Complex formation between the NS3 serine-type proteinase of the hepatitis C virus and NS4A and its importance for polyprotein maturation.

Processing of the hepatitis C virus polyprotein is mediated by host cell signalases and at least two virally encoded proteinases. Of these, the serine-type proteinase encompassing the amino-terminal one-third of NS3 is responsible for cleavage at the four sites carboxy terminal of NS3. The activity of this proteinase is modulated by NS4A, a 54-amino-acid polyprotein cleavage product essential for processing at the NS3/4A, NS4A/4B, and NS4B/5A sites and enhancing cleavage efficiency between NS5A and NS5B. Using the vaccinia virus-T7 hybrid system to express hepatitis C virus polypeptides in BHK-21 cells, we studied the role of NS4A in proteinase activation. We found that the NS3 proteinase and NS4A form a stable complex when expressed as a single polyprotein or as separate molecules. Results from deletion mapping show that the minimal NS4A domain required for proteinase activation is located in the center of NS4A between amino acids 1675 and 1686 of the polyprotein. Amino acid substitutions within this domain destabilizing the NS3-NS4A complex also impair trans cleavage at the NS4A-dependent sites. Similarly, deletion of amino-terminal NS3 sequences impairs complex formation as well as cleavage at the NS4B/5A site but not at the NS4A-independent NS5A/5B site. These results suggest that a stable NS3-NS4A interaction is important for cleavage at the NS4A-dependent sites and that amino-terminal NS3 sequences and the central NS4A domain are directly involved in complex formation.     

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.     

Cleavage of the dengue virus polyprotein at the NS3/NS4A and NS4B/NS5 junctions is mediated by viral protease NS2B-NS3, whereas NS4A/NS4B may be processed by a cellular protease.

The cleavage mechanism utilized for processing of the NS3-NS4A-NS4B-NS5 domain of the dengue virus polyprotein was studied by using the vaccinia virus expression system. Recombinant vaccinia viruses vNS2B-NS3-NS4A-NS4B-NS5, vNS3-NS4A-NS4B-NS5, vNS4A-NS4B-NS5, and vNS4B-NS5 were constructed. These recombinants were used to infect cells, and the labeled lysates were analyzed by immunoprecipitation. Recombinant vNS2B-NS3-NS4A-NS4B-NS5 expressed the authentic NS3 and NS5 proteins, but the other recombinants produced uncleaved polyproteins. These findings indicate that NS2B is required for processing of the downstream nonstructural proteins, including the NS3/NS4A and NS4B/NS5 junctions, both of which contain a dibasic amino acid sequence preceding the cleavage site. The flavivirus NS4A/NS4B cleavage site follows a long hydrophobic sequence. The polyprotein NS4A-NS4B-NS5 was cleaved at the NS4A/NS4B junction in the absence of other dengue virus functions. One interpretation for this finding is that NS4A/NS4B cleavage is mediated by a host protease, presumably a signal peptidase. Although vNS3-NS4A-NS4B-NS5 expressed only the polyprotein, earlier results demonstrated that cleavage at the NS4A/NS4B junction occurred when an analogous recombinant, vNS3-NS4A-84%NS4B, was expressed. Thus, it appears that uncleaved NS3 plus NS5 inhibit NS4A/NS4B cleavage presumably because the putative signal sequence is not accessible for recognition by the responsible protease. Finally, recombinants that expressed an uncleaved NS4B-NS5 polyprotein, such as vNS4A-NS4B-NS5 or vNS4B-NS5, produced NS5 when complemented with vNS2B-30%NS3 or with vNS2B plus v30%NS3. These results indicate that cleavage at the NS4B/NS5 junction can be mediated by NS2B and NS3 in trans.     

丙型肝炎病毒NS3/4A丝氨酸蛋白酶瞬时表达小鼠模型的建立

目的:建立丙型肝炎病毒NS3/4A丝氨酸蛋白酶体内活性评价模型。方法:利用NS4A/B是NS3/4A丝氨酸蛋白酶作用底物的特性,构建融合基因NS3/NS4A/B-SEAP,底物片段NS4A/B插在NS3/4A和人分泌性碱性磷酸酶(SEAP)之间,融合基因表达后SEAP的分泌依赖于有活性的NS3/4A在NS4A/B位点的切割。将含融合基因的质粒NS3/4A(△4AB)SEAP通过水动力转染技术转染到小鼠体内,检测小鼠血清中SEAP的活性,高活性的SEAP是该评价体系成立的证据。结果与结论:在瞬时表达NS3/4A的小鼠血清中检测到了高活性的SEAP,建立了可用于评价抗NS3/4A的小鼠体内瞬时模型。     

Both NS3 and NS4A are required for proteolytic processing of hepatitis C virus nonstructural proteins.

The proteolytic cleavages at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions of hepatitis C virus (HCV) polyprotein are effected by the virus-encoded serine protease contained within NS3. Using transient expression in HeLa cells of cDNA fragments that code for regions of the HCV polyprotein, we studied whether viral functions other than NS3 are required for proteolytic processing at these sites. We found that, in addition to NS3, a C-terminal 33-amino-acid sequence of the NS4A protein is required for cleavage at the NS3-NS4A and NS4B-NS5A sites and that it accelerates the rate of cleavage at the NS5A-NS5B junction. In addition, we show that NS4A can activate the NS3 protease when supplied in trans. Our data suggest that HCV NS4A may be the functional analog of flavivirus NS2B and pestivirus p10 proteins.     

Modulation of hepatitis C virus NS3 protease and helicase activities through the interaction with NS4A

The hepatitis C virus nonstructural 3 protein (NS3) possesses a serine protease activity in the N-terminal one-third, whereas RNA-stimulated NTPase and helicase activities reside in the C-terminal portion. The serine protease activity is required for proteolytic processing at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B polyprotein cleavage sites. NS3 forms a complex with NS4A, a 54-residue polypeptide that was shown to act as an essential cofactor of the NS3 protease. We have expressed in Escherichia coli the NS3-NS4A precursor; cleavage at the junction between NS3 and NS4A occurs during expression in the bacteria cells, resulting in the formation of a soluble noncovalent complex with a sub-nanomolar dissociation constant. We have assessed the minimal ionic strength and detergent and glycerol concentrations required for maximal proteolytic activity and stability of the purified NS3-NS4A complex. Using a peptide substrate derived from the NS5A-NS5B junction, the catalytic efficiency (kcat/Km) of NS3-NS4A-associated protease under optimized conditions was 55 000 s-1 M-1, very similar to that measured with a recombinant complex purified from eukaryotic cells. Dissociation of the NS3-NS4A complex was found to be fully reversible. No helicase activity was exhibited by the purified NS3-NS4A complex, but NS3 was fully active as a helicase upon dissociation of NS4A. On the other hand, both basal and poly(U)-induced NTPase activity and ssRNA binding activity associated with the NS3-NS4A complex were very similar to those exhibited by NS3 alone. Therefore, NS4A appears to uncouple the ATPase/ssRNA binding and RNA unwinding activities associated with NS3.     

A central region in the hepatitis C virus NS4A protein allows formation of an active NS3-NS4A serine proteinase complex in vivo and in vitro.

A virus-encoded serine proteinase mediates four site-specific cleavages in the hepatitis C virus polyprotein. In addition to the catalytic domain, which is located in the N-terminal one-third of nonstructural protein NS3, the 54-residue NS4A protein is required for cleavage at some but not all sites. Here, we provide evidence for a non-ionic detergent-stable interaction between NS4A and the NS3 serine proteinase domain and demonstrate that the central region of NS4A plays a key role in NS4A-dependent processing. Hydrophobic residues, in particular Ile-29, were shown to be important for NS4A activity, and a synthetic peptide, spanning NS4A residues 22 to 34, could substitute for intact NS4A in a cell-free trans cleavage assay. Furthermore, NS4A mutations, which abolished or inhibited processing, correlated with destabilization of the NS3-NS4A complex. These results suggest that a stable interaction exists between the central region of NS4A and the NS3 catalytic domain which is required for NS4A-dependent processing. Since NS4A is required for processing at certain serine proteinase-dependent cleavage sites, this interaction may represent a new target for development of antiviral compounds.     

Expression and purification of an active, full-length hepatitis C viral NS4A

The nonstructural protein 3 (NS3) of the hepatitis C virus (HCV) is a bifunctional protein with protease and helicase activities. Nonstructural protein 4A (NS4A) is preceded by NS3 and augments the proteolytic activity of NS3 through protein-protein interaction. The central domain of NS4A has been shown to be sufficient for the enhancement of the NS3 protease activity. However, investigations on the roles of the N-terminal and the C-terminal regions of NS4A have been hampered by the difficulty of purification of full-length NS4A, a polypeptide that contains highly hydrophobic amino acid residues. Here we report a procedure by which one can produce and purify an active, full-length NS4A using maltose-binding protein fusion method. The full-length NS4A fused to the maltose binding protein is soluble and maintains its NS3 protease-enhancing activity.     

Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins.

The cleavages at the junctions of the flavivirus nonstructural (NS) proteins NS2A/NS2B, NS2B/NS3, NS3/NS4A, and NS4B/NS5 share an amino acid sequence motif and are presumably catalyzed by a virus-encoded protease. We constructed recombinant vaccinia viruses expressing various portions of the NS region of the dengue virus type 4 polyprotein. By analyzing immune precipitates of 35S-labeled lysates of recombinant virus-infected cells, we could monitor the NS2A/NS2B, NS2B/NS3, and NS3/NS4A cleavages. A polyprotein composed of NS2A, NS2B, and the N-terminal 184 amino acids of NS3 was cleaved at the NS2A/NS2B and NS2B/NS3 junctions, whereas a similar polyprotein containing only the first 77 amino acids of NS3 was not cleaved. This finding is consistent with the proposal that the N-terminal 180 amino acids of NS3 constitute a protease domain. Polyproteins containing NS2A and NS3 with large in-frame deletions of NS2B were not cleaved at the NS2A/NS2B or NS2B/NS3 junctions. Coinfection with a recombinant expressing NS2B complemented these NS2B deletions for NS2B/NS3 cleavage and probably also for NS2A/NS2B cleavage. Thus, NS2B is also required for the NS2A/NS2B and NS2B/NS3 cleavages and can act in trans. Other experiments showed that NS2B was needed, apparently in cis, for NS3/NS4A cleavage and for a series of internal cleavages in NS3. Indirect evidence that NS3 can also act in trans was obtained. Models are discussed for a two-component protease activity requiring both NS2B and NS3.     

Differential requirements of NS4A for internal NS3 cleavage and polyprotein processing of hepatitis C virus

The NS3 protein of hepatitis C virus (HCV) possesses protease activity responsible for the proteolytic cleavage of the viral polyprotein at the junctions of nonstructural proteins downstream of NS3. The NS3 protein was also found to be internally cleaved. In this study, we demonstrated that internal cleavages occurred on the NS3 protein of genotype 1b in the presence of NS4A, both in culture cells and with a mouse model system. No internal cleavage products were detected with the NS3 and NS4A proteins of genotype 2a. Three potential cleavage sites were detected in the NS3 protein (genotype 1b), with IPT(402)|S being the major one. The internal cleavage requires the polyprotein processing activity of NS3 protease, but when supplemented in trans, the internal cleavage efficiency is reduced. In addition, several mutations in NS4A disrupted the internal cleavage of NS3 but did not affect polyprotein processing, indicating that NS4A contributes differently to these two proteolytic activities. Furthermore, Ile-25, Val-26, and Ile-29 of the NS4A protein, important for the NS4A-dependent internal cleavages, were also shown to be critical for the transforming activity of NS3, but mutations at these critical residues resulted only in a slight increase of HCV replicating efficiency. The internal cleavage-associated enhancement of the transforming activity of NS3 was reduced when a T402A substitution at the major internal cleavage site was introduced. The multiple roles of NS4A in viral multiplication and pathogenesis make NS4A an ideal molecular target for HCV therapy.     

Mechanistic role of NS4A and substrate in the activation of HCV NS3 protease

Hepatitis C virus (HCV) NS3 protease is the key enzyme for its maturation. Three hypotheses have been advanced in the literature to demonstrate the mechanism of the activation of the HCV NS3 protease. A virus-encoded protein NS4A and substrate are proposed to be involved in the activation of the HCV NS3 protease. However, the three hypotheses are not completely consistent with one another. Multiple molecular dynamics simulations were performed on various NS3 protease systems: free NS3 protease, NS3/4A, NS3/inhibitor, and NS3/4A/inhibitor complexes, to further unravel the mechanism of the activation of the NS3 protease. Simulation results suggest that the binding of NS4A induces a classic serine protease conformation of the catalytic triad of the NS3 protease. NS4A rearranges the secondary structure of both the N-terminus and catalytic site of the NS3 protease, reduces the mobility of the global structure of the NS3 protease, especially the catalytic site, and provides a rigid and tight structure, except for the S1 pocket, for the binding and hydrolysis of substrates. The binding of substrate also contributes to the activation of the NS3 protease by an induced-fit of the classic serine protease catalytic triad. However, the global structure of the NS3 protease is still loose and highly flexible without stable secondary structural elements, such as helix α0 at the N-terminus and helix α1 and β-sheet E1-F1 at the catalytic site. The structure of the NS3 protease without NS4A is not suitable for the binding and hydrolysis of substrates.     

Kinetic and structural analyses of hepatitis C virus polyprotein processing.

Recombinant vaccinia viruses were used to study the processing of hepatitis C virus (HCV) nonstructural polyprotein precursor. HCV-specific proteins and cleavage products were identified by size and by immunoprecipitation with region-specific antisera. A polyprotein beginning with 20 amino acids derived from the carboxy terminus of NS2 and ending with the NS5B stop codon (amino acids 1007 to 3011) was cleaved at the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B sites, whereas a polyprotein in which the putative active site serine residue was replaced by an alanine remained unprocessed, demonstrating that the NS3-encoded serine-type proteinase is essential for cleavage at these sites. Processing of the NS3'-5B polyprotein was complex and occurred rapidly. Discrete polypeptide species corresponding to various processing intermediates were detected. With the exception of NS4AB-5A/NS5A, no clear precursor-product relationships were detected. Using double infection of cells with vaccinia virus recombinants expressing either a proteolytically inactive NS3'-5B polyprotein or an active NS3 proteinase, we found that cleavage at the NS4A/4B, NS4B/5A, and NS5A/5B sites could be mediated in trans. Absence of trans cleavage at the NS3/4A junction together with the finding that processing at this site was insensitive to dilution of the enzyme suggested that cleavage at this site is an intramolecular reaction. The trans-cleavage assay was also used to show that (i) the first 211 amino acids of NS3 were sufficient for processing at all trans sites and (ii) small deletions from the amino terminus of NS3 selectively affected cleavage at the NS4B/5A site, whereas more extensive deletions also decreased processing efficiencies at the other sites. Using a series of amino-terminally truncated substrate polyproteins in the trans-cleavage assay, we found that NS4A is essential for cleavage at the NS4B/5A site and that processing at this site could be restored by NS4A provided in cis (i.e., together with the substrate) or in trans (i.e., together with the proteinase). These results suggest that in addition to the NS3 proteinase, NS4A sequences play an important role in HCV polyprotein processing.     

登革2型病毒非结构蛋白NS4B的原核表达、纯化及多克隆抗体制备

目的：原核表达、纯化登革2型病毒非结构蛋白NS4B,并制备其多克隆抗体,以研究其结构与功能。方法：扩增编码登革2型病毒NS4B的24-238位氨基酸残基的基因序列,并将其克隆到原核表达载体pGEX-4T-1,转化大肠杆菌BL21（DE3）,IPTG诱导表达;采用蛋白浸提方法从SDS-PAGE胶中回收融合蛋白;用纯化后的融合蛋白免疫BALB／c鼠制备多克隆抗体,采用间接免疫荧光法检测抗体效价。结果：原核表达了NS4B-GST融合蛋白,并获得了其多克隆抗体,抗体效价为1：800。结论：登革2型病毒NS4B的24-238位氨基酸残基可诱导小鼠产生具有较高效价和特异性的多克隆抗体,这为研究NS4B的结构与功能奠定了基础。     

Processing and localization of Dengue virus type 2 polyprotein precursor NS3-NS4A-NS4B-NS5.

Processing of dengue virus type 2 polyprotein precursor NS3-NS4A-NS4B-NS5 could be mediated by the catalytically active NS3 protease domain and NS2B in trans at the dibasic sites NS3-NS4A and NS4B-NS5. Subcellular localization of the unprocessed precursor NS3-NS4A-NS4B-NS5 showed that it was confined to a distinct subcellular organelle in the cytoplasm, which was distinct from the distribution of the mature NS5.     

In-cell selectivity profiling of membrane-anchored and replicase-associated hepatitis C virus NS3-4A protease reveals a common, stringent substrate recognition profile

The need to identify anti-Flaviviridae agents has resulted in intensive biochemical study of recombinant nonstructural (NS) viral proteases; however, experimentation on viral protease-associated replication complexes in host cells is extremely challenging and therefore limited. It remains to be determined if membrane anchoring and/or association to replicase-membrane complexes of proteases, such as hepatitis C virus (HCV) NS3-4A, plays a regulatory role in the substrate selectivity of the protease. In this study, we examined trans-endoproteolytic cleavage activities of membrane-anchored and replicase-associated NS3-4A using an internally consistent set of membrane-anchored protein substrates mimicking all known HCV NS3-4A polyprotein cleavage sequences. Interestingly, we detected cleavage of substrates encoding for the NS4B/NS5A and NS5A/NS5B junctions, but not for the NS3/NS4A and NS4A/NS4B substrates. This stringent substrate recognition profile was also observed for the replicase-associated NS3-4A and is not genotype-specific. Our study also reveals that ER-anchoring of the substrate is critical for its cleavage by NS3-4A. Importantly, we demonstrate that in HCV-infected cells, the NS4B/NS5A substrate was cleaved efficiently. The unique ability of our membrane-anchored substrates to detect NS3-4A activity alone, in replication complexes, or within the course of infection, shows them to be powerful tools for drug discovery and for the study of HCV biology.     

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.     

Isolation and characterization of monoclonal antibodies that inhibit hepatitis C virus NS3 protease

A series of mouse monoclonal antibodies (MAbs) to the nonstructural protein 3 (NS3) of hepatitis C virus was prepared. One of these MAbs, designated 8D4, was found to inhibit NS3 protease activity. This inhibition was competitive with respect to the substrate peptide (K(i) = 39 nM) but was significantly decreased by the addition of the NS4A peptide, a coactivator of the NS3 protease. 8D4 also showed marked inhibition of the NS3-dependent cis processing of the NS3/4A polyprotein but had virtually no effect on the succeeding NS3/4A-dependent trans processing of the NS5A/5B polyprotein in vitro. Epitope mapping of 8D4 with a random peptide library revealed a consensus sequence, DxDLV, that matched residues 79 to 83 (DQDLV) of NS3, a region containing the catalytic residue Asp-81. Furthermore, synthetic peptides including this sequence were shown to block the ability of 8D4 to bind to NS3, indicating that 8D4 interacts with the catalytic region of NS3. The data showing decreased inhibition potency of 8D4 against the NS3/4A complex suggest that 8D4 recognizes the conformational state of the protease active site caused by the association of NS4A with the protease.     

The acidic domain of hepatitis C virus NS4A contributes to RNA replication and virus particle assembly

Hepatitis C virus NS3-4A is a membrane-bound enzyme complex that exhibits serine protease, RNA helicase, and RNA-stimulated ATPase activities. This enzyme complex is essential for viral genome replication and has been recently implicated in virus particle assembly. To help clarify the role of NS4A in these processes, we conducted alanine scanning mutagenesis on the C-terminal acidic domain of NS4A in the context of a chimeric genotype 2a reporter virus. Of 13 mutants tested, two (Y45A and F48A) had severe defects in replication, while seven (K41A, L44A, D49A, E50A, M51A, E52A, and E53A) efficiently replicated but had severe defects in virus particle assembly. Multiple strategies were used to identify second-site mutations that suppressed these NS4A defects. The replication defect of NS4A F48A was partially suppressed by mutation of NS4B I7F, indicating that a genetic interaction between NS4A and NS4B contributes to RNA replication. Furthermore, the virus assembly defect of NS4A K41A was suppressed by NS3 Q221L, a mutation previously implicated in overcoming other virus assembly defects. We therefore examined the known enzymatic activities of wild-type or mutant forms of NS3-4A but did not detect specific defects in the mutants. Taken together, our data reveal interactions between NS4A and NS4B that control genome replication and between NS3 and NS4A that control virus assembly.     

Membrane Permeabilization by Small Hydrophobic Nonstructural Proteins of Japanese Encephalitis Virus

Infection with Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, may cause acute encephalitis in humans and induce severe cytopathic effects in various types of cultured cells. We observed that JEV replication rendered infected baby hamster kidney (BHK-21) cells sensitive to the translational inhibitor hygromycin B or alpha-sarcine, to which mock-infected cells were insensitive. However, little is known about whether any JEV nonstructural (NS) proteins contribute to virus-induced changes in membrane permeability. Using an inducible Escherichia coli system, we investigated which parts of JEV NS1 to NS4 are capable of modifying membrane penetrability. We found that overexpression of NS2B-NS3, the JEV protease, permeabilized bacterial cells to hygromycin B whereas NS1 expression failed to do so. When expressed separately, NS2B alone, but not NS3, was sufficient to alter bacterial membrane permeability. Similarly, expression of NS4A or NS4B also rendered bacteria susceptible to hygromycin B inhibition. Examination of the effect of NS1 to NS4 expression on bacterial growth rate showed that NS2B exhibited the greatest inhibitory capability, followed by a modest repression from NS2A and NS4A, whereas NS1, NS3, and NS4B had only trivial influence with respect to the vector control. Furthermore, when cotransfected with a reporter gene luciferase or beta-galactosidase, transient expression of NS2A, NS2B, and NS4B markedly reduced the reporter activity in BHK-21 cells. Together, our results suggest that upon JEV infection, these four small hydrophobic NS proteins have various modification effects on host cell membrane permeability, thereby contributing in part to virus-induced cytopathic effects in infected cells.     

Subcellular localization, stability, and trans-cleavage competence of the hepatitis C virus NS3-NS4A complex expressed in tetracycline-regulated cell lines

A tetracycline-regulated gene expression system and a panel of novel monoclonal antibodies were used to examine the subcellular localization, stability, and trans-cleavage competence of the hepatitis C virus (HCV) NS3-NS4A complex in inducible cell lines. The NS3 serine protease domain and the full-length NS3 protein expressed in the absence of the NS4A cofactor were diffusely distributed in the cytoplasm and nucleus. Coexpression of NS4A, however, directed NS3 to the endoplasmic reticulum (ER) or an ER-like modified compartment, as demonstrated by colocalization with 3,3'-dihexyloxacarbocyanine iodide, protein disulfide isomerase, and calnexin, as well as subcellular fractionation analyses. In addition, coexpression with NS4A dramatically increased the intracellular stability of NS3 (mean protein half-life of 26 versus 3 h) and allowed for NS4A-dependent trans-cleavage at the NS4B-NS5A junction. Deletion analyses revealed that the hydrophobic amino-terminal domain of NS4A was required for ER targeting of NS3. These results demonstrate the importance of studying HCV proteins in their biological context and define a well-characterized cell culture system for further analyses of the NS3-NS4A complex and the evaluation of novel antiviral strategies against hepatitis C.