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E Ehrenfeld

Publications and source records attributed to E Ehrenfeld.

At least 37 records · Page 2Linked to original sources
PubMed1995

Identification of nucleotide binding sites in the poliovirus RNA polymerase.

Poliovirus RNA polymerase (3Dpol) was cross-linked to [32P]ribonucleoside triphosphates (NTPs) by reduction of oxidized NTP-protein complexes. Cross-linked complexes were digested with cyanogen bromide, and resulting peptides were fractionated by reverse-phase HPLC. 32P-Labeled peptides were purified by secondary HPLC fractionation and/or additional digestion with endoproteinases Glu-C, TPCK-trypsin, or Asp-N followed by another HPLC fractionation. N-Terminal sequences of the major [32P]-peptides were determined, and approximate sizes of these peptides were obtained by SDS-polyacrylamide gel electrophoresis. Two major NTP binding sites in 3Dpol were found. One site was between Asp-266 and Met-286; possible binding residues in this fragment were Lys-276, Lys-278, or Lys-283. A second binding site was between Ala-57 and Met-74 with Lys-61 or Lys-66 as possible binding residues. Alignment of these regions on the known structure of HIV-1 reverse transcriptase allowed us to predict the position of the downstream nucleotide binding site in the conserved "fingers" subdomain present near the active site cleft of both RNA and DNA polymerases. The N-terminal nucleotide binding site is not contained within a region that is conserved among other polymerases.

Amino Acid Sequence↗
PubMed1995

Antibodies against viral nonstructural proteins in response to infection with poliovirus.

Sera from recent and past cases of poliomyelitis as well as from healthy subjects vaccinated with live attenuated poliovirus, all contain antibodies to poliovirus nonstructural proteins. Reactivity of these antisera with nonstructural protein antigens present in virus-infected HeLa cells or translated from cDNA transcripts in rabbit reticulocyte lysates was sometimes sensitive to denaturation of the antigens. The finding of antibodies to nonstructural proteins of poliovirus and other viruses should have applications in the development of diagnostic assays for virus infection and may have implications for future vaccine design.

Antibodies, Viral↗
PubMed1995

Elongation activity of poliovirus RNA polymerase derived from Sabin type 1 sequence is not temperature sensitive.

Determinants of attenuation in the Sabin type 1 strain of poliovirus are located in the 5' noncoding region, the capsid coding region and the viral RNA-dependent RNA polymerase (3Dpol) coding region. These mutations also contribute to a temperature sensitive pheno-type of virus replication. We have cloned and expressed the Sabin 1 virus 3Dpol) protein which contains three amino acid differences from the wild-type (Mahoney) sequence, as well as a wild- type polymerase containing only a single Sabin amino acid substitution at nt 6203. These enzymes have been examined and compared for temperature sensitive polymerase activity. Wild-type and mutated polymerases demonstrated identical specific activities at 30, 35 and 39 degrees C. All three showed the same kinetics of heat inactivation after pre-incubation at elevated temperatures. Thus the contribution of Sabin 3Dpol sequences to the inability of the virus to grow at elevated temperatures must lie in a function or activity of the enzyme other than RNA polymerization. A likely reaction is the initiation step of RNA chain synthesis.

Cloning, Molecular↗
PubMed1995

Inefficient complementation activity of poliovirus 2C and 3D proteins for rescue of lethal mutations.

Poliovirus (PV) 2C protein is a nonstructural polypeptide involved in viral RNA replication, whose biochemical activity(ies) in this process has not been defined. By using site-directed mutagenesis, it was shown previously that disruption of nucleotide-binding motifs present in this protein abolished viral RNA synthesis (C. Mirzayan and E. Wimmer, Virology 189:547-555, 1992; N. L. Teterina, K. M. Kean, E. Gorbalenya, V. I. Agol, and M. Girard, J. Gen. Virol. 73:1977-1986, 1992). We have tested whether PV 2C or 2BC protein provided in trans could rescue the replication of these mutated genomes. Rescuing proteins were provided either by cotransfection with helper chimeric PV-coxsackievirus genomes or by expression in cells with a vaccinia virus-T7 RNA polymerase transient-expression system. We report here that replication of mutated RNAs genomes was poorly supported in trans both by helper genomes and by expressed 2C or 2BC proteins. Similarly, very inefficient complementation was observed for two mutated genomes with lethal lesions in 3D polymerase coding sequence. Our results indicate that poliovirus RNA replication shows marked preference for proteins contributed in cis.

Amino Acid Sequence↗
PubMed1995

Host cell proteins binding to domain IV of the 5' noncoding region of poliovirus RNA.

Translation of poliovirus RNA occurs by the binding of ribosomes to an internal segment of RNA sequence within the 5' untranslated region of the viral RNA. This region is predicted to consist of six domains (I to VI) that possess complex secondary and tertiary structures. Domain IV is a large region in which alterations in the sequence or structure markedly reduce translational efficiency. In this study, we employed RNA mobility shift assays to demonstrate that a protein(s) from uninfected HeLa cell extracts, as well as from neuroblastoma extracts, interacts with the domain IV structure. A mutation in domain IV caused reduced binding of HeLa cell proteins and reduced translation both in vitro and in vivo, suggesting that the binding of at least one of these proteins plays a role in the mechanism of viral translation. UV cross-linking indicated that a protein(s) with a size of approximately 40 kDa interacted directly with the RNA. Using streptavidin beads to capture biotinylated RNA bound to proteins, we were able to visualize a number of HeLa and neuroblastoma cell proteins that interact with domain IV. These proteins have molecular masses of approximately 39, approximately 40, and approximately 42 kDa.

Animals↗
PubMed1995

Hepatitis A viruses with deletions in the 2A gene are infectious in cultured cells and marmosets.

The 2A gene of hepatitis A virus (HAV) bears no obvious similarity to the corresponding genes of other picornaviruses and has no known function. In a preliminary effort to gain information about the HAV 2A gene product, we constructed several HAV cDNAs containing deletions of 30 or 45 nucleotides in the predicted central portion of the 2A gene. These deletions did not affect the sites of protein processing, although the rates or efficiencies of polyprotein cleavage at the surrounding cleavage junctions appeared slightly reduced. Transfection of FRhK-4 cells with RNA transcripts of the deleted HAV cDNAs generated small foci of infected cells and produced infectious virus that retained the deletion mutations. In contrast, a single amino acid insertion in the 2B coding region was lethal to virus replication despite normal protein processing. Another deletion, which included the predicted 2A/2B junction and extended into the 2B coding sequence, did not support polyprotein processing or generate viable virus. One of the viable internal 2A deletions was introduced into a wild-type HAV cDNA background, and transcripts were tested for infectivity by inoculation directly into the livers of two marmosets. Both animals seroconverted, displayed elevated serum liver enzymes, and excreted infectious virus. Thus, deletion of 10 or 15 amino acid residues from the predicted central portion of the 2A protein was tolerated with only relatively minor effects on the growth of HAV in cultured cells and in marmoset liver.

Animals↗
PubMed1994

Expression of hepatitis A virus precursor protein P3 in vivo and in vitro: polyprotein processing of the 3CD cleavage site.

Hepatitis A virus (HAV) cDNAs encoding the P3 region proteins were expressed in vivo and in vitro to characterize the HAV 3D protein and to identify the cleavage site between 3C and 3D. Protein coding sequences were placed under control of a T7 promoter and an EMCV translational initiation signal. T7 RNA polymerase was provided by simultaneous infection of transfected BS-C-1 cells with a recombinant vaccinia virus vTF7-3 (T. R. Fuerst et al., Proc. Natl. Acad. Sci. USA 83, 8122-8126, 1986). Efficient synthesis and processing of P3 proteins occurred to yield 3CD (78 kDa), 3D (54 kDa), 3ABC (33 kDa), 3BC (25 kDa), and 3C (23 kDa). Similar products were produced by translation of T7 transcripts in a rabbit reticulocyte lysate in vitro. The 3C/D cleavage site was mapped by comparing the mobility of 3D in SDS-PAGE with 3D proteins engineered to begin at each of the two proposed cleavage sites; in addition, direct N-terminal sequencing of radiolabeled 3D protein from translation in vitro was performed. The results showed that 3D was formed by cleavage at the glutamine-arginine (Q/R) pair at position 1738 and 1739 of the HAV polyprotein. HAV 3D protein produced by autocatalytic cleavage of P3 precursor proteins in BS-C-1 cells is virtually completely insoluble and sediments after low-speed centrifugation. This is in contrast to the poliovirus 3D protein, produced from a similar construct, a significant portion of which remains soluble. Extracts containing the poliovirus 3D protein manifested high levels of RNA-dependent RNA polymerase activity, whereas those containing the HAV 3D protein showed no detectable activity by the same assay.

3C Viral Proteases↗
PubMed1994

Membrane rearrangement and vesicle induction by recombinant poliovirus 2C and 2BC in human cells.

Poliovirus (PV)-infected cells undergo extensive proliferation and rearrangement of intracellular smooth membranes to generate vesicles on which viral RNA replication occurs. PV proteins 2C and 2BC are known to be tightly associated with these membranous replication complexes and have been proposed to be involved in the formation of these virus-induced vesicles. We have expressed these proteins, and proteins with mutations in the putative nucleotide (NTP) binding motifs, in human cells using recombinant vaccinia viruses and T7 RNA polymerase-directed transcription. To ascertain the subcellular localization properties of these proteins in the absence of other PV proteins and to determine whether they induced ultrastructural changes, cells expressing 2C and 2BC proteins were examined by immunofluorescence (IF) microscopy, electron microscopy (EM), and immuno-EM (IEM). The cytoplasm of cells expressing either 2C or 2BC exhibited vesicles of 50-350 nm in diameter, which resembled those found in PV-infected cells. Both 2C and 2BC were associated with these vesicles. Mutations in the putative NTP binding motif did not affect vesicle induction by 2C or 2BC. Despite the membrane reorganization and vesicle formation induced by 2C and 2BC proteins, no enhanced synthesis of lipid was observed. Guanidine hydrochloride at a concentration that inhibits PV replication, did not have significant effects on the IF patterns of either 2C or 2BC. An additional prominent alteration in cells expressing 2C, but not 2BC, was the formation of extensive tubular membrane structures with a myelin-like arrangement in the lumen of the rough endoplasmic reticulum. IEM analyses showed that 2C was associated with these structures. In the presence of other PV proteins, the tubular membrane structures induced by 2C were not detected. These structures are not observed in poliovirus-infected cells, but likely indicate a novel property of 2C that induces a complex interaction with intracellular membranes.

Amino Acid Sequence↗
PubMed1994

Interaction of cellular proteins with the poliovirus 5' noncoding region.

The 5' noncoding region (NCR) of poliovirus RNA is folded into a complex structure comprised of multiple, critically spaced, stem-loop domains. Mutations in at least one of these domains markedly affects the neurovirulence of the virus. Two proteins have been identified recently which bind and apparently mediate functions of the 5' NCR in translation. We have demonstrated specific binding of three additional proteins in a Hela cell ribosomal salt wash that can be crosslinked to specific stem-loop segments of the 5' NCR. These same RNA segments inhibit translation of polio RNA in vitro, presumably by competing for protein binding. The Sabin vaccine strain of polio RNA exhibits a reduced affinity of binding for specific proteins. The determinant for this reduction appears to be a single nucleotide difference at position 480 between the neurovirulent and attenuated viral strains.

Binding, Competitive↗
PubMed1994

Identification of terminal adenylyl transferase activity of the poliovirus polymerase 3Dpol.

A terminal adenylyl transferase (TATase) activity has been identified in preparations of purified poliovirus RNA-dependent RNA polymerase (3Dpol). Highly purified 3Dpol is capable of adding [32P]AMP to the 3' ends of chemically synthesized 12-nucleotide (nt)-long RNAs. The purified 52-kDa polypeptide, isolated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renatured, retained the TATase activity. Two 3Dpol mutants, purified from Escherichia coli expression systems, displayed no detectable polymerase activity and were unable to catalyze TATase activity. Likewise, extracts from the parental E. coli strain that harbored no expression plasmid were unable to catalyze formation of the TATase products. With the RNA oligonucleotide 5'-CCUGCUUUUGCA-3' used as an acceptor, the products formed by wild-type 3Dpol were 9 and 18 nt longer than the 12-nt oligomer. GTP, CTP, and UTP did not serve as substrates for transfer to this RNA, either by themselves or when all deoxynucleoside triphosphates were present in the reaction. Results from kinetic and stoichiometric analyses suggest that the reaction is catalytic and shows substrate and enzyme dependence. The 3'-terminal 13 nt of poliovirus minus-strand RNA also served as an acceptor for TATase activity, raising the possibility that this activity functions in poliovirus RNA replication. The efficiency of utilization and the nature of the products formed during the reaction were dependent on the acceptor RNA.

Base Sequence↗
PubMed1993

Antibody response to nonstructural proteins of hepatitis A virus following infection.

The nonstructural proteins of hepatitis A virus (HAV), produced during active virus replication, are alternative antigens that could be used to differentiate disease from inactivated vaccine-induced antibodies. An assay based on immune precipitation of proteins translated from transcripts of the P2 region of viral cDNA was used to evaluate the development of antibodies after natural infection or vaccination. Antibodies against P2 proteins were found in all sera from clinical cases of hepatitis A following the acute phase. Chimpanzees vaccinated with inactivated or cell-adapted HAV had no detectable antibodies against P2 products, either before or after wild type virus challenge. A serosurvey of sera positive for total anti-HAV (HAVAB, Abbott Laboratories, North Chicago) suggested that some individuals had no detectable antibodies to the P2 antigen by immune precipitation. These results were attributed to the lower sensitivity of the immunoprecipitation assay, since antibodies to capsid proteins, as measured by immunoprecipitation, were also not detected in most of these sera.

Adult↗
PubMed1993

Primary cleavage of the HAV capsid protein precursor in the middle of the proposed 2A coding region.

Portions of the P1 and P2 hepatitis A virus (HAV) polyprotein were generated by in vitro translation of cDNA transcripts and analyzed for a primary cleavage reaction that would release the capsid protein precursor. No autocatalytic activity was observed on either side of the 2A coding sequence. Incubation of these proteins with an extract containing active HAV 3C protease, however, resulted in cleavage at a position approximately 40 amino acids downstream of the previously proposed P1-2A junction. This cleavage site likely accounts for the VP1-containing proteins of approximately 38-40 kDa observed in HAV-infected cells.

3C Viral Proteases↗
PubMed1993

Analysis of a potential myristoylation site in hepatitis A virus capsid protein VP4.

The VP4 capsid protein of several picornaviruses has been shown to be myristoylated at an N-terminal glycine residue. Myristoylation occurs after removal of an initial methionine residue or a leader peptide, resulting in the exposure of an N-terminal eight amino acid myristoylation signal including a consensus G-x-x-x-T/S motif. Analysis of the amino acid sequence of hepatitis A virus (HAV) capsid protein reveals a potential myristoylation site beginning at position 5 of the VP4 sequence. To assess the significance of this apparent myristoylation signal, mutations were engineered (G to A; T to N) to alter the consensus sequence as well as the potential cleavage site that would be required to remove the short leader. An additional mutant was constructed in which the proposed leader was deleted. Expression of these HAV sequences in BS-C-1 cells showed that leader cleavage did not occur in the wild-type or mutant proteins, although the threonine to asparagine mutation resulted in reduced translation and processing efficiency. Transfection of BS-C-1 or FRhK-4 cells with transcripts derived from the wild-type or mutagenized cDNA clones gave rise to infectious virus, with no detectable incorporation of myristate. The results indicate that HAV does not require leader cleavage and myristoylation of VP4 for growth in cultured cells.

Animals↗
PubMed1993

RNA duplex unwinding activity of poliovirus RNA-dependent RNA polymerase 3Dpol.

The ability of highly purified preparations of poliovirus RNA-dependent RNA polymerase, 3Dpol, to unwind RNA duplex structures was examined during a chain elongation reaction in vitro. Using an antisense RNA prehybridized to an RNA template, we show that poliovirus polymerase can elongate through a highly stable RNA duplex of over 1,000 bp. Radiolabeled antisense RNA was displaced from the template during the reaction, and product RNAs which were equal in length to the template strand were synthesized. Unwinding did not occur in the absence of chain elongation and did not require hydrolysis of the gamma-phosphate of ATP. The rate of elongation through the duplex region was comparable to the rate of elongation on the single-stranded region of the template. Parallel experiments conducted with avian myeloblastosis virus reverse transcriptase showed that this enzyme was not able to unwind the RNA duplex, suggesting that strand displacement by poliovirus 3Dpol is not a property shared by all polymerases.

Adenosine Triphosphate↗
PubMed1992

Nucleotide binding by the poliovirus RNA polymerase.

Cross-linking of ribonucleoside triphosphates (NTPs) to specific binding sites on the poliovirus RNA-dependent RNA polymerase has been performed by ultraviolet irradiation and by reduction of oxidized nucleotide-protein complexes. The latter method approached a cross-linking efficiency of 1 NTP/molecule of enzyme. Nucleotide competition experiments suggested that the same binding site is occupied by all NTPs. Analysis of peptides produced by proteinase Glu-C and trypsin digestion and labeled with [32P]GTP indicated that a lysine residue between Met-189 and Lys-228 in the polymerase was cross-linked to NTP. Nucleotide binding was exploited for rapid purification of the enzyme by GTP-agarose affinity chromatography. In addition, a set of cloned, modified polymerase molecules with reduced or absent polymerization activity was analyzed for binding efficiency to a GTP-agarose column. Some mutations eliminated GTP binding, whereas others generated proteins with varying affinities for GTP. Incubation of the poliovirus polymerase with high concentrations of NTP, particularly GTP, resulted in a dramatic protection against heat denaturation and activity loss. These data suggest that nucleotide binding results in an alteration of the enzyme conformation or the stabilization of an ordered conformation.

Autoradiography↗
PubMed1992

Hepatitis A virus polyprotein synthesis initiates from two alternative AUG codons.

The genomic RNA of hepatitis A virus has two potential translation initiation sites for synthesis of a 251-kDa polyprotein. It is not known which of these AUG codons, located at positions 735-737 and 741-743, is used in vitro or in vivo. Site-directed mutagenesis was carried out to eliminate each start codon independently. Transcripts from the unmodified and modified cDNA clones were used either to program an in vitro translation system or for transfection of BS-C-1 cells. In vitro and in vivo translation data revealed preferential usage of the downstream AUG located at position 741 to 743, although either site could be utilized in the absence of the other. Both modified RNAs were able to induce productive infections in BS-C-1 cells. Deletion of almost all of the 5'-untranslated region (5'UTR) of the RNA, however, stimulated selection of AUG 735-737 in vitro resulting in equal utilization of both sites, suggesting a strong influence of the 5'UTR for directing the ribosome to a specific internal initiation site.

Animals↗