Active nuclear import and export pathways regulate E2F-5 subcellular localization

Epidermal keratinocyte differentiation is accompanied by differential regulation of E2F genes, including up-regulation of E2F-5 and its concomitant association with the retinoblastoma family protein p130. This complex appears to play a role in irreversible withdrawal from the cell cycle in differentiating keratinocytes. We now report that keratinocyte differentiation is also accompanied by changes in E2F-5 subcellular localization, from the cytoplasm to the nucleus. To define the molecular determinants of E2F-5 nuclear import, we tested its ability to enter the nucleus in import assays in vitro using digitonin-permeabilized cells. We found that E2F-5 enters the nucleus through mediated transport processes that involve formation of nuclear pore complexes. It has been proposed that E2F-4 and E2F-5, which lack defined nuclear localization signal (NLS) consensus sequences, enter the nucleus in association with NLS-containing DP-2 or pRB family proteins. However, we show that nuclear import of E2F-5 only requires the first N-terminal 56 amino acid residues and is not dependent on interaction with DP or pRB family proteins. Because E2F-5 is predominantly cytoplasmic in undifferentiated keratinocytes and in other intact cells, we also examined whether this protein is subjected to active nuclear export. Indeed, E2F-5 is exported from the nucleus through leptomycin B-sensitive, CRM1-mediated transport, through a region corresponding to amino acid residues 130-154. This region excludes the DNA- and the p130-binding domains. Thus, the subcellular distribution of E2F-5 is tightly regulated in intact cells, through multiple functional domains that direct nucleocytoplasmic shuttling of this protein.     

Identification of an arginine-rich motif in human papillomavirus type 1 E1;E4 protein necessary for E4-mediated inhibition of cellular DNA synthesis in vitro and in cells

Productive infections by human papillomaviruses (HPVs) are restricted to nondividing, differentiated keratinocytes. HPV early proteins E6 and E7 deregulate cell cycle progression and activate the host cell DNA replication machinery in these cells, changes essential for virus synthesis. Productive virus replication is accompanied by abundant expression of the HPV E4 protein. Expression of HPV1 E4 in cells is known to activate cell cycle checkpoints, inhibiting G(2)-to-M transition of the cell cycle and also suppressing entry of cells into S phase. We report here that the HPV1 E4 protein, in the presence of a soluble form of the replication-licensing factor (RLF) Cdc6, inhibits initiation of cellular DNA replication in a mammalian cell-free DNA replication system. Chromatin-binding studies show that E4 blocks replication initiation in vitro by preventing loading of the RLFs Mcm2 and Mcm7 onto chromatin. HPV1 E4-mediated replication inhibition in vitro and suppression of entry of HPV1 E4-expressing cells into S phase are both abrogated upon alanine replacement of arginine 45 in the full-length E4 protein (E1;E4), implying that these two HPV1 E4 functions are linked. We hypothesize that HPV1 E4 inhibits competing host cell DNA synthesis in replication-activated suprabasal keratinocytes by suppressing licensing of cellular replication origins, thus modifying the phenotype of the infected cell in favor of viral genome amplification.     

Recombinant adenoviral vectors can induce expression of p73 via the E4-orf6/7 protein

Despite the utility of recombinant adenoviral vectors in basic research, their therapeutic promise remains unfulfilled. Most engineered adenoviral vectors use a heterologous promoter to transcribe a foreign gene. We show that adenoviruses containing the cytomegalovirus immediate-early promoter induce the expression of the proapoptotic cellular protein TAp73 via the cyclin-dependent kinase-retinoblastoma protein-E2F pathway in murine embryonic fibroblasts. Cells transduced with these vectors also expressed high levels of the adenoviral E4-orf6/7 and E2A proteins. By contrast, adenoviruses containing the ubiquitin C promoter failed to elicit these effects. E4-orf6/7 is necessary and sufficient for increased TAp73 expression, as shown by using retrovirus-mediated E4-orf6/7 expression and adenovirus with the E4-orf6/7 gene deleted. Activation of TAp73 likely occurs via E4-orf6/7-induced dimerization of E2F and subsequent binding to the inverted E2F-responsive elements within the TAp73 promoter. In addition, adenoviral vectors containing the cytomegalovirus immediate-early promoter, but not the ubiquitin C promoter, cooperated with chemotherapeutic agents to decrease cellularity in vitro. In contrast to murine embryonic fibroblasts, adenoviruses containing the ubiquitin C promoter, but not the cytomegalovirus immediate-early promoter, induced both E4-orf6/7 and TAp73 in human foreskin fibroblasts, emphasizing the importance of cellular context for promoter-dependent effects. Because TAp73 is important for the efficacy of chemotherapy, adenoviruses that increase TAp73 expression may enhance cancer therapies by promoting apoptosis. However, such adenoviruses may impair the long-term survival of transduced cells during gene replacement therapies. Our findings reveal previously unknown effects of foreign promoters in recombinant adenoviral vectors and suggest means to improve the utility of engineered adenoviruses by better controlling their impact on viral and cellular gene expression.     

Differential nuclear localization of the major adenovirus type 2 E1a proteins.

The localization in infected and transformed cells of the two major adenovirus type 2 E1a proteins, of 289 and 243 amino acid residues, was studied with antisera raised against synthetic peptides or a TrpE-E1a fusion protein. Both E1a proteins were detected only in the nucleus of infected cells as determined by immunofluorescence analysis of cells infected with wild-type virus or with the mutants pm975 or dl1500, which produce, respectively, only the 289-residue or only the 243-residue E1a protein. However, the 289-residue protein was more tightly associated with the nucleus than was the 243-residue protein, as determined by the stability of nuclear fluorescence to different fixation procedures and by the use of radioimmunoprecipitation and Western blot analysis to analyze fractions extracted from the nucleus by detergent and other treatments. The latter experiments revealed that only the 289-residue protein, and only a fraction of that protein present in the nucleus, is associated with the nuclear matrix, both in infected HeLa cells and in the transformed human cell line 293.     

Effect of cisplatin treatment on speckled distribution of a serine/arginine-rich nuclear protein CROP/Luc7A

The C-half of cisplatin resistance-associated overexpressed protein (CROP), an SR-related protein, comprises domains rich in arginine and glutamate residues (RE domain), and is rich in arginine and serine residues (RS domain). We analyzed the role of the individual domains of CROP in cellular localization, subnuclear localization, and protein-protein interaction. CROP fused with green fluorescent protein, GFP-CROP, localized exclusively to the nucleus and showed a speckled intranuclear distribution. The yeast two-hybrid system revealed that CROP interacted with SF2/ASF, an SR protein involved in RNA splicing, as well as CROP itself. The RE and RS domains were necessary for both the intranuclear speckled distribution and the protein-protein interaction. CROP was phosphorylated by mSRPK1, mSRPK2, and Clk1 in vitro, and when cells were treated with cisplatin the subnuclear distribution of GFP-CROP was changed. These results suggest that cisplatin affects RNA splicing by changing the subnuclear distribution of SR proteins including CROP.     

Human papillomavirus 18 E1^E4 protein interacts with cyclin A/CDK 2 through an RXL motif

The human papilloma virus E4 protein is highly expressed in late times of infection. Evidence to date suggests that E4 is essential for amplification of the viral genome and that it can influence cell cycle. Examination of the sequences encoding the E4 proteins from several genotypes of human papillomavirus revealed the presence of RXL-containing motifs reminiscent of the cyclin-binding motifs that have been identified in several cyclin-binding proteins. When baculovirus-produced human cyclin E and cyclin A with cdk2 were incubated in vitro with a GST-E4 fusion protein, both cyclin E and A stably interacted with the GST-E4 protein containing the full E4 sequence from HPV18. The interaction was not dependent on the presence of the kinase subunit but was dependent on the integrity of the RXL motif in E4. When incubated with cell extracts from the C33A human cervical carcinoma cell line or when expressed in C33A cells, the GST-E4 protein formed interactions with cyclin A and cdk2 and kinase activity could be demonstrated in the GST-E4 complex. In contrast to the baculovirus-produced cyclin E, cellular cyclin E failed to detectably interact with GST-E4 suggesting that the HPV18 E4 sequences are capable of interacting only with cyclin A in mammalian cells. These observations suggest that human papillomavirus E4 proteins can interact with cyclin A/cdk2, which may contribute to viral manipulation of the host cell cycle.     

CROP/Luc7A, a novel serine/arginine-rich nuclear protein, isolated from cisplatin-resistant cell line

A novel putative SR protein, designated cisplatin resistance-associated overexpressed protein (CROP), has been cloned from cisplatin-resistant cell lines by differential display. The N-half of the deduced amino acid sequence of 432 amino acids of CROP contains cysteine/histidine motifs and leucine zipper-like repeats. The C-half consists mostly of charged and polar amino acids: arginine (58 residues or 25%), glutamate (36 residues or 16%), serine (35 residues or 15%), lysine (30 residues, 13%), and aspartate (20 residues or 9%). The C-half is extremely hydrophilic and comprises domains rich in lysine and glutamate residues, rich in alternating arginine and glutamate residues, and rich in arginine and serine residues. The arginine/serine-rich domain is dominated by a series of 8 amino acid imperfect repetitive motif (consensus sequence, Ser-Arg-Ser-Arg-Asp/Glu-Arg-Arg-Arg), which has been found in RNA splicing factors. The RNase protection assay and Western blotting analysis indicate that the expression of CROP is about 2-3-fold higher in mRNA and protein levels in cisplatin-resistant ACHN/CDDP cells than in host ACHN cells. CROP is the human homologue of yeast Luc7p, which is supposed to be involved in 5'-splice site recognition and is essential for vegetative growth.     

Proliferation-dependent expression of nuclear uracil-DNA glycosylase is mediated in part by E2F-4

There are two isoforms of the prototypical human uracil-DNA glycosylase: one mitochondrial (UDG1) and one nuclear (UDG1A). Results presented here reveal a novel genetic organization of UDG1. Specifically, the UDG1 5' UTR is composed of two non-coding exons and the promoter region is located much farther upstream than previously recognized. We also examine the proliferation-dependent expression of UDG1A and demonstrate that the protein disappears rapidly as cells transit from the cell cycle into G0. Ribonuclease protection assays reveal that UDG1A mRNA levels are greatly reduced during G0 as well. To begin to characterize the mechanisms contributing to this regulation, we identified two overlapping candidate E2F binding sites (denoted A and B) in the UDG1A 5' UTR. EMSA analysis of this region shows a unique protein complex present only in extracts derived from G0 cells. In vitro studies using purified E2F-4 and mutant competitors demonstrate that binding occurs in a proliferation-dependent manner exclusively to E2F site A. Two approaches were then used to assess the in vivo role of the candidate E2F sites. First, chromatin immunoprecipitation (ChIP) analysis demonstrates that E2F-4 binds to the UDG1A 5' UTR exclusively in G0 cells. Secondly, using transient transfection analysis, we show that mutating these sites abolishes the proliferation-dependent response of UDG1A.     

Induction of apoptosis by adenovirus E4orf4 protein

Adenovirus E4orf4 protein is a multifunctional viral regulator that induces p53-independent apoptosis in transformed cells, but not in normal cells. E4orf4-induced apoptosis can occur without activation of known caspases, although E4orf4 induces caspase activity in some cell lines. The interaction of E4orf4 with a specific subpopulation of protein phosphatase 2A (PP2A) molecules that contain B subunits, but not with those that contain B subunits, is required for induction of apoptosis. This review suggests the potential use of E4orf4 in cancer therapy, and discusses whether E4orf4-induced apoptosis plays a role in the viral life cycle. Future research directions are also highlighted.     

The nuclear export signal within the E4orf6 protein of adenovirus type 5 supports virus replication and cytoplasmic accumulation of viral mRNA

During the late phase of adenovirus infection, viral mRNA is efficiently transported from the nucleus to the cytoplasm while most cellular mRNA species are retained in the nucleus. Two viral proteins, E1B-55 kDa and E4orf6, are both necessary for these effects. The E4orf6 protein of adenovirus type 5 binds and relocalizes E1B-55 kDa, and the complex of the two proteins was previously shown to shuttle continuously between the nucleus and cytoplasm. Nucleocytoplasmic transport of the complex is achieved by a nuclear export signal (NES) within E4orf6. Mutation of this signal sequence severely reduces the ability of the E1B-55 kDa-E4orf6 complex to leave the nucleus. Here, we examined the role of functional domains within E4orf6 during virus infection. E4orf6 or mutants derived from it were transiently expressed, followed by infection with recombinant adenovirus lacking the E4 region and determination of virus yield. An arginine-rich putative alpha helix near the carboxy terminus of E4orf6 contributes to E1B-55 kDa binding and relocalization as well as to the synthesis of viral DNA, mRNA, and proteins. Further mutational analysis revealed that mutation of the NES within E4orf6 considerably reduces its ability to support virus production. The same effect was observed when nuclear export was blocked with a competitor. Further, a functional NES within E4orf6 contributed to the efficiency of late virus protein synthesis and viral DNA replication, as well as total and cytoplasmic accumulation of viral late mRNA. Our data support the view that NES-mediated nucleocytoplasmic shuttling strongly enhances most, if not all, intracellular activities of E4orf6 during the late phase of adenovirus infection.