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 721 records · Page 40Linked to original sources
PubMed2005

Infectious bursal disease virus recovery from Vero cells transfected with RNA transcripts is enhanced by expression of the structural proteins in trans.

Positive sense RNA transcripts of infectious bursal disease (IBD) virus genome segments A and B have previously been shown to be infectious. In this study we demonstrate that recovery of IBD virus from the transfection of Vero cells with positive sense RNA transcripts of genome segments A and B was enhanced by expression of the viral structural proteins VP2 with VP3 or by expression of viral polyprotein VP243 from DNA plasmids in trans. Expression of individual viral proteins VP2, VP3, or VP4 alone from DNA plasmids did not enhance IBD virus recovery. Earliest virus recovery from transfection of positive sense RNA transcripts of genomic segments A and B was at 36 h and mean titers were 10(1.8) pfu/ml. IBD virus was recovered 6 hours after transfection in cells concurrently expressing either VP2 with VP3 or VP243 and mean titers were 10(8.5) pfu/ml or 10(9.2) pfu/ml, respectively. Likewise, expression of the viral polyprotein from DNA plasmid increased the permissiveness of Vero cells for infection with non-culture adapted IBD virus. The titer of recovered non-culture adapted virus from 10(3.3) pfu/ml to 10(10.3) pfu/ml with expression of the viral polyprotein. This report is the first to describe a reverse genetics model for IBD virus with high efficiency of virus recovery for non-culture adapted strains.

Animals↗
PubMed2005

Detection and characterisation of a second potyvirus from Thunberg fritillary in China.

Plants of Thunberg fritillary (Fritillaria thunbergii Miq.) from Zhejiang Province, were found to be co-infected with two distinct potyviruses. One was an isolate of the recently reported Thunberg fritillary mosaic virus (TFMV; Wei et al., (2005) Arch Virol 150: 1271-1280), while the other was a distinct virus that did not react with TFMV antiserum nor with antisera to 17 other potyviruses, except for a weak reaction with antibodies produced to soybean mosaic virus (SMV) Pinellia strain. Both viruses could be transmitted mechanically to their original host but not to any of a range of commonly used indicator plants. No local lesion host was identified that would enable the viruses to be propagated independently. The complete sequences of both viruses were determined; that of the new virus (9656 nt) had the typical genome organisation and recognised sequence motifs of a potyvirus, encoding a putative polyprotein of 351 kDa. Phylogenetic analysis, sequence comparisons, and the pattern of polyprotein cleavage sites all indicated that it was a member of the Bean common mosaic virus subgroup. The most closely related species are Soybean mosaic virus and Wisteria vein mosaic virus, with 68-69% amino acid identity between their polyproteins. This is sufficiently different for the new virus to be regarded as a distinct species, which we have tentatively named Fritillary virus Y.

Base Sequence↗
PubMed1998

Moloney murine leukemia virus protease expressed in bacteria is enzymatically active.

Replication of Moloney murine leukemia virus requires a readthrough translation mechanism to generate the Gag-Pol polyprotein. One of the final products of this polyprotein is the protease (PR), which is required to generate the mature virion proteins. The assembly of Gag and Gag-Pol polyprotein into a virion followed by activation of the viral protease is necessary to produce a mature, infectious particle. These events are believed to occur near the cell membrane just prior to the budding of the virion. We report here the autoproteolytic activity of the viral PR when a Gag-PR fusion protein is expressed in E. coli. Efficient cleavage at the p12/CA, CA/NC and NC/PR junctions was observed. Thus the Moloney murine leukemia virus PR is capable of cleaving its substrates in the absence of specific host factors.

Endopeptidases↗
PubMed1999

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.

Generally, the maximum activities of the protein kinases A (PKA) and C (PKC) show an optimum value for their substrate concentrations rather than a saturation curve; at high substrate concentrations, the kinase activity is completely abolished. The C- and N-truncated form of the non-structural protein 3 (NS3) of hepatitis C virus (HCV) (HCV-polyprotein-(1,189-1,525)) abolishes the inhibiting effect of the substrate, yielding saturable Michaelis-Menten kinetics of PKA and its catalytical domain (C subunit). In contrast, HCV-polyprotein-(1,189-1,525) activates PKC with increasing Vmax, while it abolishes the substrate inhibition of its catalytical domain (M-kinase) through a mechanism analogous to that of PKA and C subunit. PKC isoforms alpha, beta and gamma investigated are similarly activated by HCV-polyprotein-(1,189-1,525). Our data suggest that NS3 attenuates the substrate inhibition through a generalized mechanism operating mainly on the substrate level that directly results from a specific protein-protein interaction. In the case of the PKC, an additional kinase activating mechanism operates on the enzyme level. Both actions of NS3, the attenuation of the substrate inhibition and the activation of PKC, could not be explained by classical means that predict autophosphorylation to enhance the rate of substrate phosphorylation. The results are discussed in view of similar activities displayed by matchmakers and some molecular chaperones.

Alkylation↗
PubMed1999

Cloning and expression of yellowtail ascites virus segment A.

cDNA of yellowtail ascites virus (YAV) segment A encoding a polyprotein of VP2, NS, and VP3 has been cloned. Comparison of the nucleotide and the deduced amino acid sequences showed very high homology between YAV and other aquatic birnaviruses. The two small open reading frames (VP5) besides the 5' terminus of the VP2 gene were found on segment A of YAV. Proteins encoded by cDNAs from segment A and the serotype-specific epitope region on VP2 were expressed using a baculovirus vector. Western blot analysis confirmed that a polyprotein was expressed and processed into VP2 and VP3 in insect cells infected with the recombinant baculovirus containing the complete polyprotein coding region. In the case of expression in silkworm larvae, only VP3 was detected in hemocytes and fat body of silkworm larvae infected with the recombinant virus. The recombinant fusion protein consisting of VP2 epitope region and polyhedrin was expressed in insect cells and cross-reacted with a mouse monoclonal antibody against VP2 which had a neutralizing activity to YAV.

Amino Acid Sequence↗
PubMed2000

Mutational analysis of the NIa protease from pepper vein banding potyvirus.

The nuclear inclusion protein a (NIa) protease plays an important role in the life cycle of potyviruses by processing the viral polyprotein into functional proteins. For functional characterization, the NIa protease from Pepper vein banding potyvir s (PVBV) was overexpressed in Escherichia coli and purified. Using a recombinant polyprotein substrate containing the nuclear inclusion protein b (NIb)-coat protein (CP) cleavage site, a trans-cleavage assay was developed for the NIa protease. The polyprotein substrate also possessed the cleavage site between NIa and NIb, in addition to the NIb-CP site. However, no trans-cleavage by the NIa protease between NIa and NIb was detected indicating that the cleavage between NIa and NIb under natural conditions would be by a cis-cleavage reaction. Site-specific mutations of the conserved residues D81, D90, C110, T146, C151 and H167 were performed to investigate their roles in the catalytic process of the protease. Such an analysis has revealed that D81 and C151 constitute two of the catalytic triad residues in the NIa protease, D90 and C110 are not essential for catalysis, and T146 and H167 are probably involved in binding to Gln at the P1 position of the substrate.

Capsid↗
PubMed2001

Intracellular hepatitis C virus RNA-dependent RNA polymerase activity.

Studies of intracellular hepatitis C virus (HCV) RNA-dependent RNA polymerase activity (RdRp activity) have been limited by the poor replicative capacity of HCV in cell culture. We have developed a method that allows for the measurement of HCV specific RdRp activity in eukaryotic cells. This method is based on the transient expression of the HCV polymerase and its templates under the control of the T7 promoter in the presence of an infection with recombinant vaccinia virus (vTF7-3) expressing the bacteriophage T7 DNA-dependent RNA polymerase. Both negative-strand and positive-strand RNA synthesis were characterised, and the role of the other HCV non-structural proteins for polymerase activity was assessed. With this assay we were able to show that: a) Intracellular HCV RdRp activity is not restricted to, but is higher for templates containing HCV specific sequences, b) The HCV polymerase is active within the polyprotein precursor, c) Cleavage of NS5b from the polyprotein precursor does not determine template specificity, and d) HCV RdRp activity is higher in the presence of the other HCV non-structural proteins and lower within a protease-deficient polyprotein precursor. This method allows the measurement of intracellular HCV polymerase activity and may be used to test substances against the HCV polymerase in search of potential drugs for anti-HCV therapy.

HeLa Cells↗
PubMed2000

Proteolytic processing of oligopeptides containing the target sequences by the recombinant tobacco vein mottling virus NIa proteinase.

Tobacco vein mottling virus (TVMV) belongs to the potyviridae that consists of about 200 plant viruses. Potyviruses have RNA genomes of approximately 10,000 bases from which a single polyprotein is expressed from each virus upon infection. The NIa proteinase is known to process the polyprotein at seven distinct junctions between proteins. Kinetic constants were determined for the reactions of the recombinant TVMV NIa protease (27 kDa) with synthetic oligopeptides containing the sequences for the cleavage sites. For optimum activity, the substrate needs to have six amino acids (P6-P1) in the amino region and four (P1'-P4') in the carboxy region, including four conserved amino acids (V-R-F-Q) in P4-P1 positions. Mutation of any of four conserved amino acids to Gly made the substrate inert to the enzyme. Among the substrates, the oligopeptides containing the sequences for junctions, P3-6K1, NIa (VPg-Pro), and NIa-NIb were not processed by the NIa protease. Those junctions have Glu at P3, Glu at P1, and Thr at P2. The implications of high substrate specificity and size dependence in polyprotein processing and viral replication are discussed.

Amino Acid Motifs↗
PubMed1984

Polypeptides of Mason-Pfizer monkey virus. I. Synthesis and processing of the gag-gene products.

Mason-Pfizer monkey virus (M-PMV), the prototype D-type retrovirus, differs from the mammalian C-type retroviruses by preassembling core structures in the cytoplasm of infected cells during morphogenesis. Studies that define the protein composition of M-PMV virions and identify two gag-related polyprotein precursors in M-PMV infected cells are reported. The polyprotein precursor to the internal structural (gag) proteins of M-PMV was identified by immunoprecipitation from lysates of pulse-labeled, virus-infected cells with an antiserum to the major structural protein, p27. Tryptic peptide-mapping experiments have shown that this precursor (Pr78) is cleaved to yield five virion structural polypeptides--p27, pp16, p14, p12, and p10. The pp16 polypeptide represents an additional gag-gene encoded polypeptide, not described previously; it is a phosphoprotein and present in virions in a number of forms. A second gag-related polyprotein precursor, P95, is also present in infected cells although in smaller amounts. This nonglycosylated polypeptide contains all of the leucine-containing tryptic peptides of Pr78 plus three others. Studies of the rate of synthesis and half-life of this protein argue against it being the major gag-gene precursor polypeptide. The possibility that it represents a precursor to the viral protease is discussed.

Gene Products, gag↗
PubMed1987

Deletion mapping and expression in Escherichia coli of the large genomic segment of a birnavirus.

The large genomic segment of infectious bursal disease virus encodes a polyprotein in which the viral polypeptides are present in the following order: N-VP2-VP4-VP3-C. Expression in Escherichia coli of the large segment results in the processing of the polyprotein. The expression product reacts with a virus neutralizing and protective monoclonal antibody that recognizes a conformational epitope on the surface of the virus. Different regions of the large genomic segment were deleted at defined restriction sites and the truncated fragments were ligated to various expression vectors for high-level expression in E. coli. The expressed proteins were probed with three different monoclonal antibodies that recognize epitopes encoded by different regions of the large genomic segment. These deletion mapping studies suggest that VP4 is involved in the processing of the precursor polyprotein, and the conformational epitope recognized by the virus neutralizing monoclonal antibody is present within VP2.

Animals↗
PubMed1989

Molecular genetic and biochemical evidence for the involvement of the heptapeptide cleavage sequence in determining the reaction profile at two tobacco etch virus cleavage sites in cell-free assays.

Potyviruses express their genetic information from a genome length RNA as a single polyprotein, which is post-translationally processed by at least two different viral-encoded proteolytic activities. Since regulation of the expression of individual genes is not likely to occur at the transcriptional level, we sought to determine if post-translational regulation of gene expression was possible via differential proteolytic processing. Modulating the rate of cleavage at different gene product junctions could be a potential mechanism to regulate the kinetics of formation of specific gene products. We have examined the proteolytic processing of two tobacco etch virus (TEV) cleavage sites using a cell-free system in which synthetic polyprotein precursors were processed by the TEV 49-kDa proteinase. The amino acid sequences at these two sites contained both conserved and variable positions. The cleavage reaction at the TEV 50/71-kDa junction was characterized as "slow" (T1/2 = approximately 27 min) while the cleavage reaction at the 58/30-kDa junction was judged "fast" (T1/2 = approximately 5 min). Similar cleavage reactions were observed whether the cleavage site was in its natural polyprotein context or placed in a foreign position. The slow and fast cleavage sites could be interconverted by changing the nonconserved amino acid positions. The data suggest that the heptapeptide sequence proximal to a TEV cleavage site determines not only cleavage at a particular junction but influences the cleavage reaction profile in cell-free studies.

Amino Acid Sequence↗
PubMed1991

Identification of essential amino acid residues in the functional activity of poliovirus 2A protease.

Proteolytic processing of poliovirus polyprotein is carried out by the products of two viral genes, 2A and 3C. 2A protease catalyzes cleavage of the polyprotein of type 1 poliovirus at two sites, one a cis cleavage at the 2A N-terminus and the other a trans cleavage within the 3D polymerase. In addition to polyprotein cleavage activity, 2A protease also indirectly induces cleavage of the p220 component of the cap-binding protein complex, which results in selective inhibition of host protein synthesis. Molecular genetic and biochemical analyses of 2A protease were performed to test its putative homology to small trypsin-like serine proteases and to examine the roles of individual amino acids in the reaction mechanism of 2A protease. A recombinant plasmid containing poliovirus 1C, 1D, and 2A gene sequences was expressed in a cell-free transcription/translation system, resulting in synthesis of a precursor protein that underwent efficient self-processing and produced mature 2A protease. To identify residues involved in the catalytic center and/or substrate-binding loops, we generated a series of 2A mutants by site-specific mutagenesis of this plasmid. Mutants were then expressed in vitro and tested for autocatalytic cis cleavage activity, trans cleavage of the 1D/2A junction, and trans-activation of p220-specific protease. Our data suggest that the conserved His20, Asp38, and Cys109 residues recently proposed to be equivalent to the catalytic triad of known serine proteases may comprise the catalytic triad of 2A protease. Surprisingly, Asp38 could be replaced with glutamic acid and retain autocatalytic function. Other amino acid substitutions at Tyr88, Tyr89, and Thr124 suggested that these residues lie in loops involved in substrate binding. Biochemical studies with protease inhibitors indicate that 2A protease activity is blocked by inhibitors specific for serine and cysteine proteases. Overall, the results are consistent with the hypothesis that 2A proteinase is structurally similar to the trypsin-like family of serine proteases with the substitution of cysteine 109 as the active site nucleophile.

Amino Acid Sequence↗
PubMed1992

Analysis of HIV particle formation using transient expression of subviral constructs in mammalian cells.

Segments of the human immunodeficiency virus (HIV) type 1 gag and pol genes and mutants thereof were transiently expressed in mammalian cells. Expression was dependent on the presence of the rev responsive element in cis and the rev protein in trans and was readily detected by indirect immunofluorescence or Western blotting. Transfection of constructs encoding the entire gag and pol open reading frames yielded efficient release of particles banding at a density of 1.16 g of sucrose per milliliter and consisting mainly of processed gag proteins. In addition, these particles contained the p66/p51 heterodimer of reverse transcriptase (RT), had associated RT activity, and contained RNA. Electron micrographs revealed immature retrovirus-like particles budding primarily from the plasma membrane and extracellular particles with morphological characteristics of HIV. Particle production was independent of the pol open reading frame or an active HIV proteinase (PR) but without active PR, cell-associated and particle-associated proteins remained completely uncleaved and budding occurred primarily into intracellular vacuoles. A mutation preventing myristoylation of the viral polyproteins abolished particle release but did not interfere with polyprotein synthesis and did not prevent processing. Expression of gag and PR in the same reading frame yielded complete processing of polyproteins but no budding and led to increased cell toxicity. A mutation of the PR active site in this construct prevented cytotoxicity and restored particle release indicating that the observed phenotype was caused by the overexpression of PR. These particles were aberrant in size and morphology when analyzed on sucrose density gradients and by electron microscopy. Budding was arrested at an early stage and extracellular particles appeared to be released by a different mechanism. Only short C-terminal extensions were compatible with this release mechanism since expression of a similar mutant construct encoding the entire gag-pol open reading frame did not yield particles.

Animals↗
PubMed1992

Cloned DNA copies of cowpea severe mosaic virus genomic RNAs: infectious transcripts and complete nucleotide sequence of RNA 1.

Cowpea severe mosaic virus (CPSMV) is a member of the comovirus group of messenger-sense RNA viruses with bipartite genomes, of which cowpea mosaic virus (CPMV) is the type member. Full-length copies of CPSMV RNA 1 were cloned in plasmids bearing a bacteriophage T7 promoter. Previously, similar clones of CPSMV RNA 2 had been obtained. A 5'-rUAUUAAAAUUUU sequence is common to RNA 1 and RNA 2. From two RNA 1 clones and four RNA 2 clones we excised non-CPSMV sequences so as to provide templates for in vitro transcripts that have only a single guanylate preceding CPSMV RNA sequences. Transcripts from the most active RNA 1 and RNA 2 clones, when mixed, showed about 5% of the infectivity of unfractionated CPSMV RNAs from virions. The longest, 1858 codon open reading frame of the 5957 nt CPSMV RNA 1 extends from an AUG at nt 257 to a UGA termination codon at nt 5831. The calculated molecular weight of the polyprotein is 208,000. Comparisons with the available amino acid residue (aa) sequence information from the complete CPMV RNA 1 sequence and the partial sequence of red clover mottle virus RNA 1 suggest that CPSMV RNA 1 specifies the expected set of five mature proteins: 32K proteinase cofactor, 58K presumed helicase, VPg 5'-linked protein of the genomic RNAs, 24K proteinase, and 87K presumed polymerase, separated by four cleavage sites. Of the determined and deduced cleavage sites of the three RNA 1 polyproteins, only that at the 24K/87K junction has a distinct aa pair in the CPSMV polyprotein. Of the five proteins, VPg and 87K show the greatest similarity between CPSMV and CPMV, with identities of 68 and 55%, respectively. Published mutational analysis of the CPMV 24K proteinase and alignment of aa sequences from three comoviruses suggest that cysteine-168, histidine-40 and glutamic acid-77 form the catalytic triad of the CPSMV 24K proteinase. Results are discussed in the context of the resistance that some cowpea (Vigna unguiculata) lines exhibit against CPMV but not against CPSMV.

Amino Acid Sequence↗
PubMed1992

Identification of the active site residues in the nsP2 proteinase of Sindbis virus.

The nonstructural polyproteins of Sindbis virus are processed by a virus-encoded proteinase which is located in the C-terminal domain of nsP2. Here we have performed a mutagenic analysis to identify the active site residues of this proteinase. Substitution of other amino acids for either Cys-481 or His-558 completely abolished proteolytic processing of Sindbis virus polyproteins in vitro. Substitutions within this domain for a second cysteine conserved among alphaviruses, for four other conserved histidines, or for a conserved serine did not affect the activity of the enzyme. These results suggest that nsP2 is a papain-like proteinase whose catalytic dyad is composed of Cys-481 and His-558. Since an asparagine residue has been implicated in the active site of papain, we changed the four conserved asparagine residues in the C-terminal half of nsP2 and found that all could be substituted without total loss of activity. Among papain-like proteinases, the residue following the catalytic histidine is alanine or glycine in the plant and animal enzymes, and the presence of Trp-559 in alphaviruses is unusual. A mutant enzyme containing Ala-559 was completely inactive, implying that Trp-559 is essential for a functional proteinase. All of these mutations were introduced into a full-length clone of Sindbis virus from which infectious RNA could be transcribed in vitro, and the effects of these changes on viability were tested. In all cases it was found that mutations which abolished proteolytic activity were lethal, whether or not these mutations were in the catalytic residues, indicating that proteolysis of the nonstructural polyprotein is essential for Sindbis replication.

Amino Acid Sequence↗
PubMed1992

The complete nucleotide sequence of cell fusing agent (CFA): homology between the nonstructural proteins encoded by CFA and the nonstructural proteins encoded by arthropod-borne flaviviruses.

Cell fusing agent (CFA) is an RNA virus originally isolated from a line of Aedes aegypti mosquito cells. Although our characterization of the virus many years ago showed that it resembled the flaviviruses, there was no detectable serological cross-reaction with members of the genus flavivirus. Furthermore, unlike the well-studied members of the genus flavivirus, CFA did not replicate in any of several vertebrate cell lines tested. We have now determined the nucleotide sequence of the CFA genome. Comparison of the predicted amino acid sequence of the CFA polyprotein with viral protein sequences in Genbank, has made it apparent that CFA should now be assigned to the family Flaviviridae, genus flavivirus. The homology between CFA proteins and those of other flaviviruses was highest for NS5 (45%) and NS3 (34%). Little homology was found for the structural proteins. Thus, CFA is only distantly related to the other flaviviruses for which there is sequence information; nevertheless, with respect to their hydrophobicity plots, the CFA polyprotein and the polyproteins of other flaviviruses are remarkably similar. We suggest that CFA is an insect virus, which was present in the embryos from which the Ae. aegypti cell line was established. Thus, CFA seems to be the first member of the family Flaviviridae, genus flavivirus, to be identified as an insect virus.

Amino Acid Sequence↗
PubMed1987

Alternative splicing in individual Aplysia neurons generates neuropeptide diversity.

The neuron R15 is a peptidergic cell within the abdominal ganglion of Aplysia that participates in two neural circuits governing physiological and behavioral programs. We have cloned and characterized the major gene product expressed in this neuron. The R15 cDNA encodes a polyprotein precursor that is cleaved to yield a set of small neuropeptides. One peptide, R15 alpha 1, may act on different target cells to generate distinct but complementary physiological alterations that contribute to a program of cardiovascular changes in Aplysia. We have found that the RNA encoding the R15 polyprotein is spliced differently in different neurons. Our results suggest that alternative splicing of RNAs encoding polyproteins may provide a mechanism to generate distinct but overlapping sets of peptides that govern distinct but related physiological or behavioral programs.

Animals↗
PubMed1993

Mutational analysis of AUG codons of cowpea mosaic virus M RNA.

The involvement of the AUG codons at positions 115, 161, 512 and 524 in translation and infectivity of cowpea mosaic virus M RNA was studied. Mutations were introduced in each of these codons in a full length cDNA clone of M RNA and the effect of the mutations was examined by translation from in vitro transcripts of these mutant cDNAs in rabbit reticulocyte lysates and by checking the replication of these transcripts in the presence of B RNA in cowpea protoplasts and plants. It was found that AUG115, at the beginning of an open reading frame (ORF) for a putative 2-kDa protein, can be used in vitro to initiate translation, but mutation of this AUG codon in the M RNA does not affect the ability of the virus to infect cowpea plants. AUG161, located at the beginning of the large ORF, was shown to be essential for expression of the large 105-kDa polyprotein and for replication of M RNA. Translation of the second 95-kDa polyprotein was found to start at AUG512. Upon mutation of this AUG codon efficient initiation of translation occurred at AUG524. Results with M RNAs that lack AUG512 and/or 524 indicate that an intact 95-kDa polyprotein is not required for replication of M RNA but that this protein has an essential function in cell-to-cell movement of the virus.

Amino Acid Sequence↗