Catharina Svensson

Control of adenovirus major late gene expression at multiple levels

Most late adenovirus (Ad) proteins are translated from mRNAs originating from the so-called major late transcription unit (MLTU). These mRNAs are grouped into five families (designated L1 to L5), where each family consists of mRNAs that have co-terminal 3 ends. We have used mutant and wild-type Ad infections to characterize levels at which major late gene expression is regulated. Our results suggest the existence of a novel intermediate stage during a lytic infection where mRNAs from regions L1 and L4 are selectively overexpressed compared to the L2, L3, and L5 mRNAs. Our data suggest that this RNA phenotype reflects the activity of the MLTU at a transient stage immediately following initiation of viral DNA replication. Early during an Ad infection only mRNA from region L1 accumulate. To efficiently accumulate mRNA from regions L1 and L5 both viral DNA replication and late protein synthesis were required. To allow for only viral DNA replication resulted in an extensive premature transcription termination and a preferential mRNA accumulation from regions L1 and L4. The surprising production of L4 mRNA under these conditions was not due to the activation of a novel r-strand promoter located in the vicinity of region L4 or due to a control at the level of RNA transport or stability. Instead our results indicate that in the absence of efficient late protein synthesis 3 end formation occurs preferentially at the L1 and L4 poly(A) addition sites.

A novel effect of adenovirus VA RNA, on cytoplasmic mRNA abundance

Adenovirus VA RNA1 is a small RNA polymerase III transcript that enhances mRNA translation both in transfected cells and during a lytic virus infection. Here we present evidence that VA RNA1 also, in length-dependent manner, increases cytoplasmic mRNA abundance in transient expression assays in 293 cells.

The carboxy-terminal exon of the adenovirus E1A protein is required for E4F-dependent transcription activation.

The adenovirus‐2 E1A 289R transcription activator protein contains a 49 amino acid sequence (designated CR3) that has been suggested to represent the minimal domain required for E1A‐induced activation of viral early transcription. We show here that the non‐conserved carboxy‐terminal E1A exon contains two interchangeable elements that are required for efficient CR3‐dependent transactivation of the adenovirus E4 promoter in HeLa cells. These two elements do not encode independent transactivation functions and have been designated auxiliary regions (ARs) 1 and 2. The effects of AR1 and AR2 are not additive, suggesting that they contribute a mechanistically analogous function in transcription. Previous studies have suggested that two cellular transcription factors, ATF‐2 and E4F, can function together with E1A to induce transcription of the E4 promoter. The importance of respective factors for E4 transcription has not been resolved. We find that E1A activation of E4F, but not ATF‐2 (or other ATF factors), is AR1‐ and AR2‐dependent. This result suggests that E1A induction of the E4 promoter in HeLa cells is primarily mediated by E4F.

Independent transformation activity by adenovirus-5 E1A-conserved regions 1 or 2 mutants.

Two conserved regions (CR1 and CR2) on the adenovirus E1A proteins have previously been shown to be required for cooperation with the ras oncogene in the transformation of primary rodent cells. Sequences within these regions are essential for the ability of E1A to associate with the 105K product of the retinoblastoma susceptibility gene, p105-RB, as well as with other cellular proteins, including a 107K (p107) and a 300K (p300) species. In this paper, we show that CR1 mutants deficient in p300 binding and CR2 mutants with lost or reduced binding of p105-RB and/or p107 have a low, but not abolished focus formation activity. In contrast, CR1/CR2 double mutants were deficient in focus formation, suggesting that the transformation activities displayed by the single CR1 or CR2 mutants were due to an independent transformation activity by both CR1 and CR2. No strict correlation between p105-RB binding and E1A-mediated transformation was observed. The E1A enhancer repression function was found to correlate with the binding of p300 but not with E1A-mediated transformation. Complex formation between E1A and p107, similar to the p105-RB binding, required sequences within both CR1 and CR2. The CR2 sequences required for binding of p107K or p105-RB were overlapping, but not identical. Finally, a larger segment of CR2 was required for stable complex formation between E1A and phosphorylated forms of p105-RB or p107 compared to corresponding unphosphorylated species.

Glucocorticoid hormones may partially substitute for adenovirus E1A in cooperation with ras

The effects of hormonal promotion of T24-ras oncogene-transfected rat embryo fibroblasts (REF) were compared to cotransformation of these cells with adenovirus E1A and ras. Cotransfection of E1A + ras resulted in the appearance of morphologically transformed cells which were very efficiently established into cell lines. Addition of glucocorticoid hormones to T24-ras-transfected REF cells resulted in cells with a transformed morphology and a capacity to form foci. These foci were, however, inefficiently established into stable cell lines. Removal of hormone from growing cells resulted in retarded growth, suggesting that the hormone acted as a growth factor on these cells. Both E1A-transformed cells and hormone-treated ras-transformed cells showed a reduction in synthesis of high molecular weight tropomyosin isoforms and a decreased expression of surface fibronectin. Control experiments demonstrated that the effects of hormone were mediated through the glucocorticoid receptor. Our findings suggest that glucocorticoid hormones may promote the in vitro growth of ras-initiated REF cells into stably transformed cell lines, but that this ability is limited compared to that of adenovirus E1A.

Transcription activation by the transforming domain of adenovirus E1A is efficiently repressed by the last 44 amino acids of E1A

REPRESSION OF THE c-JUN trans-ACTIVATION FUNCTION BY THE ADENOVIRUS TYPE 12 E1A 52R PROTEIN CORRELATES WITH THE INHIBITION OF PHOSPHORYLATION OF THE c-JUN trans-ACTIVATION DOMAIN. D. Brocknu an, G. KrOner, C. Bury and H. Esche. The cellular transcription factor complex AP-1 mediates growth factor signals on the level ofgene expression and is considered to be decisive in cell differentiation, proliferation and transformation. AP-1 consists mainly of proteins encoded by the jun gene family (c-jun, junB, junD) and the fos gene family (c-los, fosB) including thefos-related antigensfral andfra2. The early region 1A (E1A) 52R polypeptide, a protein expressed exclusively by the in vivo oncogenic Adenovims subtype 12 (Adl2), represses the trans-activating activity of AP-1 consisting of c-Jun:c-Jun homodimers. Repression is accompanied by a direct physical interaction of the adenovirus protein with the bZIP domain of c-Jun essential for dimerization and DNA-binding. Interestingly this interaction does not lead to the prevention of the promoter bindung of c-Jun/AP-1. Moreover, the association between c-JUN and the TATA-box binding protein TBP is not disturbed by the 52R polypeptide. Down-regulation of c-Jun activity is rather due to the inhibition of the phosphorylatinn of its acidic trans-activation domain located at the amino terminal end. In vivo phosphorylation of the cJun trans-activation domain by JNK kinases enzymes belonging to the mitogen-activated protein (MAP) kinase group is necessary for the interaction of c-Jun with specific co-factors like CBP and therefore a prerequisite for the activation of specific target genes. Due to these results we propose a model in which the 52R protein represses the trans-activating activity of c-Jun by prevemijag its phosphorylation through a JNK kinase(s). (Supported by the Deutsche Forschungsgemeinschaft through SFB 354/TP3 and the Fonds der Chemischen Industrie.)

Progressive Factors in Oncogene Transfected Rodent Embryo Fibroblasts

Activated ras oncogenes are inefficient in the transformation of primary rodent embryo fibroblasts (REFs). However, in unison with viral or cellular genes such as the adenovirus E1A, polyoma large-T, mutant p53 or myc genes, transformation will occur. No common biochemical activity has been described for these cooperating oncogenes. We and others have demonstrated that treatment of T24-ras oncogene transfected REF cells with the glucocorticoid dexamethasone (DEX) facilitates transformation (Martens et al., 1988; Yamashita et al., 1988; Marshall et al., Exp. Cell. Res, in press;). During a critical period between 1–3 months after transfection, cellular growth was found to be dependent on glucocorticoid. Hormone independence invariably develops during subsequent culture. These observations raise the possibility of hormonal promotion and progression of rat embryo fibroblasts expressing activated ras genes, and may offer a convenient model for studies of promotion and progression in vitro.