Characterization of K virus and its comparison with polyoma virus.

The antigenic relationship between the two murine papovaviruses, K virus and polyoma virus, was examined by serological techniques to determine whether they shared any antigenic components. No cross-reactivity was found associated with the viral (V) antigens by the indirect immunofluorescence, neutralization, or hemagglutination-inhibition tests. The tumor (T) antigens expressed in transformed cells or cells productively infected by either K or polyoma virus did not cross-react by indirect immunofluorescence. An antigenic relationship was detected, however, among the late proteins of K virus, polyoma virus, simian virus 40, and the human papovavirus BKV, when tested with either hyperimmune sera prepared against polyoma virus and simian virus 40 or sera prepared against disrupted virions. The nucleic acids of K and polyoma viruses were compared by agarose gel electrophoresis and restriction endonuclease analysis. No nucleotide sequence homology between the genomes of these two viruses was detectable by DNA-DNA hybridization techniques under stringent conditions. The genome of K virus was found to be slightly smaller than that of polyoma virus, and the cleavage patterns of the viral DNAs with six restriction endonucleases were different. These findings indicate that there is little relationship between these two murine papovaviruses.     

Characterization of DNA-protein complex formation in nuclear extracts with a sequence from the herpes simplex virus thymidine kinase gene

The biochemical characteristics of complex formation in nuclear extracts from mock-infected and herpes simplex virus (HSV)-infected Vero and HeLa cells with a sequence downstream of and adjacent to the promoter for the HSV thymidine kinase gene were studied using the mobility shift electrophoresis assay. This region is bound by host cell proteins, as evidenced by the formation of complexes after incubation in extracts from mock-infected cells. Unique virus-specific complexes form in extracts prepared from infected cells, and these complexes contain ICP4, the major regulatory protein of HSV. Examination of the salt requirements for assembly and the stability of preformed DNA-protein complexes to added salt demonstrate the distinct nature of the complexes that form in each extract. This finding is supported by analyses of the relative association and dissociation rates of these complexes which show that complexes formed in extracts prepared from infected cells are kinetically labile. After depletion with chelators, the divalent cation requirements for complex formation were assayed by supplementation with various metal salts. Addition of Mn2+ restored binding activity in extracts from both mock-infected and infected HeLa cells. Finally, footprinting assays revealed that sequences on each strand throughout this region of the thymidine kinase gene were involved in complex formation only in extracts from mock-infected cells. These experiments suggest that one consequence of virus gene expression is to alter the interaction of cell proteins with virus DNA.     

Characterization of polyoma virus early proteins expressed from vaccinia virus recombinants

We previously reported that live recombinant vaccinia viruses (VV) encoding either the large T (LT) or middle T (MT) antigens of polyoma virus (PyV) were able to induce rejection of tumors caused by PyV-transformed cells [Lathe et al., Nature 326 (1987) 878-880]. Here we present evidence that PyV early proteins expressed by the recombinants retain the biochemical characteristics of their authentic counterparts despite the cytopathic effect of VV infection. VV-encoded LT is a nuclear phosphoprotein, with specific DNA binding, ATPase and nucleotide-binding activities. VV-expressed MT associates with cellular kinases, particularly with pp60c-src, by which it is phosphorylated in vitro. Expression levels of LT and MT reached 10(6) molecules per infected cell. The use of VV as a vector is encouraged by the high expression level obtained and because VV infection does not seem to prevent appropriate post-translational processing of proteins encoded by VV recombinants.     

Characterization of phosphatidylthreonine in polyoma virus transformed fibroblasts.

A threonine phospholipid in polyoma virus-transformed hamster embryo fibroblasts has been characterized as phosphatidylthreonine. The identification has been made by chemical and enzymatic hydrolysis. Acid hydrolysis of the phospholipid produces free threonine. Mild alcoholysis produces a water-soluble derivative having the properties of glycerophosphorylthreonine. Hydrolysis with phospholipase C produces phosphorylthreonine which on prolonged acid hydrolysis yields threonine. Phosphatidylthreonine in the cell is more accessible to reaction with fluorodinitrobenzene than is phosphatidylserine. Phosphatidylthreonine also has been found as a major aminophospholipid in two other polyoma-transformed hamster cell lines and in the BHK-21/C13 line including the PVT-3 and TS-3 lines. the latter derived from BHK cells. Only a trace amount of phosphatidylthreonine occurs in normal liver, kidney and spleen of the adult mouse, in normal liver and kidney of the adult hamster, in whole mouse and hamster embryos, and in mouse 3T3 cells and SV40-transformed 3T3 cells.     

The complex of polyoma virus middle-T antigen and pp60c-src.

We have recently proposed that the transforming protein of polyoma virus, middle-T antigen, forms a complex with pp60c-src. Here we provide additional evidence for the existence of the complex using both monoclonal antibodies specific for middle-T and antibodies raised against synthetic peptides corresponding to sequences from both middle-T and pp60c-src. The complex was retained during partial purification of middle-T and was stable to incubation under various conditions. A survey of a number of mutants of middle-T antigen showed that there was a complete correlation between the ability of middle-T to accept phosphate in the in vitro kinase reaction and the presence of a middle-T: pp60c-src complex. This result is in accord with our hypothesis that middle-T itself is not a protein kinase but rather that pp60c-src phosphorylates middle-T. All mutant forms of middle-T antigen capable of transformation had associated pp60c-src. The middle-T of two non-transforming mutants (hr-t mutants) did not have associated pp60c-src, whereas other non-transforming middle-T species did associate with pp60c-src. We propose that the complex plays an essential role in transformation by polyoma virus, but that the existence of the complex per se may not be sufficient.     

Comparison of nuclease digestion of polyoma virus nucleoprotein complex and mouse chromatin.

We digested polyoma virus nucleoprotein complex, isolated from disrupted virions, with micrococcal nuclease and DNase I. The results were compared with digestions of chromatin from mouse nuclei. The nucleosome "core" structures were similar, but the spacing of the nucleosomes in the isolated polymoma nucleoprotein complexes was irregular, whereas in mouse chromatin it was regular. The average nucleosome repeat length in each case was 190 to 200 base pairs. This figure suggests that, unless there are substantial stretches of free DNA, the polyoma nucleoprotein complex contains about 26 nucleosomes. The commonly used method of preparing the nucleoprotein complex by disruption of virions at pH 10.2 may lead to significant damage to the structure. Such damage may be more clearly revealed by the susceptibility of the DNA to nuclease digestion than by the usual criteria of sedimentation velocity and buoyant density.     

Plasmidal maintenance of composite DNA derived from polyoma related plasmid, L factor.

Recently, we reported a multicopy mammalian plasmid with a structure related to polyoma. The plasmid, named L factor, was found at a high copy number (5,000 or more per cell) in a subclone derived from mouse L cells. We attempted to utilize L factor as a plasmid vector for mammalian cells. A series of composite DNA consisting of L factor and a foreign (herpes simplex virus tk) were constructed. These DNA could be established as plasmids after transfection to several mouse cell lines, although the copy number of the re-established plasmids was considerably less than that observed for the original subclone. The composite DNA maintained the structure of the original DNA after prolonged culture and the copy number remained constant even with no selective pressure. A composite DNA, with no DNA sequence corresponding to polyoma T antigen, could also be established as a plasmid in a mouse L cell line in which polyoma T antigen is expressed. The potential use of the plasmid is discussed.     

Membrane antigens associated with infection,transformation, and tumorigenesis by polyoma virus

Conclusion Infection, transformation, or tumorigenesis by Py virus leads inter alia to modifications in the membrane antigens of the affected cells. The modifications include antigenic gains or losses or quantitative changes in both directions. Although there is a pronounced common denominator in the antigenic alterations in the three distinct Py-induced biological processes, it is nontheless possible that each of them is characterized by specific antigenic modifications. This possibility has yet to be analyzed. Specific antigenic modifications, if they occur, are probably the result of different selective processes and adaptions to these pressures.In this brief review, we have attempted to survey the literature pertinent to this aspect. While doing so, we discovered that most researchers have not considered the possibility that differences could exist between antigens of cells infected by Py, cells transformed by this virus, and Py-induced tumor cells. We feel that a comprehensive antigenic comparison between these cells utilizing well-defined reagents is an essential prerequisite to understanding of the successful immunological surveillance against Py-induced malignancy.Incumbent: The David Furman Chair of Cancer Immunobiology     

Characterization of human alpha 2-macroglobulin monomers obtained by reduction with dithiothreitol.

We compared the physicochemical characteristics of alpha 2-macroglobulin (alpha 2M) monomers produced by limited reduction and carboxamidomethylation to those of the naturally occurring monomeric alpha-macroglobulin homologue rat alpha 1-inhibitor 3 (alpha 1 I3). Unlike alpha 1 I3, alpha 2 M monomers fail to inhibit proteolysis of the high molecular weight substrate hide powder azure by trypsin. In contrast to alpha 1 I3, which remains monomeric after reacting with proteinase, alpha 2 M monomers reassociate to higher molecular weight species (dimers, trimers, and tetramers) after reacting with proteinase. Reaction of alpha 2 M monomers at molar ratios of proteinase to alpha 2M monomers as low as 0.3:1 leads to extensive reassociation and is accompanied by complete bait-region and thiolester bond cleavage. During the reaction of alpha 2M monomers with proteinases, the proteinase binds to the reassociating alpha 2M subunits but is not inhibited. Of significance, all the bound proteinase was covalently linked to the reassociated alpha 2M species. Treatment of alpha 2M monomers with methylamine results in thiolester bond cleavage but minimal reassociation. Treatment of alpha 2M monomers with methylamine followed by proteinase results in complete bait-region cleavage and is accompanied by marked reassociation of alpha 2M monomers to higher molecular weight species. However, no proteinase is associated with these higher molecular weight forms. We infer that bait-region cleavage is more important than thiolester bond cleavage in driving alpha 2M monomers to reassociate. Despite many similarities between alpha 1I3 and alpha 2M monomers, significant differences must exist with respect to proteinase orientation within the inhibitor to account for the failure of alpha 2M monomers to protect large molecular weight substrates from proteolysis by bound proteinase, in contrast to the naturally occurring monomeric homologue rat alpha 1 I3.     

Characterization and properties of a 5S-RNA-protein complex released from heated 60S ribosomal subunits

When rat liver 60S ribosomal subunits were heated in phosphate buffer in the presence of MgCl2, 5S RNA was released in the form of a nucleoprotein complex (RNPH), which was isolated either by electrophoresis in polyacrylamide gel or centrifugation through a sucrose gradient. In addition to L5 several proteins of functional significance were identified in the complex: the acidic phosphoproteins P1-P2 and, as weaker spots, L3-L4, L6-L7 and L22. Most of these proteins were also found, but only as traces, in the RNPEDTA used as a control. RNPH was able to associate with 40S subunits. Our results support the interpretation that RNPH is located at the subunits' interface, at or near the peptidyl-transferase center.     

Partial purification and properties of a DNA-binding protein from nuclei of cells infected with polyoma virus.

A DNA-binding protein has been purified from nuclei of 3T3 cells infected with polyoma virus. The assay used to detect this activity measures the amount of double-stranded DNA retained on a nitrocellulose membrane filter in the presence of binding protein. The interaction between DNA and protein is salt dependent and occurs optimally at 0.8 M NaCl. The isolated protein can bind to both circular and linear duplex DNA. Incubation of the binding protein with PM2 or polyoma DNA results in the formation of a fast sedimenting DNA structure in neutral sucrose gradients. The isolated binding protein is also capable of producing a considerable stimulation of both Escherichia coli (Pol I) and T4 DNA polymerase activities when either single-stranded or intact, native T7 DNA is used as the template. The binding protein itself is free of detectable DNA polymerase or nuclease activity.     

Characterization of slow reacting substance as a family of thiolipids derived from arachidonic acid.

Using radiolabeled cysteine and arachidonic acid as biosynthetic precursors, the slow reacting substance (SRS) produced by the rat basophilic cell line, RBL-1, has been characterized as a family of thiolipids derived from arachidonic acid.     

Characterization of the DNA-protein complex at the termini of the bacteriophage PRD1 genome.

DNA of bacteriophage PRD1 has protein P8 at its termini. Extracts of infected cells are able to derivatize P8 in vitro with labeled dGTP. Two early proteins, P1 and P8, products of genes I and VIII, respectively, are the only phage proteins necessary for the formation of the protein P8-dGMP complex. This was shown by complementation of extracts from cells infected with mutants and by use of extracts from cells carrying cloned genes I and VIII. With Escherichia coli mutants that are temperature sensitive for DNA synthesis, it was possible to show that the formation of the protein P8-dGMP complex was dependent upon the host replication apparatus. The analysis of the purified protein P8-dGMP complex by hydrolysis and enzymatic digestion showed that there is a covalent phosphodiester bond between tyrosine and 5'-dGMP.     

Characterization and detection of lysine-arginine cross-links derived from dehydroascorbic acid

Covalently cross-linked proteins are among the major modifications caused by the advanced Maillard reaction. So far, the chemical nature of these aggregates is largely unknown. L-dehydroascorbic acid (DHA, 5), the oxidation product of L-ascorbic acid (vitamin C), is known as a potent glycation agent. Identification is reported for the lysine-arginine cross-links N6-[2-[(4-amino-4-carboxybutyl)amino]-5-(2-hydroxyethyl)-3,5-dihydro-4H-imidazol-4-ylidene]-L-lysine (9), N6-[2-[(4-amino-4-carboxybutyl)amino]-5-(1,2-dihydroxyethyl)-3,5-dihydro-4H-imidazol-4-ylidene]-L-lysine (11), and N6-[2-[(4-amino-4-carboxybutyl)amino]-5-[(1S,2S)-1,2,3-trihydroxypropyl]-3,5-dihydro-4H-imidazol-4-ylidene]-L-lysine (13). The formation pathways could be established starting from dehydroascorbic acid (5), the degradation products 1,3,4-trihydroxybutan-2-one (7, L-erythrulose), 3,4-dihydroxy-2-oxobutanal (10, L-threosone), and L-threo-pentos-2-ulose (12, L-xylosone) were proven as precursors of the lysine-arginine cross-links 9, 11, and 13. Products 9 and 11 were synthesized starting from DHA 5, compound N6-[2-[(4-amino-4-carboxybutyl)amino]-5-[(1S,2R)-1,2,3-trihydroxypropyl]-3,5-dihydro-4H-imidazol-4-ylidene]-L-lysine (16) via the precursor D-erythro-pentos-2-ulose (15). The present study revealed that the modification of lysine and arginine side chains by DHA 5 is a complex process and could involve a number of reactive carbonyl species.     

Characterization of a multienzyme complex derived from a Bacillus subtilis DNA-membrane extract that synthesizes RNA and DNA precursors.

The activity of a variety of enzymes involved in the synthesis of RNA and DNA precursors was found to copurify with initiation of DNA replication activity. These enzymes included ribo- and deoxyribonucleoside kinases, kinases for their phosphorylated intermediates, and ribonucleoside diphosphate reductase. This precursor-synthesizing complex is part of a Bacillus subtilis DNA-membrane extract originally shown to contain all of the enzymes and template necessary for initiation of DNA replication (J. Laffan and W. Firshein, J. Bacteriol. 169:2819-2827, 1987). Although the complex incorporated deoxyribonucleoside triphosphates into DNA, deoxyribonucleosides were incorporated even faster, suggesting catalytic facilitation. Both ribonucleosides and deoxyribonucleosides were found by thin-layer chromatography separation to be converted by the complex into their mono-, di-, and triphosphate derivatives. Ribonucleotides were incorporated into DNA via the action of ribonucleoside diphosphate reductase. Some regulatory mechanisms of the kinase system may also be retained by the complex. Electron microscope studies revealed that the precursor-synthesizing-initiation subcomplex is contained within a particulate fraction consisting of different-size vesicles resembling liposomes and that these particles may be structurally important in maintaining the synthetic activity of the subcomplex.