The Kinetics of Synthesis of Early Viral Proteins in KB Cells Infected with Wild-type and Transformation-defective Host-range Mutants of Human Adenovirus Type 5

104

The human subgroup C adenoviral E1B 55-kDa protein cooperates with the viral E4 Orf6 protein to induce selective export of viral, late mRNAs from the nucleus to the cytoplasm. Previous studies have suggested that such preferential transport of viral mRNA and the concomitant inhibition of export of cellular mRNAs are the result of viral colonization of specialized microenvironments within the nucleus. However, neither the molecular basis of this phenomenon nor the mechanism by which the E1B 55-kDa protein acts has been elucidated. We therefore examined viral late mRNA metabolism in HeLa cells infected with a series of mutant viruses that carry insertions at various positions in the E1B protein coding sequence (P. R. Yew, C. C. Kao, and A. J. Berk, Virology 179:795-805, 1990). All the mutations examined impaired cytoplasmic accumulation of viral L2 mRNAs and reduced L2 mRNA export efficiency. However, in most cases these defects could be ascribed to reduced E1B 55-kDa protein concentration or the unexpected failure of the altered E1B proteins to enter the nucleus efficiently. The latter property, the pleiotropic defects associated with all the mutations that impaired nuclear entry of the E1B protein, and consideration of its primary sequence suggest that these insertions result in misfolding of the protein. Insertion of four amino acids at residue 143 also inhibited viral mRNA export but resulted in increased rather than decreased accumulation of the E1B 55-kDa protein in the nucleus. This mutation specifically impaired the previously described association of the E1B Human subgroup C adenoviruses, such as adenovirus type 2 (Ad2) and Ad5, encode several proteins that exhibit transforming activity, because they disrupt host cell circuits regulating cell proliferation (33, 63). These gene products include the E1B 55-kDa protein, which can cooperate with E1A proteins in stable transformation of rodent cells and is required for efficient viral replication in permissive human cells (2,50,76). The former function has been ascribed to modulation of the activity and concentration of the cellular p53 protein, a sequence-specific transcriptional activator that induces cell cycle arrest or apoptosis in response to genotoxic and other forms of stress (36, 42). The E1B 55-kDa protein inhibits p53-dependent transcription by binding to the N-terminal activation domain of the cellular protein (34, 61, 80). The E1B protein can function as a general repressor of RNA polymerase II transcription (44, 80). It is therefore believed to inhibit p53-dependent transcription as a result of binding to promoter-associated p53 protein (44). Such transcriptional repression by the E1B protein correlates closely with its transforming activity (80). Indeed, phosphorylation of C-terminal serine (490 and 491) and threonine (495) residues is necessary for transcriptional repression, transformation, and inhibition of p53-dependent apoptosis induced by the viral 243R E1A protein (68), indicating that repression of p53-dependent transcription is importa...

Section: Resultssupporting

confidence: 81%

Effects of Mutations in the Adenoviral E1B 55-Kilodalton Protein Coding Sequence on Viral Late mRNA Metabolism

González

Flint

2002

104

“…In summary, our data demonstrate that wild-type-Ad5-in-fected A549 cells display almost the same pattern of expression of E1B-55kDa, E4orf6, and E4orf6/7 proteins as that previously described for infected HeLa cells (9,39,44,52). Having established the experimental conditions needed to analyze the E1B-E4 protein interaction in infected A549 cells, we next mapped the domains in E1B-55kDa required for binding to the E4orf6 protein in vivo.…”

Section: Analysis Of the E1b-55kda-e4orf6 Protein Complex In Wildtypesupporting

confidence: 64%

Structural analysis of the adenovirus type 5 E1B 55-kilodalton-E4orf6 protein complex

Rubenwolf

Schütt

Nevels

et al. 1997

The adenovirus type 5 (Ad5) early 1B (E1B) 55-kDa (E1B-55kDa)-E4orf6 protein complex has been implicated in the selective modulation of nucleocytoplasmic mRNA transport at late times after infection. Using a combined immunoprecipitation-immunoblotting assay, we mapped the domains in E1B-55kDa required for the interaction with the E4orf6 protein in lytically infected A549 cells. Several domains in the 496-residue 55-kDa polypeptide contributed to a stable association with the E4orf6 protein in E1B mutant virus-infected cells. Linker insertion mutations at amino acids 180 and 224 caused reduced binding of the E4orf6 protein, whereas linker insertion mutations at amino acid 143 and in the central domain of E1B-55kDa eliminated the binding of the E4orf6 protein. Earlier work showing that the central domain of E1B-55kDa is required for binding to p53 and the recent observation that the E4orf6 protein also interacts with the tumor suppressor protein led us to suspect that p53 might play a role in the E1B-E4 protein interaction. However, coimmunoprecipitation assays with extracts prepared from infected p53-negative H1299 cells established that p53 is not needed for the E1B-E4 protein interaction in adenovirus-infected cells. Using two different protein-protein interaction assays, we also mapped the region in the E4orf6 protein required for E1B-55kDa interaction to the amino-terminal 55 amino acid residues. Interestingly, both binding assays established that the same region in the E4orf6/7 protein can potentially interact with E1B-55kDa. Our results demonstrate that two distinct segments in the 55-kDa protein encoding the transformation and late lytic functions independently interact with p53 and the E4orf6 protein in vivo and provide further insight by which the multifunctional 55-kDa E1B protein can exert its multiple activities in lytically infected cells and in adenovirus transformation.

“…Human HeLa and KB cells, as well as mouse 10T1/2 cells (47), were also cultured as described above. Cells were infected with wild-type (wt) or mutant virus at a multiplicity of 35 PFU per cell, as was described previously (48). For double infections, the viruses were premixed before infection at a multiplicity of 35 PFU per cell for each virus.…”

Section: Cells and Virusesmentioning

confidence: 99%

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

Querido

Marcellus

Lai

et al. 1997

216

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.