Stable association of viral protein VP1 with simian virus 40 DNA.

Mild dissociation of simian virus 40 particles releases a 110S virion core nucleoprotein complex containing histones and the three viral proteins VP1, VP2, and VP3. The association of viral protein VP1 within this nucleoprotein complex is mediated at least partially through a strong interaction with the viral DNA. Treatment of the virion-derived 110S nucleoprotein complex with 0.25% Sarkosyl dissociated VP2, VP3, and histones, leaving a stable VP1-DNA complex. The VP1-DNA complex had a sedimentation value of 30S and a density of 1.460 g/cm3. The calculated molecular weight of the complex was 7.9 x 10(6), with an average of 100 VP1 molecules per DNA. Agarose gel electrophoresis of the VP1-DNA complex demonstrated that VP1 is associated not only with form I and form II simian virus 40 DNAs but also with form III simian virus 40 DNA generated by cleavage with EcoRI.     

A cis-acting DNA signal for encapsidation of simian virus 40.

Encapsidation of simian virus 40 is a complex biological process involving DNA-protein and protein-protein interactions in the formation of a unique three-dimensional structure around the viral minichromosome. A pseudoviral system developed in our laboratory, in which the viral early and late gene products are supplied in trans (by helpers), was used to analyze the encapsidation process independent of viral gene expression. With this experimental system we have discovered a requirement for a specific DNA signal for encapsidation, ses (for simian virus 40 encapsidation signal).ses is present within a 200-bp DNA fragment, which includes, in addition to the viral origin of replication (ori), six GGGCGG repeats (GC boxes) and 26 bp of the enhancer element. Deletion of the GC boxes and the enhancer sequences almost abolished encapsidation, while DNA replication was only moderately decreased. The ability to encapsidate was not regained by reinserting a DNA fragment carrying ses in the sesdeleted plasmid 2 kbp away from the ori, suggesting that for encapsidation the two DNA elements have to be close to each other. These findings afford novel strategies for the investigation of viral encapsidation.     

Pattern of Protein Synthesis in Monkey Cells Infected by Simian Virus 40

After infection of several permanent monkey cell lines by simian virus 40 (SV40), four additional protein bands can be detected by simple sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell extracts. These bands appear only after the onset of viral deoxyribonucleic acid (DNA) synthesis, and inhibitors of DNA synthesis prevent their appearance. Three of them correspond to three previously identified capsid components, VP1, VP2, and VP3. The fourth protein band, which does not correspond to a previously identified virion component, is induced by SV40 infection of CV-1 and BSC-1 cultures but not by infection of MA-134 cultures.     

Variation in p30-related proteins in gross virus-induced tumor cell lines derived from H-2 congenic mice

The distribution of DNA topoisomers in intracellular simian virus 40 DNA was analyzed by gel electrophoresis. The results suggested that DNA extracted from 70S chromatin had a different superhelical density distribution as compared with the DNA obtained from virions or virion assembly intermediates. The heterogeneity of simian virus 40 viral DNA superhelical density at a late time after infection was partly due to increased virion production and partly due to the intrinsic heterogeneity of the superhelical density of DNA extracted from virions. Using two-dimensional gel electrophoretic analysis we also showed that simian virus 40 DNA templates used for DNA replication have a higher average superhelical density than the bulk of intracellular viral DNA.     

Identification of Virus-Induced Proteins in Cells Productively Infected with Simian Virus 40

The number and molecular weight of the structural polypeptides of highly purified simian virus 40 (SV40) were determined by polyacrylamide gel electrophoresis. Six different polypeptides were found, two of which (VP1 and VP2) comprise the bulk of the viral capsid proteins. The pattern of protein synthesis in productively infected CV-1 cells was studied by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Identification of virus-induced proteins in the infected CV-1 cells was achieved in double-labeling experiments by electrophoresis with purified labeled SV40 capsid proteins. Four of these proteins (VP1 and VP4) could be classified as components of the virion because their synthesis occurred after the onset of viral deoxyribonucleic acid (DNA) replication and because they were inhibited by arabinofuranosylcytosine (ara-C). Appearance of two other virus-induced proteins was not prevented by ara-C; one of them did not comigrate in the electrophoresis with purified virion polypeptides, and both could be detected before the onset of viral DNA synthesis. These latter two proteins were classified on the basis of these criteria as nonvirion capsid proteins (NCVP1 and NCVP2).     

Effect of alkylation on the physical properties of simian virus 40 T-antigen species.

We analyzed large and small species of T-antigen by immunoprecipitation and two-dimensional gel electrophoresis. The T-antigen species were subjected to electrophoresis either directly or after reduction and alkylation with N-ethylmaleimide. Treatment with N-ethylmaleimide improved the resolution of large-T by two-dimensional gel electrophoresis and was a requirement for the resolution of small-t antigen on two dimensional gels. Large-T did not form a discrete protein spot, but rather formed a streak from approximately pH 6.5 to 6.9 on isoelectric focusing gels. Small-t formed a sharp protein spot at approximately pH 7.2 when subjected to electrophoresis under non-equilibrium conditions which extended the pH gradient to include proteins with basic isoelectric points. Treatment with N-ethylmaleimide decreased the mobility of the T-antigen species during sodium dodecyl sulfate gel electrophoresis. We suggest that the apparent increase in molecular weight was due to the association of N-ethylmaleimide with cysteine-rich regions of these proteins. Viable deletion mutants of simian virus 40 which do not induce the synthesis of small-t but product small-t-related polypeptides were used to localize the cysteine-rich region of small-t to between 0.54 and 0.59 on the genetic map of simian virus 40.     

The distribution of large T antigen in simian virus 40 nucleoprotein complexes

Simian virus 40 large T antigen binds to two types of nucleoprotein complexes from lytically infected cells: those containing replicating virus DNA (100S complexes) and those containing nonreplicating virus DNA (70S complexes). Analysis by agarose gel electrophoresis showed that replicating DNA was found exclusively in 100S complexes, although these complexes also contained large amounts of form I and form II DNA. In contrast, no replicating DNA was found in 70S complexes, and pulse-labeled DNA in these complexes migrated as form I and form II DNA that presumably had recently completed replication. Immunoprecipitation and gel electrophoresis showed that large T antigen was associated with both types of complexes. From 21 to 62% of replicating DNA in 100S complexes was bound to T antigen. Our estimates indicated, however, that more than three-fourths of the DNA molecules in 100S complexes were nonreplicating and unassociated with T antigen. In 70S complexes, 12 to 31% of pulse-labeled DNA was bound to T antigen, but because there were more DNA molecules in the 70S complexes, they contained a greater absolute amount of T antigen.     

Properties of simian virus 40 small t antigen overproduced in bacteria

We constructed a series of bacterial plasmids which contained the Escherichia coli lac promoter fused to a simian virus 40 restriction fragment coding for small t antigen. These plasmids expressed different levels of intact viral protein depending on the length of the constructed ribosome binding site. Small t antigen synthesized by the most efficient producer, HP1, constituted 0.5 to 1% of the total cellular protein. On the basis of extensive characterization by immunoprecipitation, gel electrophoresis, isoelectric focusing, tryptic fingerprint analysis, and chromatographic properties, this plasmid-encoded protein was virtually identical to authentic simian virus 40 small t antigen. Partial purification of the HP1-encoded and authentic small t antigens revealed the presence of both monomeric and multimeric forms.     

Characterization of a metastable, partially replicated dimeric intermediate of minute virus of mice

Intracellular, replicative-form DNA of minute virus of mice was characterized by agarose gel electrophoresis, velocity sedimentation, electron microscopy, restriction endonuclease digestion, and sensitivity to the single-stranded nuclease S1. This analysis demonstrated the presence in murine cells infected with minute virus of mice of a 10.0-kilobase pair dimer replicative form, a 5-kilobase pair monomer replicative form, as well as a 5-kilobase viral single-stranded DNA species. Two additional viral DNA species that migrated in 0.5% agarose gels with apparent sizes of 8.0 and 5.5 kilobase pairs were also observed. Further investigation indicated that the 8.0-kilobase pair DNA represents a novel class of metastable, partially replicated, dimeric intermediates. This finding has important implications for the mechanism of parvovirus DNA replication.     

Presence of free viral DNA in simian virus 40-transformed nonproducer cells.

Extracts from several simian virus 40 (SV40)-transformed nonproducer cells were prepared by the hot-phenol procedure normally used to extract cellular RNA. These extracts contained SV40 infectious units. Part of the infectious units were identified as SV40 form I DNA molecules. The results of reconstruction experiments suggest that SV40 form I DNA is extractable by the hot-phenol procedure because of its fast renaturation rate. The significance of the presence of free viral DNA in nonproducer transformed cells is discussed.     

Restriction endonuclease cleavage map of the DNA of JC virus.

A physical map of the sites cleaved by the following restriction endonucleases was derived for the DNA of JC virus, a human polyomavirus: EcoRI, HpaI, and PstI (one site each); HindII (four sites); and HindIII (three sites). By agarose gel electrophoresis of fragmented DNA, the size of full-length DNA of JC virus was estimated to be 5,125 +/- 105 base pairs (98 +/- 2% of the length of simian virus 40 DNA).     

Intracellular forms of simian virus 40 nucleoprotein complexes. III. Study of histone modifications.

The modification patterns of histones present in various forms of intracellular simian virus 40 nucleoprotein complexes were analyzed by acetic acid-urea-polyacrylamide gel electrophoresis. The results showed that different viral nucleoprotein complexes contain different histone patterns. Simian virus 40 chromatin, which contains the activities for the synthesis of viral RNA and DNA, exhibits a histone modification pattern similar to that of the host chromatin. However, virion assembly intermediates and mature virions contain highly modified histones. Pulse-chase experiments with [3H]lysine showed that the newly incorporated histones in the virion assembly intermediates were already highly modified. The majority of in vivo acetylation activity of histones occurred on the 70S simian virus 40 chromatin as analyzed by pulse-labeling with [3H]acetate. These results and our previous analysis of the virion assembly pathway suggest that three stages are involved in the packaging of simian virus 40 chromatin into the mature virion: (i) modification of histones, (ii) accumulation of capsid protein around the chromatin with highly modified histones, and (iii) organization of capsid proteins into salt-resistant shells. The role of histone modification in virion assembly is discussed.     

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.     

A vaccinia virus DNase preparation which cross-links superhelical DNA.

Multiple DNA-dependent enzyme activities have been detected in highly purified preparations of a single-strand-specific nuclease from vaccinia virus. These enzyme preparations were extensively purified and characterized by using superhelical DNAs as substrates. In particular, the nuclease activity was monitored by the extent of conversion of supercoiled closed duplex DNA (DNA I) to nicked circular DNA (DNA II), which could subsequently be converted to duplex linear DNA (DNA III) by prolonged incubation with the enzyme. DNA species which were not substrates for the enzyme included relaxed closed duplex DNA, DNA II which had been prepared by nuclease S1 treatment or by photochemical nicking of DNA I, and DNA III. With plasmid pSM1 DNA as substrate, the extent of cleavage of DNA I to DNA II was found to increase with superhelix density above a threshold value of about -0.06. The linear reaction products were examined by gel electrophoresis after restriction enzyme digestion of the DNAs from plasmids pSM1 and pBR322 and of the viral DNAs from bacteriophage phi X174 (replicative form) and simian virus 40, and the map coordinate locations of the scissions were determined. These products were further examined by electron microscopy and by gel electrophoresis under denaturing conditions. Electron micrographs taken under partially denaturing conditions revealed molecules with terminal loops or hairpins such as would result from the introduction of cross-links at the cutting sites. These species exhibited snapback renaturation. The denaturing gel electrophoresis experiments revealed the appearance of new bands at locations consistent with terminal cross-linking. With pSM1 and pBR322 DNAs, this band was shown to contain DNA that was approximately twice the length of a linear single strand. The terminal regions of the cross-linked linear duplex reaction products were sensitive to nuclease S1 but insensitive to proteinase K, suggesting that the structure is a hairpin loop not maintained by a protein linker. A similar structure is found in mature vaccinia virus DNA.     

Analysis of supercoiled DNA by agarose gel electrophoresis using low-conducting sodium threonine medium

We describe a new low-ionic-strength sodium threonine (STh) medium with the advantage of avoiding relative DNA band migration changes following electrophoresis of supercoiled DNA in agarose gel when substituted for the standard conductive medium of TBE (Tris-boric acid-ethylenediaminetetraacetic acid [EDTA]) or TAE (Tris-acetic acid-EDTA) or the low-ionic-strength sodium boric acid medium. Low-ionic-strength STh medium provided better resolution, less heat generation, and prevention of relative migration order changes among linear, covalently closed circular-, and open circular-formed DNA in the range of 2-10 kilobase pairs in 1% agarose gel electrophoresis.     

Two-dimensional analysis of proteins sedimenting with simian virus 40 chromosomes.

The nonhistone proteins sedimenting in low-salt glycerol gradients with simian virus 40 chromosomes were analyzed by two-dimensional gel electrophoresis, utilizing nonequilibrium pH gradients as the first dimension and sodium dodecyl sulfate-gel electrophoresis as the second dimension. By densitometric quantitation of the radiolabeled proteins present in each fraction of the gradients, it was possible to identify sedimenting with all or a fraction of the simian virus 40 chromosomes. VP-1 sedimented with simian virus 40 chromosomes; additional evidence for its binding to chromosomes was obtained by immunochemical techniques. Four proteins (Mr 25,000, pI 6.0; Mr 32,000, pI 7.2; Mr 35,000, pI 8.5; and Mr 80,000, pI 7.2) sedimented with specific subsets of chromosomes.     

Preparation of large quantities of separated strands from simian virus 40 DNA restriction fragments by low-temperature low-salt agarose gel electrophoresis.

We have used agarose gel electrophoresis to separate complementary DNA strands obtained from simian virus 40 DNA restriction fragments produced by HindII and III or by EcoRI and HpaII digestion. By modifying existing methods we have virtually eliminated the problematic renaturation of DNA during electrophoresis. This has allowed us to recover large quantities of separated DNA strands (approximately 20 μg of DNA per 12-mm-diameter preparative tube gel). By using a combination of low temperature and low buffer concentration during electrophoresis, we have also significantly improved the resolution of DNA strands.     

Identification of mouse mammary tumor virus-specific mRNA.

Complementary DNA corresponding to the RNA genome of mouse mammary tumor virus was used to identify viral RNA contained in polysomes of a virus-producing mammary tumor cell line. Separation of polysomal mRNA by agarose gel electrophoresis, transfer of the RNA to diazobenzyloxymethyl paper, and hybridization with 32P-labeled mouse mammary tumor virus complementary DNA revealed three viral RNA size classes of 10, 8.8, and 4.4 kilobases in length, respectively.