The maior immediate-early (IE) gene region mapping at coordinates 0.71 to 0.74 in the genome of human cytomegalovirus (HCMV) gives rise to a series of overlapping spliced IE mRNAs that are all under the transcriptional control of the complex IE68 promoter-enhancer region. We show here that one of the phosphorylated nuclear proteins encoded by this region behaves as a powerful but nonspecific trans-activator of gene expression. In transient chloramphenicol acetyltransferase (CAT) assay experiments with Vero cells all relatively weak heterologous target promoters tested, including those of herpes simplex virus IE175 and delayed-early genes, adenovirus E3, the enhancerless simian virus 40 early gene, and the human beta interferon gene, were stimulated between 30and 800-fold by cotransfection with the HindIII C fragment of HCMV (Towne) DNA. In contrast, expression of the homologous HCMV IE68-CAT gene but not SV2-CAT was specifically repressed. Inactivation mapping studies of the effector DNA, together with dose-response comparisons with subclones from the region, revealed that an intact 7. 1-kilobase sequence encompassing both the IE1 and 1E2 coding regions (exons 1 to 5) in the major IE transcription complex was required for both the nonspecific trans-activation and autoregulatory responses. The IEl coding region alone (exons 1 to 4) was inactive, but both functions were restored by insertion of the IE2 coding region (exon 5) in the correct orientation downstream from the IE1 coding region. Internal deletions or inserted terminator codons in IEI (exon 4) still gave efficient trans-activation and autoregulation, whereas the insertion of terminator codons in IE2 (exon 5) abolished both activities. Finally, IE2 (exon 5) sequences only (under the direct transcriptional control of the strong simian CMV IE94 promoter) were still able to specifically down regulate IE68-CAT expression but failed to exhibit trans-activation properties. Therefore, the IE2 gene product(s) of HCMV appear likely to be key control proteins involved in gene regulation during HCMV infection. All of the major classes of DNA viruses that replicate in mammalian cell nuclei encode immediate-early (IE) gene products that behave as trans-activators of subsequent viral gene expression and are themselves under the control of complex upstream protnoters and regulatory or enhancer regions. The large T antigens of papovaviruses, the ElA gene products of adenoviruses, the IE175 (ICP4) and IE110 (ICPO) proteins of herpes simplex virus (HSV), the E2 proteins of papillomaviruses, and the rep gene products of parvoviruses all appear to fit into this classification. They also all share the characteristic of being hydrophilic nuclear phosphoproteins whose mRNAs are synthesized immediately after infection and in the absence of de novo protein synthesis. Most of these proteins are needed within their own systems for efficient synthesis of subsequent viral genes (usually acting at the transcriptional level), but they may also show various degrees of relaxed specificity...

The regulation of mRNA levels for delta 5-3 beta-hydroxysteroid dehydrogenase/delta 5----delta 4-isomerase (3 beta HSD), 17 alpha-hydroxylase/C17-20 lyase cytochrome P450 (P450(17 alpha] and cholesterol side-chain cleavage cytochrome P450 (P450scc) was studied in primary cultures of mouse Leydig cells. Treatment of Leydig cells with 8-bromo-cAMP (cAMP) was essential for expression of P450(17 alpha) mRNA, but not for 3 beta HSD. Treatment with cAMP caused a decrease in basal levels of 3 beta HSD mRNA. The addition of aminoglutethimide (AG), an inhibitor of cholesterol metabolism, to the cAMP-treated cultures resulted in increased expression of both 3 beta HSD and P450(17 alpha) mRNA levels. The addition of testosterone or the androgen agonist mibolerone to cAMP- plus AG-treated cultures reduced 3 beta HSD and P450(17 alpha) mRNA to levels comparable to those observed when cells were treated with cAMP only. The glucocorticoid dexamethasone reduced both basal and cAMP- plus AG-induced increases in 3 beta HSD mRNA, but not in P450(17 alpha) mRNA. Estradiol at a concentration of 1 microM had no effect on cAMP- plus AG-induced 3 beta HSD or P450(17 alpha) mRNA levels. The role of protein synthesis in mediating the cAMP induction of 3 beta HSD, P450(17 alpha), and P450scc was investigated. The addition of cycloheximide (10 micrograms/ml) to cAMP-treated cultures for 24 h completely suppressed both constitutive and cAMP-induced 3 beta HSD mRNA levels. Cycloheximide also repressed cAMP-induced levels of P450(17 alpha) to 12% of levels observed in the absence of cycloheximide. In sharp contrast, 24-h treatment with cycloheximide did not suppress cAMP induction of P450scc mRNA, but reduced basal levels by approximately 50%. A time course of induction by cAMP (50 microM) of P450(17 alpha) and P450scc mRNA showed very similar rates of increase in P450(17 alpha) and P450scc mRNA, with the greatest increase occurring between 12 and 24 h of treatment. The results of the study demonstrate that in normal mouse Leydig cells steady state levels of mRNA for 3 beta HSD, P450(17 alpha), and P450scc are differentially regulated. cAMP is required for maximal levels of all three mRNAs. There is high constitutive expression of 3 beta HSD and P450scc mRNA, while expression of P450(17 alpha) mRNA is absolutely dependent on cAMP stimulation. Endogenously produced testosterone negatively regulates the expression of cAMP-induced P450(17 alpha) and 3 beta HSD, while the glucocorticoid dexamethasone negatively regulates 3 beta HSD and P450scc.(ABSTRACT TRUNCATED AT 400 WORDS)

In normal mouse Leydig cells, steady state levels of mRNA of CYP11A, 3β-hydroxysteroid dehydrogenase Δ⁵- >Δ⁴-isomerase (3βHSD), and CYP17 are differentially regulated. There is high basal expression of 3βHSD and CYP11A mRNA, while expression of CYP17 mRNA is absolutely dependent on cAMP stimulation. cAMP is required for maximal expression of all three enzymes. The expression of CYP11A in normal mouse Leydig cells is repressed by glucocorticoids. Glucocorticoids also repress both basal and cAMP-induced expression of 3βHSD mRNA, but do not repress the synthesis or mRNA levels of CYP17. cAMP induction of 3βHSD mRNA can be observed only when aminoglutethimide (AG), an inhibitor of cholesterol metabolism, is added to the Leydig cell cultures. The addition of AG also markedly increases cAMP induction of CYP17 mRNA levels. Addition of testosterone or the androgen agonist, mibolerone, to cAMP plus AG treated cultures reduced 3βHSD and CYP17 mRNA levels to levels comparable to those observed when cells were treated with cAMP only. These data indicate that testosterone acting via the androgen receptor represses expression of both CYP17 and 3βHSD. The role of protein synthesis in mediating the cAMP induction of 3βHSD, CYP17 and CYP11A was examined. The addition of cycloheximide, an inhibitor of protein synthesis, to cAMP treated cultures for 24 h completely suppressed both constitutive and cAMP-induced 3βHSD mRNA levels. Cycloheximide also repressed cAMP-induced levels of CYP17 to 12% of levels observed in the absence of cycloheximide. In sharp contrast, treatment for 24 h with cycloheximide did not suppress cAMP induction of CYP11A mRNA, but reduced basal levels by approx. 50%. These data indicate that newly synthesized protein(s) are required for cAMP induction of CYP17 and 3βHSD mRNA levels, but not for CYP11A mRNA. A mouse Cyp17 genomic clone containing the entire coding region plus 10 kb of 5' flanking region has been isolated. Fragments of 5' flanking sequences were subcloned into vectors containing the CAT reporter gene and transfected into MA-10 Leydig cells. Transfected cells were treated with cAMP and expression was determined by measuring CAT activity. A cAMP responsive element was identified in a region between -245 and -346 bp relative to the transcription initiation site of Cyp17. Cotransfection into MA-10 Leydig cells of constructs containing 4.5 kb of Cyp17 5' flanking sequences together with a mouse androgen receptor expression vector demonstrate a dose dependent repression of cAMP-induced Cyp17 transcription by the androgen receptor. Studies with the mouse Cyp11a gene demonstrate that the 5' flanking region of the gene contains sequences between 2.5 and 5 kb that are necessary for expression of mouse Cyp11a in Leydig cells but not in adrenal cells.