Fate of Infecting Simian Virus 40-Deoxyribonucleic Acid in Nonpermissive Cells: Integration into Host Deoxyribonucleic Acid

Nonpermissive 3T3 cells were infected with purified superhelical simian virus 40 (SV40) deoxyribonucleic acid I (DNA I). One hour after infection, approximately 60% of the intracellular SV40 DNA was converted to relaxed forms. One day after infection, all intracellular SV40 DNA was present as slow-sedimenting material, and no SV40 DNA I was detectable. At 2 days after infection there appeared viral DNA sequences cosedimenting with cellular DNA during alkaline velocity centrifugation. Furthermore, by both alkaline equilibrium gradient centrifugation and by DNA-ribonucleic acid hybridization analysis, covalent linkage of viral DNA sequences to cellular DNA was demonstrated. Integration of SV40 DNA into cellular DNA did not appear to require DNA synthesis, although DNA synthesis followed by mitotic division of the cells enhanced the amount of viral DNA integrated. Based on data obtained by two different methods, it was calculated that 1,100 to 1,200 SV40 DNA equivalents must be integrated per cell by 48 hr after infection.     

Synthesis of Avian Oncornavirus DNA in Infected Chicken Cells

The intracellular synthesis and integration of viral DNA (vDNA) into the host cell genome was studied in cultured chicken embryo fibroblasts infected with avian sarcoma or leukemia viruses. The newly synthesized vDNA was detected by hybridization with 70S viral RNA. Extraction of infected cell DNA by the selective procedure of Hirt resulted in the enrichment of newly synthesized vDNA in the low molecular weight supernatant fraction while leaving the bulk of cellular DNA containing integrated vDNA in the high molecular weight pellet fraction. This approach led to detection of intracellular vDNA synthesis within 1 h after infection and to vDNA integration into cellular DNA within 24 h. There was a several-fold increase in the vDNA content of infected cells during the initial phase of virus infection. But only a part of this newly synthesized vDNA appeared to become covalently linked with high molecular weight cellular DNA. Most of the remaining unintegrated vDNA gradually disappeared. The sedimentation profiles of minimally sheared cellular DNA in alkaline sucrose velocity gradients suggest that vDNA is synthesized as free linear molecules of approximately 3 x 10(6) daltons which subsequently are covalently linked to host cell DNA.     

Resolution and characterization of intracytoplasmic forms of reverse transcriptase from Rauscher leukemia virus-producing cells.

We have investigated the association of viral DNA with cell DNA in chicken embryo kidney (CEK) cells productively infected with chicken embryo lethal orphan (CELO) virus and in human (HEK) cells infected with mutants ts36 and ts125 of human adenovirus type 5 under permissive and restrictive conditions. Cell and viral DNA molecules were separated after CELO virus infection of CEK cells by alkaline sucrose gradient centrifugation, network formation, and CsCl density gradient centrifugation, methods that rely on different properties of the DNA. The cell DNA was then tested for viral sequences by DNA reannealing kinetics. Between 500 and 1,000 viral genome equivalents per cell were found at 36 h postinfection associated with cell DNA purified by each method. These values greatly exceeded the amount of free viral DNA found contaminating cell DNA prepared by the same methods from uninfected cells to which CELO virus DNA had been added. Quantitative agreement in the amounts of viral DNA found associated with cell DNA purified by these different methods suggests that CELO virus DNA is integrated into chick cell DNA during lytic infection. Similar experiments in HEK cells using mutants ts36 and ts125 of adenovirus type 5 at both restrictive and permissive temperatures showed that the same proportion of viral DNA is associated with cell DNA in the absence of viral DNA replication, and this suggests that the difference in the frequency with which cells are transformed by these mutants is not due to a difference in the frequency integration.     

Integrated Simian Virus 40 DNA: Nucleotide Sequences at Cell-Virus Recombinant Junctions

DNA fragments containing the integrated viral DNA present in the simian virus 40 (SV40)-transformed rat cell lines SVRE9 and SVRE17 were cloned in procaryotic vectors, and the DNA sequences linking SV40 and cell DNA were determined. Comparison of the DNA sequences at the SV40-cell junctions in SVRE9 and SVRE17 cells with those of a previously characterized viral insertion from SV14B cells shows that no specific viral or cellular sequences occur at SV40-cell junctions and that the cellular DNA sequences adjacent to integrated SV40 DNA do not display the direct repeat structure characteristic of transposons and retrovirus proviruses.     

Temperature-Sensitive Simian Virus 40 Mutant Defective in a Late Function

A temperature-sensitive simian virus 40 (SV40) mutant, tsTNG-1, has been isolated from nitrosoguanidine-treated and SV40-infected African green monkey kidney (CV-1) cultures. Replication of virus at the nonpermissive temperature (38.7 C) was 3,000-fold less than at the permissive temperature (33.5 C). Plaque formation by SV40tsTNG-1 deoxyribonucleic acid (DNA) on CV-1 monolayers occurred normally at 33.5 C but was grossly inhibited at 38.7 C. The time at which virus replication was blocked at 38.7 C was determined by temperature-shift experiments. In shift-up experiments, cultures infected for various times at 33.5 C were shifted to 38.7 C. In shift-down experiments, cultures infected for various times at 38.7 C were shifted to 33.5 C. All cultures were harvested at 96 hr postinfection (PI). No virus growth occurred when the shift-up occurred before 40 hr PI. Maximum virus yields were obtained at 96 hr PI when the shift-down occurred at 66 hr, but only about 15% of the maximum yield was obtained when the shift-down occurred at 76 hr PI. These results indicate that SV40tsTNG-1 contains a conditional lethal mutation in a late viral gene function. Mutant SV40tsTNG-1 synthesized T antigen, viral capsid antigens, and viral DNA, and induced thymidine kinase activity at either 33.5 or 38.7 C. The properties of the SV40 DNA synthesized in mutant-infected CV-1 cells at 33.5 or 38.7 C were very similar to those of SV40 DNA made in parental virus-infected cells, as determined by nitrocellulose column chromatography, cesium-chloride-ethidium bromide equilibrium centrifugation, and by velocity centrifugation in neutral sucrose gradients. Mutant SV40tsTNG-1 enhanced cellular DNA synthesis in primary cultures of mouse kidney cells at 33.5 and 38.7 C and also transformed mouse kidney cultures at 36.5 C. SV40tsTNG-1 was recovered from clonal lines of transformed cells after fusion with susceptible CV-1 cells and incubation of heterokaryons at 33.5 C, but not at 38.7 C.     

Integrated State of Oncornavirus DNA in Normal Chicken Cells and in Cells Transformed by Avian Myeloblastosis Virus

The covalent linkage of oncornavirus-specific DNA to chicken DNA was investigated in normal chicken embryo fibroblasts (CEF) and in virus-producing leukemic cells transformed by avian myeloblastosis virus (AMV). The virus-specific sequences present in cellular DNA fractionated by different methods were detected by DNA-RNA hybridization by using 70S AMV RNA as a probe. In CEF and in leukemic cells, the viral DNA appeared to be present only in the nucleus. After cesium chloride-ethidium bromide density equilibrium sedimentation, the viral DNA was present as linear, double-stranded molecules not separable from linear chicken DNA. After extraction by the Hirt procedure, the viral DNA precipitated with the high-molecular-weight DNA. After alkaline sucrose velocity sedimentation, the viral DNA cosedimented with the high-molecular-weight cellular DNA. The results indicate that in both types of cells studied, the oncornavirus-specific DNA sequences were linked by alkali stable bonds to nuclear cellular DNA of high molecular weight and did not appear to be present in free form of any size.     

Deoxyribonucleic Acid Replication in Simian Virus 40-Infected Cells: III. Comparison of Simian Virus 40 Lytic Infection in Three Different Monkey Kidney Cell Lines

A comparative study of simian virus 40 (SV40) lytic infection in three different monkey cell lines is described. The results demonstrate that viral deoxyribonucleic acid (DNA) synthesis and infectious virus production begin some 10 to 20 hr earlier in CV-1 cells and primary African green monkey kidney (AGMK) cells than in BSC-1 cells. Induction of cellular DNA synthesis by SV40 was observed in CV-1 and AGMK cells but not with BSC-1 cells. Excision of large molecular weight cellular DNA to smaller fragments was easily detectable late in infection of AGMK cells. Little or no excision was observed at comparable times after infection of CV-1 and BSC-1 cells. The different kinds of responses of these three monkey cell lines during SV40 lytic infection suggest the involvement of cellular functions in the virus-directed induction of cellular DNA synthesis and the excision of this DNA from the genome.     

DNA binding properties of a mutant T antigen from the simian virus 40-transformed human cell line simian virus 80

We investigated whether the T antigen of the simian virus 40-transformed human cell line simian virus 80 ( SV80 ) specifically recognizes DNA sequences of its own template, i.e, the viral sequences integrated in the SV80 cellular genome. In vitro DNA binding experiments clearly indicated that, in contrast to wild-type T antigen, SV80 T antigen does not specifically bind to sites on the integrated viral DNA in SV80 cells.     

Functional implications of oligomerization of simian virus 40 large T antigen during lytic virus infection.

The formation of oligomers of simian virus 40 (SV40) large T antigen in SV40-infected and -transformed monkey cells was analyzed by sucrose density gradient centrifugation. The overall distribution of total T antigen during lytic infection showed mainly low-molecular-weight forms (monomers and dimers) in the early phase (10 h postinfection) and an increase in the number of oligomers in the late phase of the lytic cycle (36 h postinfection), indicating an accumulation of these final products. In contrast, studying the conversion of newly synthesized T antigen into oligomers by appropriate pulse-chase radiolabeling of infected cells revealed that this processing decelerates considerably during the late phase of infection. This mechanism can be reaccelerated by blocking DNA replication with aphidicolin. Since none of these results could be obtained by using synchronized SV40-transformed monkey cells (COS-1), these observations are compatible with the idea that the process of T antigen oligomerization may be involved in viral, but not in cellular, DNA synthesis.     

Retransformation of a simian virus 40 revertant cell line, which is resistant to viral and DNA infections, by microinjection of viral DNA.

We have isolated morphological transformants of cultured cells as dense foci on a monolayer of normal cells appproximately 4 weeks after microinjection of purified simian virus 40 DNA (200 to 400 molecules per cell) directly into the nucleus. Both Rat 1 (an established contact-inhibited rat embryo fibroblast line) and F1' 1--4 (a 5-fluorodeoxyuridine-selected flat revertant from the simian virus 40-transformed 14B cell line) were transformed with an efficiency of 5 to 10% of the cells injected. F1' 1--4 is not susceptible to retransformation by either viral or DNA infection (by calcium phosphate-facilitated cellular uptake), and as a result it had previously been thought to possess a host mutation preventing expression of the simian virus 40 genome.     

Unintegrated viral sequences in rat cells transformed by simian virus 40 and a temperature sensitive A gene mutant

The status of viral sequences in rat cells transformed by simian virus 40 (SV40) and its temperature sensitive A gene mutant was investigated. Agarose gel electrophoresis of cell DNA prepared from clones picked from soft-agar and blot hybridization showed that sequences specific to SV40 genome were present both as integrated and unintegrated structures in rat clones. Digestion of rat cell DNA with various endonucleases with or without recognition sites in SV40 DNA and analysis indicated that the unintegrated viral genomes existed as full-length, covalently closed circular molecules. No differences in the free viral genomes were apparent between the clones transformed by the wild type and the mutant virus. The importance of the existence of free viral genomes in nonpermissive cells is discussed.     

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.     

Simian virus 40 gene A regulation of cellular DNA synthesis. II. In nonpermissive cells.

The stimulation of host macromolecular synthesis and induction into the cell cycle of serum-deprived G0-G1-arrested mouse embryo fibroblasts were examined after infection of resting cells with wild-type simian virus 40 or with viral mutants affecting T antigen (tsA58) or small t antigen (dl884). At various times after virus infection, cell cultures were analyzed for DNA synthesis by autoradiography and flow microfluorimetry. Whereas mock-infected cultured remained quiescent and displayed either a 2N DNA content (80%) or a 4N DNA content (15%), mouse cells infected with wild-type simian virus 40, tsA58 at 33 degrees C, or dl884 were induced into active cell cycling at approximately 18 h postinfection. Although dl884-infected mouse cells were induced to cycle initially at the same rate as wild type-infected cells, they became arrested earlier after infection and also failed to reach the saturation densities of wild-type simian virus 40-infected cells. Infection with dl884 also failed to induce loss of cytoplasmic actin cables in the majority of the infected cell population. Mouse cells infected with tsA58 and maintained at 39.5 degrees C showed a transient burst of DNA synthesis as reflected by changes in cell DNA content and an increase in the number of labeled nuclei during the first 24 h postinfection; however, after the abortive stimulation of DNA synthesis at 39.5 degrees C shift experiments demonstrated that host DNA replication was regulated by a functional A gene product. It is concluded that both products of the early region of simian virus 40 DNA play a complementary role in recruiting and maintaining simian virus 40-infected cells in the cell cycle.     

Effect of rhesus or vervet cytomegalovirus infection of DNA synthesis in untreated and 5-iodo-2''-deoxyuridine-arrested cells.

Infection of permissive cells with either rhesus or vervet cytomegalovirus resulted in suppression of cellular DNA synthesis, only viral DNA was resolved by isopycnic centrifugation after 24 h postinoculation. Infection of 5-iodo-2'-deoxyuridine (IUdR)-arrested cells with either of the simian cytomegaloviruses, however, induced synthesis of cellular DNA of normal density; synthesis of cellular DNA substituted with IUdR as evidenced by resolution of a heavy DNA peak after isopycnic centrifugation was not observed. Stimulation of DNA synthesis in IUdR-arrested cells was not observed with virus inactivated with UV light.     

Demonstration of Infectious Deoxyribonucleic Acid in Transformed Cells I. Recovery of Simian Virus 40 from Yielder and Nonyielder Transformed Cells

Deoxyribonucleic acid (DNA) was extracted from virus-free simian virus 40 (SV40)-transformed hamster, mouse, and monkey cells and was inoculated into simian cells in the presence of diethylaminoethyl (DEAE)-dextran; infectious SV40 was recovered by using DNA from cell lines which fail to yield virus by the fusion technique as well as from cell lines which readily yield virus by fusion. The rescued virus was identified as SV40 by three methods: (i) neutralization of plaque formation by specific antiserum; (ii) induction of synthesis of viral-specific antigens detected by immunofluorescence; and (iii) presence of papovavirus particles seen by the electron microscope. Treatment of the transformed cell DNA with deoxyribonuclease or omission of the DEAE-dextran prevented the rescue of virus. Large amounts of transformed cell DNA were required (>10 mug/culture of 10(6) cells) to effect rescue of SV40 by passage through monkey cells. A linear response was obtained between the input of DNA with inocula between 10 and 45 mug of DNA/culture and the yield of SV40 recovered. Biological activity was demonstrable irregularly when the transformed cell DNA was assayed directly in the presence of DEAE-dextran. The DNA induced plaque formation in about 50% of the trials as well as the synthesis of SV40 tumor and viral antigens in rare simian cells. The infectious DNA appeared to be associated with cellular DNA. The infectivity was found in the pellet of precipitated DNA obtained by the Hirt technique and was inactivated by boiling for 15 min. These properties are characteristic of linear cellular DNA and not of free, circular SV40 DNA.     

Replication of Nondefective Parvoviruses: Lack of a Virion-Associated DNA Polymerase

We have examined four of the nondefective parvoviruses for an associated DNA polymerase. Virions were purified from neuraminidase-treated infected-cell lysates by isopycnic centrifugation in CsCl or from infected cell material by CaCl(2) precipitation and centrifugation through sucrose into CsCl. Preparations of bovine parvovirus or Kilham rat virus obtained by the former procedure contained DNA polymerase activity but were not free of contaminating cellular proteins. The latter method produced viral preparations free of contaminating cellular proteins, and no DNA polymerase activity was detected in light infectious particles of H-1, LuIII, bovine parvovirus, or Kilham rat virus. Examination of levels of each cellular DNA polymerase in these preparations from each step of both purification procedures revealed that DNA polymerase beta had a greater tendency to copurify with bovine parvovirus and Kilham rat virus than did DNA polymerases alpha or gamma. Disruption of infectious virions obtained by the second purification method with detergents and sonic treatment did not result in the detection of a DNA polymerase activity. The biological activity and purity of each of the four different viruses obtained by the latter procedure were determined by hemagglutination and infectivity assays, polyacrylamide gel electrophoresis, and electron microscopy. In each case, the virions banding at a density of 1.39 to 1.41 g/cm(2) in CsCl were infectious and contained only the virion structural proteins. DNA polymerase activity was not detected in any of these preparations, and we have concluded that a virion-associated DNA polymerase is not required for productive infection with the nondefective parvoviruses.     

State and organization of polyoma virus DNA sequences in transformed rat cell lines.

Polyoma virus-transformed rat cell lines were isolated as colonies growing in agar after infection of F2408 cells with low multiplicities of wild-type virus. Viral DNA present in the transformed cells was analyzed by fractionating the cellular DNA on agarose gels before and after digestion with various restriction endonucleases, followed by detection of the DNA fragments containing viral sequences using the procedure described by Southern (E. Southern, J. Mol. Biol., 98:503--515, 1975). Five lines, independently derived, were studied in detail. All five lines, when examined after a minimum number of passages in culture, contained both free and apparently integrated viral DNA. The free polyoma DNA in three of the lines was indistinguishable, by restriction enzyme analysis, from wild-type viral DNA, whereas the two other lines also contained smaller free DNA molecules which lacked parts of the wild-type genome. The integrated DNA in the five lines studies existed as head-to-tail tandem repeats of unit-length polyoma DNA covalently attached to nonviral DNA. The same five polyoma-transformed rat lines were examined after further passage in culture. Free viral DNA was then either undetectable or greatly reduced in amounts, whereas the high-molecular-weight, integrated units persisted after passage of the cells. The subclones, derived from one of the five lines selected for detailed analysis, showed some variations in the quantity and size of the free viral DNA as well as minor alterations in the pattern of the apparently integrated sequences.