Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Polyproteins”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 91 records · Page 5Linked to original sources
PubMed1999

Hyperphosphorylation of the hepatitis C virus NS5A protein requires an active NS3 protease, NS4A, NS4B, and NS5A encoded on the same polyprotein.

The nonstructural protein NS5A of hepatitis c virus (HCV) has been demonstrated to be a phosphoprotein with an apparent molecular mass of 56 kDa. In the presence of other viral proteins, p56 is converted into a slower-migrating form of NS5A (p58) by additional phosphorylation events. In this report, we show that the presence of NS3, NS4A, and NS4B together with NS5A is necessary and sufficient for the generation of the hyperphosphorylated form of NS5A (p58) and that all proteins must be encoded on the same polyprotein (in cis). Kinetic studies of NS5A synthesis and pulse-chase experiments demonstrate that fully processed NS5A is the substrate for the formation of p58 and that p56 is converted to p58. To investigate the role of NS3 in NS5A hyperphosphorylation, point and deletion mutations were introduced into NS3 in the context of a polyprotein containing the proteins from NS3 to NS5A. Mutation of the catalytic serine residue into alanine abolished protease activity of NS3 and resulted in total inhibition of NS5A hyperphosphorylation, even if polyprotein processing was allowed by addition of NS3 and NS4A in trans. The same result was obtained by deletion of the first 10 or 28 N-terminal amino acids of NS3, which are known to be important for the formation of a stable complex between NS3 and its cofactor NS4A. These data suggest that the formation of p58 is closely connected to HCV polyprotein processing events. Additional data obtained with NS3 containing the 34 C-terminal residues of NS2 provide evidence that in addition to NS3 protease activity the authentic N-terminal sequence is required for NS5A hyperphosphorylation.

Binding Sites↗
PubMed2003

In vitro proteolytic processing of the MD145 norovirus ORF1 nonstructural polyprotein yields stable precursors and products similar to those detected in calicivirus-infected cells.

The MD145-12 strain (GII/4) is a member of the genus Norovirus in the Caliciviridae and was detected in a patient with acute gastroenteritis in a Maryland nursing home. The open reading frame 1 (ORF1) (encoding the nonstructural polyprotein) was cloned as a consensus sequence into various expression vectors, and a proteolytic cleavage map was determined. The virus-encoded cysteine proteinase mediated at least five cleavages (Q(330)/G(331), Q(696)/G(697), E(875)/G(876), E(1008)/A(1009), and E(1189)/G(1190)) in the ORF1 polyprotein in the following order: N-terminal protein; nucleoside triphosphatase; 20-kDa protein (p20); virus protein, genome linked (VPg); proteinase (Pro); polymerase (Pol). A time course analysis of proteolytic processing of the MD145-12 ORF1 polyprotein in an in vitro coupled transcription and translation assay allowed the identification of stable precursors and final mapped cleavage products. Stable precursors included p20VPg (analogous to the 3AB of the picornaviruses) and ProPol (analogous to the 3CD of the picornaviruses). Less stable processing intermediates were identified as p20VPgProPol, p20VPgPro, and VPgPro. The MD145-12 Pro and ProPol proteins were expressed in bacteria as active forms of the proteinase and used to further characterize their substrate specificities in trans cleavage assays. The MD145-12 Pro was able to cleave its five mapped cleavage sites in trans and, in addition, could mediate trans cleavage of the Norwalk virus (GI/I) ORF1 polyprotein into a similar proteolytic processing profile. Taken together, our data establish a model for proteolytic processing in the noroviruses that is consistent with nonstructural precursors and products identified in studies of caliciviruses that replicate in cell culture systems.

Base Sequence↗
PubMed1988

Biochemical and mutational analysis of a plant virus polyprotein cleavage site.

The RNA genome of tobacco etch virus (TEV) is organized as a single translational unit coding for a 346,000 (346 kd) mol. wt (Mr) polyprotein. The 346 kd Mr polyprotein is cleaved by a 49 kd Mr virus-encoded proteinase at five different sites between the dipeptides Gln-Ser or Gln-Gly. These cleavage sites or gene product boundaries are defined by the heptapeptide sequence...Glu-Xaa-Xaa-Tyr-Xaa-Gln-Ser or Gly.... We have used the 54 kd Mr nuclear inclusion protein/30 kd Mr capsid protein junction as a model to examine the role of these conserved amino acids in defining a cleavage site. The 54 kd/30 kd Mr protein cleavage site sequence of 10 TEV isolates from geographically distinct locations has been deduced. The conserved amino acids are present in all isolates. To determine if these four amino acids are an absolute requirement for polyprotein substrate activity, a site-directed mutational analysis has been performed. A recombinant cDNA molecule encoding the TEV 54 kd/30 kd Mr gene product cleavage site was mutated and polyprotein substrates were synthesized and processed in a cell-free system. Single amino acid substitutions made at the different positions reveal a strong preference for the naturally conserved amino acids.

Amino Acid Sequence↗
PubMed1990

Expression of potyviral polyproteins in transgenic plants reveals three proteolytic activities required for complete processing.

All proteins encoded by the plant potyvirus, tobacco etch virus (TEV), arise by proteolytic processing of a single polyprotein. Two virus-encoded proteinases (NIa and HC-Pro) that catalyze most of the proteolytic events have been characterized previously. The two proteins that are derived from the N-terminal 87 kd region of the viral polyprotein are a 35 kd protein and HC-Pro (52 kd). It is demonstrated in this study that a third proteolytic activity is required to process the junction between these proteins. Proteolysis at the HC-Pro N terminus to separate these proteins occurred poorly, if at all, after in vitro synthesis of a 97 kd polyprotein, whereas cleavage of the HC-Pro C terminus occurred efficiently by an autoprocessing mechanism. Synthesis of the same polyprotein in transgenic tobacco plants, however, resulted in complete and accurate proteolysis at both termini of HC-Pro. A point mutation affecting an amino acid residue essential for the proteolytic activity of HC-Pro had no effect on N-terminal processing. Expression in transgenic plants of a construct with a large deletion in the 35 kd protein coding region resulted in partial inhibition of HC-Pro N-terminal cleavage, suggesting that the 35 kd protein may affect the proteolytic event but not in a catalytic role. We speculate that this cleavage event is catalyzed by either a cryptic potyviral proteinase that requires a host factor or subcellular environment for activation, or possibly a host proteinase.

Antibodies↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1993

Overexpression of the HIV-1 gag-pol polyprotein results in intracellular activation of HIV-1 protease and inhibition of assembly and budding of virus-like particles.

Some retroviruses, including HIV-1, regulate the relative amounts of gag and pol gene products by a translational frameshift mechanism. The consequences of altering the ratios of the Gag and Pol proteins were tested using vaccinia virus expression vectors, in which the gag and pol genes were fused by placing them in the same open reading frame. Immunoblotting of cell lysates indicated that a protein of approximately 160 kDa, the expected translation product of the fused gag-pol gene, was the dominant species detected with HIV-specific antiserum during the first several hours of infection with this recombinant virus. Subsequently, the full-length polyprotein diminished in amount and a series of Gag-related intermediate size proteins appeared. Later in infection, p24 and myristoylated p17 Gag proteins predominated and larger amounts of intracellularly processed reverse transcriptase, integrase, and protease were detected compared to the amounts formed with the wild-type gag-pol gene. Large numbers of budding, immature, and mature retrovirus-like particles were visualized by electron microscopy when the wild-type gag-pol gene was expressed, whereas no particles were detected in cells that expressed the fused gag-pol gene. The block to virus assembly was partially overcome by (i) inhibition of the HIV-1 protease with a peptidomimetic inhibitor, (ii) mutagenesis of the active site of the protease, or (iii) shortening of the Gag-Pol polyprotein by deletion of most of the reverse transcriptase gene. Nevertheless, budding was inefficient and the structures appeared immature and frequently aberrant. These results indicated that overproduction of the full-length Gag-Pol polyprotein and increased intracellular protease activity were both detrimental to viral assembly. Further experiments indicated that intracellular processing of Gag and Gag-Pol polyproteins occurred in the absence of particle formation when myristoylation was prevented.

Animals↗
PubMed1994

Mutational analysis of the arginine residues in the E2-E1 junction region on the proteolytic processing of the polyprotein precursor of rubella virus.

Endoproteolytic cleavage of precursors is a key step in biosynthesis of functional proteins. The structural proteins of rubella virus are initially translated as a precursor polyprotein in the order NH2-C-E2-E1-COOH and are cleaved by host signal peptidase to yield three structural proteins. Between regions corresponding to E2 and E1 in the precursor is a region of seven amino acid residues (R-R-A-C-R-R-R) that contains a motif for stop-transfer or a possible target for trypsin-like protease cleavage. Using site-directed mutagenesis, these arginine residues, as well as the signal peptide cleavage site at the N-terminus of E1, have been mutated individually or in combination. Results from in vitro transcription/translation analysis indicated that the mutated E2E1 precursor polyproteins were translocated into the microsome and glycosylated. Expression of mutated precursor polyproteins in COS cells revealed that the cleavage of E2E1 polyprotein precursor was impaired when the signal peptide cleavage site alone or both arginine clusters were altered, whereas partial cleavage was observed in the mutants in which either one of the two arginine clusters was modified. Our data suggest that although the arginine clusters do not function as a basic protease cleavage site, they contribute to maintain the proper configuration of that region for access of cellular signal peptidase.

Amino Acid Sequence↗
PubMed1995

Identification and characterization of a 65-kDa protein processed from the gene 1 polyprotein of the murine coronavirus MHV-A59.

A 65-kDa protein has been detected in mouse hepatitis virus A59 (MHV-A59)-infected DBT cells using polyclonal antibodies directed against polypeptides encoded by the 5' 1.8 kb of gene 1. The presence of this 65-kDa protein (p65) was previously predicted from immunoprecipitation studies of gene 1 expression in MHV-A59-infected DBT cells with other antisera (1). p65 was rapidly labeled in virus-infected cells at late times of infection; however, its cleavage from the polyprotein was significantly delayed compared to the amino-terminal gene 1 polyprotein cleavage product, p28. Similar to p28, p65 was cleaved from the growing polyprotein without detectable intermediate precursors. Kinetic analysis of p65 with specific antibodies indicates that p65 is immediately adjacent to p28 in the gene 1 polyprotein. The proteolytic activity responsible for the carboxy-terminal cleavage of p65, as well as the function of the p65 protein, remains to be determined.

Animals↗
PubMed1996

Effects of P2 cleavage site mutations on poliovirus polyprotein processing.

The poliovirus genome comprises a single open reading frame which is translated to give one large polyprotein. The proteolytic cascade involved in the processing of this polyprotein is not yet understood in full detail,particularly concerning the processing of P2-P3, the precursor to the viral nonstructural polypeptides, 2A, 2B, 2C, 3A, 3B, 3C, and 3D. To investigate the possibility that the cleavage events within P2 and at the 2C/3A junction occur in an ordered fashion, we used oligonucleotide-directed mutagenesis of poliovirus cDNA to modify the 3C(prn)-mediated cleavage sites. The Gin residue of the Gin-Gly sequence at the 2A/2B, 2B/2C, and 2C/3A junctions in the poliovirus polyprotein was replaced by Asn, Glu, Asp, or Lys. The effects of each of these substitutions were studied in vivo after transfection onto HeLa cells and in vitro in a cell-free translation assay, using full-length mutated RNA transcripts. Only the mutant with the Glu-Gly sequence at the 2C/3A junction was viable. Analysis of the in vitro processing profiles showed that the efficiency of the 3C protease cleavage at any of the sites in P2 was in the following order: Gin-Gly > Glu-Gly > Asn-Gly. No cleavage could be detected with the Asp-Gly or Lys-Gly sequence at any junction. Lack of 2A/2B or 2B/2C cleavage had no consequences on the cleavage efficiency at other Gin-Gly sites in the polyprotein. Abolition of cleavage at the 2C/3A junction did not prevent the generation of the 2A, 2B, and 3CD polypeptides. Thus, these polypeptides. Thus, these polypeptides could be produced independently of the generation of the P2 and P3 precursors.

Binding Sites↗
PubMed1998

Internal ribosomal entry site scanning of the poliovirus polyprotein: implications for proteolytic processing.

Based on previous studies of dicistronic polioviruses carrying two internal ribosomal entry sites (IRESes), we performed a novel experiment of IRES scanning through a polypeptide by inserting sequentially the IRES of encephalomyocarditis virus into the open reading frame (ORF) of the poliovirus polyprotein at selected 3Cpro-specific Q*G cleavage sites. No cytopathic effects were observed after transfection of HeLa cells with any of the dicistronic constructs, and no virus was recovered. In vitro translation of the dicistronic RNA transcripts in HeLa cell-free extracts revealed that multiple defects in the processing of the P2-P3 domain of the polyprotein is the primary reason for the lethal phenotypes. Surprisingly, the interruption of 3Cpro-catalyzed cleavages downstream of 2C interfered with the 2Apro-catalyzed, primary cleavage between P1 and P2. In contrast, insertion of a foreign coding sequence (V3 loop of human immunodeficiency virus type 1 gp120) into the ORF of the polyprotein at the 2C-3A junction yielded a viable virus that appeared to be genetically stable over several passages. The results of these experiments, which are generally applicable to analyses of viral polyproteins or multidomain polypeptides, suggest that processing of the P2-P3 domain by 3C-3CDpro is rapid and accurate only in the context of the unperturbed P2-P3 precursor; this is consistent with cleavages occurring in cis. Moreover, an intact 2C-3A precursor is not required for viral proliferation.

3C Viral Proteases↗
PubMed1998

Proteolytic processing of the polyprotein encoded by ORF1b of the coronavirus infectious bronchitis virus (IBV).

We present here evidence demonstrating that four previously predicted Q-S(G) cleavage sites, encoded by the IBV sequences from nucleotide 15,129 to 15,134, 16,929 to 16,934, 18,492 to 18,497, and 19,506 to 19,511, respectively, can be recognised and transcleaved by the 3C-like proteinase. Five mature products with sizes of approximately 100 kDa, 65 kDa, 63 kDa, 42 kDa and 35 kDa are released from the ORF1b polyprotein by the 3C-like proteinase-mediated cleavage at these positions. Meanwhile, expression of plasmids containing only the ORF1b region showed no autocleavage of the polyprotein encoded, suggesting that the 3C-like proteinase may be the sole proteinase involved in processing of the 1b polyprotein. These data may therefore represent a complete processing map of the polyprotein encoded by ORF1b of mRNA1.

Antibodies↗
PubMed1997

Infectious bursal disease virus polyprotein processing does not involve cellular proteases.

The larger genome segment, segment A, of infectious bursal disease virus (IBDV) encodes VP2, VP3 and VP4 as a precursor polyprotein. The viral protease, VP4, is responsible for self-processing of the polyprotein, however, there are additional secondary precursor products such as VPX whose further processing has not been defined. Expression of IBDV cDNAs in vitro with rabbit reticulocyte lysates in a coupled transcription-translation system and in the Sindbis virus expression system (with BHK-21 and Vero cell cultures) were used to study processing of the polyprotein. In both expression systems, we identified three main gene products with molecular masses of 48, 34, and 30.5 kDa corresponding to VPX, VP3, and VP4, respectively, as found in IBDV-infected Vero cell cultures, although the amount of each product was variable. A translational time course of the polyprotein gene and analyses of products specified by various sub-clones of the full-length cDNA were used to distinguish primary processing products of translation from secondary products generated by proteolytic processing during in vitro coupled transcription-translation expression. The VPX, VP3 and VP4, which are the primary processing products, first appeared after 20 min of incubation and their production was maximum by 75 min of the coupled transcription-translation reaction. Cycloheximide chases demonstrated that there is no secondary processing of VPX (or VP3 and VP4). Thus VP2, the major capsid protein in virions, was not detected either in translation products of rabbit reticulocyte lysates or in lysates of Sindbis virus recombinant-infected cell cultures indicating the absence of secondary processing of VPX to VP2 during foreign expression of the segment A cDNA. We conclude that VPX maturation to VP2 does not involve cellular proteases.

Animals↗
PubMed1985

The nonstructural proteins of Sindbis virus as studied with an antibody specific for the C terminus of the nonstructural readthrough polyprotein.

A dodecapeptide containing the sequence of the C terminus of the nonstructural polyprotein of Sindbis virus has been synthesized and used to immunize rabbits. The antisera obtained precipitated polypeptides from cells infected with the HR strains of Sindbis or with temperature-sensitive mutants ts11 or ts18. Four different polypeptides, having apparent molecular weights of approximately 250,000, 220,000, 155,000, and 72,000, were immunoprecipitated by the antipeptide antiserum. The largest of these polypeptides is sufficiently large to represent a polyprotein translated from the entire nonstructural region of the genome. These data suggest that nsP4 of molecular weight 72,000 is produced by translation of the entire nonstructural region of the genome, which requires readthrough of an opal termination codon immediately upstream of nsP4, followed by post-translational cleavage of this polyprotein. The amounts of nsP4 and its precursors found in infected cells are small relative to the amounts of other nonstructural proteins present, as would be expected if readthrough of a termination codon is required. In addition, the relative amounts of nsP4 and of its precursors differ in HR-infected or ts mutant-infected cells and differ with temperature of infection, suggesting that temperature of infection or ts lesions affect translation and processing of the precursor polyprotein.

Animals↗
PubMed1990

Synthesis and processing of the nonstructural polyproteins of several temperature-sensitive mutants of Sindbis virus.

We have examined the synthesis and processing of nonstructural polyproteins by several temperature-sensitive mutants of Sindbis virus, representing the four known RNA-minus complementation groups. Four mutants that possess mutations in the C-terminal domain of nonstructural protein nsP2 all demonstrated aberrant processing patterns when cells infected with these mutants were shifted from a permissive (30 degrees) to a nonpermissive (40 degrees) temperature. Mutants ts17, ts18, and ts24 showed severe defects in processing of nonstructural polyproteins at 40 degrees, whereas ts7 showed only a minor defect. In each case, cleavage of the bond between nsP2 and nsP3 was greatly reduced whereas cleavage between nsP1 and nsP2 occurred almost normally, giving rise to a set of polyprotein precursors not seen in wild-type-infected cells at this stage of infection. The nsP1 produced by these mutants was unstable and only small amounts could be detected in infected cells at the nonpermissive temperature. Submolar quantities of nsP2 were also present. We suggest that nsP1 and nsP2 may function as a complex and that free nsP1, and possibly nsP2, is degraded. Cleavage between nsP3 and nsP4 appeared to be normal in the mutants except in the case of ts17, where upon shift to 40 degrees P34 was unstable and nsP4 accumulated. We propose that the change in the P34/nsP4 ratio upon shift is responsible for the previously observed temperature sensitivity of subgenomic 26 S RNA synthesis in ts17 and for the failure of the mutant to regulate minus strand synthesis at 40 degrees. Other mutations tested, including ts21, which is found in the N-terminal half of nsP2, ts11, which has a mutation in nsP1, and ts6, which has a mutation in nsP4, all demonstrated nonstructural polyprotein processing indistinguishable from that in wild-type-infected cells. These results support our conclusion, based upon deletion mapping studies, that the C-terminal domain of nsP2 contains the nonstructural proteinase activity.

Animals↗
PubMed1991

The 35-kDa protein from the N-terminus of the potyviral polyprotein functions as a third virus-encoded proteinase.

The polyprotein encoded by plant potyviruses is proteolytically processed to at least eight mature products by viral-encoded proteinases. While the proteinases that catalyze processing at most of the cleavage sites have been identified, the enzyme responsible for cleavage between the 35-kDa protein and helper component-proteinase (HC-Pro), near the N-terminus of the viral polyprotein, has not been mapped or characterized previously. Polyproteins containing the 35-kDa protein and HC-Pro were synthesized in the wheat germ system using defined RNA transcripts and were demonstrated to undergo proteolysis to generate products that resemble fully processed proteins. The C-terminal half of the 35-kDa protein was found to be required for proteolysis, whereas most of the HC-Pro sequence was dispensable. Amino acid substitutions affecting three positions, each of which are conserved in the 35-kDa protein encoded by five potyviruses, were shown to inhibit protein processing. These data suggest that the 35-kDa protein functions as a proteinase to cleave at its C-terminus. A model that accounts for all proteolytic processing events in the potyviral polyprotein is presented.

Amino Acid Sequence↗
PubMed1992

The complete nucleotide sequence of pepper mottle virus genomic RNA: comparison of the encoded polyprotein with those of other sequenced potyviruses.

The complete nucleotide sequence of a pepper mottle virus isolate from California (PepMoV C) has been determined from cloned viral cDNAs. The PepMoV C genomic RNA is 9640 nucleotides excluding the poly(A) tail and contains a long open reading frame starting at nucleotide 168 and potentially encoding a polyprotein of 3068 amino acids. Comparison of the PepMoV C presumptive polyprotein with those of other sequenced members of the potyvirus group, including tobacco etch virus (TEV), tobacco vein mottling virus (TVMV), plum pox virus (PPV), and potato virus Y (PVY), allowed localization of putative protein cleavage sites. A similar analysis was used to determine the position of conserved viral protein-coding regions along the viral genomic RNA. These analyses confirm previous work indicating that genome organization is conserved among members of the genus Potyvirus. The localization of one PepMoV C gene product, the nuclear inclusion body protein a (NIa protein), was analyzed by expressing PepMoV cDNA deletion clones in bacteria and assaying for appearance of mature-sized coat protein, a cleavage product of the NIa protease. Comparative sequence analyses of the putative PepMoV polyprotein with those of TEV, TVMV, PPV, and PVY served to identify regions of the potyviral polyproteins which have diverged within the genus, as well as highly conserved protein features which may play an important functional role in the potyviral life cycle.

Amino Acid Sequence↗
PubMed1996

Carboxy-terminal sequence divergence and processing of the polyprotein antigen from Dirofilaria immitis.

A polyprotein composed of multiple units arranged in direct tandem arrays has been identified in parasitic and free living nematodes. Analysis of previously cloned units from the Dirofilaria immitis polyprotein antigen (DiPA) indicated the units were nearly identical but here we demonstrate that they segregate into two related families. The consensus repeats, DiPA-CR1 and CR2, derived for each family are 80% identical. However, the repeats at the C-terminus of the polyprotein have diverged from DiPA-CR1 and CR2. This was shown by DNA sequence and Southern blot analysis of a 1.9 kb cDNA clone that encodes 4.4 C-terminal repeats (DiPA-TR1 through TR5). DiPA-TR3 through TR5 show 27-52% amino acid identity with the consensus repeats and 31-35% amino acid identity with one another. Metabolic labeling studies have shown that cleavage of DiPA generates a protein "ladder' from 14 to > 200 kDa. RRKR, a cleavage motif of subtilisin-like proprotein convertases, was identified as the natural cleavage site. In vitro digestion experiments with proteinase K suggest a structural model for DiPA consisting of protease resistant cores joined by protease sensitive linkers containing the RRKR site. This motif is absent between DiPA-TR3 and TR4 and has been altered to KR between DiPA-TR4 and TR5. An immunoblot of D. immitis extract probed with anti-DiPA-TR4/5 serum demonstrates the absence of cleavage at these sites. These divergent repeats provide an opportunity to investigate processing of the D. immitis polyprotein in vivo.

Amino Acid Sequence↗
PubMed2005

Competitive inhibition of the dengue virus NS3 serine protease by synthetic peptides representing polyprotein cleavage sites.

The NS3 serine protease of dengue virus is required for the maturation of the viral polyprotein and consequently represents a promising target for the development of antiviral inhibitors. However, the substrate specificity of this enzyme has been characterized only to a limited extent. In this study, we have investigated product inhibition of the NS3 protease by synthetic peptides derived from the P6-P1 and the P1'-P5' regions of the natural polyprotein substrate. N-terminal cleavage site peptides corresponding to the P6-P1 region of the polyprotein were found to act as competitive inhibitors of the enzyme with K(i) values ranging from 67 to 12 microM. The lowest K(i) value was found for the peptide representing the NS2A/NS2B cleavage site, RTSKKR. Inhibition by this cleavage site sequence was analyzed by using shorter peptides, SKKR, KKR, KR, AGRR, and GKR. With the exception of the peptide AGRR which did not inhibit the protease at a concentration of 1mM, all other peptides displayed K(i) values in the range from 188 to 22 microM. Peptides corresponding to the P1'-P5' region of the polyprotein cleavage sites had no effect on enzymatic activity at a concentration of 1mM. Molecular docking data of peptide inhibitors to a homology-based model of the dengue virus type 2 NS2B(H)-NS3p co-complex indicate that binding of the non-prime site product inhibitors is similar to ground-state binding of the corresponding substrates.

Binding Sites↗