Analysis of deletion mutants indicates that the 2A polypeptide of hepatitis A virus participates in virion morphogenesis

Unlike all other picornaviruses, the primary cleavage of the hepatitis A virus (HAV) polyprotein occurs at the 2A/2B junction and is carried out by the only proteinase encoded by the virus, 3C(pro). The resulting P1-2A capsid protein precursor is subsequently cleaved by 3C(pro) to generate VP0, VP3, and VP1-2A, which associate as pentamers. An unidentified cellular proteinase acting at the VP1/2A junction releases the mature capsid protein VP1 from VP1-2A later in the morphogenesis process. Although these aspects of polyprotein processing are well characterized, the function of 2A is unknown. To study its role in the viral life cycle, we assessed the infectivity of synthetic, genome-length RNAs containing 11 different in-frame deletions in the 2A region. Deletions in the N-terminal 40% of 2A abolished infectivity, whereas deletions in the C-terminal 60% resulted in viruses with a small-focus replication phenotype. C-terminal deletions in 2A had no effect on RNA replication kinetics under one-step growth conditions, nor did they have an effect on capsid protein synthesis and 3C(pro)-mediated processing. However, C-terminal deletions in 2A altered the VP1/2A cleavage, resulting in accumulation of uncleaved VP1-2A precursor in virions and possibly accounting for a delay in the appearance of infectious particles with these mutants, as well as a fourfold decrease in specific infectivity of the virus particles. When the capsid proteins were expressed from recombinant vaccinia viruses, the N-terminal part of 2A was required for efficient cleavage of the P1-2A precursor by 3C(pro) and assembly of structural precursors into pentamers. These data indicate that the N-terminal domain of 2A must be present as a C-terminal extension of P1 for folding of the capsid protein precursor to allow efficient 3C(pro)-mediated cleavages and to promote pentamer assembly, after which cleavage at the VP1/2A junction releases the mature VP1 protein, a process that appears to be necessary to produce highly infectious particles.     

Cleavage specificity of purified recombinant hepatitis A virus 3C proteinase on natural substrates.

Hepatitis A virus (HAV) 3C proteinase expressed in Escherichia coli was purified to homogeneity, and its cleavage specificity towards various parts of the viral polyprotein was analyzed. Intermolecular cleavage of the P2-P3 domain of the HAV polyprotein gave rise to proteins 2A, 2B, 2C, 3ABC, and 3D, suggesting that in addition to the primary cleavage site, all secondary sites within P2 as well as the 3C/3D junction are cleaved by 3C. 3C-mediated processing of the P1-P2 precursor liberated 2A and 2BC, in addition to the structural proteins VP0, VP3, and VP1-2A and the respective intermediate products. A clear dependence on proteinase concentration was found for most cleavage sites, possibly reflecting the cleavage site preference of 3C. The most efficient cleavage occurred at the 2A/2B and 2C/3A junctions. The electrophoretic mobility of processing product 2B, as well as cleavage of the synthetic peptide KGLFSQ*AKISLFYT, suggests that the 2A/2B junction is located at amino acid position 836/837 of the HAV polyprotein. Furthermore, using suitable substrates we obtained evidence that sites VP3/VP1 and VP1/2A are alternatively processed by 3C, leading to either VP1-2A or to P1 and 2A. The results with regard to intermolecular cleavage by purified 3C were confirmed by the product pattern derived from cell-free expression and intramolecular processing of the entire polyprotein. We therefore propose that polyprotein processing of HAV relies on 3C as the single proteinase, possibly assisted by as-yet-undetermined viral or host cell factors and presumably controlled in a concentration-dependent fashion.     

Disruption of TLR3 signaling due to cleavage of TRIF by the hepatitis A virus protease-polymerase processing intermediate, 3CD

Toll-like receptor 3 (TLR3) and cytosolic RIG-I-like helicases (RIG-I and MDA5) sense viral RNAs and activate innate immune signaling pathways that induce expression of interferon (IFN) through specific adaptor proteins, TIR domain-containing adaptor inducing interferon-β (TRIF), and mitochondrial antiviral signaling protein (MAVS), respectively. Previously, we demonstrated that hepatitis A virus (HAV), a unique hepatotropic human picornavirus, disrupts RIG-I/MDA5 signaling by targeting MAVS for cleavage by 3ABC, a precursor of the sole HAV protease, 3C(pro), that is derived by auto-processing of the P3 (3ABCD) segment of the viral polyprotein. Here, we show that HAV also disrupts TLR3 signaling, inhibiting poly(I:C)-stimulated dimerization of IFN regulatory factor 3 (IRF-3), IRF-3 translocation to the nucleus, and IFN-β promoter activation, by targeting TRIF for degradation by a distinct 3ABCD processing intermediate, the 3CD protease-polymerase precursor. TRIF is proteolytically cleaved by 3CD, but not by the mature 3C(pro) protease or the 3ABC precursor that degrades MAVS. 3CD-mediated degradation of TRIF depends on both the cysteine protease activity of 3C(pro) and downstream 3D(pol) sequence, but not 3D(pol) polymerase activity. Cleavage occurs at two non-canonical 3C(pro) recognition sequences in TRIF, and involves a hierarchical process in which primary cleavage at Gln-554 is a prerequisite for scission at Gln-190. The results of mutational studies indicate that 3D(pol) sequence modulates the substrate specificity of the upstream 3C(pro) protease when fused to it in cis in 3CD, allowing 3CD to target cleavage sites not normally recognized by 3C(pro). HAV thus disrupts both RIG-I/MDA5 and TLR3 signaling pathways through cleavage of essential adaptor proteins by two distinct protease precursors derived from the common 3ABCD polyprotein processing intermediate.     

Maturation of the Hepatitis A Virus Capsid Protein VP1 Is Not Dependent on Processing by the 3Cpro Proteinase

Most details of the processing of the hepatitis A virus (HAV) polyprotein are known. Unique among members of the family Picornaviridae, the primary cleavage of the HAV polyprotein is mediated by 3Cpro, the only proteinase known to be encoded by the virus, at the 2A/2B junction. All other cleavages of the polyprotein have been considered to be due to 3Cpro, although the precise location and mechanism responsible for the VP1/2A cleavage have been controversial. Here we present data that argue strongly against the involvement of the HAV 3Cpro proteinase in the maturation of VP1 from its VP1-2A precursor. Using a heterologous expression system based on recombinant vaccinia viruses directing the expression of full-length or truncated capsid protein precursors, we show that the C terminus of the mature VP1 capsid protein is located near residue 764 of the polyprotein. However, a proteolytically active HAV 3Cpro that was capable of directing both VP0/VP3 and VP3/VP1 cleavages in vaccinia virus-infected cells failed to process the VP1-2A precursor. Using site-directed mutagenesis of an infectious molecular clone of HAV, we modified potential VP1/2A cleavage sites that fit known 3Cpro recognition criteria and found that a substitution that ablates the presumed 3Cpro dipeptide recognition sequence at Glu764-Ser765 abolished neither infectivity nor normal VP1 maturation. Altered electrophoretic mobility of VP1 from a viable mutant virus with an Arg764 substitution indicated that this residue is present in VP1 and that the VP1/2A cleavage occurs downstream of this residue. These data indicate that maturation of the HAV VP1 capsid protein is not dependent on 3Cpro processing and may thus be uniquely dependent on a cellular proteinase.     

In vitro determination of dengue virus type 2 NS2B-NS3 protease activity with fluorescent peptide substrates

The NS2B-NS3(pro) polyprotein segment from the dengue virus serotype 2 strain 16681 was purified from overexpressing E. coli by metal chelate affinity chromatography and gel filtration. Enzymatic activity of the refolded NS2B-NS3(pro) protease complex was determined in vitro with dansyl-labeled peptide substrates, based upon native dengue virus type 2 cleavage sites. The 12mer substrate peptides and the cleavage products could be separated by reversed-phase HPLC, and were identified by UV and fluorescence detection. All of the peptide substrates (representing the DEN polyprotein junction sequences at the NS2A/NS2B, NS2B/NS3, NS3/NS4A and NS4B/NS5 sites) were cleaved by the recombinant protease NS2B-NS3(pro). No cleavage was observed with an enzymatically inactive S135A mutant of the NS3 protein, or with a modified substrate peptide of the NS3/NS4A polyprotein site that contained a K2093A substitution. Enzymatic activity was dependent on the salt concentration. A 50% decrease of activity was observed in the presence of 0.1 M sodium chloride. Our results show that the NS3 protease activity of the refolded NS2BNS3(pro) protein can be assayed in vitro with high specificity by using cleavage-junction derived peptide substrates.     

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.     

Foot-and-mouth disease virus 2A oligopeptide mediated cleavage of an artificial polyprotein.

We describe the construction of a plasmid (pCAT2AGUS) encoding a polyprotein in which a 19 amino acid sequence spanning the 2A region of the foot-and-mouth disease virus (FMDV) polyprotein was inserted between the reporter genes chloramphenicol acetyl transferase (CAT) and beta-glucuronidase (GUS) maintaining a single, long open reading frame. Analysis of translation reactions programmed by this construct showed that the inserted FMDV sequence functioned in a manner similar to that observed in FMDV polyprotein processing: the CAT2AGUS polyprotein underwent a cotranslational, apparently autoproteolytic, cleavage yielding CAT-2A and GUS. Analysis of translation products derived from a series of constructs in which sequences were progressively deleted from the N-terminal region of the FMDV 2A insertion showed that cleavage required a minimum of 13 residues. The FMDV 2A sequence therefore provides the opportunity to engineer either whole proteins or domains such that they are cleaved apart cotranslationally with high efficiency.     

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.     

Insertion of a Sequence Encoding Light Chain 3 of Microtubule-Associated Proteins 1A and 1B in a Pestivirus Genome: Connection with Virus Cytopathogenicity and Induction of Lethal Disease in Cattle

Pestiviruses represent the first RNA viruses for which recombination with cellular protein-coding sequences has been reported. As a result of such recombinations cytopathogenic (cp) pestiviruses can develop from noncytopathogenic (noncp) viruses. In the case of bovine viral diarrhea virus (BVDV), the generation of cp mutants is linked to the induction of the lethal syndrome mucosal disease (MD) in cattle. The cp BVDV JaCP was isolated from an animal which had come down with MD. The genome of JaCP contains a novel kind of cellular insertion (LC3*) which is flanked by duplicated pestivirus sequences. Neither insertion nor duplication is present in the genome of the accompanying noncp virus JaNCP. As part of the viral polyprotein, the insertion in the JaCP genome is translated into a polypeptide almost identical to a fragment of light chain 3, a subunit of the microtubule-associated proteins 1A and 1B from the rat. Transient-expression studies revealed that the LC3* sequence is able to induce an additional cleavage of the viral polyprotein. The respective cleavage occurs directly downstream of the LC3*-encoded sequence and is not dependent on the NS3 serine protease. Insertion of LC3* into an infectious noncp pestivirus cDNA clone without duplicated viral sequences resulted in recovery of a defective cp virus able to replicate only in the presence of a noncp helper virus. In contrast, introduction of both insertion and duplication led to an autonomously replicating cp virus.     

Foot-and-mouth disease virus 2A protease mediates cleavage in attenuated Sabin 3 poliovirus vectors engineered for delivery of foreign antigens.

Poliovirus vectors are being studied as potential vaccine delivery systems, with foreign genetic sequences incorporated as part of the viral genome. The foreign sequences are expressed as part of the viral polyprotein. Addition of proteolytic cleavage sites at the junction of the foreign polypeptide and the viral proteins results in cleavage during polyprotein processing. The ability of foot-and-mouth disease virus (FMDV) 2A to mediate proteolytic cleavage in the context of poliovirus vectors was studied. The results demonstrate that FMDV 2A is able to generate cleavage of the foreign antigen from the viral polyprotein. A second cleavage event between the FMDV 2A peptide and the foreign protein was also observed.     

Molecular cloning, expression and purification of protein 2A of hepatitis A virus

Expression of the protein 2A of Hepatitis A virus (HAV), spanning amino acids 764 through 981 of the viral polyprotein results in a strong inhibition of cap-dependent translation (Maltese et al., 2000). However, the molecular mechanism responsible has remained unclear, in part because the HAV 2A protein was not available in amounts large enough to allow biological or structural studies. To address this issue, a cDNA representation of the sequences encoding HAV 2A was generated by PCR, using primers that introduced an AUG triplet, and a sequence coding for 6 histidine residues at the 5'- and 3'-termini of the genomic sequence, respectively. The cDNA fragment was introduced by cassette exchange in the inducible expression vector pQE-60, and the construct was propagated in bacteria E. coli M15 which constitutively expresses the lac repressor. Upon induction with IPTG (1 mM), HAV 2A was visualized by SDS-PAGE of bacterial lysates as a prominent band M(r) = 21 kDa. The identity of the polypeptide was confirmed by both MALDI-TOF peptide mapping and direct amino acid sequencing. The His-tagged HAV 2A was extracted from bacterial pellets under totally denaturing conditions (6 M urea), subjected to Ni(++)-Sepharose affinity chromatography, allowed to refold while still attached to the matrix, and eluted with 250 mM Imidazole. Contaminant material was partly removed by differential ammonium sulfate precipitation. The protein was further concentrated (Vivaspin centrifugal concentrator), the insoluble material (if present) was discarded, and the homogeneity of the dispersion was ascertained by light scattering. SDS-PAGE revealed that in addition to the main protein (Mr = 21 kDa), a second one of apparent Mr = 14 kDa was always present in variable amounts. The proportion of the latter tended to increase with aging of the preparation. Edman degradation analysis proved that the 14 kDa protein resulted from the cleavage of HAV 2A at a so far undetected scissile bond Gly856/Val857 of the viral polyprotein. A first attempt to crystallize the protein by the hanging drop procedure yielded only small crystals containing exclusively the 14 kDa derivative of HAV 2A. Western blot analysis of HeLa cell extracts that had been incubated with the His-tagged HAV 2A so purified failed to reveal any change in the electrophoretic mobility of the eukaryotic initiation factor (eIF) 4G I.     

Detection of a genome-linked protein (VPg) of hepatitis A virus and its comparison with other picornaviral VPgs.

The nucleotide sequence corresponding to the P3 region of the hepatitis A virus (HAV) polyprotein genome was determined from cloned cDNA and translated into an amino acid sequence. Comparison of the amino acid sequences of the genome-linked proteins (VPgs) of other picornaviruses with the predicted amino acid sequence of HAV was used to locate the primary structure of a putative VPg within the genome of HAV. The sequence of HAV VPg, like those of other picornaviral VPg molecules, contains a tyrosine residue as a potential binding site for HAV RNA in position 3 from its N terminus. The potential cleavage sites to generate VPg from a putative HAV polyprotein are between glutamic acid and glycine at the N terminus and glutamic acid and serine or glutamine and serine at the C terminus. A synthetic peptide corresponding to 10 amino acids of the predicted C terminus of HAV VPg induced anti-peptide antibodies in rabbits when it was conjugated to thyroglobulin as a carrier. These antibodies were specific for the peptide and precipitated VPg, linked to HAV RNA, from purified HAV and from lysates of HAV-infected cells. The precipitation reaction was blocked by the synthetic peptide (free in solution or coupled to carrier proteins) and prevented by pretreatment of VPg RNA with protease. Thus, our predicted amino acid sequence is colinear with the nucleotide sequence of the VPg gene in the HAV genome. From our results we concluded that HAV has the typical organization of picornavirus genes in this part of its genome. Similarity among hydrophobicity patterns of amino acid sequences of different picornaviral VPgs was revealed in hydropathy plots. Thus, the VPg of HAV appears to be closely related to VPg1 and VPg2 of foot-and-mouth disease virus. In contrast, HAV VPg has a unique isoelectric point (pI = 7.15) among the picornavirus VPgs.     

Genetic variation of the poliovirus genome with two VPg coding units.

Amongst the picornaviruses, poliovirus encodes a single copy of the genome-linked protein, VPg wheras foot-and-mouth disease virus uniquely encodes three copies of VPg. We have previously shown that a genetically engineered poliovirus genome containing two tandemly arranged VPgs is quasi-infectious (qi) that, upon genome replication, inadvertently deleted one complete VPg sequence. Using two genetically marked viral genomes with two VPg sequences, we now provide evidence that this deletion occurs via homologous recombination. The mechanism was abrogated when the second VPg was engineered such that its nucleotide sequence differed from that of the first VPg sequence by 36%. Such genomes also expressed a qi phenotype, but progeny viruses resulted from (i) random deletions yielding single VPg coding sequences of varying length lacking the Q*G cleavage site between the VPgs and (ii) mutations in the AKVQ*G cleavage sites between the VPgs at either the P4, P1 or P1' position. These variants present a unique genetic system defining the cleavage signals recognized in 3Cpro-catalyzed proteolysis. We propose a recognition event in the cis cleavages of the polyprotein P2-P3 region, and we present a hypothesis why the poliovirus genome does not tolerate two tandemly arranged VPg sequences.     

Probing the substrate specificity of the dengue virus type 2 NS3 serine protease by using internally quenched fluorescent peptides

The NS3 (dengue virus non-structural protein 3) serine protease of dengue virus is an essential component for virus maturation, thus representing an attractive target for the development of antiviral drugs directed at the inhibition of polyprotein processing. In the present study, we have investigated determinants of substrate specificity of the dengue virus NS3 protease by using internally quenched fluorogenic peptides containing Abz (o-aminobenzoic acid; synonymous to anthranilic acid) and 3-nitrotyrosine (nY) representing both native and chimaeric polyprotein cleavage site sequences. By using this combinatorial approach, we were able to describe the substrate preferences and determinants of specificity for the dengue virus NS2B(H)-NS3pro protease. Kinetic parameters (kcat/K(m)) for the hydrolysis of peptide substrates with systematic truncations at the prime and non-prime side revealed a length preference for peptides spanning the P4-P3' residues, and the peptide Abz-RRRRSAGnY-amide based on the dengue virus capsid protein processing site was discovered as a novel and efficient substrate of the NS3 protease (kcat/K(m)=11087 M(-1) x s(-1)). Thus, while having confirmed the exclusive preference of the NS3 protease for basic residues at the P1 and P2 positions, we have also shown that the presence of basic amino acids at the P3 and P4 positions is a major specificity-determining feature of the dengue virus NS3 protease. Investigation of the substrate peptide Abz-KKQRAGVLnY-amide based on the NS2B/NS3 polyprotein cleavage site demonstrated an unexpected high degree of cleavage efficiency. Chimaeric peptides with combinations of prime and non-prime sequences spanning the P4-P4' positions of all five native polyprotein cleavage sites revealed a preponderant effect of non-prime side residues on the K(m) values, whereas variations at the prime side sequences had higher impact on kcat.     

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.     

Molecular views of viral polyprotein processing revealed by the crystal structure of the hepatitis C virus bifunctional protease-helicase

BACKGROUND: Hepatitis C virus (HCV) currently infects approximately 3% of the world's population. HCV RNA is translated into a polyprotein that during maturation is cleaved into functional components. One component, nonstructural protein 3 (NS3), is a 631-residue bifunctional enzyme with protease and helicase activities. The NS3 serine protease processes the HCV polyprotein by both cis and trans mechanisms. The structural aspects of cis processing, the autoproteolysis step whereby the protease releases itself from the polyprotein, have not been characterized. The structural basis for inclusion of protease and helicase activities in a single polypeptide is also unknown. RESULTS: We report here the 2.5 A resolution structure of an engineered molecule containing the complete NS3 sequence and the protease activation domain of nonstructural protein 4A (NS4A) in a single polypeptide chain (single chain or scNS3-NS4A). In the molecule, the helicase and protease domains are segregated and connected by a single strand. The helicase necleoside triphosphate and RNA interaction sites are exposed to solvent. The protease active site of scNS3-NS4A is occupied by the NS3 C terminus, which is part of the helicase domain. Thus, the intramolecular complex shows one product of NS3-mediated cleavage at the NS3-NS4A junction of the HCV polyprotein bound at the protease active site. CONCLUSIONS: The scNS3-NS4A structure provides the first atomic view of polyprotein cis processing. Both local and global structural rearrangements follow the cis cleavage reaction, and large segments of the polyprotein can be folded prior to proteolytic processing. That the product complex of the cis cleavage reaction exists in a stable molecular conformation suggests autoinhibition and substrate-induced activation mechanisms for regulation of NS3 protease 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.