Association of adenovirus early-region 1A proteins with cellular polypeptides.

Extracts from adenovirus-transformed human 293 cells were immunoprecipitated with monoclonal antibodies specific for the early-region 1A (E1A) proteins. In addition to the E1A polypeptides, these antibodies precipitated a series of proteins with relative molecular weights of 28,000, 40,000, 50,000, 60,000, 80,000, 90,000, 110,000, 130,000, and 300,000. The two most abundant of these polypeptides are the 110,000-molecular-weight protein (110K protein) and 300K protein. Three experimental approaches have suggested that the 110K and 300K polypeptides are precipitated because they form stable complexes with the E1A proteins. The 110K and 300K polypeptides do not share epitopes with the E1A proteins, they copurify with a subset of the E1A proteins, and they bind to the E1A proteins following mixing in vitro. The 110K and 300K polypeptides are not adenoviral proteins, but are encoded by cellular DNA. Both the 12S and the 13S E1A proteins bind to the 110K and 300K species, and these complexes are found in adenovirus-transformed and -infected cells.     

Homologous sequences in adenovirus E1A and human papillomavirus E7 proteins mediate interaction with the same set of cellular proteins.

Studies of adenovirus E1A oncoprotein mutants suggest that the association of E1A with the retinoblastoma protein (pRB) is necessary for E1A-mediated transformation. Mutational analysis of E1A indicates that two regions of pRB are required for E1A to form stable complexes with the retinoblastoma protein. In addition to pRB binding, these regions are necessary for E1A association with several other cellular proteins, including p130, p107, cyclin A, and p33cdk2. Here we show that short synthetic peptides containing the pRB-binding sequences of E1A are sufficient for interaction with p107, cyclin A, and p130. The E7 protein of human papillomavirus type 16 contains an element that binds to pRB and appears to be functionally homologous to the E1A sequences. Peptides containing this region of the E7 protein were able to interact with p107, cyclin A, and p130 in addition to pRB. These findings suggest that the common mechanism of transformation used by these viral oncogenes involves their association with a set of polypeptides.     

Differential distribution of the adenovirus E1A proteins and colocalization of E1A with the 70-kilodalton cellular heat shock protein in infected cells.

Five distinct localization patterns were observed for the adenovirus E1A proteins in the nuclei of infected HeLa cells: diffuse, reticular, nucleolar, punctate, and peripheral. The variable distribution of E1A was correlated with the time postinfection and the cell cycle stage of the host cell at the time of infection. All staining patterns, with the exception of peripheral E1A localization, were associated with the early phase of infection since only the diffuse, reticular, nucleolar, and punctate staining patterns were observed in the presence of hydroxyurea. Because the E1A proteins (12S and 13S) stimulate the expression of the cellular heat shock 70-kilodalton protein (hsp70), we examined the intracellular distribution of hsp70 in the adenovirus-infected cells. Whereas hsp70 was predominantly cytoplasmic in the cells before infection, after adenovirus infection most of the protein was now found within the nucleus. Specifically, hsp70 was found within the nucleoli as well as exhibiting reticular, diffuse, and punctate nuclear staining patterns, analogous to those observed for the E1A proteins. Double-label indirect immunofluorescence of E1A and hsp70 in infected cells demonstrated a colocalization of these proteins in the nucleus. Translocation of hsp70 to the nucleus was dependent upon both adenovirus infection and expression of the E1A proteins. The localization of hsp70 was unaltered by infection with an E1A 9S cDNA virus which does not synthesize a functional E1A gene product. Moreover, the discrete nuclear localization patterns of E1A and the colocalization of E1A with hsp70 were not observed in adenovirus-transformed 293 cells which constitutively express E1A and E1B. E1A displayed exclusively diffuse nuclear staining in 293 cells; however, localization of E1A into the discrete nuclear patterns occurred after adenovirus infection of 293 cells. Immunoprecipitation of labeled infected-cell extracts with a monoclonal antibody directed against the E1A proteins resulted in precipitation of small amounts of hsp70 along with E1A. These data indicate that the adenovirus E1A proteins colocalize with, and possibly form a physical complex with, cellular hsp70 in infected cells. The relevance of this association, with respect to the function of these proteins during infection and the association of other oncoproteins with hsp70, is discussed.     

Different functional domains of the adenovirus E1A gene are involved in regulation of host cell cycle products.

We have analyzed the cell cycle effects that different domains of the adenovirus E1A proteins have on quiescent primary BRK cells. Studies with deletion mutants that in combination removed all but the N-terminal 85 amino acids common to both the 12S and 13S proteins suggest that this region may be sufficient for the induction of synthesis of proliferating cell nuclear antigen and the stimulation of DNA synthesis. A second domain also common to the N-terminal exon of the 12S and 13S proteins was required for the induction of mitosis and stimulation of proliferation of primary BRK cells. A virus containing a mutation in this region was still able to stimulate DNA synthesis efficiently. A third domain, unique to the 13S protein, was required for the accelerated activation of the cellular thymidylate synthase gene in a manner similar to the 13S-dependent stimulation of adenovirus early region genes.     

Inhibition of the cellular response to interferons by products of the adenovirus type 5 E1A oncogene.

Expression of the E1A oncogene of adenovirus type 5 inhibits the response of interferon (IFN)-inducible constructs to Type I (alpha,beta) and II (gamma) IFNs in transient transfection assays. In human cell lines stably expressing E1A mRNA and protein acquisition of an antiviral state and the induction of a number of genes in response to alpha- and gamma-IFNs is inhibited. A short IFN-stimulable response element (ISRE) present in the 5' flanking region of a number of genes mediates induction by alpha- and gamma-IFNs. In cells expressing E1A there is a substantial reduction in the levels of the ISRE-binding factors E and M, inducible by alpha-IFN, and of factor G, inducible by gamma-IFN. In E1A-expressing cells the E alpha subunit of factor E is activated normally in response to alpha-IFN; the defect is in the production or activation of the E gamma subunit. The inhibitory activity of E1A is lost upon deletion of the CR1 domain. The induction of HLA class II genes by gamma-IFN, which involves a different DNA response element(s), and of beta-IFN mRNA in response to double-stranded RNA are also inhibited by E1A. An essential component(s) of a number of signalling pathways must, therefore, be subject, directly or indirectly, to inhibition by E1A.     

Sequence-specific interactions between cellular DNA-binding proteins and the adenovirus origin of DNA replication.

The adenovirus origin of DNA replication contains three functionally distinct sequence domains (A, B, and C) that are essential for initiation of DNA synthesis. Previous studies have shown that domain B contains the recognition site for nuclear factor I (NF-I), a cellular protein that is required for optimal initiation. In the studies reported here, we used highly purified NF-I, prepared by DNA recognition site affinity chromatography (P. J. Rosenfeld and T. J. Kelly, Jr., J. Biol. Chem. 261:1398-1408, 1986), to investigate the cellular protein requirements for initiation of viral DNA replication. Our data demonstrate that while NF-I is essential for efficient initiation in vitro, other cellular factors are required as well. A fraction derived from HeLa cell nuclear extract (BR-FT fraction) was shown to contain all the additional cellular proteins required for the complete reconstitution of the initiation reaction. Analysis of this complementing fraction by a gel electrophoresis DNA-binding assay revealed the presence of two site-specific DNA-binding proteins, ORP-A and ORP-C, that recognized sequences in domains A and C, respectively, of the viral origin. Both proteins were purified by DNA recognition site affinity chromatography, and the boundaries of their binding sites were defined by DNase I footprint analysis. Additional characterization of the recognition sequences of ORP-A, NF-I, and ORP-C was accomplished by determining the affinity of the proteins for viral origins containing deletion and base substitution mutations. ORP-C recognized a sequence between nucleotides 41 and 51 of the adenovirus genome, and analysis of mutant origins indicated that efficient initiation of replication is dependent on the presence of a high-affinity ORP-C-binding site. The ORP-A recognition site was localized to the first 12 base pairs of the viral genome within the minimal origin of replication. These data provide evidence that the initiation of adenovirus DNA replication involves multiple protein-DNA interactions at the origin.     

Membrane proteins involved in pollen-pistil interactions.

Self-incompatibility (SI) is a widespread mechanism in angiosperms which prevents self-fertilization. This mechanism relies on cell-cell interactions between pollen and pistil. Among the different SI systems that have been reported, two have been particularly investigated: the gametophytic system of Solanaceae and the sporophytic system of Brassicaceae. In these two families, although the molecular bases of SI response are different, secreted and/or membrane-anchored proteins are required for self-pollen rejection. Interestingly, these proteins exhibit two functions: recognition and a catalytic activity. In this review article, we present recent advances which permit a better understanding of how these proteins control the male/female recognition event associated with the SI response.     

Two regions of the adenovirus early region 1A proteins are required for transformation.

Regions of the adenovirus type 5 early region 1A (E1A) proteins that are required for transformation were defined by using a series of deletion mutants. Deletion mutations collectively spanning the entire protein-coding region of E1A were constructed and assayed for their ability to cooperate with an activated ras oncogene to induce transformation in primary baby rat kidney cells. Two regions of E1A (amino acids 1 to 85 and 121 to 127) were found to be essential for transformation. Deletion of all or part of the region from amino acids 121 to 127 resulted in a total loss of transforming ability. An adjacent stretch of amino acids (residues 128 to 139), largely consisting of acidic residues, was found to be dispensable for transformation but appeared to influence the efficiency of transformation. Amino acids 1 to 85 made up a second region of the E1A protein that was essential for transformation. Deletion of all or part of this region resulted in a loss of the transforming activity. Even a mutation resulting in a single amino acid change at position 2 of the polypeptide chain was sufficient to eliminate transformation. Deletion of amino acids 86 to 120 or 128 to 289 did not eliminate transformation, although some mutations in these regions had lowered efficiencies of transformation. Foci induced by transformation-competent mutants could be expanded into cell lines that retained their transformed morphology and constitutively expressed the mutant E1A proteins.     

Lytic and transforming functions of individual products of the adenovirus E1A gene.

To distinguish the individual roles of the 13S, 12S, and 9S adenovirus E1A gene products, we isolated the corresponding cDNA clones and recombined them into both plasmids and viruses. Only the expected E1A mRNA products were made from the corresponding 12S and 13S viruses. The 9S mRNA was detected when the 9S virus was coinfected with the 13S virus but not when either virus was infected alone. The 13S virus formed plaques equally well in 293 cells, HeLa cells, and A549 cells, a human lung oat cell carcinoma line. Plaque titers of the 12S virus were much reduced in HeLa and A549 cells compared with 293 cells, although the 12S virus is multiplicity-dependent leaky in both HeLa and A549 cells. A549 cells were significantly more permissive than HeLa cells for growth of the 12S virus. In A549 cells even at low multiplicities of infection the final yield of 12S virus eventually approached the maximum yield from 293 cells. Expression from the adenovirus early region 2 and early region 3 promoters in HeLa cells was activated in the presence of a 13S cDNA E1A region but not in the presence of a 12S E1A cDNA region. Although defective for lytic growth in HeLa cells, the 12S virus immortalized BRK cells at very high efficiency, whereas infection of these cells with 13S virus, as with wild-type E1A virus, resulted mainly in cell death. The 13S product does have an immortalization function, however, revealed in the absence of adenovirus lytic functions when a plasmid containing the E1A 13S cDNA region was transfected into BRK cells. The 9S virus failed to immortalize infected BRK cells or to interfere with focus formation when coinfected with the 12S virus.     

Interactions between cell growth-regulating domains in the products of the adenovirus E1A oncogene.

Among the various biological activities expressed by the products of the adenovirus E1A gene are the abilities to induce cellular DNA synthesis and proliferation in quiescent primary baby rat kidney cells. The functional sites for these activities lie principally within two regions of the E1A proteins: an N-terminal region and a small second region of approximately 20 amino acids further downstream. To study the biological functions of the first domain, we constructed an in-frame deletion of amino acid positions 23 through 107 of the E1A products. This deletion did not impede the ability of the E1A products to transactivate the adenovirus early region 3 promoter in a transient-expression assay in HeLa cells. The ability to induce DNA synthesis in quiescent baby rat kidney cells was, however, lost in the absence of these sequences. Deletion of the small second region induced a form of S phase in which DNA synthesis occurred in the apparent absence of controls required for the cessation of DNA synthesis and progression through the remainder of the cell cycle. These cells did not appear to accumulate in or before G2, and many appeared to have a DNA content greater than that in G2. The functions of both domains are required for production of transformed foci in a ras cooperation assay. Focus formation occurred, however, even when the two domains were introduced on two separate plasmids. This complementation effect appeared to require expression of both of the mutant proteins and did not appear to result merely from recombination at the DNA level.     

Cellular targets for transformation by the adenovirus E1A proteins

Three cellular proteins, including species of 300,000 daltons and 107,000 daltons as well as p105-RB, the product of the retinoblastoma susceptibility gene, stably interact with the adenovirus E1A proteins. To help determine the functional basis of these interactions, the regions of E1A that participate in these interactions were mapped using a series of deletion mutants. The 300,000 dalton and the 107,000 dalton proteins interacted with sequences within amino acids 1 to 76 and 121 to 127, respectively. Interaction with the third cellular protein, p105-RB, required the presence of sequences from two noncontiguous regions of the E1A polypeptide chain, amino acids 30 to 60 and 121 to 127. The regions of E1A that are required for these interactions coincided precisely with the regions of E1A that are required for its transforming function. These results suggest that the interactions with these cellular proteins are fundamental to the transforming activity of E1A.     

Functional interactions between papillomavirus E1 and E2 proteins.

DNA replication of papillomaviruses requires the viral E1 and E2 proteins. These proteins bind cooperatively to the viral origin of replication (ori), which contains binding sites for both proteins, forming an E1-E2-ori complex which is essential for initiation of DNA replication. To map the domains in E2 that are involved in the interaction with E1, we have used chimeric bovine papillomavirus (BPV)/human papillomavirus type 11 (HPV-11) E2 proteins. The results from this study show that both the DNA binding domain and the transactivation domain from BPV E2 independently can interact with BPV E1. However, the roles of these two interactions are different: the interaction between E1 and the activation domain of E2 is necessary and sufficient for cooperativity in binding and for DNA replication; the interaction between E1 and the DNA binding domain of E2 is required only when the binding sites for E1 and E2 are adjacent to each other, and the function of this interaction appears to be to facilitate the interaction between E1 and the transactivation domain of E2. These results indicate that the cooperative binding of E1 and E2 to the BPV ori takes place via a novel two-stage mechanism where one interaction serves as a trigger for the formation of the second, productive, interaction between the two proteins.