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Biomedical subjects

E Ehrenfeld

Publications and source records attributed to E Ehrenfeld.

At least 73 records · Page 4Linked to original sources
PubMed1990

The p220 component of eukaryotic initiation factor 4F is a substrate for multiple calcium-dependent enzymes.

Eukaryotic initiation factor 4F (eIF-4F) is a multisubunit protein that functions in the first step of the binding of capped mRNAs to the small ribosomal subunit. Its largest polypeptide component, p220, is cleaved following poliovirus infection. This is thought to inactivate eIF-4F function, thereby preventing cap-dependent initiation of translation of cellular mRNAs. In this report, we show that p220 in extracts of uninfected HeLa cells is specifically lost in the presence of calcium. The responsible activities have been partially purified and identified as the calcium-dependent, neutral, cysteine proteases calpains I and II. In addition, a third calcium-dependent activity was resolved from the calpains and also results in the loss of p220. This activity has properties similar to a transglutaminase and copurifies with tissue transglutaminase through several chromatographic steps. None of these calcium-dependent activities appears to mediate p220 cleavage in poliovirus-infected cells.

Calcium↗
PubMed1990

Eukaryotic initiation factor 3 is required for poliovirus 2A protease-induced cleavage of the p220 component of eukaryotic initiation factor 4F.

After cultured cells are infected with poliovirus, cellular mRNA fails to bind to ribosomes, and synthesis of the majority of cellular proteins ceases. The defective step has been localized to the cap-dependent activity of the eukaryotic translation initiation factor 4F. Inactivation of this factor correlates with the cleavage of its largest subunit, p220, into characteristic products observed in infected cells. This cleavage is mediated by the poliovirus protease 2Apro. Previous work suggests that 2Apro does not catalyze the reaction directly, suggesting that one or more cellular proteins is required for the degradation of p220. To identify such a protein, we have developed an assay in which cleavage of a p220 substrate in the presence of poliovirus 2Apro is dependent upon the addition of HeLa cell proteins. By using this assay, we show that another factor, eukaryotic translation initiation factor 3, is required for 2Apro-dependent cleavage of p220.

Cysteine Endopeptidases↗
PubMed1989

Detection of hepatitis A virus RNA and capsid antigen in individual cells.

The replication of hepatitis A virus (HAV) RNA and the production of HAV VP1 protein were examined in cultures of BS-C-1 cells under one-step growth conditions by in situ hybridization and immunofluorescence. Individual cells that had undergone active viral RNA replication were detectable at 24 h post-infection. During subsequent days, increasing numbers of cells began replicating viral RNA, so that by seven days post-infection, all cells had accumulated significant amounts of viral RNA. The results show that the protracted replication cycle of HAV in cultured cells represents a slow recruitment of infected cells into a replication mode, rather than an inherently slow virus reproduction in all cells. With the reagents utilized in this study, nucleic acid hybridization was more sensitive than antigen detection by immunofluorescence or immunoblot analysis.

Animals↗
PubMed1989

Effects of mutations in poliovirus 3Dpol on RNA polymerase activity and on polyprotein cleavage.

A series of short insertion mutations was introduced into the poliovirus gene for 3Dpol at a number of different locations. When substituted for wild-type sequences in a full-length, infectious cDNA and tested for infectivity, all 3D mutants were nonviable. The mutant cDNAs were introduced into a bacterial plasmid designed to direct the expression of poliovirus 3CD, a viral protein composed of contiguous protease and RNA polymerase sequences. Bacteria transformed with these plasmids all expressed similar amounts of 3CD, and all mutant proteins cleaved themselves to generate wild-type 3Cpro and mutant 3Dpol polypeptides with approximately the same efficiency as wild-type 3CD. The released mutant 3Dpol proteins were all defective in RNA-dependent RNA polymerase activity in vitro. Uncleaved 3CD is a protease required for processing the viral capsid protein precursor, P1. In an in vitro assay of P1 cleavage activity, some of the mutant 3CD proteins expressed in Escherichia coli showed normal activity, while others were clearly inactive. Thus, alterations in the sequence and/or folding of different regions of the 3D protein have differential effects on its various activities.

Amino Acid Sequence↗
PubMed1988

Enzymatic activity of poliovirus RNA polymerase synthesized in Escherichia coli from viral cDNA.

Plasmids have been constructed that contain DNA sequences that direct the expression of the poliovirus RNA-dependent RNA polymerase, in the form of recombinant fusion proteins. Inclusion of an additional gene for the poliovirus protease results in cleavage of the fusion protein to yield a 52-kDa, enzymatically active, polymerase protein, apparently identical to the functional enzyme isolated from virus-infected HeLa cells. A large amount of polymerase protein accumulates as particulate or insoluble material in bacteria, and this protein has little or no activity. However, significant amounts of soluble, active enzyme are recovered, such that the resulting specific activity of crude bacterial extracts is greater than that obtained from virus-infected HeLa cells. Purification of the enzyme from Escherichia coli is readily accomplished, and yields a preparation that will copy poliovirion RNA as template, in the presence of oligo(U) primer. The availability of cloned DNA sequences encoding catalytically active RNA polymerase will allow genetic manipulations to initiate structure-function studies of this enzyme.

Cloning, Molecular↗
PubMed1988

Expression of hepatitis A virus capsid sequences in insect cells.

A cDNA coding for hepatitis A virus (HAV) VP1 and portions of the flanking VP3 and P2 sequences was inserted into the genome of Autographa californica nuclear polyhedrosis virus under the control of the polyhedrin promoter and translational start codon. Cells infected with recombinant virus produced high levels of a 55 kDa protein, identified as containing HAV VP1 by reactivity with anti-VP1 serum. The expressed protein also reacted on immunoblots with human HAV convalescent sera as well as sera from rabbits immunized with intact HAV. This protein was found predominantly in the cytoplasm of infected insect cells, probably as an insoluble aggregate.

Animals↗
PubMed1988

Antigenic and immunogenic properties of a hepatitis A virus capsid protein expressed in Escherichia coli.

We have constructed a recombinant plasmid producing, in bacteria, a hepatitis A virus (HAV) capsid protein that may be useful as a subunit vaccine. HAV VP1 coding sequences were fused in-frame to NH2-terminal Escherichia coli TrpE coding sequences under the control of the tryptophan promoter. In the absence of exogenous tryptophan, E. coli containing this recombinant plasmid produced high levels of an 88-kilodalton fusion protein that was recognized in immunoblots by antibodies to TrpE and HAV. The TrpE/HAV VP1 protein was gel-purified and used to immunize rabbits. The resulting antiserum reacted with denatured HAV VP1 in immunoblots but did not react with intact virus. However, subsequent inoculation of an immunized animal with a subimmunogenic dose of inactivated, whole HAV resulted in the rapid appearance of a stable, virus-neutralizing antibody response.

Animals↗
PubMed1988

Relationship of p220 cleavage during picornavirus infection to 2A proteinase sequencing.

Infection of HeLa cells by poliovirus results in an abrupt inhibition of host cell protein synthesis. It is thought that the mechanism of this inhibition involves proteolytic cleavage of the p220 component of the cap-binding protein complex, thereby causing functional inactivation of the cap-binding protein complex and preventing capped (cellular) mRNAs from binding ribosomes. Current data suggest that the viral proteinase 2A indirectly induces p220 cleavage via alteration or activation of a second proteinase of cellular origin. We present evidence that translation of poliovirus proteinase 2A sequences in vitro activates p220 cleavage. We have also aligned published picornavirus 2A amino acid sequences for maximum homology, and we show that the picornaviruses can be divided into two classes based on the presence or absence of a highly conserved 18-amino acid sequence in the carboxy-terminal portion of 2A. This conserved 2A sequence is homologous with the active site of the cysteine proteinase 3C common to all picornaviruses. We show that picornaviruses which contain the putative 2A active site sequence (e.g., enteroviruses and rhinoviruses) will induce cleavage of p220 in vivo. Conversely, we show that two cardioviruses (encephalomyocarditis virus and Theiler's encephalomyelitis virus) do not encode this putative proteinase sequence in the 2A region and do not induce cleavage of p220 in vivo. The foot-and-mouth disease virus (FMDV) 2A sequence represents an apparent deletion and consists of only 16 amino acids, most homologous with the carboxy terminus of the cardiovirus 2A sequence. It does not contain the putative cysteine proteinase active site. However, FMDV infection induces complete cleavage of BK cell p220, and translation of FMDV RNA in vitro induces an activity that cleaves HeLa cell p220. The data predict that an alternate FMDV viral protease is responsible for the induction of p220 cleavage.

Amino Acid Sequence↗
PubMed1988

Leader protein of foot-and-mouth disease virus is required for cleavage of the p220 component of the cap-binding protein complex.

Suppression of host protein synthesis in cells infected by poliovirus and certain other picornaviruses involves inactivation of the cap-binding protein complex. Inactivation of this complex has been correlated with the proteolytic cleavage of p220, a component of the cap-binding protein complex. Since picornaviral RNA is not capped, it continues to be translated as the cap-binding protein complex is inactivated. The cleavage of p220 can be induced to occur in vitro, catalyzed by extracts from infected cells or by reticulocyte lysates translating viral RNA. Expression of polioviral protease 2A is sufficient to induce p220 cleavage, and the presence in 2A of an 18-amino-acid sequence representing a putative cysteine protease active site correlates with the ability of different picornaviruses to induce p220 cleavage. Foot-and-mouth disease virus (FMDV) infection induces complete cleavage of p220, yet the FMDV genome codes for a 2A protein of only 16 amino acids, which does not include the putative cysteine protease active site. Using cDNA plasmids encoding various regions of the FMDV genome, we have determined that the leader protein is required to initiate p220 cleavage. This is the first report of a function for the leader protein, other than that of autocatalytic cleavage from the FMDV polyprotein.

Aphthovirus↗
PubMed1988

An internal 5'-noncoding region required for translation of poliovirus RNA in vitro.

A truncated poliovirus RNA that contains the entire 5'-noncoding region as well as some capsid protein-coding sequences was produced from cloned cDNA inserted into an SP6 transcription vector and subsequently was translated in a mixed rabbit reticulocyte-HeLa cell lysate. Deletions or modifications of regions of the 5'-noncoding sequences had significant effects upon the efficiency of translation. The presence of a 60-nucleotide sequence located at positions 567 to 627 appeared to be essential for active ribosome binding and translation of this uncapped RNA.

Cell-Free System↗
PubMed1987

The cap-binding protein complex in uninfected and poliovirus-infected HeLa cells.

In poliovirus-infected HeLa cells, the mechanism of protein synthesis initiation factor recognition of m7G cap groups on mRNA is impaired. Translation of capped host cell mRNAs is inhibited, whereas translation of uncapped poliovirus mRNA proceeds exclusively. The site of this defect has been localized to the cap-binding protein complex (CBPC). To elucidate the specific structural and functional defects of the CBPC following poliovirus infection, the CBPC and/or its polypeptide components were purified from uninfected and poliovirus-infected HeLa cells. The CBPC from uninfected cells consisted of tightly associated 24- and 220-kDa polypeptides; minor amounts of polypeptides of 40, 44, and 80 kDa also consistently co-purified with the p24/p220 cores. No evidence of a 50-kDa, eIF-4A-related polypeptide subunit of the CBPC was obtained. The CBPC from poliovirus-infected cells had undergone major structural alterations. The 220-kDa component was absent; antigenically related (100-130 kDa) degradation products were present instead. The 24-kDa component co-purified with the p220 degradation products, but other components were missing. The association of the infected cell CBPC components was quite labile compared with that demonstrated by the components of CBPC from uninfected cells. Differential stimulation of capped, but not uncapped mRNAs in a cell-free translation assay was demonstrated by unmodified CBPC. Conversely, modified CBPC from poliovirus-infected cells differentially stimulated in vitro translation of uncapped poliovirus mRNA but not capped mRNAs. The implications of these results for the mechanism of cap-independent translation are briefly discussed.

Carrier Proteins↗
PubMed1987

Formation of poliovirus RNA polymerase 3D in Escherichia coli by cleavage of fusion proteins expressed from cloned viral cDNA.

The poliovirus polymerase 3D was synthesized in Escherichia coli by cleavage of fusion proteins expressed from cloned viral cDNA inserted into several plasmid expression vectors. Cleavage was accomplished by the action of viral protease 3C sequences expressed in the same bacteria, either from a second plasmid or from the same plasmid, cloned so as to produce contiguous sequences in the same protein. In the case of two plasmids, protease 3C functioned in trans to cleave the fusion protein at or very near the normal Gln/Gly cleavage site. When protease and polymerase sequences were produced in the same protein, the protease sequences acted in the precursor form to release the polymerase from itself. Thus, cleavage can occur to generate polymerase 3D both as an intermolecular reaction and, very likely, also as an intramolecular event.

Cloning, Molecular↗
PubMed1987

Host factor-induced template modification during synthesis of poliovirus RNA in vitro.

Poliovirus RNA polymerase, 3Dpol, transcribes poliovirus RNA in vitro in the presence of a host factor (HF) preparation from uninfected HeLa cells to yield heterogeneous-size product RNAs. The products include some molecules larger than the template that represent self-primed elongations of the template from a 3'-terminal hairpin. We showed that transcription proceeded through the formation of a modified RNA intermediate that was generated by nucleolytic cleavage of the template by HF in the absence of nucleoside triphosphates. Cleavage resulted in the loss of the original poly(A) 3' end and the generation of new, heterogeneous 3' ends that formed self-priming structures that could then be elongated by 3Dpol or reverse transcriptase. The two stages of the reaction, (i) cleavage to yield self-priming templates and (ii) subsequent chain elongation to yield heterogeneous-size products up to nearly dimer length, could be separated. RNAs whose original 3' ends were chemically oxidized so as to prevent chain elongation showed no reduction in template activity after preincubation with HF. We conclude that an HF preparation that contains a low level of nuclease activity is sufficient to activate RNA templates for transcription by 3Dpol to generate up to apparent dimer-length products. This reaction likely has little relevance to the mechanism of poliovirus RNA replication in vivo. It is likely that numerous other factors or activities, in addition to 3Dpol, would also result in transcription of poliovirus RNA in vitro.

Carrier Proteins↗
PubMed1987

Poliovirus snapback double-stranded RNA isolated from infected HeLa cells is deficient in poly(A).

A portion of poliovirus double-stranded RNA (25 to 50%) isolated from infected HeLa cells contains hairpin loops at one end of the duplex structure. These structures rapidly reformed double-stranded molecules after denaturation and appeared as molecules of up to two times genome length upon electrophoresis in denaturing agarose gels. A second form of poliovirus double-stranded RNA was readily denaturable into genome length strands. When the hairpin RNA was treated with S1 nuclease, subsequent denaturation resulted in formation of strands of up to genome length. Hairpin molecules contained very little, if any, poly(A) sequences, suggesting that the hairpin forms after nucleolytic removal of the 3' end of plus-strand templates. We conclude that the hairpin double-stranded RNA found in infected cells is likely generated by intracellular nicking and self-priming and that it does not represent an intermediate in the process of RNA replication.

HeLa Cells↗
PubMed1987

Restriction of translation of capped mRNA in vitro as a model for poliovirus-induced inhibition of host cell protein synthesis: relationship to p220 cleavage.

Poliovirus infection of HeLa cells results in a rapid inhibition of host protein synthesis by a mechanism that does not affect the translation of poliovirus RNA. It has been suggested that this virus-induced translational control results from inactivation of the cap-binding protein complex, and it has been shown that the 220-kilodalton component(s) (p220) of the cap-binding protein complex is cleaved in infected HeLa cells to form antigenically related polypeptides of 100 to 130 kilodaltons. We have previously described an activity in infected cells that specifically restricts translation of capped mRNA in rabbit reticulocyte lysates. Here, we describe further refinements and characterization of restriction assay. We determined that the assay is a good in vitro model for study of host cell shutoff by several criteria: (i) translation was inhibited in both instances at the step involving mRNA binding to ribosomes; (ii) translation of capped mRNA was specifically inhibited, whereas translation of poliovirus RNA was not; (iii) restriction activity appeared in infected cells with kinetics which parallel host cell shutoff; and (iv) restriction activity, like the specific inhibition of host translation, appeared in cells infected in the presence of guanidine-HCl. The restricting activity was partially purified from poliovirus-infected cells and was compared with the virus-induced p220 cleavage activity. Both activities copurified through numerous cell fractionation and biochemical fractionation procedures. However, specific restriction of capped mRNA translation in reticulocyte lysates occurred without complete cleavage of the endogenous p220.

Animals↗
PubMed1986

Two-dimensional gel analyses of the 24-kDa cap binding protein from poliovirus-infected and uninfected HeLa cells.

The 24-kDa cap binding protein (CBP) from uninfected, mock-infected and poliovirus-infected HeLa cell extracts was isolated by m7GTP affinity chromatography and examined by isoelectric focusing followed by SDS-PAGE. Two major species (pI 6.7 and 7.1) and two minor species (pI 6.5 and 6.8) were found in all cases. Preparations from postribosomal supernate (S200) and the supernate from 0.5 M KCl washed ribosomes (RSW) also demonstrated these same four species. We conclude that there are no detectable differences between ribosome-associated and soluble 24-kDa CBP and that the 24-kDa CBP is not detectably altered by poliovirus infection. We also report the presence of a previously undescribed 16-kDa polypeptide(s) doublet that copurifies with the 24-kDa CBP from uninfected, mock-infected and poliovirus-infected HeLa cells.

Carrier Proteins↗
PubMed1986

Cleavage of the cap binding protein complex polypeptide p220 is not effected by the second poliovirus protease 2A.

Poliovirus protein 2A contains a short amino acid sequence that also occurs in the putative active site of the known viral proteinase, 3C, previously shown to be responsible for glutamine/glycine cleavages in the poliovirus polyprotein precursor. Experimental evidence indicates that 2A is a second viral proteinase that mediates the cleavage of two tyrosine/glycine cleavages in the generation of virus-specific proteins. Since poliovirus inhibition of host cell protein synthesis correlates with the specific cleavage of the 220,000-Da component of the cap binding protein complex, we have tested whether viral protein 2A contains the p220 cleavage activity. The results show that 2A does not copurify with p220 cleavage activity, partially purified fractions containing high p220 cleavage activity contain no detectable 2A sequences in the form of either mature or precursor protein, and anti-2A serum or IgG does not inhibit p220 cleavage in vitro.

Amino Acid Sequence↗