Chromatin assembly. Relationship of chromatin structure to DNA sequence during simian virus 40 replication

The accessibility of five specific DNA sequences to six different single site restriction endonucleases was evaluated in replicating and mature simian virus 40 chromosomes isolated by three different methods. Electron microscopic and gel electrophoretic analysis of the DNA digestion products demonstrated that DNA accessibility in chromatin was established within 400 base pairs of replication forks and remained essentially unchanged during production of mature chromosomes and their subsequent re-entry into the replication pool. Saturating amounts of each enzyme reproducibly cut a fraction of the chromosomes, ranging from 13 to 49%. This is consistent with a nearly random phasing of chromatin structure. Examples in which all chromosomes were either cleaved or intact were never observed. Although variation in the accessibility of DNA sites near the origin of replication could be interpreted as preferred phasing in about 25% of the chromosomes, the finding that two isoschizomers, Hpa II and Msp I, did not cut chromosomes to the same extent precludes an unambiguous interpretation of the extents of cleavage of individual restriction enzymes. Since the extent of DNA cleavage observed at each restriction site was essentially indistinguishable in replicating as compared to mature chromosomes, the accessibility of DNA sequences near the origin is not obviously related to replication. Furthermore, the accessibility of DNA sites on one arm of a single replication fork was the same as the homologous sites on the other arm, consistent with a nearly random phasing of chromatin structure on both arms. This suggests that chromatin assembly occurs independently on the 2 sibling molecules of a single replicating chromosome.     

Structure of simian virus 40-adeno-associated virus recombinant genomes.

The structures of recombinant genomes formed by recombination between simian virus 40 (SV40) and adeno-associated virus 2 (AAV) DNAs after either DNA cotransfection or coinfection by virions were characterized. Two types of structures were found. Group A structures, found after cotransfection and in one of seven recombinants arising from coinfection, represented a simple deletion of SV40 sequences replaced by a slightly shorter AAV sequence. Group B structures were found in six of seven recombinants arising after virion coinfection. All contained either the left or right terminal sequences (approximately 250 to 450 bases) of the AAV genome adjacent to the SV40 origin of DNA replication. Only 350 to 650 bases (including the origin) remained of the SV40 sequence. The joined SV40-AAV sequences were present in the recombinant genome as a tandem repeat of a size that can be packaged into SV40 capsids.     

Selectivity of interferon action in simian virus 40-transformed cells superinfected with simian virus 40.

Treatment of African green monkey kidney CV-1 cells with human alpha interferons before infection with simian virus 40 (SV40) inhibited the accumulation of SV40 mRNAs and SV40 T-antigen (Tag). This inhibition persisted as long as the interferons were present in the medium. SV40-transformed human SV80 cells and mouse SV3T3-38 cells express Tag, and interferon treatment of these cells did not affect this expression. SV80 and SV3T3-38 cells which had been exposed to interferons were infected with a viable SV40 deletion mutant (SV40 dl1263) that codes for a truncated Tag. Exposure to interferons inhibited the accumulation of the truncated Tag (specified by the infecting virus) but had no significant effect on the accumulation of the endogenous Tag (specified by the SV40 DNA integrated into the cellular genome). The level of Tag in SV40-transformed mouse SV101 cells was not significantly decreased by interferon treatment. SV40 was rescued from SV101 cells and used to infect interferon-treated and control African green monkey kidney Vero cells. Tag accumulation was inhibited in the cells which had been treated with interferons before infection. Our data demonstrate that even within the same cell the interferon system can discriminate between expression of a gene in the SV40 viral genome and expression of the same gene integrated into a host chromosome.     

Structure of simian virus 40-phiX174 recombinant genomes isolated from single cells.

Three simian virus (SV40)-phi X174 recombinant genomes were isolated from single BSC-1 monkey cells cotransfected with SV40 and phi X174 RF1 DNAs. The individual cell progenies were amplified, cloned, and mapped by a combination of restriction endonuclease and heteroduplex analyses. In each case, the 600 to 1,000 base pairs of phi X174 DNA (derived from different regions of the phi X174 genome) were present as single inserts, located in either the early or late SV40 regions; the deletion of SV40 DNA was greater than the size of the insert; and the remaining portions of the hybrid genome were indistinguishable from wild-type SV40 DNA, as judged by both mapping and biological tests. Hence, apart from the deletion which accommodates the phi X174 DNA insert, no other rearrangements of SV40 DNA were detected. The restriction map of a SV40-phi X174 recombinant DNA isolate before molecular cloning was indistinguishable from those of two separate cloned derivatives of that isolate, indicating that the species cloned was the major amplifiable recombinant structure generated by a single recombinant-producing cell. The relative simplicity of the SV40-phi X174 recombinant DNA examined is consistent with the notion that most recombinant-producing BSC-1 cells support single recombination events generating only one amplifiable recombinant structure.     

Restriction enzyme cleavage of ultraviolet-damaged simian virus 40 and pBR322 DNA

Cleavage of specific DNA sequences by the restriction enzymes EcoRI, HindIII and TaqI was prevented when the DNA was irradiated with ultraviolet light. Most of the effects were attributed to cyclobutane pyrimidine dimers in the recognition sequences; the effectiveness of irradiation was directly proportional to the number of potential dimer sites in the DNA. Combining EcoRI with dimer-specific endonuclease digestion revealed that pyrimidine dimers blocked cleavage within one base-pair on the strand opposite to the dimer but did not block cleavage three to four base-pairs away on the same strand. These are the probable limits for the range of influence of pyrimidine dimers along the DNA, at least for this enzyme. The effect of irradiation on cleavage by TaqI seemed far greater than expected for the cyclobutane dimer yield, possibly because of effects from photoproducts flanking the tetranucleotide recognition sequence and the effect of non-cyclobutane (6-4)pyrimidine photoproducts involving adjacent T and C bases.     

Cold-sensitive growth of simian virus 40 in semipermissive variants of CV1 cells.

Two cell clones were isolated from the simian line CV1, permissive for simian virus 40 (SV40), by selection at low temperature with the tsA239 mutant of SV40. These clones exhibited cold-sensitive semipermissivity to both SV40 virions and SV40 DNA. On the basis of virus yields, their resistance to viral DNA was increased approximately 15 times over that of CV1 cells when the incubation temperature was lowered from 38.5 to 33.5 degrees C. A further 30- to 40-fold resistance increase was exhibited at both temperatures upon infection with SV40 virions. Partial characterization of these clones indicated that the cold sensitivity affected an early function in viral growth, between viral uncoating and the appearance of T-antigen positivity, with a burst-size decrease in all cells at the restricted temperature. This conditional defect appeared to be superimposed upon a temperature-independent uncoating defect, presumably carried in a CV1 subpopulation from which the two clones were ultimately selected.     

Stimulation of non-histone chromosomal protein synthesis in simian virus 40-infected simian cells.

The pattern of synthesis of non-histone chromosomal proteins in simian virus (SV) 40-infected African green monkey kidney cells was analyzed by polyacryl-amide gel electrophoresis to see whether the changes in chromosomal protein metabolism are involved in the viral-induced synthesis of cellular DNA and mRNA. During the prereplicative phase of infection, the rate of histone synthesis was decreased until 15 h postinfection, whereas that of non-histone protein synthesis was increased after 5 h postinfection and reached a maximum at 10 to 15 h postinfection when viral-induced synthesis of cellular DNA and mRNA began to be observed. Stimulation of non-histone protein synthesis was also observed in the infected cells treated with cytosine arabinoside and was dependent on the multiplicity of infection. Stimulation occurred in almost all species of non-histone proteins. These results suggest that the stimulation of non-histone protein synthesis is caused by an early SV40 function and occurs prior to the viral-induced synthesis of cellular DNA and mRNA. During the replicative phase of infection, a marked increase in the rate of synthesis was observed in the non-histone proteins with molecular weights of about 48,000, 35,000, and 23,000, which were subsequently found to be SV40 capsid proteins.     

Characterization of simian cells tranformed by temperature-sensitive mutants of simian virus 40.

Seven lines derived from primary African green monkey kidney cells, which had survived lytic infection by wild-type simian virus 40 (SV40) or temperature-sensitive mutants belonging to the A and B complementation groups, were established. These cultures synthesize SV40 tumor (T) antigen constitutively and have been passaged more than 60 times in vitro. The cells released small amounts of virus even at high passage levels but eventually became negative for the spontaneous release of virus. Virus rescued from such "nonproducer" cells by the transfection technique exhibited the growth properties of the original inoculum virus. Four of the cell lines were tested for the presence of altered growth patterns commonly associated with SV40-induced transformation. Although each of the cell lines was greater than 99% positive for T antigen, none of the cultures could be distinguished from primary or stable lines of normal simian cells on the basis of morphology, saturation density in high or low serum concentrations, colony formation on plastic or in soft agar, hexose transport, or concanavalin A agglutinability. However, the cells could be distinguished from the parental green monkey kidney cells by a prolonged life span, the presence of T antigen, a resistance to the replication of superinfecting SV40 virus or SV40 viral DNA, and, with three of the four lines, an ability to complement the growth of human adenovirus type 7. These properties were expressed independent of the temperature of incubation. These results indicate that the presence of an immunologically reactive SV40 T antigen is not sufficient to ensure induction of phenotypic transformation and suggest that a specific interaction between viral and cellular genes and/or gene products may be a necessary requirement.     

Prereplicative events involving simian virus 40 DNA in permissive cells.

Simian virus 40 DNA molecules were found to be unable to replicate for 9 h after infection, even in cells that were already replicating the DNA of preinfecting simian virus 40; after 9 h, the ability of the DNA to replicate began to rise sharply. The kinetics of activation indicated that each DNA molecule undergoes a series of slow consecutive reactions, not involving T-antigen, before it can replicate. These pre-replicative molecular transformations probably involve configurational changes; their nature and their relation to the initiation of viral DNA synthesis is discussed. Observation of the replicative behavior of one viral DNA in the presence of another was made possible by the use of two different mutants with distinguishable DNAs: a viable deletion mutant containing DNA insensitive to TAqI restriction enzyme was used to provide viral functions required for replication, and a tsA mutant with TaqI-sensitive DNA was introduced at various times as a probe to determine the ability of the DNA to replicate under different conditions.     

Salt-resistant association of simian virus 40 T antigen with simian virus 40 DNA in nucleoprotein complexes.

Treatment of nucleoprotein complexes (NPCs) from simian virus 40 (SV40)-infected TC7 cells with NaCl (1 or 2 M) or guanidine-hydrochloride (1 or 2 M) resulted in a significant fraction of T antigen still associated with SV40 (I) DNA. Immunoprecipitation of the salt-treated NPCs with SV40 anti-T serum indicated that T antigen is preferentially associated with SV40 (I) DNA rather than with SV40 (II) DNA. Treatment of the NPCs with 4 M guanidine-hydrochloride, however, resulted in a substantial decrease in the amount of SV40 (I) and (II) DNA associated with T antigen. As the temperature was increased to 37 degrees C during incubation of NPCs with NaCl or guanidine-hydrochloride, there was a decrease in the amount of SV40 (I) and (II) DNA immunoprecipitated with SV40 anti-T serum. In the absence of salt, temperature had no effect on the association of T antigen with the SV40 DNA in the NPCs. Treatment of NPCs from SV40 wildtype or tsA58-infected cells grown at the permissive temperature with 1 or 2 M NaCl indicated that tsA T antigen has the same sensitivities as wild-type T antigen to high salt treatment when bound to DNA in NPCs. Characterization of the proteins associated with SV40 (I) DNA after high salt treatment revealed that, in addition to T antigen, a certain amount of viral capsid proteins VP1 and VP3 remained associated with the DNA. Complexes containing SV40 (I) DNA had a sedimentation value of 53S after 1 M NaCl treatment and 43S after 2 M NaCl treatment.     

Inhibition of simian virus 40 DNA synthesis in Yaba virus-preinfected cells.

The effect of Yaba virus preinfection on DNA synthesis in SV40-infected Jinet cells was studied. Time-course synthesis studies were conducted using the incorporation of labeled thymidine. Yaba virus preinfection resulted in the inhibition of SV40 DNA synthesis when the elapsed time between Yaba virus and SV40 infections was three days. This inhibition was demonstrated by hybridization studies and sedimentation analysis. In addition, the usual stimulation of cellular DNA synthesis induced by SV40 infection was inhibited. This inhibition occurred at a time in Yaba virus infection when no cytoplasmic Yaba virus-specific DNA synthesis occurred.     

Domain structure of the simian virus 40 core origin of replication.

The simian virus 40 core origin of replication consists of nucleotides 5211 through 31. These 64 base pairs contain three functional domains with strict sequence requirements and two spacer regions with relaxed sequence specificity but precise positional constraints. The early domain extends for 10 contiguous base pairs between nucleotides 5211 and 5220. A 9-base pair spacer from sequences 5221 through 5229 separates the early domain from the 23-base pair central palindrome that directs the binding of T antigen. The late end of the core between nucleotides 12 and 31 also contains spacer and sequence-specific functions that are not yet completely mapped. We propose that the sequence-specific domains are interaction sites for viral and cellular proteins, determinants of DNA conformation, or both. The spacers would position these signals at required distances and rotations relative to one another.     

Differential increase in topoisomerase II in simian virus 40-infected cells.

The time course of expression of topoisomerase I, topoisomerase II, and simian virus 40 (SV40) large tumor (T) antigen was determined in whole-cell extracts of uninfected versus SV40-infected TC7 cells. After a minor increase, the level of topoisomerase I remained fairly constant throughout the time course in both uninfected and SV40-infected cells. In contrast, the level of topoisomerase II increased markedly in SV40-infected cells but not in uninfected cells following the appearance of SV40 T antigen.     

Association of simian virus 40 T antigen with replicating nucleoprotein complexes of simian virus 40.

An immunoprecipitation assay was established for simian virus 40 T-antigen-bound nucleoprotein complexes by means of precipitation with sera from hamsters bearing simian virus 40-induced tumors. About 80% of simian virus 40 replicating nucleoprotein complexes in various stages of replication were immunoprecipitated. In contrast, less than 21% of mature nucleoprotein complexes were immunoprecipitated. Pulse-chase experiments showed that T antigen was lost from most of the nucleoprotein complexes concurrently with completion of DNA replication. T antigen induced by dl-940, a mutant with a deletion in the region coding for small T antigen, was also associated with most of the replicating nucleoprotein complexes. Once bound with replicating nucleoprotein complexes at the permissive temperature, thermolabile T antigen induced by tsA900 remained associated with the complexes during elongation of the replicating DNA chain at the restrictive temperature. These results suggest that simian virus 40 T antigen (probably large T antigen) associates with nucleoprotein complexes at or before initiation of DNA replication and that the majority of the T antigen dissociates from the nucleoprotein complexes simultaneously with completion of DNA replication.