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.     

The replicative capacities of large E1B-null group A and group C adenoviruses are independent of host cell p53 status

Recent reports suggest that an early region 1B (E1B) 55, 000-molecular-weight polypeptide (55K)-null adenovirus type 5 (Ad5) mutant (dl1520) can replicate to the same extent as wild-type (wt) Ad5 in cells either deficient or mutated in p53, implicating p53 in limiting viral replication in vivo. In contrast, we show here that the replicative capacity of Ad5 dl1520 is wholly independent of host cell p53 status, as is the replicative capacity of comparable Ad12 E1B 54K-null adenoviruses (Ad12 dl620 and Ad12 hr703). Furthermore, we show that there is no requirement for complex formation between p53 and Ad5 E1B 55K or Ad12 E1B 54K for a productive infection, such that wt Ad5 and wt Ad12 will both replicate in cells which are null for p53. In addition, we find that these Ad5 and Ad12 mutant viruses induce S phase irrespective of the p53 status of the cell and that, therefore, S-phase induction does not correlate with the replicative capacity of the virus. Interestingly, the replicative capacities of the large E1B-null adenoviruses correlated positively with the ability to express E1B 19K and were related to the ability to repress premature adenovirus-induced apoptosis. Infection of primary human cells indicated that Ad5 dl1520, wt Ad5, and wt Ad12 replicated better in cycling normal human skin fibroblasts (HSFs) than in quiescent HSFs. Thus, the cell cycle status of the host cell, upon infection, also influences viral yield.     

Regulation of p53 levels by the E1B 55-kilodalton protein and E4orf6 in adenovirus-infected cells

The adenovirus type 5 243R E1A protein induces p53-dependent apoptosis in the absence of the 19- and 55-kDa E1B polypeptides. This effect appears to result from an accumulation of p53 protein and is unrelated to expression of E1B products. We now report that in the presence of the E1B 55-kDa polypeptide, the 289R E1A protein does not induce such p53 accumulation and, in fact, is able to block that induced by E1A 243R. This inhibition also requires the 289R-dependent transactivation of E4orf6 expression. E4orf6 is known to form complexes with the E1B 55-kDa protein and to function both in the transport and stabilization of viral mRNA and in shutoff of host cell protein synthesis. We demonstrated that the block in p53 accumulation is not due to the generalized shutoff of host cell metabolism. Rather, it appears to result from a mechanism targeted specifically to p53, most likely involving a decrease in the stability of p53 protein. The E1B 55-kDa protein is known to interact with both E4orf6 and p53, and as demonstrated recently by others, we showed that E4orf6 also binds directly to p53. Thus, multiple interactions between all three proteins may regulate p53 stability, resulting in the maintenance of low levels of p53 following virus infection.     

p53/E1b58kDa complex regulates adenovirus replication

We have explored a role for the adenovirus (Ad5) E1b58kDa/p53 protein complex in adenovirus replication. This was done by using virus mutants containing different defects in the E1b58kDa gene and cell lines that express either a wild-type p53 protein or a mutant p53 protein. We find that infection of wild-type p53-containing cells with wild-type Ad5 causes a shutoff of p53 and alpha-actin protein synthesis by distinct mechanisms, but neither occurs in mutant p53 cells. Our data also indicate that the shutoff is dependent on formation of the p53/E1b complex and may also involve another virus protein, E4ORF6. Following from these observations we asked whether failure to form the complex resulted in impaired adenovirus replication. Our experiments showed that neither wild-type Ad5 nor the E1b mutant dl338 could replicate in cells expressing a mutant p53 protein, but that wild-type adenovirus replicated well in wild-type p53-expressing cells. Collectively, our data suggest that the interaction between p53 and the E1b58kDa protein is necessary for efficient adenovirus replication. This is the first time such a direct link between the complex and virus replication has been demonstrated. These data raise serious questions about the usefulness of E1b-defective viruses in tumor therapy.     

The 23-kilodalton E1 phosphoprotein of bovine papillomavirus type 1 is nonessential for stable plasmid replication in murine C127 cells

The 23-kDa protein encoded by the 5' segment of the E1 open reading frame of bovine papillomavirus type 1 (BPV1) was previously ascribed a negative regulatory function for the replication of viral plasmid DNA. However, results from recent functional and biochemical studies do not readily support this genetic assignment. Therefore, we have reassessed the role of this protein in papillomavirus DNA replication by using a mutant of BPV1 which is unable to express this E1 protein. This mutant viral DNA was found to replicate extrachromosomally with stability and copy number per cell similar to those of wild-type plasmid DNA. Thus, the absence of expression of the 23-kDa E1 protein did not lead to deregulated viral plasmid replication. We conclude that the 23-kDa E1 protein is nonessential for stable plasmid replication.     

Analysis of synthesis, stability, phosphorylation, and interacting polypeptides of the 34-kilodalton product of open reading frame 6 of the early region 4 protein of human adenovirus type 5

The 34-kDa early-region 4 open reading frame 6 (E4orf6) product of human adenovirus type 5 forms complexes with both the cellular tumor suppressor p53 and the viral E1B 55-kDa protein (E1B-55kDa). E4orf6 can inhibit p53 transactivation activity, as can E1B-55kDa, and in combination these viral proteins cause the rapid turnover of p53. In addition, E4orf6-55kDa complexes play a critical role at later times in the regulation of viral mRNA transport and shutoff of host cell protein synthesis. In the present study, we have further characterized some of the biological properties of E4orf6. Analysis of extracts from infected cells by Western blotting indicated that E4orf6, like E1A and E1B products, is present at high levels until very late times, suggesting that it is available to act throughout the infectious cycle. This pattern is similar to that of E4orf4 but differs markedly from that of another E4 product, E4orf6/7, which is present only transiently. Synthesis of E4orf6 is maximal at early stages but ceases completely with the onset of shutoff of host protein synthesis; however, it was found that unlike E4orf6/7, E4orf6 is very stable, thus allowing high levels to be maintained even at late times. E4orf6 was shown to be phosphorylated at low levels. Coimmunoprecipitation studies in cells lacking p53 indicated that E4orf6 interacts with a number of other proteins. Five of these were shown to be viral or virally induced proteins ranging in size from 102 to 27 kDa, including E1B-55kDa. One such species, of 72 kDa, was shown not to represent the E2 DNA-binding protein and thus remains to be identified. Another appeared to be the L4 100-kDa nonstructural adenovirus late product, but it appeared to be present nonspecifically and not as part of an E4orf6 complex. Apart from p53, three additional cellular proteins, of 84, 19, and 14 kDa were detected by using an adenovirus vector that expresses only E4orf6. The 19-kDa species and a 16-kDa cellular protein were also shown to interact with E4orf6/7. It is possible that complex formation with these viral and cellular proteins plays a role in one or more of the biological activities associated with E4orf6 and E4orf6/7.     

E1B 55-kilodalton-associated protein: a cellular protein with RNA-binding activity implicated in nucleocytoplasmic transport of adenovirus and cellular mRNAs

The adenovirus type 5 (Ad5) early 1B 55-kDa protein (E1B-55kDa) is a multifunctional phosphoprotein that regulates viral DNA replication and nucleocytoplasmic RNA transport in lytically infected cells. In addition, E1B-55kDa provides functions required for complete oncogenic transformation of rodent cells in cooperation with the E1A proteins. Using the far-Western technique, we have isolated human genes encoding E1B-55kDa-associated proteins (E1B-APs). The E1B-AP5 gene encodes a novel nuclear RNA-binding protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) family that is highly related to hnRNP-U/SAF-A. Immunoprecipitation experiments indicate that two distinct segments in the 55-kDa polypeptide which partly overlap regions responsible for p53 binding are required for complex formation with E1B-AP5 in Ad-infected cells and that this protein interaction is modulated by the adenovirus E4orf6 protein. Expression of E1B-AP5 efficiently interferes with Ad5 E1A/E1B-mediated transformation of primary rat cells. Furthermore, stable expression of E1B-AP5 in Ad-infected cells overcomes the E1B-dependent inhibition of cytoplasmic host mRNA accumulation. These data suggest that E1B-AP5 might play a role in RNA transport and that this function is modulated by E1B-55kDa in Ad-infected cells.     

A method of limited replication for the efficient in vivo delivery of adenovirus to cancer cells

Replication-deficient viral vectors are currently being used in gene transfer strategies to treat cancer cells. Unfortunately, viruses are limited in their ability to diffuse through tissue. This makes it virtually impossible to infect the majority of tumor cells in vivo and results in inadequate gene transfer. This problem can be addressed by allowing limited viral replication. Limited viral replication facilitates greater penetration of virions into tissue and can improve gene transfer. We have developed a strategy of limited viral replication using AdRSVlaclys, a chemically modified E1-deleted adenovirus, to codeliver an exogenous plasmid encoding the adenovirus E1 region. This system allows one round of viral replication. We examined the effect of this limited adenovirus replication in vitro and in vivo. In culture, codelivery of virus and pE1 resulted in a large increase in infected cells when compared with control cells exposed to virus and pUC19. In experiments on nude mice bearing HeLa ascites tumors, intraperitoneal injection of AdRSVlaclys/pE1 resulted in a significantly higher percentage of infected HeLa cells as compared with the PBS controls (p < 0.05) or the AdRSVlaclys/pUC19 controls (p < 0.01). These data demonstrate that the transcomplementation of replication-deficient adenovirus with exogenous E1 DNA leads to limited replication, and this controlled replication enhances gene transfer efficiency of adenovirus in vivo.     

E1B 55K sequesters WT1 along with p53 within a cytoplasmic body in adenovirus-transformed kidney cells

WT1 encodes a tumor suppressor that is expressed in cells of the developing kidney and is inactivated in Wilms tumor, a pediatric kidney cancer. The adenovirus E1B 55K gene product contributes to the transformation of primary baby rat kidney (BRK) cells by binding and inactivating the product of the p53 tumor suppressor. We have previously demonstrated that WT1 and p53 are present within a protein complex in vivo. We now show that WT1 is physically associated with E1B 55K in adenovirus-transformed cells, an interaction that is mediated by the first two zinc fingers of WT1. Immunodepletion of p53 abrogates the coimmunoprecipitation of E1B 55K and WT1, consistent with the presence of a trimeric protein complex containing these three proteins. In the presence of E1B 55K, WT1 which is normally localized in the nucleus, is retained within a very high molecular weight complex and sequestered in the characteristic perinuclear cytoplasmic body that contains E1B 55K and p53. Expression of E1B 55K in osteosarcoma cells that undergo apoptosis following expression of WT1 inhibits WT1-mediated cell death. We conclude that E1B 55K may target WT1 along with p53, resulting in the functional inactivation of both tumor suppressor gene products by this viral oncoprotein.     

Novel T cell antigen 4-1BB associates with the protein tyrosine kinase p56lck1

4-1BB is a 30-kDa inducible T cell Ag, and is expressed predominantly as a 55-kDa dimer on both CD4+ and CD8+ T lymphocytes. The cytoplasmic tail of 4-1BB contains the sequence Cys-Arg-Cys-Pro, which is similar to the sequence Cys-X-Cys-Pro, which mediates the binding of the CD4 and CD8 molecules to the protein tyrosine kinase p56lck. An anti-4-1BB mAb, 53A2, was used to determine whether 4-1BB may associate with p56lck. The 53A2 mAb specifically recognized 4-1BB on a CD8+ T cell line, CTLL-2, and coimmunoprecipitated a 56-kDa protein along with 4-1BB. Peptide mapping indicated that the 56-kDa phosphoprotein was identical to p56lck. The coimmunoprecipitation of p56lck with 4-1BB also occurred in nonlymphoid cells such as insect (Sf-21) and HeLa cells when the two recombinant proteins were coexpressed. Analysis of mutant p56lck recombinant proteins showed that two cysteine residues critical for p56lck-CD4 (or -CD8) complex formation are also required for the p56lck-4-1BB interaction. These studies establish that 4-1BB physically associates with p56lck.     

Calcium and S100B regulation of p53-dependent cell growth arrest and apoptosis

In glial C6 cells constitutively expressing wild-type p53, synthesis of the calcium-binding protein S100B is associated with cell density-dependent inhibition of growth and apoptosis in response to UV irradiation. A functional interaction between S100B and p53 was first demonstrated in p53-negative mouse embryo fibroblasts (MEF cells) by sequential transfection with the S100B and the temperature-sensitive p53Val135 genes. We show that in MEF cells expressing a low level of p53Val135, S100B cooperates with p53Val135 in triggering calcium-dependent cell growth arrest and cell death in response to UV irradiation at the nonpermissive temperature (37.5 degreesC). Calcium-dependent growth arrest of MEF cells expressing S100B correlates with specific nuclear accumulation of the wild-type p53Val135 conformational species. S100B modulation of wild-type p53Val135 nuclear translocation and functions was confirmed with the rat embryo fibroblast (REF) cell line clone 6, which is transformed by oncogenic Ha-ras and overexpression of p53Val135. Ectopic expression of S100B in clone 6 cells restores contact inhibition of growth at 37.5 degreesC, which also correlates with nuclear accumulation of the wild-type p53Val135 conformational species. Moreover, a calcium ionophore mediates a reversible G1 arrest in S100B-expressing REF (S100B-REF) cells at 37.5 degreesC that is phenotypically indistinguishable from p53-mediated G1 arrest at the permissive temperature (32 degreesC). S100B-REF cells proceeding from G1 underwent apoptosis in response to UV irradiation. Our data support a model in which calcium signaling and S100B cooperate with the p53 pathways of cell growth inhibition and apoptosis.