Nucleoprotein complexes of minute virus of mice have a distinct structure different from that of chromatin.

We studied the structure of viral nucleoprotein complexes extracted from the nuclei of mouse cells infected with the immunosuppressive strain of the minute virus of mice (MVMi). Two types of complex were detected, with sedimentation coefficients of about 110 and 40S. The complexes sedimenting at 110S contained single-stranded MVMi DNA as well as a second form of viral DNA which apparently had a heat-sensitive secondary structure. The 110S peak also contained proteins which coelectrophoresed with the MVMi capsid proteins. Complexes sedimenting at 40S contained the double-stranded replicative form of MVMi DNA. These complexes sedimented faster than did the pure replicative form DNA (15S), but more slowly than cellular chromatin fragments containing DNA of the same length. They incorporated labeled deoxynucleoside triphosphate in vitro into the replicative form DNA. We investigated the structure of MVMi nucleoprotein complexes in the following ways. Nuclei of MVMi-infected cells were digested with staphylococcal nuclease, and the resulting DNA fragments were electrophoresed, transferred to nitrocellulose, and hybridized first with labeled MVMi DNA and then with cellular DNA. A nucleosomal repeat pattern was seen with the cellular DNA probe but not with the MVMi DNA probe. The DNA in MVMi nucleoprotein complexes was cross-linked with psoralen, purified, denatured, and examined with an electron microscope. Bubbles, indicating the presence of proteins, were seen in the MVMi DNA. The length of the DNA in the bubbles was 90 +/- 29 nucleotides. On the other hand, nucleosomes protected 160 base pairs from cross-linking by psoralen. The MVMi nucleoprotein complexes thus have a distinct structure which is different from that of chromatin.     

Parental adenovirus DNA accumulates in nucleosome-like structures in infected cells.

Micrococcal-nuclease digestion of adenovirus 2(ad 2) infected HeLa cell nuclei early after infection has been used to investigate the nucleoprotein nature of parental viral DNA. Viral DNA is more susceptible to nuclease digestion than cellular DNA. The pattern of digestion products changes as digestion proceeds from an indistinct pattern 1 hour post infection(pi) to a nucleosome-like pattern at 6 hours pi. The major differences between viral and cellular nucleoprotein products were i) a subnucleosome fraction from viral DNA and ii) the repeat size of DNA in viral nucleosomes was 165 base pairs and in cellular nucleosomes, 195 base pairs. Up to 50% viral DNA in nuclei 6 hours pi seems to be in nucleosome-like structures. Such patterns are not seen on digestion of partially-uncoated virus or isolated cores.     

Intracellular Forms of Adenovirus DNA III. Integration of the DNA of Adenovirus Type 2 into Host DNA in Productively Infected Cells

KB cells productively infected with human adenovirus type 2 contain an alkalistable class of viral DNA sedimenting in a broad zone between 50 and 90S as compared to 34S for virion DNA. This type of DNA is characterized as viral by DNA-DNA hybridization. It is extremely sensitive to shear fragmentation. Extensive control experiments demonstrate that the fast-sedimenting viral DNA is not due to artifactual drag of viral DNA mechanically trapped in cellular DNA or to association of viral DNA with protein or RNA. Furthermore, the fast-sedimenting DNA is found after infection with multiplicities between 1 and 1,000 PFU/cell and from 6 to 8 h postinfection until very late in infection (24 h). Analysis in dye-buoyant density gradients eliminates the possibility that the fast-sedimenting viral DNA represents supercoiled circular molecules. Upon equilibrium centrifugation in alkaline CsCl density gradients, the fast-sedimenting viral DNA bands in a density stratum intermediate between that of cellular and viral DNA. In contrast, the 34S virion DNA isolated and treated in the same manner as the fast-sedimenting DNA cobands with viral marker DNA. After ultrasonic treatment of the fast-sedimenting viral DNA, it shifts to the density positions of viral DNA and to a lesser extent to that of cellular DNA. The evidence presented here demonstrates that the 50 to 90S viral DNA represents adenovirus DNA covalently integrated into cell DNA.     

Intracellular DNA of the parvovirus minute virus of mice is organized in a minichromosome structure.

Minute virus of mice (MVM) nucleoprotein complexes were leached from infected cell nuclei in the presence of a hypotonic buffer. Detailed biochemical analyses performed on the extracted complexes revealed nucleoprotein complexes sedimenting together with virions at 110S and defective particles sedimenting at 50S. In contrast to the virions, the nucleoprotein complexes were found to be sensitive to treatment with DNase, Sarkosyl, and heparin. They were found to be composed of replicative forms of MVM DNA and cellular histones. After extensive micrococcal nuclease digestion performed on purified nucleoprotein complexes, a viral nucleosomes core containing a DNA segment of about 140 base pairs in length was identified. These complexes when visualized by electron microscopy revealed the existence of beaded structures (minichromosomes) having 26 and 52 beads per monomer and dimer molecules, respectively. We suggest that the organization of the intracellular viral DNA in a minichromosome structure is an essential step in the virus growth cycle.     

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.     

Parental adenovirus type 2 genomes recovered early or late in infection possess terminal proteins.

At both early (3 h) and late (18 h) times after infection of KB cells with adenovirus 2, more than 90% of parental nuclear viral genomes exist as complexes which contain terminally linked proteins. Density shift experiments employing 5-bromo-2'-deoxyuridine indicate that these parental DNA molecules remain complexed with terminal proteins after DNA replication. The persistent linkage of proteins to the termini of intranuclear viral DNA suggests that these proteins have an essential role in adenovirus replication.     

Transcription map for adenovirus type 12 DNA.

The regions of the adenovirus type 12 genome which encode l- and r-strand-specific cytoplasmic RNA were mapped by the following procedure. Radioactive, intact, separated complementary strands of the viral genome were hybridized to saturating amounts of unlabeled late cytoplasmic RNA. The segments of each DNA strand complementary to the RNA were then purified by S1 nuclease digestion of the hybrids. The arrangement of the coding regions of each strand was deduced from the pattern of hybridization of these probes to unlabeled viral DNA fragments produced by digestion with EcoRI, BamHI, and HindIII.. The resulting map is similar, if not identical, to that of adenovirus type 2. The subset of the late cytoplasmic RNA sequences which are expressed at early times were located on the map by hybridizing labeled, early cytoplasmic RNA to both unlabeled DNA fragments and unlabeled complementary strands of specific fragments. Early cytoplasmic RNA hybridized to the r-strand to EcoRI-C and BamHI-B and to the l-strand of BamHI-E. Hybridization to BamHI-C was also observed. The relative rates of accumulation of cytoplasmic RNA complementary to individual restriction fragments was measured at both early and late times. Early during infection, most of the viral RNA appearing in the cytoplasm was derived from the molecular ends of the genome. Later (24 to 26 h postinfection) the majority of the newly labeled cytoplasmic RNA was transcribed from DNA sequences mapping between 25 and 60 map units on the genome.     

Intracellular forms of simian virus 40 nucleoprotein complexes. I. Methods of isolation and characterization in CV-1 cells.

A new method was developed for isolation of intracellular forms of simian virus 40 (SV40) nucleoprotein complexes from SV40-infected CV-1 cells late in the infectious cycle. In contrast to the Triton extraction method, which yields only a 60-70S complex, this new procedure yielded three forms of SV40 nucleoprotein complexes: complex I, complex II, and the nature virion (V). The three nucleoprotein complexes differed in physical as well as biochemical properties. Complex I, which is only a small portion of the total SV42 nucleoprotein complexes late during infection, was active in synthesizing both SV40-specific DNA and RNA. Pulse-labeling experiments suggest the following metabolic pathway: I leads to II leads to V. Conversion of complex I to II occurred shortly after the completion of SV40 DNA replication and resulted in the inactivation of the biosynthetic activities of I.     

Characterization of Polyoma DNA-Protein Complexes I. Electrophoretic Identification of the Proteins in a Nucleoprotein Complex Isolated from Polyoma-Infected Cells

A study was undertaken to examine polyoma DNA-protein complexes. A biophysical characterization of the complexes was made, and the proteins found in such complexes were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A comparison was made between a 52S nucleoprotein complex isolated from nuclei of 26-h polyoma-infected cells and a 28S virion core complex ejected out of mature virus particles. It was found that both complexes were reduced to a 20S viral DNA component plus free protein after incubation in 1 M NaCl or Sarkosyl. Treatment of the complexes with either Pronase or 0.5 M NaCl resulted in only partial removal of proteins from the viral DNA. After fixation in formaldehyde, the 52S nucleoprotein complex had a buoyant density of 1.45 g/cm³, and the virion core complex had a buoyant density of 1.59 g/cm³. Sodium dodecyl sulfate-polyacrylamide gel profiles of purified polyoma virion proteins, used as a reference marker, demonstrated three capsid proteins, V1 to V3, as well as four histones, V4 to V7, which constituted about 7% of the total virion protein. Electrophoretic analysis of the proteins comprising the 52S nucleoprotein complex revealed that the same seven proteins present in the mature virion were also found in this complex. However, the ratios of the proteins in the complex were quite different from that of the mature virion, with the four histones comprising 48% of the total complex protein. A pulse-chase experiment of the nucleoprotein complex demonstrated that the 26-h complex was chased into mature virions.     

Intracellular forms of simian virus 40 nucleoprotein complexes. IV. Micrococcal nuclease digestion.

The structures of DNAs present in various intracellular forms of simian virus 40 (SV40) nucleoprotein complexes were analyzed by micrococcal nuclease digestion. The results showed that the 70S SV40 chromatin was completely sensitive to nuclease digestion, whereas CsCl gradient-purified mature virion was completely resistant. Virion assembly intermediates with different degrees of virion maturation showed intermediate resistance, and three products were found: nucleosomal DNA fragments, representing the fraction of intermediates that were sensitive to nuclease; linear SV40 genome-sized DNA, representing the more mature intermediates that contained one or limited defects in the capsid shell; and supercoiled SV40, which was derived from mature virions. These digestion products, however, remained associated with capsid shells after nuclease digestion. These results were consistent with the model in which maturation of the SV40 virion is achieved through the organization of capsid proteins that accumulate around SV40 chromatin. Mild digestion of SV40 nucleoprotein complexes with micrococcal nuclease revealed the difference in nucleosome repeat length between SV40 chromatin and virion assembly intermediates. A novel DNA fragment of about 75 nucleotides was observed early in nuclease digestion.     

Adenovirus chromatin structure at different stages of infection.

We investigated the structure of adenovirus deoxyribonucleic acid (DNA)-protein complexes in nuclei of infected cells by using micrococcal nuclease. Parental (infecting) DNA was digested into multimers which had a unit fragment size that was indistinguishable from the size of the nucleosomal repeat of cellular chromatin. This pattern was maintained in parenteral DNA throughout infection. Similar repeating units were detected in hamster cells that were nonpermissive for human adenovirus and in cells pretreated with n-butyrate. Late in infection, the pattern of digestion of viral DNA was determined by two different experimental approaches. Nuclear DNA was electrophoresed, blotted, and hybridized with labeled viral sequences; in this procedure all virus-specific DNA was detected. This technique revealed a diffuse protected band of viral DNA that was smaller than 160 base pairs, but no discrete multimers. All regions of the genome were represented in the protected DNA. To examine the nuclease protection of newly replicated viral DNA, infected cells were labeled with [3H]thymidine after blocking of cellular DNA synthesis but not viral DNA synthesis. With this procedure we identified a repeating unit which was distinctly different from the cellular nucleosomal repeat. We found broad bands with midpoints at 200, 400, and 600 base pairs, as well as the limit digest material revealed by blotting. High-resolution acrylamide gel electrophoresis revealed that the viral species comprised a series of closely spaced bands ranging in size from less than 30 to 250 base pairs.     

Adenoviral heterogeneous nuclear RNA is associated with the host nuclear matrix during splicing

After infection of HeLa cells with adenovirus type 2, virus-specific heterogeneous nuclear RNA is quantitatively associated with a higher ordered structure, the nuclear matrix. Analysis of this matrix-associated RNA by S₁ nuclease mapping showed that precursors as well as processed messenger RNAs from the late region L4 were present. By irradiation of intact cells with ultraviolet light, proteins tightly associated with heterogeneous nuclear RNA can be induced to cross-link with the RNA. Characterization of the cross-linked RNA-protein complexes showed that all viral polyadenylated RNAs (precursors, products and processing intermediates) could be cross-linked to two host proteins, earlier found to be involved in the association of host-specific heterogeneous nuclear RNA to the nuclear matrix (van Eekelen &; van Venrooij, 1981). Our results thus further support the concept that the nuclear matrix may function in the localization and the structural organization of (viral) heterogeneous nuclear RNA during its processing.     

Adenovirus DNA is associated with the nuclear matrix of infected cells.

Viral DNA was found to be tightly associated with the nuclear matrix from HeLa cells lytically infected with human adenovirus type 5. The bound viral DNA, like cell DNA, was resistant to nonionic detergent and to extraction with high-salt (2 M NaCl) solution. However, whereas over 95% of the cell DNA was recovered in the matrix fraction, the amount of associated viral DNA varied during infection. Throughout the lytic cycle, the amount of matrix-associated adenovirus type 5 DNA increased until it reached a plateau level at 20 to 24 h after infection. At this stage, the matrix-bound DNA represented 87% of the total viral DNA; after this stage, additional newly synthesized viral DNA accumulated as non-matrix-associated DNA. DNase digestion studies revealed that all viral DNA sequences were equally represented in the matrix-bound DNA both early and late in infection; thus, unlike cell DNA, there seem to be no preferred attachment sites on the viral genome. An enrichment of viral DNA relative to cell DNA was found in the matrix-associated DNA after extensive DNase I digestion. This finding, together with an in situ hybridization study, suggests that the viral DNA is more intimately associated with the nuclear matrix than is cell DNA and probably does not exist in extended loops.