In vitro synthesis of adenovirus type 5 T antigens. II. Translation of virus-specific RNA from cells transformed by fragments of adenovirus type 5 DNA.

Virus-specific cytoplasmic RNA was isolated from rat cell lines transformed by fragments of adenovirus type 5 DNA, and the RNAs were translated in cell-free systems derived from wheat germ or rabbit reticulocytes. RNA was isolated from cell lines transformed by the following fragments: XhoI-C (leftmost 15.5%), HindIII-G (leftmost 8%), and HpaI-E (leftmost 4.5%). In addition, the adenovirus type 5-transformed human embryonic kidney line 293.C31 was investigated. The products were immunoprecipitated with serum from tumor-bearing hamsters and analyzed by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels. The results show that all transformed cells investigated contain early region 1a-specific RNAs which can be translated into proteins with molecular weights of 34,000 (34K), 36K, 40K, and 42K. Transformed cells that also contain an intact early region 1b synthesized RNA which can be translated into proteins with molecular weights of 19K and 65K. Minor proteins of 15K, 16K, 17.5K, 18K, 25K, and 29K were also observed, but these proteins could not be mapped unambiguously. Cells transformed by the 8% HindIII-G apparently lack RNA encoding the 65K protein, but they do contain RNA coding for the 19K protein.     

Differentiation of human epidermal cells transformed by SV40

Human epidermal cells were transformed with DNA from wild-type SV40 virus or with DNA from a temperature-sensitive A mutant (tsA209). The SV40-transformed cells differed from nontransformed cells in their morphologic appearance, growth properties, and expression of certain characteristics associated with differentiation. The transformed cells were more variable in size and shape than their nontransformed counterparts and were less stratified and less keratinized. While the growth properties of the cells were similar under optimal growth conditions, the transformed cells could be propagated under stringent growth conditions that did not support the growth of nontransformed human epidermal cells. The transformants still required a 3T3 feeder layer for growth, remained anchorage dependent as assayed in soft agar, and were not tumorigenic in athymic nude mice. The expression of certain differentiated functions of the human epidermal cell, the presence of keratins and cross-linked envelopes, was decreased in the transformed cells, and these functions could be restored at the nonpermissive temperature in the tsA209 transformed cells.     

Stimulation of cellular RNA synthesis in mouse-kidney cell cultures infected with SV40 virus

In confluent primary mouse-kidney cell cultures, abortive infection with SV40 has been demonstrated to cause an increase in the bulk of cellular RNA (mainly rRNA). However, the increase in the rate of rRNA synthesis is not involved in the initiation of the virus-induced cellular DNA replication since after actinomycin D treatment (0.05 μg/ml, from 6 to 9 h p.i.) the onset of cellular DNA replication takes place at a time when the rate of rRNA synthesis is still depressed.     

The location of SV40 specific sequences in the heterogeneous nuclear RNA of transformed mouse cells

Summary Rapidly labelled nuclear RNA from an SV40 transformed mouse 3T3 line was isolated, and different molecular size classes pooled. The fractions were treated with Ehrlich ascites exonuclease III for various lengths of time, and the digestion of the 3 ends of the RNA measured by the appearance of TCA-soluble material. The resulting partially degraded RNA populations were then hybridised to SV40 DNA. The data suggest that a high proportion of the viral specific sequences are located near the 3 ends of heterogeneous nuclear RNA.     

Altered patterns of keratin synthesis in human epidermal keratinocytes transformed by SV40

Transformation of human epidermal keratinocytes by the oncogenic virus SV40 is a stage-specific process in which normal patterns of differentiation are progressively altered over time following infection. Within the context of this scheme, we examined the keratins produced by the infected cells. Immunofluorescence studies indicated that viral infection led to the formation of variant cells visibly lacking the normal keratin cytoskeleton after about 10-15 serial passages (60-90 cell generations) post infection. Analyses of variant cell formation in clonal populations grown on palladium islands revealed that the variants were derived within 2-3 cell divisions from cells containing an apparently normal keratin cytoskeleton, but that variant formation depended upon cell density. Immunoprecipitation of 35S-methionine labelled keratins from the infected keratinocytes revealed a gradual loss of the normal 46, 50, 56 and 58Kd keratin species over a period of many months after infection. The loss of the normal keratins was accompanied by the appearance of at least two species in the 48-52Kd size range not present in uninfected cells and the enhancement of a third, 40Kd, protein quite early after infection. Analysis of the altered keratin patterns on two-dimensional acrylamide gels using either isoelectric focusing (IEF) or non-equilibrium pH gradient electrophoresis (NEPHG) along the first dimension showed that the infected cells produced basic keratins which increased in relative abundance as cells became more transformed with serial passage including at least five isoelectric forms not seen in uninfected cells. Translation of poly A+ RNAs from the infected cells indicated that the altered keratin synthesis probably reflects changes in the translatable mRNA pool.