Isolation of viral specific RNA from SV40 infected cells by viral DNA chemically linked to a cellulose matrix.

SV40 DNA fragments chemically attached to neutral cellulose powder with a water-soluble carbodiimide have been used to isolate late lytic viral specific RNA from virus infected cells. Exhaustive hybridization to SV40 DNA reveals that virtually all of the isolated RNA molecules contain SV40 specific sequences. Comparison with SV40 cRNA prepared with purified Escherichia coli RNA polymerase and a SV40 DNA I template suggests that the purity of the isolated SV40 specific RNA is very close to 100%. The background level for the nonspecific binding of RNA to a purified cellulose matrix is very low. Retention of nonspecific RNA by SV40 DNA-cellulose is only 1.5% of the viral specific RNA isolated under saturating conditions for the column. Sedimentation in neutral sucrose suggests that the major 16S viral specific RNA has been isolated largely intact.     

Cell-free translation of simian virus 40 16S and 19S L-strand-specific mRNA classes to simian virus 40 major VP-1 and minor VP-2 and VP-3 capsid proteins.

Simian virus 40 capsid proteins VP-1, VP-2, and VP-3 have been synthesized in wheat germ and reticulocyte cell-free systems in response to either poly(A)-containing mRNA from the cytoplasm of infected cells or viral RNA purified by hybridization to simian virus 40 DNA linked to Sepharose. All three viral polypeptides synthesized in vitro are specifically immunoprecipitated with anti-simian virus 40 capsid serum. VP-2 and VP-3 are related by tryptic peptide mapping to each other but not to VP-1. The most abundant class of L-strand-specific viral mRNA, the 16S species, codes for the major capsid protein. The relatively minor 19S class directs the cell-free synthesis of VP-1, VP-2, and VP-3. Whether the 19S RNA represents more than one distinct species of mRNA is not yet clear. VP-1 mRNA can be isolated from the cytoplasm, detergent-washed nuclei, and the nuclear wash fraction. The mRNA from the nuclear wash fraction is enriched for VP-2 mRNA when compared to other viral or cellular polypeptides.     

Biosynthesis of fibronectin by human embryonal fibroblasts transformed by SV-40 virus

Fibronectin biosynthesis by human embryonic fibroblasts transformed with virus SV-40 was studied in intact cells and in a cell-free protein synthesizing system on free and membrane-bound polyribosomes isolated from these cells. It was found that fibronectin release from transformed fibroblasts into the culturing medium was decreased 4.5-fold, while its per cent content--2-fold. The amount of fibronectin precipitated by antibodies in the course of an immunoprecipitation reaction in transformed cells appeared to be somewhat higher than in normal cells, although when expressed on a per cent basis this content was decreased only 1.5-fold. However, the content of fibronectin monomer with Mr = 220 kD exceeded that in normal fibroblast cell material 1.6 times. Study on fibronectin biosynthesis in a cell-free system revealed that in transformed cells 45% of fibronectin is synthesized on free polyribosomes as compared to 13% in normal fibroblasts. It is assumed that the decreased fibronectin biosynthesis in human fibroblasts transformed with virus SV-40 results in spatial uncoupling of polyribosomes and membrane structures responsible for protein transport from the cell, as a result of which a significant part of fibronectin synthesized by transformed fibroblasts undergoes intracellular degradation.     

SV40 RNA: Filter hybridization for rapid isolation and characterization of rare RNAs

Modifications of the filter hybridization method for the measurement and isolation of simian virus 40 (SV40) RNA from transformed cells are described. The modified method used small (0.02 cm²) nitrocellulose filters with > 30 μg/cm² SV40 DNA applied following formaldehyde denaturation. The small volume and high DNA densities allowed hybridization to be completed in 2 h and washing after hybridization to be completed in 6 h. The washing reduced background to 1 × 10^?5 of input radioactivity without using nucleases. The efficiency of hybridization after washing was 40% or greater. These procedures have allowed the quantification of proportions of SV40 RNA in labeled RNA from transformed lines and the characterization of SV40 RNAs by electrophoresis and cell-free translation. A 3.7-kb SV40 RNA from SV80 cells was discovered in this work.     

GnRH analogues containing SV-40 virus T-antigen nuclear localization sequence

To improve the efficiency of anticancer drugs due to their delivery to intracellular targets a set of GnRH analogues containing nuclear localization signal (NLS) of SV-40 virus large T-antigen have been synthesized. NLS was attached to the parent molecule via ε-amino group of D-Lysine in position 1 or 6 of peptide sequence using orthogonal protection strategy. The biological activity studies revealed that incorporation of NLS moiety significantly increases cytotoxic activity of palmitoyl-containing GnRH analogues in vitro. The influence of tested peptides on tumor cells does not accompanied by the destruction of cell membrane, as confirmed in experiments with normal fibroblasts, used as a control.     

Nucleic acid hybridization using DNA covalently coupled to cellulose.

We describe a method for linking RNA and DNA covalently to finely divided cellulose through a diazotized aryl amine, which reacts primarily with guanine and uracil (thymine) residues of single strands. The high efficiency of coupling and high capacity of the cellulose for nucleic acid make possible a product with as much as 67 mug of nucleic acid per mg of cellulose. The product is especially suitable for hybridization experiments where very low backgrounds are important, and it is stable in 99% formamide at 80 degrees C so that hybridized nucleic acid can be recovered easily. Full length linear Simian Virus 40 (SV40) DNA, produced by cleavage of SV40(I) DNA with S1 nuclease, can be coupled to diazo cellulose with an efficiency of 80-90%, and is effective in hybridization experiments with SV40 DNA, complementary RNA synthesized in vitro from SV40(I) DNA with E. coli RNA polymerase, and the SV40-specific fraction of total RNA from SV40-infected and transformed cells. In these experiments an excess of cellulose-bound DNA was used, and the efficiency of hybridization was about 90% when ribonuclease treatment of the hybrids was omitted.     

Attempts to Detect Deoxyribonucleic Acid from Agrobacterium tumefaciens and Bacteriophage PS8 in Crown Gall Tumors by Complementary Ribonucleic Acid/Deoxyribonucleic Acid-Filter Hybridization

Labeled ribonucleic acid (RNA) complementary to Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA (cRNA) were used in a systematic study of the sensitivity of cRNA/deoxyribonucleic acid (DNA)-filter hybridization for detection of small amounts of phage or bacterial DNA immobilized on filters. A. tumefaciens cRNA of specific activity 10(6) to 2 x 10(6) counts per min per mug reacted to a significant extent when the DNA-filter contained 1% A. tumefaciens DNA in a salmon DNA background, but 0.1% A. tumefaciens DNA was not detectable. PS8 phage cRNA of the same specific activity reacted to a significant extent when the DNA-filter contained as little as 0.01% PS8 DNA in a salmon DNA background. Both kinds of cRNA were found to bind to tobacco crown gall tumor DNA-filters. Similar reaction was found with control normal callus DNA-filters but not with tobacco seedling DNA-filters. The "hybrids" formed by cRNA with normal callus and tumor DNA-filters had low thermal stability. Attempts to purify the tumor and normal callus DNA prior to immobilization on the filter resulted in elimination of this spurious binding. No evidence was found for bacterial or phage DNA in crown gall tumor DNA.     

Studies of Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses II. Relationship of Adenovirus 2 Deoxyribonucleic Acid and Simian Virus 40 Deoxyribonucleic Acid in the Ad2+ND1 Genome

A nondefective adenovirus 2 (Ad2)-simian virus 40 (SV40) hybrid virus, Ad2(+)ND(1), has been plaque-isolated from an Ad2-SV40 hybrid population. This virus, unlike the defective Ad-SV40 hybrid populations previously described, replicates without the aid of nonhybrid adenovirus helper. Consequently, the hybrid virus deoxyribonucleic acid (DNA) can be obtained free of nonhybrid adenovirus DNA. The DNA of the Ad2(+)ND(1) virus was shown by ribonucleic acid (RNA)-DNA hybridization to consist of nucleotide sequences complementary to Ad2- and SV40-specific RNA. Techniques of equilibrium density and rate zonal centrifugation were employed to demonstrate that these Ad2 and SV40 nucleotide sequences were linked together in the same DNA molecules by alkali-resistant bonds. Calibration curves were established relating the amount of tritium-labeled SV40-specific RNA (prepared in vitro or in vivo) bound to given amounts of SV40 DNA in a hybridization reaction, and these curves were employed to determine the equivalent amount of SV40 DNA in the Ad2(+)ND(1) molecule. From the results obtained, it was estimated that 1% of the Ad2(+)ND(1) DNA consists of SV40 nucleotide sequences.     

Nucleotide Sequence Relationships of Avian RNA Tumor Viruses: Measurement of the Deletion in a Transformation-Defective Mutant of Rous Sarcoma Virus

Stocks of cloned helper-independent Rous sarcoma virus (RSV) spontaneously segregate transformation-defective (td) mutants that appear to have an RNA genome composed of smaller subunits than those of the patent virus. Differential hybridization and competitive hybridization techniques involving reactions between viral RNA and proviral sequences in host cell DNA (under conditions of initial DNA excess) were used to measure the extent of the deletion in a td mutant of Prague strain (Pr) of RSV (Pr RSV-C). Viral 60 to 70S RNA sequences labeled to 1 to 5 x 10(7) counts per min per mug with (125)I were characterized with respect to their properties in hybridization reactions and used to reinforce data obtained with [(3)H]RNA of lower specific activity. By these techniques, about 13% +/- 3% of the sequences Pr RSV-C that formed hybrids with DNA from virus-induced sarcomas appeared to be deleted from the genome of td Pr RSV-C. Studies comparing hybridization of RNA from Pr RSV-C and td Pr RSV-C with RSV-related sequences in normal cells, and competition experiments with RNA from the endogenous chicken oncornavirus Rous-associated virus type 0 (RAV-0) provided evidence that the majority, if not all, of the RNA sequences of Pr RSV-C deleted from its transformation-defective mutant are not represented in normal chicken DNA. Competition studies with a leukosis virus, RAV-7, indicated this virus also lacks a genome segment of about the same size as the deletion in the td mutant. Finally, the genome of all three "exogenous" viruses was found to lack a small segment (about 12%) of sequences present in the endogenous provirus of RAV-O.     

Preparation of AzoDNA and its use in affinity chromatography

A limited coupling reaction between 4-diazobenzoic acid ([¹⁴C]carboxyl) and sheared single-stranded DNA was employed to prepare a ligand capable of bonding covalently with aminopentane Sepharose C1-4B. The ligand AzoDNA demonstrated small changes in ultraviolet absorbance spectra yet, unlike the parent DNA, had a distinct fluorescence emission peak at 400 nm when excited at 292 nm in neutral or alkaline solutions. On hydroxyapatite thermal chromatography the AzoDNA eluted as single-stranded DNA, while following catalytic reduction, the associated fluorescence and [¹⁴C]azobenzoate radioactivity were removed in large part from the derivatized DNA. In the coupling reaction, prior derivatization of the ligand DNA was required for covalent bonding to aminopentane Sepharose C1-4B and, at optimal polydeoxynucleotide concentrations, about 75 μg was bound/ml of packed gel. DNA:DNA hybridization reactions were accomplished using AzoDNA aminopentane Sepharose C1-4B gels with 50% of the hybridized polynucleotide strands being eluted at temperatures approximating the T_m values measured optically. The use of the AzoDNA gel was extended to the hybridization of adenovirus 2 and vaccinia complementary RNA. The viral complementary RNAs were specifically bound to matrices containing the homologous AzoDNA and eluted under conditions consistent with destabilization of RNA:DNA hybrids. These applications indicate the potential utility of AzoDNA-extensor arm affinity chromatography for the isolation of specific viral RNA molecules.     

Virus-Specific Deoxyribonucleic Acid in Simian Virus 40-Exposed Hamster Cells: Correlation with S and T Antigens

Several homologous hamster embryonic cell lines, transformed in association with simian virus (SV) 40 infection, were examined for the presence of deoxyribonucleic acid (DNA) complementary to SV40 ribonucleic acid (RNA) made in vitro. The methods employed permitted the detection of 10(-5) mug of viral DNA in 100 mug of cellular DNA, corresponding to one-fifth of an SV40 DNA molecule per cell. Those lines which contained both the SV40 surface (S) and tumor (T) antigens also contained DNA complementary to SV40 RNA synthesized in vitro. In contrast, neither of two lines which contained S, but not T, antigen contained detectable DNA complementary to SV40 RNA. These findings suggest that the production of S antigen does not depend upon the persistence of SV40 DNA in transformed cells.     

Simian Virus 40-Host Cell Interactions II. Cytoplasmic and Nucleolar Accumulation of Simian Virus 40 Virion Protein

We have used immunofluorescence in parallel with transmission and scanning electron microscopy to characterize the unusual cytoplasmic and nucleolar accumulation of Simian virus 40 (SV40) virion protein (C antigen) at restrictive temperatures (39 to 41 C) in monkey cells infected with a temperature-sensitive mutant of SV40 defective in virion assembly, tsB11. Cytoplasmic and nucleolar accumulation of C antigen did not occur in wild-type-infected cells at any temperature. Wild-type- and tsBll-infected cells were not distinguishable at 33 C by immunofluorescence or electron microscopy. Temperature-shift experiments using metabolic inhibitors of DNA (cytosine arabinonucleoside, 20 mug/ml), RNA (actinomycin D, 5 mug/ml), and protein synthesis (cycloheximide, 2 x 10(-4) to 10 x 10(-4) M) were used to investigate the requirements for ongoing DNA, RNA, and protein synthesis in the distribution of virion protein between the nucleus, nucleolus, and cytoplasm. The transport of C antigen from the nucleolus and cytoplasm into the nucleus was complete after a temperature shift-down (41 and 39 to 33 C). Limited virus particle formation occurred after the shift-down in the presence of actinomycin D and cycloheximide, indicating some of the 39 to 41 C synthesized virion protein could be used for capsid assembly at 33 C in the absence of further virion protein synthesis. Nucleolar and cytoplasmic accumulations of C antigen occurred in the absence of drugs after a shift-up (33 to 39 C and 41 C) indicating a continuous requirement for the tsB11 mutant function. Furthermore, the virion protein synthesized at 33 C remained confined to the nucleus when the cells were shifted to 39 and 41 C in the presence of actinomycin D or cycloheximide. In the presence of cytosine arabinonucleoside, however, the virion protein accumulated in large aggregates in the nucleus and nucleolus after the shift-up, but did not migrate into the cytoplasm as it did in drug-free tsB11-infected control cells. Colchicine (10(-3) M) had no effect on the abnormal accumulation of C antigen during shift-up or shift-down experiments suggesting that microtubular transport plays little if any role in the abnormal transport of tsB11 virion protein from cytoplasm to nucleus. Although virus particles were never observed by electron microscopy and V antigen was not detected by immunofluorescence at 39 or 41 C in tsB11-infected cells, dense amorphous accumulations were formed in the nucleoli and cytoplasm. We suggest that the tsB11 function is continuously required for the normal transport of SV40 virion protein between the cytoplasm, nucleolus, and nucleus and for the assembly of capsids and virions. Several possible mechanisms for the altered tsB11 function or protein are discussed. One of the virion proteins may also be involved in some presently undetermined nucleolar function during SV40 productive infection.     

The control of human WI-38 cell proliferation by extracellular calcium and its elimination by SV-40 virus-induced proliferative transformation.

The proliferative activity of diploid human WI-38 cells in sparse cultures depends on the extracellular concentration of free (or physiologically available) calcium, and cultivation in a medium having a calcium concentration of 0.1 mM or less gradually, but reversibly, arrest their proliferative development in the prereplicative (G1) phase of the cell cycle. Calcium's proliferative control of this cell type is eliminated by proliferative and morphological transformation by the oncogenic SV-40 virus, and the proliferative activity of SV-WI-38 cells in sparse cultures is unaffected by variation of the extracellular free calcium concentration between 0.00 and 1.25 mM.     

Luliberin analogues containing the nuclear localization sequence of the SV-40 virus T-antigen

With the goal of improving the efficacy of anticancer drugs due to the directed delivery to intracellular targets, a set of luliberin analogues containing the nuclear localization signal (NLS) of the SV-40 virus large T-antigen has been synthesized. The NLS was attached to the parent molecule via the D-lysine ɛ-amino group introduced into position 1 or 6 of the peptide sequence using the orthogonal protection strategy. Biological tests demonstrated that this modification supported in vitro a significant increase in the cytotoxic activity of luliberin analogues containing palmitoyl residues. The influence of the studied peptides on tumor cells was not associated with the distortion of the membrane integrity, which was confirmed by experiments with normal fibroblasts used as a control.     

Mutagenesis by simian virus 40. I. detection of mutations in Chinese hamster cell lines using different resistance markers.

The mutagenic action of SV40 in permanent lines of Chinese hamster cells (CHO-K1 and V79) was investigated with the aid of different resistance markers. The markers studied had resistance to 8-azaguanine (25 and 30 mug/ml), aminopterin (3.3--5.5X10(-3) mug/ml), colchicine (6.5 and 7.0X10(-2) mug/ml) and 5-bromodeoxyuridine (50--120 mug/ml), respectively. After virus infection the mutation frequencies were increased by one (azaguanine, aminopterin) and two (colchicine) orders of magnitude as compared with spontaneous mutation frequencies. In contrast, it was not possible to enhance the frequency of mutation to BUdR resistance. On the other hand, the ability to proliferate in HAT medium was induced in three of five BUdR-resistant cell clones by infection with SV40. The resistance induced by SV40 was stable when isolated clones were cultured under non-selective conditions. Mechanisms are proposed that may be responsible for the mutagenic action of SV40.     

Endocytosis of simian virus 40 into the endoplasmic reticulum

The endocytosis of SV-40 into CV-1 cells we studied using biochemical and ultrastructural techniques. The half-time of binding of [35S]methionine-radiolabeled SV-40 to CV-1 cells was 25 min. Most of the incoming virus particles remained undegraded for several hours. Electron microscopy showed that some virus entered the endosomal/lysosomal pathway via coated vesicles, while the majority were endocytosed via small uncoated vesicles. After infection at high multiplicity, one third of total cell-associated virus was observed to enter the ER, starting 1-2 h after virus application. The viruses were present in large, tubular, smooth membrane networks generated as extentions of the ER. The results describe a novel and unique membrane transport pathway that allows endocytosed viral particles to be targeted from the plasma membrane to the ER.     

Properties of the genome in normal and SV-40 transformed WI-38 human diploid fibroblasts. III. Turnover of nonhisone chromosomal proteins and their phosphate groups.

The turnover of nonhistone chromosomal proteins and their phosphate groups was compared in normal and in SV-40 virus transformed WI-38 human diploid fibroblasts. Cells were pulse labelled with tryptophan-³H and ³²P for 30 minutes and the specific activities of tryptophan-³H and ³²P in the various molecular weight classes of nonhistone chromosomal proteins were determined during the first four hours following termination of labelling. While a rapid turnover of high molecular weight nonhistone polypeptides (142, 000 to 200, 000 Daltons) is evident after one hour in SV_40 transformed cells, the specific activities of these nonhistone chromosomal polypeptides are not significantly decreased in normal cells. In contrast, a rapid turnover of low molecular weight (30, 000 to 51, 000 Daltons) nonhistone chromosomal proteins occurs during the first hour in normal WI-38 cells with no corresponding decrease in the specific activities of these proteins in SV-40 transformed cells. There is no apparent net turnover of phosphate groups on nonhistone chromosomal proteins in either normal or SV-40 transformed cells four hours following pulse labelling. Rather, during the first four hours significant fluctuations are observed in the ³²P specific activities of defined molecular weight fractions. Taken together with previous reports of differences in the composition, synthesis and phosphorylation of nonhistone chromosomal proteins in normal and SV-40 transformed human diploid cells the present results further indicate the complex nature of the alterations in these proteins which accompany viral transformation.     

Synthesis of reovirus ribonucleic acid in L cells

Kudo, Hajime (The Wistar Institute of Anatomy and Biology, Philadelphia, Pa.), and A. F. Graham. Synthesis of reovirus ribonucleic acid in L cells. J. Bacteriol. 90:936-945. 1965.-There is no inhibition of protein or deoxyribonucleic acid (DNA) synthesis in L cells infected with reovirus until the time that new virus starts to form about 8 hr after infection. At this time, both protein synthesis and DNA synthesis commence to be inhibited. Neither the synthesis of ribosomal ribonucleic acid (RNA) nor that of the rapidly labeled RNA of the cell nucleus is inhibited before 10 hr after infection. Actinomycin at a concentration of 0.5 mug/ml does not inhibit the formation of reovirus, although higher concentrations of the antibiotic do so. Pulse-labeling experiments with uridine-C(14) carried out in the presence of 0.5 mug/ml of actinomycin show that, at 6 to 8 hr after infection, two species of virus-specific RNA begin to form and increase in quantity as time goes on. One species is sensitive to ribonuclease action and the other is very resistant. The latter RNA is probably double-stranded viral progeny RNA, and it constitutes approximately 40% of the RNA formed up to 16 hr after infection. The function of the ribonuclease-sensitive RNA is not yet known. Synthesis of both species of RNA is inhibited by 5 mug/ml of actinomycin added at early times after infection. Added 6 to 8 hr after infection, when virus-specific RNA has already commenced to form, 5 mug/ml of actinomycin no longer inhibit the formation of either species of RNA.