Abortive adenovirus infection and host range determinants.
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Adenoviruses, Human↗
Biomedical subjects
L A Lucher
Publications and source records attributed to L A Lucher.
Altered expression of adenovirus 12 DNA-binding protein but not DNA polymerase during abortive infection of hamster cells.
Replication of human adenovirus type 12 DNA is blocked in abortively infected baby hamster kidney cells. The activity and accumulation of adenovirus 12 DNA polymerase is equivalent in infected hamster and human cell extracts. However, the accumulation of adenovirus type 12 DNA-binding protein is approximately 120-fold lower in extracts from infected hamster cells when compared to infected permissive human cells. This difference in accumulation is not due to replication of viral DNA during productive infection, since this difference is observed in the presence of hydroxyurea. The DNA-binding protein from infected hamster cells retains the ability to bind denatured DNA-cellulose. An adenovirus 5 early region 1 transformed hamster cell line competent to complement the adenovirus 12 DNA replication defect also stimulates accumulation of the DNA-binding protein even when the cells are treated with hydroxyurea. Thus, the reduced expression of the viral DNA-binding protein may play a role in the mechanism of abortive infection of hamster cells by adenovirus 12.
Adenovirus Early Proteins↗
Formation in vitro of the pTP-dCMP initiation complex of human adenovirus type 12.
We report the covalent addition of [32P]dCMP to a protein from group A adenovirus 12 (Ad12)-infected human (KB) cells in vitro, using crude extracts. Synthesis of the 60K protein-dCMP complex required a DNA template containing a terminally located adenovirus replication origin; the protein-dCMP bond was alkali-labile but acid-stable. We therefore conclude that this product is the Ad12 terminal protein precursor (pTP)-dCMP initiation complex for DNA replication. Synthesis of Ad12 pTP-dCMP was specific for dCTP but was stimulated by dATP. In contrast to Ad2, the Ad12 initiation reaction required ATP. Antipeptide antiserum targeted to Ad DNA polymerase inhibited Ad12 pTP-dCMP synthesis in vitro, providing evidence that Ad DNA polymerase catalyse dCMP addition to pTP during initiation.
Adenoviruses, Human↗
Extracts of hamster cells abortively infected with human adenovirus type 12 are competent to support initiation of viral DNA replication.
Baby hamster kidney (BHK-21) cells do not allow replication of human adenovirus type 12 (Ad12) DNA during abortive infection by this virus. However, we have determined that crude extracts of BHK-21 cells abortively infected with Ad12 support in vitro the initiation reaction of Ad12 DNA replication. Synthesis of the Ad12 pTP-dCMP initiation complex by BHK extracts is two- to five-fold less than when crude infected human (KB) cell extracts are used in the reaction. Combining infected KB cytoplasmic and uninfected BHK nuclear extracts in the reaction indicates that the decreased efficiency is probably due to a lesser ability of hamster nuclear extracts to support the initiation reaction, rather than to decreased synthesis of Ad12 pTP and DNA polymerase during abortive infection, or to the presence of an inhibitor in BHK cells.
Adenoviridae↗
A microscale analytical batch chromatographic method for detecting soluble viral DNA-binding proteins in crude extracts.
We describe a micro-method for determining the presence in crude cellular extracts of soluble proteins which can bind to immobilized DNA, using the DNA-binding protein of human adenovirus as an example. Batch chromatography of radiolabeled proteins is performed in microcentrifuge tubes containing 50 microliters packed volume of commercially available denatured calf thymus DNA-cellulose. Eluted single-stranded DNA-binding proteins are then visualized by fluorography following gel electrophoresis. The batch procedure gives yields of adenovirus DNA-binding protein which are comparable to those obtained with a mini-column of similar adsorbent volume. The scale of the procedure makes it convenient for simultaneously analyzing multiple samples.
Adenoviruses, Human↗
Adenovirus type 12 tumour antigen synthesis differs during infection of permissive and non-permissive cells.
Synthesis of the adenovirus type 12 E1A and E1B tumour antigens was compared in productively infected human (KB) cells and in abortively infected baby hamster kidney (BHK) cells. By the use of anti-peptide antibodies, the E1A tumour antigens were easily detectable in infected KB extracts as early as 6 h post-infection, but were not detectable in infected BHK extracts until 12 h post-infection. The level of E1A tumour antigens detected in BHK extracts was 10 to 15% of that detected in KB extracts. The level of the E1B 163R (19K) tumour antigen was also lower in BHK extracts: 5 to 10% of that detected in KB extracts. Stability of the E1A tumour antigens was not significantly different in the two infected cell species, indicating that the lower E1A level during abortive infection was due to a lower rate of synthesis of these proteins. These data suggest that early protein synthesis is not the same in abortively infected cells as it is in productively infected cells.
Adenovirus Early Proteins↗
Use of the water-soluble fluor sodium salicylate for fluorographic detection of tritium in thin-layer chromatograms and nitrocellulose blots.
We have determined that sodium salicylate, a water-soluble fluor which we use routinely for fluorography with polyacrylamide gels, is also useful for fluorography with thin-layer media. Detection of 3H-labeled material applied to thin-layer chromatography plates, or nitrocellulose membranes, can be enhanced up to 150-fold after treatment with an aqueous solution of 2 M sodium salicylate, while detection of 35S-labeled material is enhanced only about 2-fold. We demonstrate the utility of sodium salicylate fluorography in detecting 3H-labeled palmitic acid following thin-layer chromatography and 3H-labeled proteins following blotting to nitrocellulose.
Autoradiography↗
Reactions catalyzed by purified L-glutamine: keto-scyllo-inositol aminotransferase, an enzyme required for biosynthesis of aminocyclitol antibiotics.
Dialyzed extracts of the gentamicin producer Micromonospora purpurea catalyze reactions which represent transaminations proposed for 2-deoxystreptamine biosynthesis. To determine whether these transaminations were catalyzed by a single aminotransferase or by multiple enzymes, we purified and characterized an L-glutamine:keto-scyllo-inositol aminotransferase from M. purpurea. This enzyme was purified 130- to 150-fold from late-log-phase mycelia of both wild-type M. purpurea and a 2-deoxystreptamine-less idiotroph. The cofactor pyridoxal phosphate was found to be tightly bound to the enzyme, and spectral analysis demonstrated its participation in the transamination reactions of this enzyme. The major physiological amino donor for the enzyme appears to be L-glutamine; the keto acid product derived from glutamine was characterized as 2-ketoglutaramate, indicating that the alpha amino group of glutamine participates in the transamination. We found that crude extracts contained omega-amidase activity, which may render transaminations with glutamine irreversible in vivo. The substrate specificity of the aminotransferase was shown to be restricted to deoxycyclitols, monoaminocyclitols, and diaminocyclitols, glutamine, and 2-ketoglutaramate, which contrasts with the broader substrate specificity of mammalian glutamine aminotransferase. The appearance of the enzyme in late-log phase, coupled with its narrow substrate specificity, indicates that it participates predominantly in 2-deoxystreptamine biosynthesis rather than in general metabolism. The enzyme catalyzes reactions which represent both transamination steps of 2-deoxystreptamine biosynthesis. Although copurification of two aminotransferases cannot be ruled out, our data are consistent with the participation of a single aminotransferase in the formation of both amino groups of 2-deoxystreptamine during biosynthesis by M. purpurea. We propose that this aminotransferase participates in a key initial step in the biosynthesis of most aminocyclitol antibiotics.
Amidohydrolases↗
Biosynthesis and properties of the adenovirus 2 L1-encoded 52,000- and 55,000-Mr proteins.
The adenovirus type 2 L1 region, which is located at 30.7 to 39.2 map units on the viral genome, is transcribed from the major late promoter during both early and late stages of virus replication, and a 52,000-Mr (52K) protein-55K protein doublet has been translated in vitro on L1-specific RNA. To investigate the biosynthesis and properties of the L1 52K and 55K proteins, we prepared antibody against a synthetic peptide encoded near the predicted N terminus. As determined by immunoprecipitation and immunoblot analysis, the antipeptide antibody recognized major 52K and 55K proteins synthesized in adenovirus type 2-infected cells that appeared to be identical to the 52K-55K doublet translated in vitro. The immunoprecipitated 52K and 55K proteins were very closely related, as shown by a peptide map analysis. Both L1 proteins were phosphorylated, and they were phosphorylated at similar sites. No precursor-product relationship was detected between the 52K and 55K proteins by a pulse-chase analysis. Biosynthesis of the L1 52K and 55K proteins began about 6 to 7 h postinfection, after biosynthesis of the early region 1A and early region 1B 19K (175R) T antigens, and reached a maximum rate at about 15 h; the maximum rate was maintained until at least 25 h postinfection. At all times, the 55K protein appeared to be synthesized at a severalfold-higher level than the 52K protein. Both proteins were quite stable and accumulated until late times after infection. Viral DNA replication was not essential for formation of the L1 proteins. Thus, the L1 52K-55K gene appears to be regulated in a manner different from the classical early and late viral genes but similar to the protein encoded by the i-leader (Symington et al., J. Virol. 57:849-856, 1986). The L1 proteins were detected in the cell nucleus by immunofluorescence microscopy with antipeptide antibody and were found to be primarily associated with the nuclear membrane by an immunoblot analysis of subcellular fractions.
Adenoviridae Infections↗
Biosynthesis of adenovirus type 2 i-leader protein.
The i-leader is a 440-base-pair sequence located between 21.8 and 23.0 map units on the adenovirus type 2 genome and is spliced between the second and third segments of the major tripartite leader in certain viral mRNA molecules. The i-leader contains an open translational reading frame for a hypothetical protein of Mr about 16,600, and a 16,000-Mr polypeptide (16K protein) has been translated in vitro on mRNA selected with DNA containing the i-leader (A. Virtanen, P. Aleström, H. Persson, M. G. Katze, and U. Pettersson, Nucleic Acids Res. 10:2539-2548, 1982). To determine whether the i-leader protein is synthesized during productive infection and to provide an immunological reagent to study the properties and functions of the i-leader protein, we prepared antipeptide antibodies directed to a 16-amino acid synthetic peptide which is encoded near the N terminus of the hypothetical i-leader protein and contains a high acidic amino acid and proline content. Antipeptide antibodies immunoprecipitated from extracts of adenovirus type 2-infected cells a major 16K protein that comigrated with a 16K protein translated in vitro. Partial N-terminal amino acid sequence analysis by Edman degradation of radiolabeled 16K antigen showed that methionine is present at residue 1 and leucine is present at residues 8 and 10, as predicted from the DNA sequence, establishing that the 16K protein precipitated by this antibody is indeed the i-leader protein. Thus, the i-leader protein is a prominent species that is synthesized during productive infection. The i-leader protein is often seen as a doublet on polyacrylamide gels, suggesting that either two related forms of i-leader protein are synthesized in infected cells or that a posttranslational modification occurs. Time course studies using immunoprecipitation analysis with antipeptide antibodies revealed that the E1A 289R T antigen and the E1B-19K (175R) T antigen are synthesized beginning at 2 to 3 and 4 to 5 h postinfection, respectively, whereas the i-leader protein is synthesized starting at about 8 h postinfection and continues unabated until at least 25 h postinfection. The i-leader protein is very stable, as determined by pulse-chase labeling experiments, and accumulates continuously from 8 to 25 h postinfection, as shown by immunoblot analysis. The synthesis of i-leader protein does not depend upon viral DNA replication. Thus, the i-leader protein is a viral gene product of unknown function and high stability that is made in large quantities at intermediate times of productive infection.(ABSTRACT TRUNCATED AT 400 WORDS)
Adenoviridae Infections↗
Posttranslational modification at the N terminus of the human adenovirus type 12 E1A 235R tumor antigen.
The adenovirus E1A transforming region, which encodes immortalization, partial cell transformation, and gene activation functions, expresses two early mRNAs, 13S and 12S. Multiple-T antigen species with different electrophoretic mobilities are formed from each mRNA, presumably by unknown posttranslational modifications. The adenovirus type 12 (Ad12) 13S and 12S mRNAs encode E1A T antigens of 266 and 235 amino acid residues (266R and 235R), respectively. To study possible posttranslational processing at the N and C termini and to distinguish between the Ad12 266R and 235R T antigens, we prepared antibodies targeted to synthetic peptides encoded at the common C (peptide 204) and N (peptide 202) termini of the 266R and 235R T antigens and at the unique internal domain of the 266R T antigen (peptide 206). The specificity of each anti-peptide antibody was confirmed by immunoprecipitation of the 266R and 235R T antigens produced in Escherichia coli. Immunoprecipitation analysis of the E1A T antigens synthesized in Ad12-infected KB cells revealed the following. Antibody to the common C terminus recognized three T antigens with apparent Mrs of 43,000, 42,000, and 39,000 (43K, 42K, and 39K). All three forms were phosphorylated and were present in both the nucleus and the cytoplasm. The 43K and 42K T antigens were rapidly synthesized during a 10-min pulse with [35S]methionine in Ad12-infected cells. The 43K T antigen had a half-life of 20 min, the 42K T antigen had a longer half-life of about 40 min, and the 39K T antigen became the predominant E1A T antigen. Antibodies to the unique region immunoprecipitated the 43K T antigen but not the 42K and 39K T antigens. Antibody to the N terminus immunoprecipitated the 43K and 42K T antigens but not the 39K T antigen, suggesting that the 39K T antigen possessed a modified N terminus. Partial N-terminal amino acid sequence analysis showed that the 43K and 42K T antigens contain methionine at residues 1 and 5, as predicted from the DNA sequence, whereas no methionine was released from the 39K T antigen during the first six cycles of Edman degradation. We propose that the short-lived 43K T antigen is the primary product of the 13S mRNA, the 266R T antigen; the somewhat more stable 42K T antigen is the primary product of the 12S mRNA, the 235R T antigen.(ABSTRACT TRUNCATED AT 400 WORDS)
Adenovirus Early Proteins↗
Phosphorylation in vitro of Escherichia coli-produced 235R and 266R tumor antigens encoded by human adenovirus type 12 early transformation region 1A.
The tumor (T) antigens encoded by the human adenovirus early transforming region 1A (E1A) are gene regulatory proteins whose functions can immortalize cells. We have recently described the synthesis in Escherichia coli and the purification of the complete T antigens encoded by the adenovirus type 12 (Ad12) E1A 12S mRNA (235-residue [235R] T antigen) and 13S mRNA (266R T antigen). In this study, we show that the Ad12 E1A T antigens are extensively phosphorylated in Ad12-infected mammalian cells but are not phosphorylated in E. coli. Inasmuch as posttranslational phosphorylation at specific amino acid sites may be important for biological activity, we have studied the phosphorylation of the E. coli-produced T antigens in vitro by using a kinase activity isolated from cultured human KB cells. The kinase was purified about 300-fold and appears to be a cyclic AMP-independent, Ca2+-independent protein kinase requiring only ATP and Mg2+ for activity. To determine which amino acids are phosphorylated and whether phosphorylation in vitro occurs at the same amino acid sites that are phosphorylated in vivo, the Ad12 E1A T-antigen species synthesized by infected cells were metabolically labeled with 32Pi and compared with the E. coli-produced E1A T antigens labeled in vitro with [gamma-32P]ATP by using the partially purified kinase. Partial V8 proteolysis analysis gave similar patterns for in vivo- and in vitro-phosphorylated T antigen. Two-dimensional maps of tryptic phosphopeptides and of chymotryptic phosphopeptides suggested that mainly the same amino acid sites are phosphorylated in vitro and in vivo and that phosphorylation occurred at multiple sites distributed throughout the T-antigen molecule. Serine was the only amino acid that was phosphorylated both in vivo and in vitro, and, surprisingly, most serines appeared to be phosphorylated. The feasibility of faithfully phosphorylating T antigens in vitro suggests that the E. coli-produced Ad12 E1A 235R and 266R T antigens may prove useful for molecular studies on T-antigen function.
Adenoviruses, Human↗
Antibody directed to a synthetic peptide encoding the NH2-terminal 16 amino acids of the adenovirus type 2 E1B-53K tumor antigen recognizes the E1B-20K tumor antigen.
A peptide, H2N-Glu-Arg-Arg-Asn-Pro-Ser-Glu-Arg-Gly-Val-Pro-Ala-Gly-Phe-Ser-Gly-(Cys )COOH, containing the amino acid sequence at the NH2 terminus of the adenovirus type 2 (Ad2) E1B-coded large T antigen (E1B-53K) has been synthesized. Anti-peptide antibody was generated in rabbits and used to immunoprecipitate Ad T antigens from Ad2 early infected cell extracts. In addition to the expected E1B-53K T antigen, anti-peptide antibody precipitated the Ad2 E1B-20K T antigen that was previously shown to be related to E1B-53K (M. Green, K.H. Brackmann, M.A. Cartas, and T. Matsuo, J. Virol. 42, 30-41, 1982). Anti-peptide prepared against the COOH terminus of the E1B-53K T antigen or against the NH2 terminus of the E1B-19K T antigen did not precipitate the E1B-20K T antigen. These data suggest that the Ad2 E1B-20K T antigen initiates translation at nucleotide 2016 in reading frame 3, as does E1B-53K. The viral mRNA that encodes the E1B-20K T antigen has not been identified.
Adenoviridae↗
Antibodies to synthetic peptides targeted to the transforming genes of human adenoviruses: an approach to understanding early viral gene function.
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Adenoviridae↗
Synthesis in Escherichia coli of human adenovirus type 12 transforming proteins encoded by early region 1A 13S mRNA and 12S mRNA.
Human adenovirus (Ad)-encoded early region 1A (E1A) tumor (T) antigens have been implicated in the positive regulation of viral early genes, the positive and negative regulation of some cellular genes, and cell immortalization and transformation. To further study the Ad E1A T antigens and to facilitate their purification, we have cloned cDNA copies of the Ad12 E1A 13S mRNA and 12S mRNA downstream of a hybrid Escherichia coli trp-lac (tac) promoter. Up to 8% of the protein synthesized in E. coli cells transformed by each of the two different Ad12 E1A cDNA constructs were immunoprecipitated as a Mr 47,000 protein by antibody to a synthetic peptide encoded in the Ad12 E1A DNA sequence. Both proteins produced in E. coli appear to be authentic and complete Ad12 E1A T antigens because they possess (i) the Ad12 E1A NH2-terminal amino acid sequence predicted from the DNA sequence; (ii) the Ad12 E1A COOH-terminal sequence, as shown by immunoprecipitation with anti-peptide antibody; and (iii) a molecular weight and an acidic isoelectric point similar to that of the E1A T antigens synthesized in Ad12-infected and transformed mammalian cells. The T antigens were purified to near homogeneity in yields of 100-200 micrograms per g wet weight of transformed E. coli cells.
Adenoviruses, Human↗
Identification of adenovirus 12-encoded E1A tumor antigens synthesized in infected and transformed mammalian cells and in Escherichia coli.
A 16-amino acid peptide, H2N-Arg-Glu-Gln-Thr-Val-Pro-Val-Asp-Leu-Ser-Val-Lys-Arg-Pro-Arg-Cys-COOH (peptide 204), targeted to the common C-terminus of human adenovirus 12 (Ad12) tumor antigens encoded by the E1A 13S mRNA and 12S mRNA, has been synthesized. Antibody prepared in rabbits against peptide 204 immunoprecipitated two proteins of apparent Mr 47,000 and 45,000 from extracts of [35S]methionine-labeled Ad12-early infected KB cells and a 47,000 protein from extracts of the Ad12-transformed hamster cell line, HE C19. Immunoprecipitation analysis of infected and transformed cells labeled with 32Pi showed that both major Ad12 E1A T antigens are phosphoproteins. Immunofluorescence microscopy of Ad12-early infected KB cells with antipeptide antibody showed the site of E1A protein concentration to be predominantly nuclear. E1A proteins were detected by immunofluorescence at 4 to 6 h postinfection and continued to increase until at least 18 h postinfection. Antipeptide 204 antibody was used to analyze the proteins synthesized in Escherichia coli cells transformed by plasmids containing cDNA copies of the Ad12 E1A 13S mRNA or 12S mRNA under the control of the tac promoter (D. Kimelman, L. A. Lucher, M. Green, K. H. Brackmann, J. S. Symington, and M. Ptashne, Proc. Natl. Acad. Sci. U.S.A., in press). A major protein of ca. 47,000 was immunoprecipitated from extracts of each transformed E. coli cell clone. Two-dimensional gel electrophoretic analysis of immunoprecipitates revealed that the T antigens synthesized in infected KB cells, transformed hamster cells, and transformed E. coli cells possess very similar molecular weights and acidic isoelectric points of 5.2 to 5.4.
Adenoviruses, Human↗
Human adenovirus 2 E1B-19K and E1B-53K tumor antigens: antipeptide antibodies targeted to the NH2 and COOH termini.
The human adenovirus 2 (Ad2) transforming region is located in the left 11.1% of the viral genome and encodes two early transcription units, E1A and E1B. Based on the amino acid sequence deduced from the Ad2 E1B DNA sequence (Gingeras et al., J. Biol. Chem. 257:13475-13491, 1982), we have prepared antibodies against synthetic peptides, 8 to 16 amino acids in length, encoded at the NH2 and COOH termini of the major E1B-19K and E1B-53K tumor antigens. The antipeptide antibodies immunoprecipitated the targeted E1B-19K or E1B-53K tumor antigens from extracts of Ad2-infected cells. The specificity of the peptide competition studies. Antipeptide antibodies directed to the NH2 and COOH termini immunoprecipitated the E1B-19K and E1B-53K tumor antigens from two Ad2-transformed rat cell lines, F17 and F4, providing evidence that identical tumor antigens are synthesized in Ad2-infected and Ad2-transformed cells. These results show that the E1B-19K and E1B-53K T antigens are not processed proteolytically at either the NH2 or COOH terminus. Our data provide strong evidence at the protein level that the E1B-19K and E1B-53K tumor antigens partially overlap in DNA sequence, with the E1B-19K initiating translation at the first ATG at nucleotide 1711 in translation reading frame 1 and the E1B-53K tumor antigen initiating translation at the second ATG at nucleotide 2016 in reading frame 3. This confirms the results of others on the N-terminal amino acid sequence of E1B-19K and theoretical deductions based on the DNA sequence. Our findings prove that the large E1B-53K T antigen initiates translation at the second ATG at nucleotide 2016 and not at equally plausible initiation codons located farther downstream at nucleotides 2202 and 2235. Thus, the E1B-53K T antigen is another example of a protein which initiates translation at an internal ATG rather than at the 5'-proximal ATG.
Adenoviruses, Human↗
Viral transformation of baby hamster kidney cells affects the oxygen sensitivity of nitroxide reduction.
In order to explore and expand the potential applications of nitroxides, it is important to understand how nitroxides are metabolized by living systems. We studied the oxygen sensitivity of a BHK (baby hamster kidney) cell line and BHK cells that had been transformed by adenovirus to determine if the process of adenoviral transformation affects nitroxide metabolism. When the BHK cells are transformed by adenovirus, they reduce nitroxides essentially independently of oxygen concentration. In addition, the rate of reduction of the nitroxides decreases by a factor of 10 compared to the rate of reduction in BHK cells. These data suggest to us that the process of viral transformation has affected dramatically the metabolism of these cells; one possible interpretation is that the viral transformed cells lost the ability to perform oxidative phosphorylation. We believe that this is a metabolic event which could be exploited by magnetic resonance imaging.
Animals↗