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.     

Adenovirus E1A 12S protein induces DNA synthesis and proliferation in primary epithelial cells in both the presence and absence of serum.

Infection of primary baby rat kidney (BRK) cells with an adenovirus that carries an E1A 12S cDNA in place of the normal E1A region (adenovirus 5 [Ad5] 12S) resulted in the induction of cellular DNA synthesis and proliferation of the epithelial cells in the population, even in the absence of serum. Increased cellular DNA synthesis was first detectable by 12 h after infection and was maintained at a 10- to 20-fold higher level than in mock-infected cells. By 5 days after infection there was a 10-fold-greater number of 12S virus-infected BRK cells. These infected BRK cells retained many of their normal epithelial cell characteristics and were not transformed. The expression of the E1A 12S protein(s) occurred early after infection. There was no induction of adenoviral gene expression or viral DNA replication in these cells. The early effects of a fully transforming gene product(s) were also examined. The Ad5-simian virus 40 hybrid virus, Ad5.SVR4, in which the early region of simian virus 40 has replaced the E1 region of Ad5, was used to infect BRK cells. The kinetics of expression of the T antigens were similar to those of the 12S polypeptides. Infection with Ad5.SV4 also resulted in the induction of cellular DNA synthesis and cell proliferation at levels similar to those observed with the 12S virus. However, infection with Ad5.SVR4 resulted in cells that had lost some of their epithelial cell characteristics and were fully transformed. Thus, although the early cellular events induced by the two genes were similar, they did not yield the same final cellular phenotype.     

Rapid intracellular turnover of adenovirus 5 early region 1A proteins

The half-life of the adenovirus 5 early region 1A (E1A) proteins was examined in productively infected and transformed cells. In HeLa cells infected with adenovirus 5, the half-life of the E1A proteins was approximately 30 min; in the transformed 293 cells, the constitutively expressed E1A proteins had a half-life of approximately 120 min. In HeLa cells, the E1A proteins produced by an adenovirus mutant that expresses only the 13S mRNA had a half-life of about 35 min; E1A proteins produced by a mutant that express only the 12S mRNA had a half-life of about 80 min. This difference in the stability of these two classes of E1A proteins helps explain why the steady-state level of the 12S class is usually equal to or greater than that of the 13S class, despite the fact that the concentration of the 13S mRNA is about four times greater than the concentration of the 12S mRNA.     

Adenovirus early region 1A modulation of interferon antiviral activity.

Human adenovirus type 5 (Ad5) is a DNA virus which replicates as efficiently in human A549 cells treated with human interferon-alpha 2 (IFN) as in untreated cells. Vesicular stomatitis virus (VSV), on the other hand, is a negative-strand RNA virus which is very sensitive to the effects of IFN treatment in A549 cells. The IFN-mediated inhibition of VSV replication was not observed in cells coinfected with Ad5. Abrogation of IFN-mediated antiviral activity was maximal when Ad5 infection preceded VSV infection by at least 36 h, but did not require adenovirus DNA synthesis for manifestation. Coinfection experiments with VSV and deletion variants of adenovirus demonstrated that neither virus-associated RNA synthesis nor expression of adenovirus early regions E1B, E2A, E3, or E4 are required for abrogation of IFN-mediated inhibition of VSV replication. However, expression of early region E1A was essential, suggesting that E1A products can modulate, either directly or indirectly, IFN activity in adenovirus-infected cells.     

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.     

Cells transformed by adenovirus type 12 but not by type 5 are dependent on insulin or insulin-like growth factor I for their proliferation

We have investigated the responsiveness to growth factors (GFs) of primary baby rat kidney (BRK) cells transformed by the E1 region of adenovirus 5 or 12. The in vitro growth of non-oncogenic adenovirus 5-transformed BRK cells is largely independent of serum GFs, whereas growth of highly oncogenic adenovirus 12-transformed cells is strictly dependent on GFs present in serum. For the growth of adenovirus 12 E1-transformed BRK cells serum can be replaced by insulin or insulin-like growth factor-I but not by epidermal growth factor. To maintain the in vitro growth of adenovirus 12-transformed cells physiological levels of insulin-like growth factor-I, but not of insulin, are sufficient. Similar results have been found with adenovirus-transformed primary murine cells and with transformants of an established rat cell line, NRK 49F. This indicates that the observed GF responsiveness is not dependent of the cell type used but is determined by the serotype of the adenovirus-transforming region. Using hybrid E1 regions consisting of E1A of one serotype and E1B of the other, we show that the pattern of GF-responsiveness correlates with the origin of the E1A region. The differences in the GF-responsiveness of the adenovirus 5-transformed and adenovirus 12-transformed cells will be discussed in terms of the oncogenicity of these cells.     

A new human cellular protein AUP1. III. The intracellular localization of AUP1 protein in different human and rat cell lines

Previously, we cloned a full-length cDNA of human Aup1 and showed that AUP1 may represent a new cellular target for the two adenovirus oncoproteins, E1A Ad5 and E4ORF3. In this study, we generated a polyclonal anti-AUP1 antibody and examined the subcellular localization of AUP1 in MCF7 cells, HeLa cells, H1299 cells, 293 cells, BRK1 cells and transfectants expressing adenoviruse E1 genes. Double staining of AUP1 and various markers for cytoplasmic structures showed that the pattern of AUP1 distribution in the cytoplasm was puctuate and diffuse and without any colocalization with Golgi apparatus or endoplasmic reticulum. Additional studies with ectopically expressed AUP1, fused with red fluorescent protein (RFP) in H1299 and McG7 human cell lines and BRK1 rat cell line, showed cytoplasmic localization of RFP-AUP1. Western blot analysis revealed that AUP1 was expressed at similar levels in all tested cell lines and had the same molecular weight as the rat protein (45 kDa). Taken together, these results suggest that AUP1 is a cytoplasmic protein that is expressed in all cell lines we examined.     

Expression of adenovirus E1B mutant phenotypes is dependent on the host cell and on synthesis of E1A proteins.

Adenovirus mutants containing genetic alterations in the gene encoding the E1B 19,000-molecular-weight (19K) tumor antigen induce the degradation of host cell chromosomal DNA (deg phenotype) and enhanced cytopathic effect (cyt phenotype) after infection of HeLa and KB cells. The deg and cyt phenotypes are a consequence of viral early gene expression in the absence of the E1B 19K protein. The role of the E1A proteins in induction of the cyt and deg phenotypes was investigated by constructing E1A-E1B double mutant viruses. Viruses were constructed to express the individual E1A 13S, 12S, or 9S cDNA genes in the presence of a mutation in the gene encoding the E1B 19K tumor antigen. Expression of either the 13S or 12S E1A proteins in the absence of functional E1B 19K protein produced the deg and cyt phenotypes. In contrast, a virus which expressed exclusively the 9S E1A gene product in the absence of the E1B 19K gene product did not induce the deg and cyt phenotypes, even at high multiplicities of infection. Therefore, both the 13S and 12S E1A gene products could directly or indirectly cause the deg and cyt phenotypes during infection of HeLa cells with an E1B 19K gene mutant virus. Furthermore, the deg phenotype was found to be host cell type specific, occurring in HeLa and KB cells but not in growth-arrested human WI38 cells. These results indicate that expression of the E1A trans-activating and transforming proteins is necessary for the induction of the cyt and deg phenotypes and that host cell factors also play a role.     

A block in release of progeny virus and a high particle-to-infectious unit ratio contribute to poor growth of enteric adenovirus types 40 and 41 in cell culture.

The fastidious enteric adenovirus (FEAd) types 40 (Ad40) and 41 (Ad41) are found in stool specimens of infants and young children in association with gastroenteritis. Although they can be isolated routinely from clinical specimens by using 293 cells, they are propagated with variable success in cell lines which support the replication of other adenovirus serotypes. HeLa cells are generally considered to be nonpermissive for the replication of FEAds, but in this study, Ad40 and Ad41 grew to comparable titers in individual 293 and HeLa cells. However, virus was not efficiently released from infected HeLa cells and thus did not undergo multiple cycles of infection in HeLa cell cultures. The block in virus release was not overcome in KB18 cells which, like 293 cells, constitutively express proteins encoded by the E1B region of a subgroup C adenovirus (in this case Ad2). Moreover, it was apparent from these studies that Ad40 and Ad41 have particle-to-infectious unit ratios several orders of magnitude greater than that for Ad5, even in 293 cells which express the E1A and E1B proteins of Ad5 and are considered to be permissive for replication of the FEAds. Neither the block in release of progeny virus nor the high particle-to-infectious unit ratio is explained solely by the defect in expression of the E1B 55K protein identified by Mautner et al. (V. Mautner, N. MacKay, and V. Steinthorsdottir, Virology 171:619-622, 1989; V. Mautner, N. MacKay, and K. Morris, Virology 179:129-138, 1990).     

Fastidious human adenovirus type 40 can propagate efficiently and produce plaques on a human cell line, A549, derived from lung carcinoma.

Human adenovirus type 40 (Ad40) cannot propagate in conventional established human cell lines such as KB or HeLa cells. However, it has been shown that Ad40 DNA replicates in KB18 cells which express Ad2 E1B genes, suggesting that Ad40 is defective in the E1B gene function in KB or HeLa cells. We show here that Ad40 can propagate and produce plaques on A549 cells which do not contain Ad E1B genes. Our experiments show that the levels of replication of Ad40 DNA and production of infectious Ad40 virus in A549 cells are the same as or higher than those in 293 or KB18 cells. Dot blot analysis shows that the levels of Ad40 E1A and E1B mRNAs expressed in A549 cells at early to intermediate times postinfection are at least 10-fold higher than those in KB or KB18 cells. Northern (RNA) blot analysis shows that large E1B mRNA species (approximately 24S to 26S) are synthesized prior to the onset of DNA replication in A549 cells. No E1B mRNA species are synthesized in KB or KB18 cells at early times postinfection, and no differences in the expression of E1B mRNAs are seen between KB and KB18 cells. The experiment suggests that A549 cells have a cellular factor(s) which activates Ad40 E1B mRNA synthesis and that the E1B mRNA synthesis helps Ad40 propagation. In contrast, Ad40 can propagate in KB18 cells by using Ad2 E1B gene products that are constitutively expressed in this cell line. Furthermore, this result shows that Ad40 cannot propagate in KB cells because of the failure in the expression of E1B genes at early times postinfection.     

The release of growth arrest by microinjection of adenovirus E1A DNA.

The induction of DNA synthesis in growth-arrested mouse fibroblasts (NIH 3T3) was studied by microinjection of different constructs of adenovirus DNA using SV40 DNA and plasmid DNA as positive and negative controls. The E1A region of adenovirus types 2 and 12 appears to be sufficient to induce cellular DNA synthesis after growth arrest in approximately 30% of the cells and both 13S and 12S cDNA constructs mediate this effect. The presence of the E1A protein products as assayed by immunofluorescence does not strictly correlate with the induction of DNA synthesis in microinjected cells in contrast to the SV40 large T-antigen. Microinjection of truncated fragments of the Ad12 E1A region suggests, however, that the protein products of 12S and 13S may be involved in the induction process. A sequence comparison of the SV40 T-antigen and the adenovirus E1A products identified a region of significant homology providing a basis for a hypothesis concerning the evolution of T-antigen genes in DNA viruses.     

一种新的重组腺病毒的构建及其对人肿瘤细胞的影响

腺病毒E1A基因诱导细胞凋亡．E1B19K基因及E1B55K基因抑制细胞凋亡,前者被克隆到腺病毒转移载体pCA13的HCMVIE启动子下游．构建成转移载体pCAE1A。采用lipofectin法将PCAE1A和含腺病毒基因组（E1、E3区缺失）的质粒pBHG11共转染293细胞,7～10d后得到重组病毒v5Ad4。用v5Ad4感染人肺腺癌细胞系A549,结果表明v5Ad4有明显杀伤和裂解肿瘤细胞功能。在人胚肺正常二倍体细胞中,v5Ad4没有表现出可见的细胞毒效应。     

The early region 1B 55-kilodalton oncoprotein of adenovirus relieves growth restrictions imposed on viral replication by the cell cycle.

The E1B 55-kDa oncoprotein of adenovirus enables the virus to overcome restrictions imposed on viral replication by the cell cycle. Approximately 20% of HeLa cells infected with an E1B 55-kDa mutant adenovirus produced virus when evaluated by electron microscopy or by assays for infectious centers. By contrast, all HeLa cells infected with a wild-type adenovirus produced virus. The yield of E1B mutant virus from randomly cycling HeLa cells correlated with the fraction of cells in S phase at the time of infection. In synchronously growing HeLa cells, approximately 75% of the cells infected during S phase with the E1B mutant virus produced virus, whereas only 10% of the cells infected during G1 produced virus. The yield of E1B mutant virus from HeLa cells infected during S phase was sevenfold greater than that of cells infected during G1 and threefold greater than that of cells infected during asynchronous growth. Cells infected during S phase with the E1B mutant virus exhibited severe cytopathic effects, whereas cells infected with the E1B mutant virus during G1 exhibited a mild cytopathic effect. Viral DNA synthesis appeared independent of the cell cycle because equivalent amounts of viral DNA were synthesized in cells infected with either wild-type or E1B mutant virus. The inability of the E1B mutant virus to replicate was not mediated by the status of p53. These results define a novel property of the large tumor antigen of adenovirus in relieving growth restrictions imposed on viral replication by the cell cycle.