Vector Expression of Adenovirus Type 5 E1a Proteins: Evidence for E1a Autoregulation

Abstract

We transiently expressed adenovirus type C E1a proteins in wild-type or mutant form from plasmid vectors which have different combinations of E1a and simian virus 40 enhancer elements and which contain the DNA replication origin of SV40 and can replicate in COS 7 cells. We measured the levels of E1a mRNA encoded by the vectors and the transition regulation properties of the protein products. Three vectors encoded equivalent levels of E1a mRNA in COS 7 cells: (i) a plasmid encoding the wt 289-amino acid E1a protein (this complemented the E1a deletion mutant dl312 for early region E2a expression under both replicative and nonreplicative conditions); (ii) a vector for the wt 243-amino acid E1a protein (this complemented dl312 weakly and only under conditions of high multiplicities of dl312); (iii) a mutant, pSVXL105, in which amino acid residues-38 through 44 of the 289-amino acid E1a protein (which includes two highly conserved residues) are replaced by 3 novel amino acids (this also complemented dl312 efficiently). A fourth vector, mutant pSVXL3 with which linker substitution shifts the reading frame to encode a truncated 70-amino acid fragment from the amino terminus of the 289-amino acid protein, was unable to complement dl312. Surprisingly, pSVXL3 overexpressed E1a mRNA approximately 30-fold in COS 7 cells in comparison with the other vectors. The pSVXL3 overexpression could be reversed by cotransfection with a wt E1a vector. We suggest that wt E1a proteins regulate the levels of their own mRNAs through the recently described transcription repression functions of the 289- and 243-amino acid E1a protein products and that pSVXL3 fails to autoregulate negatively.

trans Activation of Type 1 Interferon Promoters by Simian Virus 40 T Antigen

“…The truncated SVo,B-37 plasmid was generated by cleavage of SVo, with AvaII-BamHI, and the 1,690-bp IFN-P promoter and CAT-coding fragment were subcloned into a 2,866-bp AccI-BamHI vector fragment derived from SV2CAT. Plasmids encoding Ela proteins (pSVEla), the 289-amino-acid (289aa) Ela protein (pSVN20), and the 243aa Ela protein (pSVF12) were described previously (42).…”

“…Since 293 cells express adenoviral early proteins which also possess trans-activating or repressing (2,19,32,46) activity, the relative contributions of TAg and adenovirus Ela proteins to IFN promoter activation were assessed by cotransfection experiments in HeLa cells. The IFN-a1 and IFN-P hybrid genes were cotransfected by electroporation with either the pSVEla expression plasmid, encoding both the 289aa and 243aa Ela proteins, pSVN20 and pSVF12 encoding, respectively, the 289aa and 243aa Ela proteins (42), or pwtSV. The results of these experiments (Table 2) illustrate that although basal and induced CAT levels were lower in HeLa cells than in 293 cells, TAg coexpression augmented constitutive levels of CAT activity from the IFN-al and IFN-3 promoters; a similar response was observed with IFN-CAT genes containing the SV40 enhancer fragment.…”

Notes: The c-fos Cyclic AMP-Responsive Element Conveys Constitutive Expression to a Tissue-Specific Promoter

“…To test this possibility, an expression plasmid containing the adenovirus type 5 ElA region, pSVElA (24), was cotransfected with HCA and HCAfl into C2 and HeLa cells (Fig. 5).…”

“…Assays were performed as described in the legend to Fig.2. For lanes labeled +, 0.8 ,ug of the pSVElA plasmid containing the adenovirus type 5 ElA genes(24) was cotransfected with S ,ug of the HCA promoter contructs. The total transfected DNA was 10 ,ug.…”