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At least 523 records · Page 29Linked to original sources
PubMed1990

Analysis of the in vitro cleavage products of the tomato black ring virus RNA-1-encoded 250K polyprotein.

Tomato black ring virus RNA-1 was translated in a rabbit reticulocyte lysate. The primary translation product of Mr 250K, which corresponds to its whole coding capacity, was synthesized within 45 min and, during further incubation in the translation medium, was proteolytically processed. Essentially, four cleavage products (P190, P120, P60 and P50) were detected and located within P250 by pulse-chase and immunoprecipitation experiments. P190 is an intermediate cleavage product which is further cleaved to form P60 and P120. P120, which contains the region that has been assigned to the virus protease and the virus polymerase, was not further cleaved in vitro.

Kinetics↗
PubMed1991

Proteolytic processing of Semliki Forest virus-specific non-structural polyprotein.

The processing and stability of the non-structural (ns) proteins of Semliki Forest virus were studied in vivo. Virus-specific proteins from infected cells were identified by immunoprecipitation with monospecific antisera. The complete ns precursor, P1234, translated within 7 to 9 min of the start of translation, was processed before the completion of translation into P123 and nsP4. Pulse-chase experiments showed that the mature ns proteins were relatively stable for at least 2 h. Interestingly, the decrease in the amount of the P34 precursor during chase is accompanied by an increase only in the amount of nsP3, which could explain the observed lower amount of nsP4 in infected cells. In cells infected with SFV RNA- mutants ts4 and ts6 maintained at the restrictive temperature, nsP4 but no nsP1, nsP2 or nsP3 accumulated in addition to the ns precursor proteins P1234, P123, P12 and P34. Translation in vitro of mRNAs from a cDNA clone, encoding P1234 with a deletion in the carboxy-terminal proteinase domain of nsP2, did not yield nsP1, nsP2 or nsP3 but only nsP4, indicating that cleavage at the nsP3/4 sites is independent. Evidently, nsP4 is produced by a nascent cleavage of the growing P1234, catalysed by its own proteinase activity; the proteolytic cleavages at the nsP1/2 and nsP2/3 sites are catalysed by the proteinase moiety of nsP2.

Animals↗
PubMed1997

Classification of hepatitis C virus variants in six major types based on analysis of the envelope 1 and nonstructural 5B genome regions and complete polyprotein sequences.

The phylogenetic status of recently described isolates of hepatitis C virus (HCV) from Vietnam, Thailand and Indonesia (previously classified as types 7, 8, 9, 10 and 11) was re-analysed by the neighbour-joining method instead of the unweighted pair-group method with arithmetic mean (UPGMA) that was first used by the discoverers of these strains. The analysis of complete amino acid sequences and of nucleotide sequences of the envelope 1 (672 nt) and nonstructural 5B (1092 nt) genomic regions permitted the re-assignment of the type 7, 8, 9 and 1 1 isolates to type 6, and that of type 10 strains to type 3. Finally, this study made possible the classification of the previously described HCV strains (including these South-East Asian isolates) in six major types and at least 30 subtypes. It confirms that analysis of the E 1 and NS5B genomic regions using the neighbour-joining method is a reliable tool for the assignment of most new isolates.

Genome, Viral↗
PubMed2005

Yellow fever virus NS2B-NS3 protease: characterization of charged-to-alanine mutant and revertant viruses and analysis of polyprotein-cleavage activities.

A series of 46 charged-to-alanine mutations in the yellow fever virus NS2B-NS3 protease, previously characterized in cell-free and transient cellular expression systems, was tested for their effects on virus recovery. Four distinct plaque phenotypes were observed in cell culture: parental plaque-size (13 mutants), reduced plaque-size (17 mutants), small plaque-size (8 mutants) and no plaque-formation (8 mutants). No mutants displayed any temperature sensitivity based on recovery of virus after RNA transfection at 32 versus 37 degrees C. Most small plaque-mutants were defective in growth efficiency compared with parental virus. However not all small plaque-mutants had defective 2B/3 cleavage, with some showing selective defects at other non-structural protein cleavage sites. Revertant viruses were recovered for six mutations that caused reduced plaque sizes. Same-site and second-site mutations occurred in NS2B, and one second-site mutation occurred in the NS3 protease domain. Some reversion mutations ameliorated defects in cleavage activity and plaque size caused by the original mutation. These data indicate that certain mutations that reduce NS2B-NS3 protease cleavage activity cause growth restriction of yellow fever virus in cell culture. However, for at least two mutations, processing defects other than impaired cleavage activity at the 2B/3 site may account for the mutant phenotype. The existence of reversion mutations primarily in NS2B rather than NS3, suggests that the protease domain is less tolerant of structural perturbation compared with the NS2B protein.

Amino Acid Substitution↗
PubMed2006

A link between translation of the hepatitis C virus polyprotein and polymerase function; possible consequences for hyperphosphorylation of NS5A.

Hyperphosphorylation of NS5A is thought to play a key role in controlling hepatitis C virus (HCV) RNA replication. Using a tetracycline-regulable baculovirus delivery system to introduce non-culture-adapted HCV replicons into HepG2 cells, we found that a point mutation in the active site of the viral polymerase, NS5B, led to an increase in NS5A hyperphosphorylation. Although replicon transcripts lacking elements downstream of NS5A also had altered NS5A hyperphosphorylation, this did not explain the changes resulting from polymerase inactivation. Instead, two additional findings may be related to the link between polymerase activity and NS5A hyperphosphorylation. Firstly, we found that disabling polymerase activity, either by targeted mutation of the polymerase active site or by use of a synthetic inhibitor, stimulated translation from the replicon transcript. Secondly, when the rate of translation of non-structural proteins from replicon transcripts was reduced by use of a defective encephalomyocarditis virus internal ribosome entry site, there was a substantial decrease in NS5A hyperphosphorylation, but this was not observed when non-structural protein expression was reduced by simply lowering replicon transcript levels using tetracycline. Therefore, one possibility is that the point mutation within the active site of NS5B causes an increase in NS5A hyperphosphorylation because of an increase in translation from each viral transcript. These findings represent the first demonstration that NS5A hyperphosphorylation can be modulated without use of kinase inhibitors or mutations within non-structural proteins and, as such, provide an insight into a possible means by which HCV replication is controlled during a natural infection.

Cell Line, Tumor↗
PubMed2004

The C-terminal domains of the gingipain K polyprotein are necessary for assembly of the active enzyme and expression of associated activities.

The Porphyromonas gingivalis lysine-specific cysteine protease (gingipain K, Kgp) is expressed as a large precursor protein consisting of a leader sequence, a pro-fragment, a catalytic domain with a C-terminal IgG-like subdomain (IgSF) and a large haemagglutinin/adhesion (HA) domain. In order to directly study the role of these non-catalytic domains in pro-Kgp processing and maturation in P. gingivalis, the wild-type form of the gene was replaced with deletion variants encoding C-terminally truncated proteins, including KgpDeltaHA3/4 (Delta1292-1732 aa), KgpDeltaHA2-4 (Delta1157-1732 aa), KgpDeltaHA1-4 (Delta738-1732 aa), KgpDeltaC-term/HA (Delta681-1732 aa) and KgpDeltaIg/C-term/HA (602-1732 aa). Northern blot and reverse transcription polymerase chain reaction (RT-PCR) analysis revealed that all truncated variants of the kgp gene were transcribed in P. gingivalis. Despite high levels of kgpDeltaC-term/HA and kgpDeltaIg/C-term/HA transcripts, no Kgp-specific antigen was detected in cultures of these mutants as determined by Western blot analysis with monoclonal antibodies specific for the Kgp catalytic domain. Furthermore, only barely measurable amounts of Kgp-specific activity were detected in these two mutants. The remaining mutants expressed significant Kgp activity, however, at lower levels when compared with the parental strain. The decreased activity most probably resulted from altered folding and/or hindered secretion of the protein. The kgp gene truncation was also demonstrated to alter the distribution of the gingipain protein between membrane-associated and -secreted forms. While both gingipain K activity and the protein were cell membrane-associated in the parental strain, the mutants released significant amounts of both protein and activity into the media. Taken together, these results suggest that the C-terminal HA domains of Kgp are not only essential for full expression of gingipain activity, but also for proper processing of the multiprotein complex assembly on the P. gingivalis outer membrane. Moreover, our results indicate that the immunoglobulin-like subdomain is indispensable for proper folding and expression of the gingipains.

Adhesins, Bacterial↗
PubMed1980

Analysis of Semliki-Forest-virus structural proteins to illustrate polyprotein processing of alpha viruses.

The four structural proteins of Semliki Forest virus were purified in an amount of 30-50 nmol by preparative sodium dodecylsulfate/polyacrylamide gel electrophoresis. Each protein was subjected to N-terminal structural analysis by degradation in a liquid-phase sequencer. About 20 residues were determined for each of the two membrane glycoproteins E1 and E2. The amino acid sequence of E1 but not that of E2 showed extensive homology to the corresponding proteins of the closely related Sindbis virus. Both E1 and E2 seem to lack a signal sequence at the N terminus, since the proportion of polar amino acids in this region deviates from the proportion in the known hydrophobic signal sequences. The envelope glycoprotein E3 and the capsid protein did not yield any significant result on Edman degradation, suggesting that they have blocked N-terminal amino groups.

Amino Acid Sequence↗
PubMed1982

Proteolytic maturation of the turnip-yellow-mosaic-virus polyprotein coded in vitro occurs by internal catalysis.

The genomic RNA of turnip yellow mosaic virus is translated in vitro into two major high-molecular-weight proteins, the larger of which (Mr 195 000) undergoes post-translational cleavage. The mechanism of formation of the primary cleavage products (Mr 120 000 and Mr 78 000) of the 195 000-Mr protein has been examined. The fact that cleavage partly occurs at a rate insensitive to dilution of the 195 000-Mr protein is suggestive of an intramolecular mechanism of proteolytic maturation.

Genes, Viral↗
PubMed1999

Modulation of major histocompatibility complex protein expression by human gamma interferon mediated by cysteine proteinase-adhesin polyproteins of Porphyromonas gingivalis.

Cysteine proteinases have been emphasized in the virulence of Porphyromonas gingivalis in chronic periodontitis. These hydrolases may promote the degradation of extracellular matrix proteins and disrupt components of the immune system. In this study it was shown that purified Arg-gingipain and Lys-gingipain inhibited expression of class II major histocompatibility complex (MHC) proteins in response to the stimulation of endothelial cells with human gamma interferon (IFN-gamma). Treatment with the cysteine proteinases resulted in a rapid shift in the apparent molecular size of IFN-gamma from 17 to 15 kDa, as shown by Western blot analysis, a response which also occurred in the presence of serum. Further, glycosylated natural IFN-gamma from human leukocytes and unglycosylated recombinant IFN-gamma from Escherichia coli were both digested by the cysteine proteinases. Immunoblot analysis indicated that cleavage within the carboxyl terminus of recombinant IFN-gamma correlated with the loss of induction of MHC class II expression as monitored by analytical flow cytometry. No hydrolysis of MHC class II molecules or human IFN-gamma receptor by these proteinases was detected by Western blot analysis. These findings suggest that P. gingivalis cysteine proteinases may alter the cytokine network at the point of infection through the cleavage of IFN-gamma. Degradation of IFN-gamma could have important consequences for the recruitment and activation of leukocytes and therefore may contribute significantly to the destruction of the periodontal attachment.

Adhesins, Bacterial↗
PubMed2002

Recombinant Dirofilaria immitis polyprotein that stimulates murine B cells to produce nonspecific polyclonal immunoglobulin E antibody.

Nonspecific immunoglobulin E (IgE) production is an event characteristically observed in parasitic helminth infections, but its mechanisms are still unclear. To define these mechanisms, we prepared a recombinant Dirofilaria immitis protein (rDiAg) and assessed its effect on nonspecific IgE production. rDiAg preferentially induced nonspecific IgE production, without eliciting specific IgE production, as well as a Th2-type cytokine profile (high interleukin-4 [IL-4] and IL-10 production but low gamma interferon production) in BALB/c mice. rDiAg significantly elicited the proliferative response of naive B cells. This response was not abolished by polymyxin B, an inhibitor of lipopolysaccharide (LPS), and rDiAg normally expanded splenic B cells from LPS nonresponder C3H/HeJ mice. Thus, the mitogenic effect of rDiAg was not due to LPS contamination. rDiAg also enhanced levels of CD23 expression on splenic B cells. Splenic B cells produced marked levels of IgE when cultured with the combination of rDiAg and IL-4 (rDiAg-IL-4), whereas peritoneal B cells produced negligible levels of IgE. rDiAg-IL-4-induced IgE production by splenic B cells was synergistically increased by coculture with peritoneal B cells. rDiAg-driven IL-10 secretion was higher in peritoneal B cells than in splenic B cells. IgE production by splenic B cells cocultured with peritoneal B cells was decreased to a level comparable to that by splenic B cells in the presence of a neutralizing anti-IL-10 monoclonal antibody. Collectively, these results suggest that rDiAg-induced polyclonal expansion and IgE class switching of splenic B cells contribute to nonspecific IgE production and that these responses are enhanced by peritoneal B-cell-derived IL-10.

Animals↗
PubMed2006

Human immunodeficiency virus type 1 Gag polyprotein modulates its own translation.

The full-length viral RNA of human immunodeficiency virus type 1 (HIV-1) functions both as the mRNA for the viral structural proteins Gag and Gag/Pol and as the genomic RNA packaged within viral particles. The packaging signal which Gag recognizes to initiate genome encapsidation is in the 5' untranslated region (UTR) of the HIV-1 RNA, which is also the location of translation initiation complex formation. Hence, it is likely that there is competition between the translation and packaging processes. We studied the ability of Gag to regulate translation of its own mRNA. Gag had a bimodal effect on translation from the HIV-1 5' UTR, stimulating translation at low concentrations and inhibiting translation at high concentrations in vitro and in vivo. The inhibition was dependent upon the ability of Gag to bind the packaging signal through its nucleocapsid domain. The stimulatory activity was shown to depend on the matrix domain of Gag. These results suggest that Gag controls the equilibrium between translation and packaging, ensuring production of enough molecules of Gag to make viral particles before encapsidating its genome.

5' Untranslated Regions↗
PubMed1974

Cleavage of mengovirus polyproteins in vivo.

The synthesis of mengovirus-specific proteins in vivo was studied by labeling the viral proteins with radioactive amino acids under conditions in which host protein synthesis was almost completely inhibited. Pulse-chase experiments enabled the kinetic analysis of the cleavages of certain viral protein precursors and the formation of others. The pattern of cleavages of mengovirus precursor polypeptides is similar to that of encephalomyocarditis virus. The major difference between the two viruses seems to be in the molar concentration in which the various primary products are produced. The molar ratio of the A protein, which is the precursor of the capsid proteins, to that of the primary products F and C, is approximately 1.5 to 2.0: 1: 1. Possible explanations for the unequal appearance of the structural and nonstructural proteins are discussed.

Amino Acids↗
PubMed1978

Polyproteins related to the major core protein of mouse mammary tumor virus.

The mouse mammary tumor virus (MuMTV) contains several low-molecular-weight proteins which, together with the genomic RNA, constitute the core structure of the virion. The most abundant protein in the core is the 27,000-dalton protein (p27), and, by analogy to the type C viruses, this protein probably forms the core shell. In mouse mammary tumor cell lines (GR and Mm5MT) producing MuMTV the major p57 antigenic specificity resides in a large protein, which migrates in polyacrylamide gels as a doublet of 77,000 and 75,000 daltons (p 77/75). A series of lower-molecular-weight proteins, p61, p48, p38, and p34, is also present in small amounts and is probably derived by proteolytic cleavage of the p 77/75. These proteins have been identified by immunoprecipitation with monospecific antiserum, and their sequence relatedness to p27 has been determined by an analysis of the peptides after trypsin digestion. After a 15-min pulse with [35S]-methionine, all of the p27-related proteins in these cell lines were labelled and, during a subsequent chase, progressively disappeared. The p27 was labeled poorly during the pulse, but the amount of label in this protein increased during the chase. A quantitation of these experiments suggested that the majority of the p27-related proteins were quite rapidly turned over in these cell lines. Hence, if p27 is derived by a progressive proteolytic cleavage mechanism, then the process is inefficient in the GR cells and only moderately efficient in the Mm5MT cells. When MuMTV was isolated from the culture medium of these cells harvested at 5-min intervals, the major p27-related protein was p34. The p27 accounted for only 29% of the anti-p27 serum immunoprecipitable proteins compared to 95% in virus isolated from an 18-h harvest. Incubation of the rapid-harvest virus at 37 degrees C for 2 h resulted in some conversion of p34 to p27. These results suggest that some of the p27 in MuMTV is formed in the virions by proteolytic cleavage of p34.

Animals↗
PubMed1981

Replication-defective Friend murine leukemia virus particles containing uncleaved gag polyproteins and decreased levels of envelope glycoprotein.

An erythroleukemia cell clone, 7C, which failed to produce reverse transcriptase-containing virions or infectious virus, was found to produce noninfectious virus particles by gradient banding of [3H]leucine- and [3H]uridine-labeled virions. The RNA from the 7C virus was shown to consist of the normal 70S size component, which converted to 35S upon heat denaturation. In contrast, the 7C virion proteins showed multiple defects. Analysis of the virion proteins by gel electrophoresis demonstrated that the pr65 gag precursor was incorporated into the 7C virus and that the processing of this precursor was severely diminished. Polymerase proteins pr180gag-pol and pr120pol were also detected in virions, and a third possible polymerase protein, p70, was reduced in size compared to its normal counterpart, p80. Incorporation of the viral gp70 glycoprotein into particles was also reduced 10-fold, despite synthesis and incorporation of gp70 into the 7C cell membrane in normal amounts. Pulse-chase analysis of the synthesis of the viral gag and env proteins in 7C cells showed greatly reduced amounts of pr180gag-pol, pr65gag, p80gag, and p42gag, whereas pr90env, gp70, and spleen focus-forming virus-specific gp55 were synthesized and processed normally. These results suggested that at least one defect in 7C virus was impaired cleavage of gag or pol proteins or both, most likely due to a lack of the appropriate viral protease, and that this lack of cleavage might affect incorporation of gp70 into virus particles.

Animals↗
PubMed1982

Carboxy-terminal analysis of poliovirus proteins: termination of poliovirus RNA translation and location of unique poliovirus polyprotein cleavage sites.

The carboxy-terminal amino acids of a number of poliovirus proteins were determined by carboxypeptidase A analysis. The nonstructural proteins P3-2, P3-4b and their precursor. P3-1b, were found to be coterminal with a sequence of -Ser-Phe-COOH. As these proteins are coded for at the extreme 3' end of the viral RNA, it is possible to establish the termination site of translation at nucleotide 7,361, 73 nucleotides before the start of the polyadenylic acid tract of the RNA. Two additional nonstructural proteins, P2-X and its precursor, P2-3b, were also found to be coterminal with a sequence of -Phe-Gln-COOH. This result confirms the existence of at least one Gln-Gly proteolytic cleavage site. These Gln-Gly cleavage sites are predicted from the nucleotide sequence to be ubiquitous throughout the poliovirus genome. The only exceptions are the cleavage sites at the carboxy termini of the structural protein VP4 and VP1. Carboxypeptidase A analysis of VP1 establishes a terminal sequence of -Thr-Tyr-COOH, and similar analysis of VP4 shows Asn to be the terminal amino acid residue, observations that prove the existence of the exceptional C-terminal amino acids. In none of the analyzed cases has C-terminal trimming after cleavage been observed.

Amino Acid Sequence↗
PubMed1983

P85: a gag-mos polyprotein encoded by ts110 Moloney murine sarcoma virus.

Antibody to a synthetic peptide (anti-C3 serum) with the predicted sequence of the C terminus of the Moloney murine sarcoma virus (strain 124) v-mos gene was used in immunoprecipitation experiments with cytoplasmic extracts of a clone of NRK cells infected with ts110 Moloney murine sarcoma virus, termed 6m2 cells. ts110 Moloney murine sarcoma virus codes for two viral proteins of 85,000 and 58,000 M(r), termed P85 and P58, respectively, in nonproducer 6m2 cells maintained at 33 degrees C. Anti-C3 serum specifically recognized [(3)H]leucine-labeled P85, but not P58, from infected cells maintained at 33 degrees C, whereas antiserum prepared against murine leukemia virus p12 recognized both proteins. Normal serum and anti-C3 serum pretreated with excess C3 peptide did not precipitate P85. Immunoprecipitation experiments after metabolic labeling of 6m2 cells with (32)P(i) showed that P85 is phosphorylated. Both anti-C3 and anti-p12 sera specifically detected (32)P-labeled P85. Cell-free translation of ts110 murine sarcoma virus/murine lukemia virus RNA produces P85, P58, and helper virus protein Pr63(gag). Anti-C3 serum specifically precipitated P85 but neither P58 nor Pr63(gag). We conclude from these studies that P85 is a product of both the gag and mos genes of ts110 murine sarcoma virus, and, therefore, it is referred to as P85(gag-mos). We have not detected any other v-mos gene product in ts110-infected cells.

Animals↗