The adenovirus E4-6/7 protein directs nuclear localization of E2F-4 via an arginine-rich motif.

E2F transcription factors are key participants in the regulation of proliferation, apoptosis, and differentiation in mammalian cells. E2Fs are negatively regulated by members of the retinoblastoma protein (pRb) family. During adenovirus (Ad) infection, viral proteins that displace pRb family members from E2Fs and recruit E2F complexes to viral and cellular promoter regions are expressed. This recruitment of E2F involves the induction of stable E2F binding to inverted E2F binding sites in the Ad E2a and cellular E2F-1 promoters and induces both viral and cellular gene expression. E2F-4 has abundant E2F activity within cells, and the majority of E2F-4 in asynchronous cells is found in the cytoplasm. Upon expression of the adenovirus E4-6/7 protein, a significant portion of E2F-4 is translocated to the nucleus, and its activity constitutes the majority of Ad-induced nuclear E2F DNA binding activity. This redirection of E2F-4 from cytoplasm to the nucleus requires an N-terminal arginine-rich nuclear localization sequence within E4-6/7. The directed targeting of E4-6/7 to the nucleus is important for the function of this protein in the context of viral infection. This function of E4-6/7 has a redundant component as well as nonredundant components in cooperation with the adenovirus E1A oncoproteins to deregulate and usurp host cell E2F function.

[Expression of epidermal growth factor-adenovirus E4orf4 fusion protein in tumor cells and its cytotoxicity].

BACKGROUND & OBJECTIVE: Human epidermal growth factor (EGF), an important growth factor, may stimulate cell growth and proliferation. EGF receptor (EGFR) expresses on the surface of normal cells, and abnormally over-expresses on many kinds of tumor cells, especially on solid tumor cells. Adenovirus early region 4 open reading frame 4 protein (E4orf4) is a novel cytotoxin that can specifically induce p53-independent apoptosis in tumor cells. Based on the targeting of EGF and cytotoxicity of E4orf4, we proposed to design a novel fusion protein at molecular level by recombining EGF and E4orf4 to target and then kill tumor cells. METHODS: EGF and E4orf4 coding sequences were amplified by polymerase chain reaction (PCR), and then genetically fused by overlapping PCR. EGF-E4orf4 fragment was cloned into the yeast expression vector. Recombinant plasmid DNA was transformed into the yeast Pichia pastoris. Fusion proteins were purified by SP Sepharose ion exchange chromatography. Cytotoxicity of EGF-E4orf4 on cultured BT325 and MDA-MB-231 cells was detected by MTT assay, and cell apoptosis was measured by flow cytometry. RESULTS: The fusion fragment has 805 base pairs, which consists of Kozak consensus sequence, and the sequences encoding alpha-factor signal peptide, EGF, flexible linker, and E4orf4. Recombinant plasmids pAO-EGF-E4orf4, and pAO-3EGF-E4orf4 were obtained, the latter contained 3 expression cassettes. Apparent molecular weight of fusion protein was 20 ku. Immunoblot analysis showed that the fusion protein was immunoreactive with rabbit-anti-human EGF polyclonal antibody. EGF-E4orf4 in high concentrations (5, and 0.5 microg/ml) inhibited growth of BT325 and MDA-MB-231 tumor cells as compared with controls. Apoptosis was induced in 15.4%-28.2% of MDA-MB-231 cells by EGF-E4orf4 at the dosage of 10-25 microg/3 x 10(5) cells. CONCLUSIONS: Fusion protein EGF-E4orf4 may enter cells mediated by EGFR, and thus inhibit growth of tumor cells.

Induction of the cellular E2F-1 promoter by the adenovirus E4-6/7 protein.

The adenovirus type 5 (Ad5) E4-6/7 protein interacts directly with different members of the E2F family and mediates the cooperative and stable binding of E2F to a unique pair of binding sites in the Ad5 E2a promoter region. This induction of E2F DNA binding activity strongly correlates with increased E2a transcription when analyzed using virus infection and transient expression assays. Here we show that while different adenovirus isolates express an E4-6/7 protein that is capable of induction of E2F dimerization and stable DNA binding to the Ad5 E2a promoter region, not all of these viruses carry the inverted E2F binding site targets in their E2a promoter regions. The Ad12 and Ad40 E2a promoter regions bind E2F via a single binding site. However, these promoters bind adenovirus-induced (dimerized) E2F very weakly. The Ad3 E2a promoter region binds E2F very poorly, even via a single binding site. A possible explanation of these results is that the Ad E4-6/7 protein evolved to induce cellular gene expression. Consistent with this notion, we show that infection with different adenovirus isolates induces the binding of E2F to an inverted configuration of binding sites present in the cellular E2F-1 promoter. Transient expression of the E4-6/7 protein alone in uninfected cells is sufficient to induce transactivation of the E2F-1 promoter linked to chloramphenicol acetyltransferase or green fluorescent protein reporter genes. Further, expression of the E4-6/7 protein in the context of adenovirus infection induces E2F-1 protein accumulation. Thus, the induction of E2F binding to the E2F-1 promoter by the E4-6/7 protein observed in vitro correlates with transactivation of E2F-1 promoter activity in vivo. These results suggest that adenovirus has evolved two distinct mechanisms to induce the expression of the E2F-1 gene. The E1A proteins displace repressors of E2F activity (the Rb family members) and thus relieve E2F-1 promoter repression; the E4-6/7 protein complements this function by stably recruiting active E2F to the E2F-1 promoter to transactivate expression.

Inactivation of p53 but not p73 by adenovirus type 5 E1B 55-kilodalton and E4 34-kilodalton oncoproteins.

The adenovirus E1B 55-kDa and E4 34-kDa oncoproteins bind and inactivate the p53 tumor suppressor gene product, resulting in cell transformation. A recently discovered cellular protein, p73, shows extensive similarities to p53 in structure and function. Here we show that the simultaneous transient expression of E1B 55-kDa and E4 34-kDa proteins is sufficient to drastically shorten the intracellular half-life of p53, leading to strongly reduced steady-state p53 levels. Concomitantly, the E1B 55-kDa and E4 34-kDa proteins act synergistically to inactivate the transcriptional activity of p53. Mutational analysis suggests that physical interactions between the E1B 55-kDa protein and p53 and between the E1B 55-kDa and E4 34-kDa proteins are both required for p53 degradation. In contrast, the ability of p53 to interact with the cellular mdm2 oncoprotein or with its cognate DNA element appears to be dispensable for its destabilization by adenovirus gene products. The adenovirus E1B 55-kDa protein did not detectably interact with p73 and failed to inhibit p73-mediated transcription; also, the E1B 55-kDa and E4 34-kDa proteins did not promote p73 degradation. When five amino acids near the amino termini were exchanged at corresponding positions between p53 and p73, this rendered p53 resistant and p73 susceptible to complex formation and inactivation by the E1B 55-kDa protein. Our results suggest that while p53 inactivation is a central step in virus-induced tumor development, efficient transformation can occur without targeting p73.