Biomedical subjects

D F Summers

Publications and source records attributed to D F Summers.

At least 19 recordsLinked to original sources

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↗

PubMed1992

Influenza A virus NP protein expressed in insect cells by a recombinant baculovirus is associated with a protein kinase activity and possesses single-stranded RNA binding activity.

Influenza A virus NP protein, the phosphoprotein associated with viral RNA in ribonucleoprotein (RNP) complexes, has been expressed at high levels (approximately 100 mg/liter cells) in insect (Sf9) cells by a baculovirus recombinant, and was localized almost entirely in the nuclei of these cells. NP was purified by immuno-affinity chromatography, and purified NP was shown to autophosphorylate and to phosphorylate casein in a cAMP-independent reaction. Furthermore, purified NP was able to bind to ssRNA as demonstrated by a mobility shift of ssRNA in non-denaturing gels. The binding of NP to ssRNA caused a diminution of its kinase activity in proportion to binding.

Animals↗

PubMed1992

A cis-acting element within the hepatitis A virus 5'-non-coding region required for in vitro translation.

Every picornavirus studied thus far has a sequence within the 5'-non-coding region that is required for internal ribosome binding and translation of the polyprotein. In an attempt to identify this region in hepatitis A virus we constructed a truncated hepatitis A virus (HAV) cDNA clone that contains the entire 736 bp 5' non-coding region (5'-NCR) and 754 base pairs of the viral capsid coding region (P1) under control of the SP6 promoter. In vitro transcription and translation of this transcript in a rabbit reticulocyte lysate yielded a protein product of about 29 kDa as analyzed by autoradiography following sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). A series of mutations of this construct have defined a minimal sequence between bases 347 and 734 in the 5'-NCR that is required for efficient in vitro translation. The deleted constructs (D 523-734 and D 632-734) showed a reduced ability to translate in the rabbit reticulocyte lysate system in comparison with the full-length 5'-NCR construct, pH1489. The translation of these deleted constructs was artificially restored by the addition of a 5'-terminal methylated cap structure, m7GpppG, to the RNA. This increase in translational efficiency could be competed away with cap analog (m7GDP) thus indicating that this region is required for cap-independent internal ribosome binding for HAV translation.

Base Sequence↗

PubMed1992

Serological approaches to distinguish immune response to hepatitis A vaccine and natural infection.

Currently, the immune status of an individual exposed to hepatitis A virus (HAV) is determined by assays which measure antibodies against the capsid proteins. These assays indicate exposure to the viral capsid that could result from either infection or from vaccination. Recent data indicate that proteins from the non-structural genome region of the virus (P2 or P3), which are only produced during active virus replication, generate antibodies after clinical disease. A sub-genomic cDNA segment of HAV corresponding to the P2 region was used for in vitro transcription-translation followed by immune precipitation of the translated products under non-denaturing conditions. Serial serum specimens from experimentally infected chimpanzees and humans naturally infected with hepatitis A verified the development of antibodies to P2 proteins following infection. A serosurvey of individuals positive for antibodies to the HAV capsid (HAV AB assay, Abbott Laboratories) revealed that 50-60% of children and 16-32% of adults had no detectable antibodies to the P2 antigen by immune precipitation. These results may reflect subclinical infections resulting in a lower level of antibodies against the non-structural antigens or may represent a greater sensitivity of the competitive assay (HAV AB) used to detect capsid antibodies compared to the immunoprecipitation assay used to detect non-structural antigens.

Adult↗

PubMed1992

Host antibody response to viral structural and nonstructural proteins after hepatitis A virus infection.

Subgenomic hepatitis A virus (HAV) RNA sequences were translated in vitro to produce proteins representing the structural (P1) and nonstructural (P2 and P3) domains of the viral polyprotein. These proteins were used as antigens to detect the presence of antibodies in sera from acute and convalescent humans and an experimentally infected chimpanzee. All infected individuals tested had antibodies that recognized uncleaved P1 proteins as well as nonstructural proteins. Antibodies in sera from infected individuals recognized conformation-dependent epitopes that were sensitive to SDS and heat treatment. Time-course studies of the experimentally infected chimpanzee showed that antibodies to the HAV proteins were detectable between 24 and 31 days after infection and persisted for greater than 6 months. Human sera remained positive for antibodies to both structural and nonstructural antigens for at least 2 1/2 years. The data suggest that HAV nonstructural proteins could be used as serologic markers for HAV diagnosis and for evaluating field trials of inactivated vaccines.

Animals↗

PubMed1992

Polyprotein processing in cis and in trans by hepatitis A virus 3C protease cloned and expressed in Escherichia coli.

To determine the P3 region protein-processing sites cleaved by the hepatitis A virus 3C protease, a nested set of constructs containing a portion of 3A (3A* [the asterisk denotes an incomplete protein]), 3B and 3C and various amounts of 3D, fused in frame to Escherichia coli TrpE-coding sequences under control of the tryptophan promoter, was made. Additional plasmids that encoded a portion of 2C (2C*) and the P3 proteins, including complete or incomplete 3D sequences, were constructed. After induction, E. coli containing these recombinant plasmids produced high levels of fusion proteins as insoluble aggregates. 3C-mediated cleavage products were identified by comparison of expression with a matching set of plasmids, containing an engineered mutation in 3C. Cleavage products were detected by immunoblot analyses by using antisera against the TrpE protein, against 3D*, and against 3CD*. Scissile bonds were determined by N-terminal amino acid sequencing of the proteins formed by cleavage. The results showed that when a portion of 2C was present, the primary cleavage by the 3C protease was between 2C and 3A, and the cleavage site was QG, as predicted by J. I. Cohen, J. R. Ticehurst, R. H. Purcell, A. Buckler-White, and B. M. Baroudy, J. Virol. 61:50-59, 1987. Very little further cleavage of the released P3 protein was detected. When the fusion protein contained no 2C and included only 3A*-to-3D sequences, efficient cleavage occurred between 3B and 3C, at the QS pair, also as predicted by Cohen et al. (J. Virol. 61:50-59, 1987). The latter proteins were also cleaved between 3C and 3D, but less efficiently than between 3B and 3C. Extracts of bacteria expressing proteins from 3A* to 3D also cleaved a radiolabelled hepatitis A virus substrate containing VP1*2ABC* sequences in trans.

3C Viral Proteases↗

PubMed1991

Influenza A virus in vitro transcription: roles of NS1 and NP proteins in regulating RNA synthesis.

To study the mechanisms by which the influenza A virus RNA-dependent RNA polymerase switches from transcription to replication we have devised a riboprobe protection technique with which we analyzed the 3' end sequence of (+)-strand RNA products of an in vitro transcription reaction containing purified virion-RNP complexes in the presence and the absence of the putative regulatory proteins NP and NS1. We found that the addition of these proteins did not result in the synthesis of full-length (+)-strand RNA products resulting from read-through of the polyadenylation signal or replication. Because NS1 and NP are both phosphoproteins we searched for protein kinase activity that might play a role in regulating RNA synthesis. We showed that virion RNP complexes phosphorylated NS1 but possessed no autophosphorylating activity. Soluble NP protein derived from RNP complexes did not phosphorylate NS1, but did phosphorylate casein. When NP protein was dephosphorylated, however, it no longer phosphorylated casein. We also showed that NS1 was an ssRNA-binding protein which binds nonspecifically to all ssRNA, and that this activity is not dependent on its state of phosphorylation.

Animals↗

PubMed1991

Proteolytic activity of hepatitis A virus 3C protein.

Although the genome organization and overall structure of hepatitis A virus are similar to those of other picornaviruses, nothing is known about the protein-processing pathways used by this virus to generate its capsid and nonstructural proteins from the polyprotein precursor. RNA transcripts of cloned hepatitis A virus cDNAs representing parts of the P2 and P3 regions of the genome were translated in rabbit reticulocyte lysates in vitro, and the translation products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis before and after immunoprecipitation with specific antisera. Pulse-chase experiments demonstrated rapid cleavage at the P2-P3 junction, followed by further but incomplete processing at the 3C-3D junction. Mutation of the 3C coding sequence eliminated all cleavages. Efforts to demonstrate intermolecular cutting of the P2-P3 cleavage site by active 3C or 3CD sequences were unsuccessful; thus, it is likely that this cleavage occurs by intramolecular reaction, in cis.

3C Viral Proteases↗

PubMed1991

The 5'-terminal nucleotides of hepatitis A virus RNA, but not poliovirus RNA, are required for infectivity.

A series of plasmids containing hepatitis A virus (HAV) cDNA was constructed such that positive-strand HAV RNA could be transcribed with T7 RNA polymerase. The plasmids differed in the number of 5'-terminal nucleotides representing the junctions between vectors and HAV sequences that were present in the transcripts. When these transcripts were used to transfect cultured BS-C-1 cells, it was found that only those transcripts that contained all of the 5'-terminal HAV nucleotides, in addition to one or more nucleotides from the vector, were capable of initiating an infectious cycle leading to production of progeny virus. Transcripts that contained one 5'-terminal nucleotide from the vector sequence but were missing two uridylate residues corresponding to the first two nucleotides of HAV sequences, or were missing U and C residues corresponding to nucleotides 2 and 3 of the HAV sequence, were not infectious. A similar plasmid containing poliovirus cDNA was engineered to produce transcripts similarly lacking the first two uridylate residues of the poliovirus RNA sequence. These transcripts were infectious.

Antigens, Viral↗

PubMed1990

A minor microtubule-associated protein is responsible for the stimulation of vesicular stomatitis virus transcription in vitro.

An activity found in bovine brain microtubule-associated proteins (MAPs) which stimulated vesicular stomatitis virus (VSV) transcription in vitro co-eluted from a gel filtration column (Sepharose CL-6B) with a minor MAP subset of an apparent Mr of 100,000 to 250,000. The activity did not appear to be closely associated with MAPs 1, MAPs 2, tau or tubulin. Anti-MAPs 1 and anti-MAPs 2 IgG did not reduce or abolish the stimulatory activity. The bovine brain MAPs stimulatory activity was similar to that found in HeLa cell extracts: both were heat-resistant, and both co-purified with the MAPs fraction of cell extracts; also amounts of each which gave maximum stimulation when used separately did not give additional stimulation when combined. Fractions from the Sepharose CL-6B column that contained the greatest amount of stimulatory activity exhibited little or no cAMP-dependent or -independent kinase activity. The MAPs stimulatory activity required the presence of both L and NS polymerase subunits.

Animals↗

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↗

PubMed1988

Preparative elution of proteins blotted to Immobilon membranes.

Conditions for the preparative elution of proteins from Immobilon membranes after transfer of proteins to this matrix from sodium dodecyl sulfate-polyacrylamide gels have been established. Proteins were completely eluted from the membrane at room temperature by short incubation in 50 mM Tris-HCl, pH 9.0, containing 2% SDS and 1% Triton X-100. Good protein recoveries were also obtained in the same buffer containing 1% Triton X-100 only. The efficiency of elution was practically independent of the molecular weight of proteins, the method allowed for the precise excision of protein bands, and the proteins eluted from the matrix were not degraded. In some cases it was possible to recover enzymatic activity of the eluted proteins.

Electrophoresis, Polyacrylamide Gel↗

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

Purification, thioredoxin renaturation, and reconstituted activity of the three subunits of the influenza A virus RNA polymerase.

The virion-associated RNA polymerase and the structural nucleoprotein of influenza A virus were separated by sodium dodecyl sulfate/PAGE, electroblotted to a polyvinylidine membrane, and eluted with good recovery from the membrane. After renaturation by incubating with Escherichia coli thioredoxin, these proteins were active in a reconstituted in vitro transcription reaction with purified genomic RNAs. All four proteins (i.e., the three subunits of the RNA polymerase as well as the structural nucleoprotein) were required for activity. The RNA products were plus-strand, mRNA-sized species.

Bacterial Proteins↗

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↗

PubMed1987

Fluorescent staining of proteins transferred to nitrocellulose allowing for subsequent probing with antisera.

A sensitive staining method for protein blots on nitrocellulose is described. It is based on the coupling of a fluorochrome, dichlorotriazynylaminofluorescein, to protein which yields products colorless in visible light but colored when protein blots are illuminated with long-range ultraviolet light. The coupling of a fluorochrome does not affect the antigenic properties of proteins and the stained blots can be subsequently probed with antisera. Thus, the method allows for the unambiguous identification of antigenic proteins transferred to nitrocellulose from sodium dodecyl sulfate-polyacrylamide gels.

Antibodies↗

PubMed1986

Stimulation of vesicular stomatitis virus in vitro RNA synthesis by microtubule-associated proteins.

Microtubule-associated proteins purified from bovine brains stimulated the in vitro transcription and replication reactions of vesicular stomatitis virus. The products of these reactions were intact messenger or genome-sized RNA species. A preparation from HeLa cells containing tubulin and microtubule-associated proteins also stimulated vesicular stomatitis virus transcription in vitro. This observation is in accord with previous studies, which suggested that a host cell factor was involved with the function of the vesicular stomatitis virus RNA polymerase, and others that indicated that several animal viruses displayed an association with host cell cytoskeletal elements during their replication cycles. We show evidence in this report of a host cell protein that seems to have a functional role in interacting with the virion polymerase.

Animals↗

PubMed1985

Ultrastructural localization of L and NS enzyme subunits on vesicular stomatitis virus RNPs using gold sphere-staphylococcal protein A-monospecific IgG conjugates.

Colloidal gold spheres were coated with staphylococcal protein A and were used to determine the location of NS and L proteins on vesicular stomatitis virus (VSV) ribonucleoprotein (RNP) complexes using monospecific anti-NS and anti-L IgG preparations. Conjugates using either anti-NS or anti-L demonstrated that these enzyme subunits were uniformly distributed along the entire length of the RNP complex. Under saturating conditions of IgG concentrations, it was observed that there were at least 60-70 molecules of NS protein and 30-35 molecules of L protein labeled per RNP complex.

HeLa Cells↗