disrupted. Correspondingly, the HCV RNA titer was reduced by approximately 50%. A scrambled peptide used as a control did not have any effects. These findings suggest that the membrane association of NS5B facilitates HCV RNA synthesis. However, a related transmembrane domain derived from bovine viral diarrhea virus could not replace the HCV NS5B transmembrane segment. This finding suggests that the C-terminal 21 amino acids not only have a membrane-anchoring function but also may perform additional functions for RNA synthesis in vivo.     

Probing the substrate specificity of hepatitis C virus NS3 serine protease by using synthetic peptides.

We probed the substrate specificity of a recombinant noncovalent complex of the full-length hepatitis C virus (HCV) NS3 serine protease and NS4A cofactor, using a series of small synthetic peptides derived from the three trans-cleavage sites of the HCV nonstructural protein sequence. We observed a distinct cleavage site preference exhibited by the enzyme complex. The values of the turnover number (k(cat)) for the most efficient NS4A/4B, 4B/5A, and 5A/5B peptide substrates were 1.6, 11, and 8 min(-1), respectively, and the values for the corresponding Michaelis-Menten constants (Km) were 280, 160, and 16 microM, providing catalytic efficiency values (k(cat)/Km) of 92, 1,130, and 8,300 M(-1) s(-1). An alanine-scanning study for an NS5A/5B substrate (P6P4') revealed that P1 Cys and P3 Val were critical. Finally, substitutions at the scissile P1 Cys residue by homocysteine (Hcy), S-methylcysteine (Mcy), Ala, S-ethylcysteine (Ecy), Thr, Met, D-Cys, Ser, and penicillamine (Pen) produced progressively less efficient substrates, revealing a stringent stereochemical requirement for a Cys residue at this position.     

Steady-state cleavage kinetics for dengue virus type 2 ns2b-ns3(pro) serine protease with synthetic peptides

The N-terminal part of the NS3 protein from dengue virus contains a trypsin-like serine protease responsible for processing the nonstructural region of the viral polyprotein. Enzymatic activity of the NS2B-NS3(pro) precursor incorporating a full-length NS2B cofactor of dengue virus type 2 was examined by using synthetic dodecamer peptide substrates encompassing native cleavage sequences of the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 polyprotein junctions. Cleavage of the dansylated substrates was monitored by a HPLC-based assay and kinetic parameters for K(1M), k(cat) and k(cat)/K(m) were obtained. The data presented here show that NS2B-NS3(pro) expressed in recombinant E. coli can be renatured to an active protease which reacts in the absence of microsomal membranes with all 4 substrate peptides, albeit the molecule does not exhibit autoproteolytic processing at the NS2B/NS3 site. A marked difference in cleavage efficiency was found for the NS2B/NS3 substrate and the remaining 3 peptides based on the NS2A/NS2B, NS3/NS4A and NS4A/NS5 cleavage sites.     

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-beta (TRIF). The TRIF cleavage site has remarkable homology with the viral NS4B/5A substrate, although an 8-residue polyproline track extends upstream from the P(6) 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 3(10) helix close to the protease active site. Thus, a polyproline II interaction with the 3(10) 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 3(10) helix may be a site of interaction with other viral proteins.     

Identification of Peptide Leads to Inhibit Hepatitis C Virus: Inhibitory Effect of Plectasin Peptide Against Hepatitis C Serine Protease

The emerging of hepatitis C virus (HCV) resistant strains has been considered as a main drawback of the available drugs. Since HCV has a large inactive surface, we would like to hypothesis that the mutation occur in HCV is minimal and causing less resistance against inhibition. In this study, a short peptide inhibitor of HCV namely plectasin was identified by HCV NS3-4A serine protease assay. Plectasin peptide showed considerable inhibition against HCV NS3-4A serine protease. Enzymatic activity of the recombinant NS3-4Apro was analysed by fluorescence release from several fluorogenic peptide substrates which resembling the dibasic cleavage site sequences of the flavivirus polyprotein precursor. Of all amc-labelled peptides, Pyr-RTKR-amc was the most efficiently cleaved substrate with the lowest Km value of 20 µM. The kinetic assay showed that plectasin peptide inhibited NS3-4Apro activity with an IC₅₀ value of 4.3 μM compared to the aprotinin as a standard proteases inhibitor with an IC₅₀ of 6.1 μM. From the results, plectasin peptide also demonstrated a dose-dependent inhibition of HCV replication with a considerable reduction in RLuc activity at 15 µM using HCV replicon- containing Huh-7 cells. Our study has identified a unique natural peptide that can be used to highlight novel structures for the development of drug derivatives with high efficacy of HCV NS3-4A protease inhibitors.     

Functional profiling of recombinant NS3 proteases from all four serotypes of dengue virus using tetrapeptide and octapeptide substrate libraries

Regulated proteolysis by the two-component NS2B/NS3 protease of dengue virus is essential for virus replication and the maturation of infectious virions. The functional similarity between the NS2B/NS3 proteases from the four genetically and antigenically distinct serotypes was addressed by characterizing the differences in their substrate specificity using tetrapeptide and octapeptide libraries in a positional scanning format, each containing 130,321 substrates. The proteases from different serotypes were shown to be functionally homologous based on the similarity of their substrate cleavage preferences. A strong preference for basic amino acid residues (Arg/Lys) at the P1 positions was observed, whereas the preferences for the P2-4 sites were in the order of Arg > Thr > Gln/Asn/Lys for P2, Lys > Arg > Asn for P3, and Nle > Leu > Lys > Xaa for P4. The prime site substrate specificity was for small and polar amino acids in P1' and P3'. In contrast, the P2' and P4' substrate positions showed minimal activity. The influence of the P2 and P3 amino acids on ground state binding and the P4 position for transition state stabilization was identified through single substrate kinetics with optimal and suboptimal substrate sequences. The specificities observed for dengue NS2B/NS3 have features in common with the physiological cleavage sites in the dengue polyprotein; however, all sites reveal previously unrecognized suboptimal sequences.     

Calcium-dependent calpain proteases are implicated in processing of the hepatitis C virus NS5A protein

The nonstructural 5A (NS5A) protein of the hepatitis C virus (HCV) is a multifunctional phosphoprotein that is implicated in viral replication and HCV-mediated pathogenesis. We report here that the NS5A protein from the HCV genotype 1a is processed into shorter distinct forms when expressed in mammalian cells (Vero, HepG2, HuH-7, and WRL68) infected with an NS5A-expressing HSV-1-based amplicon vector or when transiently transfected with NS5A-expressing plasmids in the absence of exogenous apoptotic stimuli. Inhibitor studies combined with cell-free cleavage assays suggest that calcium-dependent calpain proteases, in addition to caspase-like proteases, are involved in NS5A processing. Interestingly, His-tagging experiments indicated that all the detectable NS5A-cleaved products are N-terminal forms of the protein. Additionally, immunofluorescence studies showed that, despite proteolytic cleavage, the NS5A protein exhibits a cytoplasm-perinuclear localization similar to that of the full-length protein. Thus, our results are consistent with recent data that demonstrated that NS5A is capable of perturbing intracellular calcium homeostasis and suggest that NS5A is both an inducer and a substrate of the calcium-dependent calpain protease(s). This may imply that cleavage of NS5A by calpain(s) could play a role in the modulation of NS5A function.     

Performance of random forest when SNPs are in linkage disequilibrium

Background

Proteases of human pathogens are becoming increasingly important drug targets, hence it is necessary to understand their substrate specificity and to interpret this knowledge in practically useful ways. New methods are being developed that produce large amounts of cleavage information for individual proteases and some have been applied to extract cleavage rules from data. However, the hitherto proposed methods for extracting rules have been neither easy to understand nor very accurate. To be practically useful, cleavage rules should be accurate, compact, and expressed in an easily understandable way.

Results

A new method is presented for producing cleavage rules for viral proteases with seemingly complex cleavage profiles. The method is based on orthogonal search-based rule extraction (OSRE) combined with spectral clustering. It is demonstrated on substrate data sets for human immunodeficiency virus type 1 (HIV-1) protease and hepatitis C (HCV) NS3/4A protease, showing excellent prediction performance for both HIV-1 cleavage and HCV NS3/4A cleavage, agreeing with observed HCV genotype differences. New cleavage rules (consensus sequences) are suggested for HIV-1 and HCV NS3/4A cleavages. The practical usability of the method is also demonstrated by using it to predict the location of an internal cleavage site in the HCV NS3 protease and to correct the location of a previously reported internal cleavage site in the HCV NS3 protease. The method is fast to converge and yields accurate rules, on par with previous results for HIV-1 protease and better than previous state-of-the-art for HCV NS3/4A protease. Moreover, the rules are fewer and simpler than previously obtained with rule extraction methods.

Conclusion

A rule extraction methodology by searching for multivariate low-order predicates yields results that significantly outperform existing rule bases on out-of-sample data, but are more transparent to expert users. The approach yields rules that are easy to use and useful for interpreting experimental data.     

Efficient rescue of hepatitis C virus RNA replication by trans-complementation with nonstructural protein 5A

Studies of Hepatitis C virus (HCV) RNA replication have become possible with the development of subgenomic replicons. This system allows the functional analysis of the essential components of the viral replication complex, which so far are poorly defined. In the present study we wanted to investigate whether lethal mutations in HCV nonstructural genes can be rescued by trans-complementation. Therefore, a series of replicon RNAs carrying mutations in NS3, NS4B, NS5A, and NS5B that abolish replication were transfected into Huh-7 hepatoma cells harboring autonomously replicating helper RNAs. Similar to data described for the Bovine viral diarrhea virus (C. W. Grassmann, O. Isken, N. Tautz, and S. E. Behrens, J. Virol. 75:7791-7802, 2001), we found that only NS5A mutants could be efficiently rescued. There was no evidence for RNA recombination between helper and mutant RNAs, and we did not observe reversions in the transfected mutants. Furthermore, we established a transient complementation assay based on the cotransfection of helper and mutant RNAs. Using this assay, we extended our results and demonstrated that (i) inactivating NS5A mutations affecting the amino-terminal amphipathic helix cannot be complemented in trans; (ii) replication of the helper RNA is not necessary to allow efficient trans-complementation; and (iii) the minimal sequence required for trans-complementation of lethal NS5A mutations is NS3 to -5A, whereas NS5A expressed alone does not restore RNA replication. In summary, our results provide the first insight into the functional organization of the HCV replication complex.     

Reporter substrates for assessing the activity of the hepatitis C virus NS3-4A serine protease in living cells

We describe a versatile system for monitoring the activity of the NS3-4A serine protease of the hepatitis C virus (HCV) in mammalian cells. The system relies on coexpression of the protease and of an artificial substrate containing a reporter domain and an intracellular targeting sequence separated by a NS3-4A-specific cleavage site. We constructed two different substrates suitable for different applications. The first substrate secretory alkaline phosphatase-1 (SEAP-1) harbors the NS3-4A cleavage site inserted between the SEAP and a membrane anchor featuring an endoplasmic reticulum retention sequence. The arrangement of this substrate is such that SEAP is secreted in the extracellular medium depending on the NS3 protease activity. We show that SEAP-1 can be used to evaluate the activity of NS3-4A inhibitors in living cells. In the second substrate (CD8-1), SEAP is replaced by the extracellular domain of the lymphocyte surface antigen CD8 alpha. The arrangement of this substrate is such that the CD8 alpha domain is transported to the cell surface upon NS3-4Ap cleavage and remains associated with the plasma membrane as an integral membrane protein. We show that CD8-1 can be used for selecting cells capable of supporting HCV replication.     

Effect of naturally occurring active site mutations on hepatitis C virus NS3 protease specificity

A comparison of the DNA sequences from all available genotypes of HCV indicate that the active site residues of the NS3 protease are strictly conserved with the exception of positions 123 and 168, which border the S(4) subsite. In genotype 3, the canonic arginine and aspartic acid have been replaced with threonine and glutamine, respectively. To determine if these differences contribute to an altered specificity, we characterized single-chain NS3 proteases from strains 1a, 1b, and 3a with peptide substrates and product inhibitors on the basis of the natural cleavage junction sequences, in addition to polyprotein substrates derived from the 1a strain. No statistically significant differences in specificity were observed. To demonstrate that the active sites were actually different, we generated and evaluated peptide substrates with unnatural extended side-chains. These studies confirmed that there are measurable differences between the NS3 proteases of genotypes 1 and 3. Specifically, a 5-fold difference in K(i) was observed between the proteases from genotypes 1 and 3 when a D-Glu occupied P(5), and a 30-fold difference was seen when this position contained a D-homoglutamate. The contribution of residues 123 and 168 toward the altered specificity was then evaluated individually by site-directed mutagenesis. These mutants showed that potency differences within this series could be attributed to the residue that occupied position 123 of the protease. Modeling these unnatural substrate/mutant protease interactions, on the basis of cocrystal structures of enzyme-substrate complexes, provides a structural basis for these observations. Proteins 2001;43:82-88.     

Increasing rate of cleavage at boundary between non-structural proteins 4B and 5A inhibits replication of hepatitis C virus

In hepatitis C virus, non-structural proteins are cleaved from the viral polyprotein by viral encoded proteases. Although proteolytic processing goes to completion, the rate of cleavage differs between different boundaries, primarily due to the sequence at these positions. However, it is not known whether slow cleavage is important for viral replication or a consequence of restrictions on sequences that can be tolerated at the cleaved ends of non-structural proteins. To address this question, mutations were introduced into the NS4B side of the NS4B5A boundary, and their effect on replication and polyprotein processing was examined in the context of a subgenomic replicon. Single mutations that modestly increased the rate of boundary processing were phenotypically silent, but a double mutation, which further increased the rate of boundary cleavage, was lethal. Rescue experiments relying on viral RNA polymerase-induced error failed to identify second site compensatory mutations. Use of a replicon library with codon degeneracy did allow identification of second site compensatory mutations, some of which fell exclusively within the NS5A side of the boundary. These mutations slowed boundary cleavage and only enhanced replication in the context of the original lethal NS4B double mutation. Overall, the data indicate that slow cleavage of the NS4B5A boundary is important and identify a previously unrecognized role for NS4B5A-containing precursors requiring them to exist for a minimum finite period of time.     

Hepatitis C virus-encoded nonstructural protein NS4A has versatile functions in viral protein processing.

A transient protein expression system in COS-1 cells was used to study the role of hepatitis C virus (HCV)-encoded NS4A protein on HCV nonstructural polyprotein processing. By analyzing the protein expression and processing of a deletion mutant polypeptide, NS delta 4A, which encodes the entire putative HCV nonstructural polyprotein except the region encoding NS4A, the versatile functions of NS4A were revealed. Most of the NS3 processed from NS delta 4A was localized in the cytosol fraction and was degraded promptly. Coproduction of NS4A stabilizes NS3 and assists in its localization in the membrane. NS4A was found to be indispensable for cleavage at the 4B/5A site but not essential for cleavage at the 5A/5B site in NS delta 4A. The functioning of NS4A as a cofactor for cleavage at the 4B/5A site was also observed when 30 amino acids around this site was used as a substrate and a serine proteinase domain of 167 amino acids, from Gly-1049 to Ser-1215, was used as an enzyme protein, suggesting that possible domains for the interaction of NS4A were in those regions of the enzyme protein (NS3) and/or the substrate protein. Two proteins, p58 and p56, were produced from NS5A. For the production of p58, equal or excess molar amounts of NS4A relative to NS delta 4A were required. Deletion analysis of NS4A revealed a minimum functional domain of NS4A of 10 amino acids, from Gly-1678 to Ile-1687.