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At least 595 records · Page 33Linked to original sources
PubMed1999

Activity of a purified His-tagged 3C-like proteinase from the coronavirus infectious bronchitis virus.

Previous studies in vitro of the processing of cloned polyprotein fragments from the coronavirus infectious bronchitis virus (IBV) large open reading frame (ORF1), confirmed the activity of a predicted 3C-like proteinase (3CLP) domain and suggested that the proteinase is released autocatalytically from the polyprotein in the form of a 35 kDa protein, 3CLpro, capable of further cleavages in trans. In order to identify such cleavages within the ORF1 polyprotein mediated by 3CLpro, the proteinase was expressed in bacteria, purified and used in trans cleavage assays with polyprotein fragments lacking the 3CLP domain as targets. The proteinase was expressed as a polyprotein fragment which was able to process during expression in bacterial cells, releasing mature 3CLpro. A histidine (His6) tag was introduced close to the C-terminus of the proteinase to aid purification. Processing demonstrated by the tagged proteinase was indistinguishable from that of the wild-type enzyme indicating that the site chosen for the tag was permissive. From these studies we were able to demonstrate trans cleavages consistent with the use of most of the previously predicted or identified sites within the open reading frame of gene 1. This tentatively completes the processing map for the ORF1 region with respect to 3CLpro.

Animals↗
PubMed1999

Identification and characterization of a histone binding site of the non-structural protein 3 of hepatitis C virus.

BACKGROUND: Chronic hepatitis resulting from the hepatitis C virus (HCV) infection leads to cirrhosis in at least half the infected patients and increases the risk of hepatocellular carcinoma. There are indications that this pathogenic effect may result from the disturbance of intracellular signal cascades caused by the interaction with viral antigens. Although a great amount of data has been accumulated about functional regions in HCV proteins, relatively little is known about their intracellular targets. Previously, we have demonstrated that the full-length non-structural protein 3 of HCV (NS3) (Borowski P, Heiland M, Feucht H, Laufs R. Characterisation of non-structural protein 3 of hepatitis C virus as modulator of protein phosphorylation mediated by PKA and PKC. Evidences for action on the level of substrate and enzyme. Arch Virol 1999a; 144) and its NH2- and COOH-terminal truncated form (Borowski P, Heiland M, Oehlmann K, Becker B, Kornetzky L, Feucht HH, Laufs R. Non-structural protein 3 of hepatitis C virus inhibits phosphorylation mediated by cAMP-dependent protein kinase. Eur J Biochem 1996;237:611-618) associate to stable complexes with core histones H2B and H4. The changes of the properties of histones as substrate for cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) were found as a direct consequence of the interaction. OBJECTIVE: In the present study we further these observations, localize the histone binding domain of NS3 and investigate the mechanisms by which NS3 affects the functions of the histones in vitro. STUDY DESIGN: HCV protein exhibiting the mentioned histone binding activity was produced in a bacterial expression system, purified and binding to histones was biochemically characterized. The region of NS3 involved in the interaction with histones was defined by proteolytic fragmentation, microsequencing and a specific histone binding assay. Furthermore, a functional test to quantify the interaction of histones with DNA was established and the binding of DNA to histone as a function of NS3 concentration was analysed by means of graphical methods. RESULTS: The investigated fragment of HCV polyprotein consisting of amino acid residues 1189-1525 (HCV-polyprotein-(1189-1525)) displayed significant histone binding activity. The binding occurred at a molar ratio 1:1 of histone to HCV-polyprotein-(1189-1525) and was mediated by a linear stretch of amino acids located between the residues 1343 and 1379 of the HCV polyprotein. To demonstrate that HCV-polyprotein-(1189-1525) affects the binding of DNA to histones we used two independent methods: overlay assay and binding assay on Sepharose beads. Graphic analysis of the binding kinetics revealed an uncompetitive type of inhibition. CONCLUSIONS: Our results provide the first evidence that NS3 binds and affects the functions of core histones. The mechanism by which the NS3 interferes with the histone functions involves conformational changes of histone molecule.

Binding Sites↗
PubMed1999

Complete nucleotide sequence of bean pod mottle virus RNA1: sequence comparisons and evolutionary relationships to other comoviruses.

The complete nucleotide sequence of bean pod mottle comovirus (BPMV) RNA 1 was determined. It is 5983 nucleotides long, excluding the poly(A) tail, and encodes a polyprotein of 1850 amino acid (aa) residues. Multiple alignments of the deduced aa sequence of BPMV polyprotein with those of cowpea mosaic virus (CPMV), red clover mottle virus (RCMV) and cowpea severe mosaic virus (CPSMV) indicated that BPMV RNA1 encodes the predicted set of five mature proteins: the equivalent of CPMV 32K protease cofactor, 58K putative helicase, VPg, 24K protease and 87K putative RNA-dependent RNA polymerase. Of the four proposed cleavage sites in BPMV RNA1 polyprotein, the one at the 32K/58K site (Q/A) is distinct for BPMV polyprotein and those at the 58K/VPg and VPg/24K junctions (Q/S and Q/M, respectively) are identical in all four comovirus polyproteins. Sequence comparison and phylogenetic analysis revealed that BPMV RNA1 is more closely related to CPSMV than to CPMV or to RCMV.

Amino Acid Sequence↗
PubMed1999

Modulation of the RNA binding and protein processing activities of poliovirus polypeptide 3CD by the viral RNA polymerase domain.

To study the role of the RNA polymerase domain (3D) in the proteinase substrate recognition and RNA binding properties of poliovirus polypeptide 3CD, we generated recombinant 3C and 3CD polypeptides and purified them to near homogeneity. By using these purified proteins in in vitro cleavage assays with structural and non-structural viral polyprotein substrates, we found that 3CD processes the poliovirus structural polyprotein precursor (P1) 100 to 1000 times more efficiently than 3C processes P1. We also found that trans-cleavage of other 3CD molecules and sites within the non-structural P3 precursor is more efficiently mediated by 3CD than 3C. However, 3C and 3CD appear to be equally efficient in the processing of a non-structural polyprotein precursor, 2C3AB. Four mutated 3CD polyproteins with site-directed lesions in the 3D domain of the proteinase were analyzed for their ability to process viral polyprotein precursors and to form a ternary complex with RNA sequences encoded in the 5' terminus of the viral genome. Analysis of mutated 3CD polypeptides revealed that specific mutations within the 3D amino acid sequences of 3CD confer differential effects on 3CD activity. All four mutated 3CD proteins tested were able to process the P1 structural precursor with wild type or near wild type efficiency. However, three of the mutated enzymes demonstrated an impaired ability to process some sites within the P3 non-structural precursor, relative to wild type 3CD. One of the mutant 3CD polypeptides, 3CD-3DK127A, also displayed a defect in its ability to form a ternary ribonucleoprotein complex with poliovirus 5' RNA sequences.

3C Viral Proteases↗
PubMed2005

Comparison of antibody- and cell-mediated immune responses after intramuscular hepatitis C immunizations of BALB/c mice.

Current treatments for hepatitis C infection have limited efficacy, and there is no vaccine available. The goal of this study was to compare the immune response to several immunization combinations against hepatitis C virus (HCV). Six groups of mice were immunized at weeks 0, 4, and 8 with different combinations of a candidate HCV vaccine consisting of 100 microg recombinant HCV core/E1/E2 (rHCV) DNA plasmid and/or 25 microg rHCV polyprotein and 50 microL Montanide ISA- 51. Four weeks after the last injection, all groups of mice were sacrificed and blood samples and spleens were collected for measuring the levels of specific HCV antibodies (total IgG, IgG1, and IgG2a). Cell proliferation and intracellular interferon-gamma were also measured. Among the groups of immunized mice, only the mice immunized with rHCV DNA plasmid, rHCV polyprotein, and montanide (group D) and mice immunized with rHCV polyprotein and montanide (group F) demonstrated a significant increase in the total IgG titer after immunization. IgG1 was the predominant antibody detected in both groups D and F. No IgG2a was detected in any of the groups. Proliferation assays demonstrated that splenocytes from group D and group C (rHCV DNA primed/rHCV polyprotein boost) developed significant anti-HCV proliferative responses. The combination of an rHCV DNA plasmid, rHCV polyprotein, and montanide induced a high antibody titer with a predominance of IgG1 antibodies and recognized the major neutralization epitopes in HVR1. In contrast, group C did not show an increase in anti-HCV antibodies, but did show a proliferative response.

Adjuvants, Immunologic↗
PubMed1989

Nucleotide sequence of potato virus Y (N Strain) genomic RNA.

The complete nucleotide sequence of the genomic RNA of the potyvirus potato virus Y strain N (PVYn) was obtained from cloned cDNAs. This sequence is 9704 nucleotides long and can encode a polyprotein of 3063 amino acids. The positions of the cleavage sites at the N terminus of the capsid and cytoplasmic inclusion proteins have been determined. Other putative protein cleavage sites have been deduced by searching for consensus sequences and by analogy with the polyprotein of the tobacco vein mottling virus and of the tobacco etch virus. Comparison of the PVY polyprotein sequence with that of other potyvirus polyproteins shows similarities in genome organization and a high level of identity along most of the polyprotein, except for the putative proteins flanking the helper component. A search for specific protein motifs has revealed the existence of a potential metal-binding site at the putative N terminus of the helper component in potyviruses. The possible functions of this structure are discussed.

Amino Acid Sequence↗
PubMed1991

The complete nucleotide sequence of pea seed-borne mosaic virus RNA.

The complete nucleotide sequence of the RNA genome of pea seed-borne mosaic virus (PSbMV) was determined from cloned cDNA and by direct sequencing of viral RNA. The PSbMV genomic sequence was determined to be 9924 nucleotides in length excluding the poly(A) tract. The RNA contained an open reading frame (ORF) of 9618 nucleotides with the potential to encode a polyprotein with a calculated Mr of 364000 (364K). The ORF was flanked by a 5' untranslated leader sequence of 143 nucleotides and a 3' untranslated region of 163 nucleotides. A comparison of the PSbMV polyprotein with the polyproteins of the potyviruses tobacco etch virus (TEV), tobacco vein mottling virus (TVMV), plum pox virus (PPV) and potato virus Y (PVY) showed that PSbMV had a similar genome organization. The polyproteins had a high level of amino acid identity except in the N-terminal region, which varied in both sequence and length. Putative proteolytic cleavage sites were identified in the polyprotein of PSbMV by comparison with those identified for other potyviruses. The cleavage site between the 6K protein and the 49K proteinase is proposed to occur at the C-terminal side of glutamic acid and not at the C-terminal side of glutamine as in other potyviruses. In addition to the five proteolytic cleavage sites for the 49K proteinase identified previously, a sixth putative cleavage site was identified internally in the 49K proteinase of PSbMV, as well as in the 49K proteinases of TEV, TVMV, PPV, PVY and soybean mosaic virus. Cleavage at this site in the 49K proteinases of TEV, TVMV and PPV would result in an N-terminal protein of 22K to 24K, which is similar in size to the size determined for their VPgs.

Amino Acid Sequence↗
PubMed1997

The sequence and genomic organization of a GB virus A variant isolated from captive tamarins.

Recently, gene fragments of several novel variants of GB virus A were isolated from the serum of distinct monkey species that had not been experimentally inoculated with an infectious agent. These variants appeared to be species-specific in that sequences isolated within a species were virtually identical, though sequences were strikingly different when compared between each species. In the present study, the nucleotide sequence of one of these variants, GBV-Alab, was extended to near-genome length. Similar to the other GB viruses, GBV-Alab appears to encode a single large polyprotein of 2967 amino acids that is post-translationally cleaved by cellular and viral proteases into the individual viral proteins. The structural proteins are found at the N-terminal end of the polyprotein, while the nonstructural proteins are found at the C teminus. Amino acid sequence comparisons of the large polyprotein demonstrate that GBV-Alab is 74% identical to GBV-A and 48% identical to GBV-C, sharing only marginal identity with GBV-B and HCV-1 at 27%. Examination of the GBV-Alab polyprotein reveals that structural motifs are conserved for a protease, a helicase and a replicase. Phylogenetic analysis of the polyprotein confirms previous results that GBV-Alab is a member of the Flaviviridae, distinct from GBV-B and HCV, though more closely related to GBV-A and GBV-C.

Amino Acid Sequence↗
PubMed2004

Coordinate expression and independent subcellular targeting of multiple proteins from a single transgene.

A variety of conventional methods allow the expression of multiple foreign proteins in plants by transgene stacking or pyramiding. However, most of these approaches have significant drawbacks. We describe a novel alternative, using a single transgene to coordinate expression of multiple proteins that are encoded as a polyprotein capable of dissociating into component proteins on translation. We demonstrate that this polyprotein system is compatible with the need to target proteins to a variety of subcellular locations, either cotranslationally or posttranslationally. It can also be used to coordinate the expression of selectable marker genes and effect genes or to link genes that are difficult to assay to reporter genes that are easily monitored. The unique features of this polyprotein system are based on the novel activity of the 2A peptide of Foot-and-mouth disease virus (FMDV) that acts cotranslationally to effect a dissociation of the polyprotein while allowing translation to continue. This polyprotein system has many applications both as a research tool and for metabolic engineering and protein factory applications of plant biotechnology.

Chloroplasts↗
PubMed1993

Assembly of functional Sindbis virus RNA replication complexes: requirement for coexpression of P123 and P34.

A vaccinia virus transient expression system was used to determine which of the Sindbis virus (SIN) proteins and/or polyproteins are necessary for the formation of active replication complexes and, in particular, to analyze the role of nsP4, the putative polymerase, versus P34 in RNA replication. We generated vaccinia virus recombinants in which the cDNA for the entire SIN nonstructural coding region as well as cDNA copies of the individual nonstructural proteins (nsPs) and several intermediate polyproteins were placed downstream of the promoter for T7 RNA polymerase and the encephalomyocarditis virus 5' untranslated region. The proteins expressed by the vaccinia virus recombinants comigrate with authentic proteins synthesized in SIN-infected cells, and the polyproteins appear to be processed to the individual proteins of the correct size. To examine the replication efficiencies of different protein combinations, a vaccinia virus recombinant was designed to express an engineered substrate RNA which could serve as a template for replication and subgenomic mRNA transcription by the SIN nsPs. Expression of the entire SIN nonstructural coding region resulted in the synthesis of high levels of both genomic and subgenomic RNAs derived from the engineered template. No RNA replication could be detected during coexpression of the four individual nsPs, although the proteins were indistinguishable, in terms of electrophoretic mobility, from those synthesized in SIN-infected cells. Coexpression of polyproteins P12, P23, and/or P34 with the individual nsPs also did not result in detectable levels of RNA replication. However, when P123 and P34 were coexpressed, efficient RNA replication and subgenomic mRNA transcription of the substrate RNA was observed. Coexpression of nsP4 with P123 resulted in the synthesis of only minus-strand RNAs. These studies show that expression of both P123 and P34 is necessary for establishment of functional RNA replication and transcription complexes and raise the possibility that the polyproteins themselves may be functional components of these complexes. In addition, these data indicate that an nsP4 moiety expressed independently with an additional N-terminal methionine is capable of functioning in minus-strand but not plus-strand RNA synthesis.

Animals↗
PubMed1994

The tobacco etch potyvirus 6-kilodalton protein is membrane associated and involved in viral replication.

The tobacco etch potyvirus (TEV) genome encodes a polyprotein that is processed by three virus-encoded proteinases. Although replication of TEV likely occurs in the cytoplasm, two replication-associated proteins, VPg-proteinase (nuclear inclusion protein a) (NIa) and RNA-dependent RNA polymerase (nuclear inclusion protein b) (NIb), accumulate in the nucleus of infected cells. The 6-kDa protein is located adjacent to the N terminus of NIa in the TEV polyprotein, and, in the context of a 6-kDa protein/NIa (6/NIa) polyprotein, impedes nuclear translocation of NIa (M. A. Restrepo-Hartwig and J. C. Carrington, J. Virol. 66:5662-5666, 1992). The 6-kDa protein and three polyproteins containing the 6-kDa protein were identified by affinity chromatography of extracts from infected plants. Two of the polyproteins contained NIa or the N-terminal VPg domain of NIa linked to the 6-kDa protein. To investigate the role of the 6-kDa protein in vivo, insertion and substitution mutagenesis was targeted to sequences coding for the 6-kDa protein and its N- and C-terminal cleavage sites. These mutations were introduced into a TEV genome engineered to express the reporter protein beta-glucuronidase (GUS), allowing quantitation of virus amplification by a fluorometric assay. Three-amino-acid insertions at each of three positions in the 6-kDa protein resulted in viruses that were nonviable in tobacco protoplasts. Disruption of the N-terminal cleavage site resulted in a virus that was approximately 10% as active as the parent, while disruption of the C-terminal processing site eliminated virus viability. The subcellular localization properties of the 6-kDa protein were investigated by fractionation and immunolocalization of 6-kDa protein/GUS (6/GUS) fusion proteins in transgenic plants. Nonfused GUS was associated with the cytosolic fraction (30,000 x g centrifugation supernatant), while 6/GUS and GUS/6 fusion proteins sedimented with the crude membrane fraction (30,000 x g centrifugation pellet). The GUS/6 fusion protein was localized to apparent membranous proliferations associated with the periphery of the nucleus. These data suggest that the 6-kDa protein is membrane associated and is necessary for virus replication.

Amino Acid Sequence↗
PubMed1995

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

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

Amino Acid Sequence↗
PubMed1995

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.

3C Viral Proteases↗
PubMed1996

Processing of the equine arteritis virus replicase ORF1b protein: identification of cleavage products containing the putative viral polymerase and helicase domains.

The replicase open reading frame lb (ORF1b) protein of equine arteritis virus (EAV) is expressed from the viral genome as an ORF1ab fusion protein (345 kDa) by ribosomal frameshifting. Processing of the ORF1b polyprotein was predicted to be mediated by the nsp4 serine protease, the main EAV protease. Several putative cleavage sites for this protease were detected in the ORF1b polyprotein. On the basis of this tentative processing scheme, peptides were selected to raise rabbit antisera that were used to study the processing of the EAV replicase ORF1b polyprotein (158 kDa). In immunoprecipitation and immunoblotting experiments, processing products of 80, 50, 26, and 12 kDa were detected. Of these, the 80-kDa and the 50-kDa proteins contain the putative viral polymerase and helicase domains, respectively. Together, the four cleavage products probably cover the entire ORF1b-encoded region of the EAV replicase, thereby representing the first complete processing scheme of a coronaviruslike ORF1b polyprotein. Pulse-chase analysis revealed that processing of the ORF1b polyprotein is slow and that several large precursor proteins containing both ORF1a- and ORF1b-encoded regions are generated. The localization of ORF1b-specific proteins in the infected cell was studied by immunofluorescence. A perinuclear staining was observed, which suggests association with a membranous compartment.

Amino Acid Sequence↗
PubMed1998

Template-dependent initiation of Sindbis virus RNA replication in vitro.

Recent insights into the early events in Sindbis virus RNA replication suggest a requirement for either the P123 or P23 polyprotein, as well as mature nsP4, the RNA-dependent RNA polymerase, for initiation of minus-strand RNA synthesis. Based on this observation, we have succeeded in reconstituting an in vitro system for template-dependent initiation of SIN RNA replication. Extracts were isolated from cells infected with vaccinia virus recombinants expressing various SIN proteins and assayed by the addition of exogenous template RNAs. Extracts from cells expressing P123C>S, a protease-defective P123 polyprotein, and nsP4 synthesized a genome-length minus-sense RNA product. Replicase activity was dependent upon addition of exogenous RNA and was specific for alphavirus plus-strand RNA templates. RNA synthesis was also obtained by coexpression of nsP1, P23C>S, and nsP4. However, extracts from cells expressing nsP4 and P123, a cleavage-competent P123 polyprotein, had much less replicase activity. In addition, a P123 polyprotein containing a mutation in the nsP2 protease which increased the efficiency of processing exhibited very little, if any, replicase activity. These results provide further evidence that processing of the polyprotein inactivates the minus-strand initiation complex. Finally, RNA synthesis was detected when soluble nsP4 was added to a membrane fraction containing P123C>S, thus providing a functional assay for purification of the nsP4 RNA polymerase.

Animals↗
PubMed2000

Identification of mouse hepatitis virus papain-like proteinase 2 activity.

Mouse hepatitis virus (MHV) is a 31-kb positive-strand RNA virus that is replicated in the cytoplasm of infected cells by a viral RNA-dependent RNA polymerase, termed the replicase. The replicase is encoded in the 5'-most 22 kb of the genomic RNA, which is translated to produce a polyprotein of >800 kDa. The replicase polyprotein is extensively processed by viral and perhaps cellular proteinases to give rise to a functional replicase complex. To date, two of the MHV replicase-encoded proteinases, papain-like proteinase 1 (PLP1) and the poliovirus 3C-like proteinase (3CLpro), have been shown to process the replicase polyprotein. In this report, we describe the cloning, expression, and activity of the third MHV proteinase domain, PLP2. We show that PLP2 cleaves a substrate encoding the first predicted membrane-spanning domain (MP1) of the replicase polyprotein. Cleavage of MP1 and release of a 150-kDa intermediate, p150, are likely to be important for embedding the replicase complex in cellular membranes. Using an antiserum (anti-D11) directed against the C terminus of the MP1 domain, we verified that p150 encompasses the MP1 domain and identified a 44-kDa protein (p44) as a processed product of p150. Pulse-chase experiments showed that p150 is rapidly generated in MHV-infected cells and that p44 is processed from the p150 precursor. Protease inhibitor studies revealed that unlike 3CLpro activity, PLP2 activity is not sensitive to cysteine protease inhibitor E64d. Furthermore, coexpression studies using the PLP2 domain and a substrate encoding the MP1 cleavage site showed that PLP2 acts efficiently in trans. Site-directed mutagenesis studies confirmed the identification of cysteine 1715 as a catalytic residue of PLP2. This study is the first to report enzymatic activity of the PLP2 domain and to demonstrate that three distinct viral proteinase activities process the MHV replicase polyprotein.

Animals↗
PubMed1983

Association of the transforming proteins of the ST and GA strains of feline sarcoma virus and their in vitro associated protein kinase activities with cellular membranes.

The translation products of the Snyder-Theilen (ST) and Gardner-Arnstein (GA) strains of feline sarcoma virus (FeSV), termed gag-fes proteins, are high molecular weight polyproteins containing different amounts of the amino terminus of the feline leukemia virus (FeLV) gag gene-coded precursor protein linked to a similar sarcoma virus-specific polypeptide. Both polyproteins are phosphoproteins with indistinguishable in vitro associated tyrosine-specific protein kinase activities. The polyproteins are extremely hydrophobic proteins which are intimately associated with the plasma membrane fraction of transformed cells. Approximately 10% of the proteins are modified by glycosylation and expressed on the cell surface where they are accessible to lactoperoxidase-mediated radio-iodination and trypsinization. Cell surface localization of the polyproteins does not appear to be necessary for transformation. However, preliminary evidence suggests that the amount of FeLV p30 sequences at the amino end of the proteins may have some effect on the intracellular distribution of the gag-fes polyproteins and on the phenotype of the transformed cell.

Animals↗
PubMed2000

Cloning, sequencing, and expression of the hepatitis E virus (HEV) nonstructural open reading frame 1 (ORF1).

Hepatitis E virus (HEV) causes enterically transmitted epidemic and sporadic viral hepatitis affecting millions of people in the developing world. Different geographical isolates of HEV show a high degree of homology at the nucleotide and amino acid levels. The approximately 7.2 kb RNA genome has three open reading frames of which ORF1 is predicted to code for the viral nonstructural polyprotein. The expression, processing and properties of the nonstructural ORF1 polyprotein have not been reported so far. In this study, the complete HEV ORF1 was reconstructed from overlapping fragments amplified by polymerase chain reaction (PCR) of total RNA isolated from the bile fluid of a rhesus monkey experimentally infected with HEV isolate from an epidemic. The complete assembled ORF1 was sequenced using HEV specific primers. The ORF1 polyprotein was expressed in E. coli, in a cell free translation system and in HepG2 cells, and was characterized by western blotting and immunoprecipitation using acute phase patient serum as well as polyclonal antibodies raised against defined parts of the ORF1 polyprotein. The nonstructural polyprotein of HEV was expressed as a 186 kDa protein. No processing was observed into discrete units, either in-vitro based on a kinetic analysis, or in HepG2 cells based on immunoprecipitation.

Animals↗