Simian virus 40-specific proteins in HeLa cells infected with nondefective adenovirus 2-simian virus 40 hybrid viruses.

The synthesis of simian virus 40 (SV40)-specific proteins in HeLa cells infected with the nondefective adenovirus 2 (Ad2)-SV40 hybrid viruses, Ad2+ND2, Ad2+ND3, Ad2+ND4, and Ad2+ND5, was investigated. Infected-cell proteins were labeled with radioactive amino acids late after infection, when host protein synthesis was shut off, and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. All polypeptides normally seen in Ad2-infected cells were found in cells infected by the hybrid viruses. In addition to the Ad2-specific proteins, cells infected with Ad2+ND2 contain two SV40-specific proteins with apparent molecular weights of 42,000 and 56,000, cells infected with Ad2+ND4 contain one protein with an apparent molecular weight of 56,000, and cells infected with Ad2+ND5 contain one protein with an apparent molecular weight of 42,000. Cells infected with Ad2+ND3 do not contain detectable amounts of proteins not seen during Ad2 infection. Pulse-chase experiments demonstrate that the SV40-specific proteins induced by Ad2+ND2, Ad2+ND4, and Ad2+ND5 are metabolically unstable. These proteins are not present in purified virions. Two nonstructural Ad2-specific proteins have been demonstrated in Ad2 and hybrid virus-infected cells which have a smaller apparent molecular weight after a short pulse than after a pulse followed by a chase. The molecular weight increase during the chase may be caused by the addition of carbohydrate to a polypeptide backbone.     

Simian virus 40-transformed human cells that express large T antigens defective for viral DNA replication.

Many types of human cells cultured in vitro are generally semipermissive for simian virus 40 (SV40) replication. Consequently, subpopulations of stably transformed human cells often carry free viral DNA, which is presumed to arise via spontaneous excision from an integrated DNA template. Stably transformed human cell lines that do not have detectable free DNA are therefore likely to harbor harbor mutant viral genomes incapable of excision and replication, or these cells may synthesize variant cellular proteins necessary for viral replication. We examined four such cell lines and conclude that for the three lines SV80, GM638, and GM639, the cells did indeed harbor spontaneous T-antigen mutants. For the SV80 line, marker rescue (determined by a plaque assay) and DNA sequence analysis of cloned DNA showed that a single point mutation converting serine 147 to asparagine was the cause of the mutation. Similarly, a point mutation converting leucine 457 to methionine for the GM638 mutant T allele was found. Moreover, the SV80 line maintained its permissivity for SV40 DNA replication but did not complement the SV40 tsA209 mutant at its nonpermissive temperature. The cloned SV80 T-antigen allele, though replication incompetent, maintained its ability to transform rodent cells at wild-type efficiencies. A compilation of spontaneously occurring SV40 mutations which cannot replicate but can transform shows that these mutations tend to cluster in two regions of the T-antigen gene, one ascribed to the site-specific DNA-binding ability of the protein, and the other to the ATPase activity which is linked to its helicase activity.     

Simian virus 40 rapidly lowers cAMP levels in mouse cells

The addition of SV40 to contact inhibited $\text{Balb}\text{3T3}$ cells causes a 2-fold decrease in intracellular cAMP levels. The levels reach a minimum 3 hours after virus addition, and after a few hours begin to rise toward normal. No significant changes in cAMP levels are observed after cells are exposed to UV-inactivated virus or are mock-infected. This is the earliest known effect of SV40 infection. We propose that SV40 induces host DNA synthesis by lowering cAMP levels.     

Simian virus 40 tumor-specific proteins: subcellular distribution and metabolic stability in HeLa cells infected with nondefective adenovirus type 2-simian virus 40 hybrid viruses.

HeLa cells infected with adenovirus type 2 (Ad2)-simian virus 40 (SV40) hybrid viruses produce several SV40-specific proteins. These include the previously reported 28,000-dalton protein of Ad2+ND1, and 42,000- and 56,000-dalton proteins of Ad2+ND2, the 56,000-dalton protein of Ad2+ND4, and the 42,000-dalton protein of Ad2+ND5. In this report, we extend the list of SV40-specific proteins induced by Ad2+ND4 to include proteins of apparent molecular weights of 28,000 42,000, 60,000, 64,000, 72,000, 74,000, and a doublet of 95,000. Cell fractionation studies demonstrate that the SV40-specific proteins are detectable in the nuclear, cytoplasmic, and plasma membrane fractions. By pulse-chase and cell fractionation experiments, three classes of SV40-specific proteins can be distinguished with regard to metabolic stability: (i) unstable in the cytoplasmic but stable in the nuclear and plasma membrane fractions; (ii) stable in the nuclear, cytoplasmic, and plasma membrane fractions; and (iii) unstable in all subcellular fractions. Immunoprecipitation of infected cell extracts demonstrates that most of the above proteins share antigenic determinants with proteins expressed in hamsters bearing SV40-induced tumors. Only the 42,000-dalton protein of Ad2+ND5 is not immunoprecipitable.     

Simian virus 40 (SV40) T-antigen mutations in tumorigenic transformation of SV40-immortalized human uroepithelial cells.

pSV2Neo, a plasmid that contains the wild-type simian virus 40 (SV40) origin of replication (ori), is widely used in mammalian cell transfection experiments. We observed that pSV2Neo transforms two nontumorigenic SV40-immortalized human uroepithelial cell lines (SV-HUC and CK/SV-HUC2) to G418 resistance (G418r) at a frequency lower than that at which it transforms SV-HUC tumorigenic derivatives (T-SV-HUC). Transient expression studies with the chloramphenicol transferase assay showed that these differences could not be explained by differences in Neo gene expression. However, when we replaced the SV40 ori in pSV2Neo with a replication-defective ori to generate G13.1Neo and G13.1'Neo, the G418r transformation frequency of the SV40-immortalized cell lines was elevated. Because SV40 T antigen stimulates replication at its ori, we tested plasmid replication in these transfected cell lines. The immortalized cell lines that showed low G418r transformation frequencies after transfection with pSV2Neo showed high levels of plasmid replication, while the T-SV-HUC that showed high G418r transformation frequencies failed to replicate pSV2Neo. To determine whether differences in the status of the T-antigen gene contributed to the phenomenon, we characterized the T-antigen gene in these cell lines. The results showed that the T-SV-HUC had sustained mutations in the T-antigen gene that would interfere with the ability of the T antigen to stimulate replication at its ori. Most T-SV-HUC contained a super-T-antigen replication-defective ori that apparently resulted from the partial duplication of SV40 early genes, but one T-SV-HUC had a point mutation in the ori DNA-binding domain of the T-antigen gene. These results correlate with the high G418r transformation frequencies with pSV2Neo in T-SV-HUC compared with SV-HUC and CK/SV-HUC2. Furthermore, these results suggest that alterations in SV40 T antigen may be important in stabilizing human cells immortalized by SV40 genes that contain the wild-type SV40 ori, thus contributing to tumorigenic transformation. This is the first report of a super T antigen occurring in human SV40-transformed cells.     

Simian virus 40 DNA extracted from infected cells with sodium deoxycholate no longer reflects its in vivo superhelix density.

When simian virus 40 DNA is extracted from infected cells with low concentrations of sodium deoxycholate, which selectively extract non-encapsidated simian virus 40 DNA, the DNA has a lower average number of superhelical turns than the DNA extracted from purified viral particles. During extraction, a partial deproteinization of the DNA by a concentration of detergent that did not inactivate a nicking-closing activity led to the removal of some superhelical turns. The DNA extracted in this way no longer reflected its in vivo number of superhelical turns.     

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.     

Complementation of translational defect for growth of human adenovirus type 2 in Simian cells by a Simian virus 40-induced factor

The defective step which leads human adenovirus type 2 infection of African green monkey kidney cells (clone C14) to be abortive and its complementation in simian virus 40-transformed cells (clone T22) were studied by comparing the synthesis and function of macromolecules in these cell lines. Neither a quantitative nor a qualitative difference was detected in virus DNA replication and in virus mRNA synthesis in these cells, while a definite difference was observed in protein synthesis. The capsid proteins, such as hexon or penton, were synthesized in T22 cells but not in C14 cells. Inability of polyribosomes to synthesize the capsid proteins in C14 cells infected with adenovirus type 2 may not be due to a defect in elongation of nascent polypeptides or their release, since nascent polypeptides pulse-labelled with [³H]leucine were completely released from polyribosomes after the chase. The electrophoretic analysis of proteins synthesized in vitro with polyribosomes from either infected T22 or C14 cells using the pH 5 enzyme and S100 fraction from T22 cells revealed that hexon was synthesized with polyribosomes from T22 cells but not from C14 cells, thereby suggesting that the defect is not ascribed to a component in the pH 5 enzyme and S100 fraction, but resides in polyribosomes. The analysis of late adenovirus mRNA associated with polyribosomes in the infected T22 and C14 cells by hybridization competition or by sedimentation revealed that all the species of virus mRNA were present in the cytoplasm of these cells; however, certain species of virus mRNA larger than 20 S were absent in polyribosomes of the infected C14 cells. Sedimentation analysis of late adenovirus mRNA following separation on poly(U)-Sepharose or by membrane filtration gave the same results. These results suggest that the defect of C14 cells to support growth of adenoviruses is due to the inability of ribosomes to associate with certain species of late virus mRNA to form polyribosomes and suggest that a factor complementing this defect is induced by simian virus 40.     

Phosphorylation patterns of tumour antigens in cells lytically infected or transformed by simian virus 40.

The phosphorylation sites of simian virus 40 (SV40) large tumor (T) antigens have been analyzed by partial proteolysis peptide mapping and phosphoamino acid analysis of the resulting products. At least four sites were found to be phosphorylated. An amino-terminal part of the molecule contained both phosphoserine and phosphothreonine. One phosphothreonine residue was located in the proline-rich carboxy-terminal end of the molecule, either at position 701 or at position 708. The mutant dl 1265, which is defective in adenovirus helper function, lacked this phosphorylation site. In addition, the carboxy-terminal part of the molecule contained phosphoserine at a more central position. T-antigen-associated proteins of SV40-transformed cell (nonviral T; 51,000 to 55,000 daltons) also contained multiple phosphorylation sites involving at least two serine residues in mouse antigens and an additional threonine residue in rat, human, and monkey antigens. The latter residue and at least one phosphoserine residue were located near one terminus of the human NVT molecule. We did not find any evidence for phosphorylation of tyrosine residues in any of the multiple species of either large T or nonviral T molecules. Several forms of large T antigens were extracted from both SV40-transformed and SV40-infected permissive and nonpermissive cells, and their phosphorylation patterns were compared. No evidence was found for a different phosphorylation pattern of T antigen in transformed cells.     

Simian virus 40 T antigens and J domains: analysis of Hsp40 cochaperone functions in Escherichia coli

The N-terminal exon of DNA tumor virus T antigens represents a J domain that can direct interaction with the host-encoded Hsp70 chaperones. We have taken advantage of rapid Hsp40 cochaperone assays with Escherichia coli to assess simian virus 40 (SV40)-encoded J-domain loss of function. We found a strong correlation between loss of cochaperone function in E. coli and defective SV40 growth, suggesting that the major role of the J domain in DNA tumor viruses is to provide cochaperone function. We also report the expression of native SV40 virus T antigens in E. coli. Our results show that small t antigen, but not large T antigen (LT) or LT truncation TN125 or TN136, can functionally replace under limited growth conditions DnaJ (Hsp40) function in vivo. In addition, purified small t antigen can efficiently stimulate E. coli DnaK's (Hsp70) ATPase in vitro, thus behaving like a bona fide cochaperone. Furthermore, small t amino acids 83 to 174, which are adjacent to the viral J domain, can replace the E. coli DnaJ J-domain glycine-phenylalanine-rich domain, immediately adjacent to the J-domain sequences, even in the absence of significant amino acid similarity to their DnaJ counterpart. Taken together, our studies demonstrate that functionally related Hsp40 proteins from mammalian viral systems can be rapidly studied in bacteria and exploited to probe the universally conserved Hsp70 chaperone machine mechanism.     

Formation of varicella-zoster virus antigens in infected Vero cells

The formation of varicella-zoster (V-Z) virus-associated antigens was studied in V-Z virus-infected Vero cells by means of indirect immunofluorescence. Early antigen (EA) was first detected inside V-Z virus-infected Vero cells 4 to 6 hr after infection, whereas surface membrane antigen (SMA) was expressed on the outer surface of infected cells 2 to 3 hr later than EA, and intranuclear late antigen (LA) was detected several hours later than SMA antigen. EA expression was not inhibited by cytosine arabinoside (Ara-C) treatment, whereas LA formation was completely blocked by Ara-C. The presence of two components of SMA early SMA (ESMA) and late SMA (LSMA), was suggested by this difference in susceptibility to Ara-C. The formation of all viral antigens, EA, SMA, and LA, was blocked by inhibitors of RNA and protein synthesis.     

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.     

Simian virus 40 maturation in cells harboring mutants deleted in the agnogene

The predominant leader region of the late 16 S mRNAs of simian virus 40 encodes a histone-like, 61-amino acid, DNA-binding protein called the agnoprotein or LP1. To test the hypothesis that this protein facilitates assembly of viral minichromosomes into virions, we have studied the synthesis of virions in cells infected with mutants deleted in this region of the SV40 genome. We found that 220 S mature virions, indistinguishable from those of wild type, were produced in cells infected with these mutants. As in wild-type-infected cells, no assembly intermediates other than 75 S chromatin were observed. However, data obtained from both steady-state and pulse-chase labeling experiments indicated that cells infected with agnogene deletion mutants produced virions more slowly than cells infected with wild-type virus. Taken together with data showing that similar levels of virion proteins were present in the wild-type- and mutant-infected cells, these findings strongly suggest that LP1 plays a role in expediting virion assembly.     

Envelope antigens of Sindbis virus in cells infected with temperature-sensitive mutants.

Indirect fluorescent-antibody studies of living and fixed chick cells infected with temperature-sensitive mutants of Sindbis virus suggest that functional envelope glycoprotein E1 must be inserted through the plasma membrane before E2. PE2 and E2 do not affect the insertion of E1. The experiments also suggest that normal PE2, a glycosylated precursor to E2, reacts with anti-E2 serum; the abnormal PE2 made by a temperature-sensitive PE2 cleavage-defective mutant did not. Abnormal E1 proteins made by E1-defective mutants also failed to react with anti-E1 serum.