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

Abstract

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 transforned 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 la-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 lb 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.

Control of adenovirus early gene expression: accumulation of viral mRNA after infection of transformed cells

“…Furthermore, all proteins which we detected corresponded in size and map location to the proteins expressed during lytic infection of human cells with Ad5. Recently similar results have been described by Jochemsen et al (13).…”

Adenovirus E1A/E1B Transformed Amniotic Fluid Cells Support Human Cytomegalovirus Replication

“…These cells do support virus entry and IE1 gene expression, but are known to be non-permissive for viral DNA replication [ 27 ]. Interestingly, these cells are transformed by the same adenoviral E1 gene functions as CAP cells [ 25 , 31 ], indicating that the expression of E1A and E1B in CAP cells does not generally block HCMV DNA replication. Taken together, these results provided proof that HCMV DNA replication was initiated in CAP cells.…”

“…Human cells, however, are not easily transformed by E1A and E1B. Transformation of human embryonal kidney cells with E1A and E1B of adenovirus type 5 (Ad5) resulted in one single clone [ 25 ]. These HEK293 cells have been widely used for research purposes as well as for commercial applications (reviewed in [ 26 ]), yet they do not support the full HCMV replication cycle [ 27 , 28 ].…”

Identification of genes differentially expressed as result of adenovirus type 5- and adenovirus type 12-transformation

“…Expression of E1A in cell lines immortalised by the AccI-H fragment corresponding to the 4.7% of the Ad12 genome (Ad12 E1A-TC) or the HpaI-E fragment corresponding to the left-terminal 4.5% of the Ad5 genome (Ad5 E1A-TC) is lower than that shown by cell lines transformed by the entire E1 region, in agreement with previous observations that the E1B region acts to increase the frequency and stability of transformation by E1A [ 16 ]. The lowest levels of E1A expression are in the Ad5 E1A immortalised cell-lines; probably due to the absence of the poly (A) signal of E1A in the HpaI-E fragment of the Ad5 genome used to immortalise this cell line [ 17 ].…”

“…All four Ad-transformed rat cell lines contained integrated copies of the respective transforming (E1) region and expressed the transforming E1A and E1B proteins ((K. Raska, personal communication and GEB, unpublished results). In addition to the cell lines used in the real-time PCR analysis, the expression levels of targets at the protein level was also investigated using BRK cells immortalised with Ad12 E1A only (Ad12 E1A-TC; the AccIH cell line) and Ad5 E1A only (Ad5 E1A-TC; the HpaIE cell line [ 17 ]). Expression levels were also assessed in untransformed Fisher rat embryonic fibroblasts (3Y1 cell line).…”