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.     

Complete interaction of cellular 56,000- and 32,000-Mr proteins with simian virus 40 small-t antigen in productively infected cells.

Two cellular proteins are found to be complexed with simian virus 40 small-t antigen in cellular extracts. The complex is a relatively unstable but dynamic one which can dissociate and reform in extracts. In extracts of permissive monkey kidney cells, the small-t antigen appeared to be present in excess, whereas the cellular proteins were nearly entirely committed to the complex in permissive monkey kidney cells.     

DNA polymerase activities in growing cells infected with simian virus 40.

Growing CV1 cells were infected with simian virus 40 (SV40), and the levels of DNA polymerases-alpha, -beta, and -gamma were analyzed in the cytoplasm, nuclear Triton wash, and nucleus. In the cytoplasmic fraction, the amount of alpha-, beta-, or gamma-polymerase remained unaltered after SV40 infection. The activity of DNA polymerase-alpha increased five- to sixfold in the nuclear Triton wash and threefold in the nuclei and then remained enhanced only inside the nuclei. That of DNA polymerases-beta and gamma increased mostly in the nuclei after infection. These results suggest that DNA polymerase-alpha could be the major enzyme involved in SV40 DNA replication.     

Virus-specific RNA synthesis in interferon-treated mouse cells productively infected with Moloney murine leukemia virus.

Mouse cells productively infected with Moloney murine leukemia virus were treated with interferon, and intracellular virus-specific RNA was studied by hybridization with complementary DNA. The steady-state concentration of virus-specific RNA in interferon-treated cells was somewhat greater than that in untreated cells, and the rates of virus-specific RNA synthesis were approximately equal in treated and untreated cells.     

T antigen banding on chromosomes of simian virus 40 infected muntjac cells.

Chromosomes were prepared from mitotic munjac cells 48 to 72 h after infection with SV40 virus. When stained for SV40 T antigen by indirect immunofluorescence, all chromosomes within an infected cell were fluorescent, indicating the presence of T antigen. Furthermore, the chromosomes were not uniformly stained but appeared to have regions of high and low fluorescence intensity. A variety of controls showed that the banding patterns are specific and highly reproducible and may indeed reflect the binding sites of T antigen. The bright, fluorescent bands T antigen were found to correspond to bands visualized by trypsin-Giesma staining (G-bands) and also by quinacrine staining (Q-bands). Current knowledge of chromosome banding indicates that Q-bands reflect the distribution of AT-rich regions along the chromosome. From the DNA sequence of SV40, it is known that one of the T antigen binding sites contains AT-rich sequences; thus, T antigen banding might be due to the base-specific binding of T antigen to chromatin. In addition, these bands have been implicated as centers for chromosome condensation and units in control of DNA replication. While the functional significance of T antigen binding has yet to be determined, the SV40-muntjac system provides an unusual opportunity to study the interaction of a known regulatory protein with mammalian chromosomes.     

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.     

Collagenase production by immortalized human aortic endothelial cells infected with simian virus 40.

Human aortic endothelial cells, isolated at autopsy from a 52-year-old male dying from lung cancer, were treated with simian virus 40 (SV40). One colony was isolated from the infected endothelial cell culture 4 weeks after infection. The cells expressed SV40 large T antigen and p53 protein (p53) in their nuclei but lacted the characteristics of a transformed phenotype. The cells grew well in a monolayer over the 97th passage and exhibited Factor VIII-related antigen, Ulex europaeus 1 agglutinin (UEA-1) as endothelial cell markers, and a well-developed fibronectin network. The amount of prostacyclin synthesized by the cells was less than the amount synthesized by normal aortic or umbilical cord vein endothelial cells. The cells produced relatively large amounts of procollagenase, and 12-o-tetradecanoyl-phorbol-13-acetate (TPA) augmented the ability of the cells to produce this enzyme. These immortalized human aortic endothelial cells, which have some characteristics of normal endothelial cells and, like capillary endothelial cells, have the ability to produce collagenase, will probably prove useful for studies of atherosclerosis and angiogenesis.     

Kinetics of DNA and histone messenger RNA synthesis in CV-1 monkey kidney cells infected with simian virus 40.

Histone messenger RNA has been identified in CV-1 monkey kidney cells and its synthesis during the simian virus 40 (SV40) productive cycle has been correlated with the synthesis of cellular DNA and viral DNA. In cultures of CV-1 cells that have reached confluence, infection with SV40/5 (a high-yield clone of SV40) promotes an increase in the rate of cellular DNA synthesis followed by a decline. During this decline the rate of viral DNA synthesis continues to rise and eventually surpasses that of cellular DNA.The synthesis of histone mRNA rises concomitantly with the increase in the synthesis of cellular DNA. This occurs in a fashion similar to that observed when confluent CV-1 cultures are stimulated by the addition of fresh serum to the growth medium. However, whereas in cells stimulated with serum the synthesis of histone mRNA closely parallels that of cellular DNA, in cells infected with SV40, histone mRNA synthesis continues at a high rate even after the decline of cellular DNA synthesis. The rate of histone mRNA synthesis thus appears to he coupled to the total (cellular plus viral) DNA synthesis and not to the synthesis of the host DNA alone. The high rate of synthesis of the F1 histone at late times after infection suggests that histone genes are transcribed co-ordinately.     

Cellular proteins associated with simian virus 40 early gene products in newly infected cells.

Immunoprecipitates of extracts of simian virus 40-infected permissive monkey kidney cells contained two proteins with molecular weights of 56,000 and 32,000 (52K and 32K) in addition to the known viral early gene products. Immunoprecipitates of cells infected with the 0.54-0.59 deletion mutants that lack the viral 17K gene product did not contain thhe 56K and 32K proteins. The additional proteins appeared in immunoprecipitates of deletion mutant extracts if unlabeled extracts of wild-type-infected cells were added before addition of antiserum. The proteins can also be identified in uninfected cells by co-precipitation with unlabeled viral proteins. Thus, it appears that the 56K and 32K proteins are cellular products that associate with the viral proteins, the 17K in particular, and are indirectly immunoprecipitated by anti-tumor serum.     

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.     

DNA polymerase alpha from the nuclear matrix of cells infected with simian virus 40.

The nuclear matrix prepared from normal, simian virus 40 (SV40)-infected, and SV40-transformed cells contained DNA polymerase activities. Approximately 12% of the total DNA polymerase activities in isolated nuclei remained with the nuclear matrix. alpha-polymerase was the major matrix DNA polymerase activity as judged by sensitivity to various inhibitors: aphidicolin, dideoxy-TTP, and N-ethylmaleimide. Approximately 2-4 fold higher DNA polymerase activity was detected in matrices obtained from lytically infected and virus-transformed cells than that found in normal cells. In lytically infected cells, 30-50% of the matrix-bound DNA polymerase activity solubilized by sonication co-sedimented with majority of the matrix T-antigen, and was co-precipitated with anti-T sera. The results suggest that alpha-polymerase and viral T-antigen may form a functional complex in the matrix.     

Inhibition of viral protein synthesis in monkey cells treated with interferon late in simian virus 40 lytic cycle.

We have investigated the effect of interferon on SV40 gene expression late in the lytic cycle, after early functions have been expressed and viral DNA replication has been initiated. Whereas pretreatment with interferon prior to infection reduces the amount of early SV40 RNA, post-infection treatment does not inhibit viral RNA synthesis. Viral 19S and 16S RNA species are found undiminished in quantity and poly(A) content. Despite the apparent normalcy of viral RNA classes, however, there is a marked reduction in the synthesis of their protein products, both T antigen and capsid polypeptides. The association of viral RNA with heavy polyribosomes is strongly reduced. On the other hand, there is no degradation of nonviral polyribosomes and the synthesis of most cellular proteins continues. These experiments demonstrate that late in infection, interferon treatment results in an inhibition of viral mRNA translation.     

Characterization of T antigen in cells infected with a temperature-sensitive mutant of simian virus 40.

T antigen induced in African green monkey kidney cells by a temperature-sensitive mutant of simian virus 40, defective in a function required for cell transformation, was characterized. The number of T antigen-positive cells estimated by an immunofluorescent techniques was almost equal at permissive (32.5 C) and restrictive (38.5 C) temperatures, but was slightly reduced when the infected cells were incubated at a higher temperature (40.5 C). However, a complement fixation test indicated that the amount of T antigen induced by the mutant is not significantly different from that induced by wild-type virus at 40.5 C. These results suggest that the T antigen-inducing ability of the mutant is not defective. Two distinct molecular species of T antigen were induced by the mutant at the permissive temperature, whereas only one form was observed at the restrictive temperature. The larger molecular form (14 to 15S) induced by the mutant at the permissive temperature was more heat labile than that induced by wild-type virus, suggesting that the mutated gene product is a component of the larger molecular form.     

Effect of cytosine arabinoside on viral-specific protein synthesis in cells infected with herpes simplex virus.

The relationship between viral DNA and protein synthesis during herpes simplex virus type 1 (HSV-1) replication in HeLa cells was examined. Treatment of infected cells with cytosine arabinoside (ara-C), which inhibited the synthesis of HSV-1 DNA beyond the level of detection, markedly affected the types and amounts of viral proteins made in the infected cell. Although early HSV-1 proteins were synthesized normally, there was a rapid decline in total viral protein synthesis beginning 3 to 4 h after infection. This is the time that viral DNA synthesis would normally have been initiated. ara-C also prevented the normal shift from early to late viral protein synthesis. Finally, it was shown that the effect of ara-C on late protein synthesis was dependent upon the time after infection that the drug was added. These results suggest that inhibition of progeny viral DNA synthesis by ara-C prevents the "turning on" of late HSV-1 protein synthesis but allows early translation to be "switched off."     

Collagenase production by immortalized human aortic endothelial cells infected with simian virus 40

Human aortic endothelial cells, isolated at autopsy from a 52-year-old male dying from lung cancer, were treated with simian virus 40 (SV40). One colony was isolated from the infected endothelial cell culture 4 weeks after infection. The cells expressed SV40 large T antigen and p53 protein (p53) in their nuclei but lacted the characteristics of a transformed phenotype. The cells grew well in a monolayer over the 97th passage and exhibited Factor Vlll-related antigen, Ulex europaeus 1 agglutinin (UEA-1) as endothelial cell markers, and a well-developed fibronectin network. The amount of prostacyclin synthesized by the cells was less than the amount synthesized by normal aortic or umbilical cord vein endothelial cells. The cells produced relatively large amounts of procollagenase, and 12-o-tetradecanoyl-phorbol-13-acetate (TPA) augmented the ability of the cells to produce this enzyme. These immortalized human aortic endothelial cells, which have some characteristics of normal endothelial cells and, like capillary endothelial cells, have the ability to produce collagenase, will probably prove useful for studies of atherosclerosis and angiogenesis.