A 49-Kilodalton Isoform of the Adenovirus Type 5 Early Region 1B 55-Kilodalton Protein Is Sufficient To Support Virus Replication

Abstract

The adenovirus type 5 (Ad5) early region 1B 55-kDa (E1B-55K) protein is a multifunctional regulator of cell-cycle-independent virus replication that participates in many processes required for maximal virus production. As part of a study of E1B-55K function, we generated the Ad5 mutant H5pm4133, carrying stop codons after the second and seventh codons of the E1B reading frame, thereby eliminating synthesis of the full-length 55K product and its smaller derivatives. Unexpectedly, phenotypic studies revealed that H5pm4133 fully exhibits the characteristics of wild-type (wt) Ad5 in all assays tested. Immunoblot analyses demonstrated that H5pm4133 and wt Ad5 produce very low levels of two distinct polypeptides in the 48-to 49-kDa range, which lack the amino-terminal region but contain segments from the central and carboxy-terminal part of the 55K protein. Genetic and biochemical studies with different Ad5 mutants show that at least one of these isoforms consists of two closely migrating polypeptides of 433 amino acid residues (433R) and 422R, which are produced by translation initiation at two downstream AUG codons of the 55K reading frame. Significantly, a virus mutant producing low levels of the 433R isoform alone replicated to levels comparable to those of wt Ad5, demonstrating that this polypeptide provides essentially all functions of E1B-55K required to promote maximal virus growth in human tumor cells. Altogether, these results extend previous findings that the wt Ad5 E1B region encodes a series of smaller isoforms of E1B-55K and demonstrate that very low levels of at least one of these novel proteins (E1B-433R) are sufficient for a productive infection.Products of early region 1B (E1B) from subgroup C adenovirus type 5 (Ad5) are required for productive infection of human cells and for complete transformation of primary mammalian cells in cooperation with the E1A gene products (reviewed in reference 6). Most of these activities can be assigned to two different polypeptides of 176 amino acid residues (176R) (E1B 19-kDa protein [E1B-19K]) and 496R (E1B-55K). Both proteins are produced by translation of two overlapping reading frames starting from two separate initiation codons of the 22S transcript (28, 48) (Fig. 1). The 19-kDa product is also translated from three alternatively spliced transcripts of 13S, 14S, and 14.5S, which additionally direct the synthesis of three smaller 55K-related proteins (156R, 93R, and 84R) containing the amino-terminal 79 residues of E1B-55K (12, 45). E1B-156R also shares an identical carboxy terminus with 55K, while the 93R and 84R E1B polypeptides contain unique carboxy termini.It has been well established that E1B-19K and E1B-55K promote efficient viral replication via a number of different mechanisms. In the early phase of the infection, both E1B proteins counteract antiproliferative processes induced by the host cell, including activation of p53-dependent and -independent apoptosis, induction of cell cycle arrest, and stimulation of cellular DNA damage responses (reviewed in r...

SUMO modification of E1B-55K oncoprotein regulates isoform-specific binding to the tumour suppressor protein PML

“…Consequently, we decided to use human hepatocytic cells (HepaRG) (Gripon et al, 2002;Cerec et al, 2007), as they express PML in the well-defined physiological amounts (Condemine et al, 2006) and represent a suitable system to analyse endogenously PML components during viral infection Lukashchuk and Everett, 2010). Cells were infected with wild-type virus (H5pg4100), E1B-55K null mutant H5pm4149 (Kindsmu¨ller et al, 2009) or previously described H5pm4102/H5pm4101 virus harbouring mutations within the SCM (K104R)/NES (L83/87/91A) of E1B-55K (Kindsmu¨ller et al, 2007). Representative results of IF analysis is shown in Figure 4A.…”

“…H5pm4149 carries stop codons in the E1B-55K open reading frame and does not express the E1B-55K protein (Kindsmu¨ller et al, 2009). All viruses were propagated and titered as fluorescent forming units in HEK293 monolayer cultures .…”

“…Primary antibodies specific for Ad proteins used included E1B-55K mouse mAb 2A6 (Sarnow et al, 1982b), E1B-55K rat mAb 7C11 (Kindsmu¨ller et al, 2009) and E1B-55K rat mAb 4E8 (Kindsmu¨ller et al, 2009). Primary antibodies specific for cellular and epitopically expressed proteins included PML rabbit pAb H-238 (Santa Cruz Biotechnology, Inc., Heidelberg, Germany), PML rabbit pAb NB 100-59787 (Novus Biologicals, Ltd., Cambridge, UK), PML mouse mab 5E10 (Stuurman et al, 1992), rabbit pAb a-SAF-A (Herrmann et al, 2005), flag mouse mAb flag-M2 (Sigma-Aldrich, Inc., Mu¨nchen, Germany) and b-actin mouse mab AC-15 (SigmaAldrich, Inc.).…”

“…Proteins bound to the antibody-coupled protein A-sepharose were precipitated by centrifugation, washed three times, boiled for 3 min at 95 1C in 2Â Laemmli buffer and analysed by immunoblotting. For immunoblotting of steady-state protein levels/immunoprecipitation solutions, equal amounts of total protein were separated by SDS-PAGE, transferred to nitrocellulose membranes (Schleicher and Schu¨ll/Whatman) and incubated as described earlier (Kindsmu¨ller et al, 2009). The bands were visualized by enhanced chemiluminescence as recommended by the manufacturer (Pierce, Thermo Fisher Scientific, Bonn, Germany) on X-ray films (CEA RP, medical X-ray film).…”

The Adenoviral Oncogene E1A-13S Interacts with a Specific Isoform of the Tumor Suppressor PML To Enhance Viral Transcription

“…H5dl312 lacks a large segment of the E1A gene and therefore does not produce E1A products (64). H5pm4150 carries a frameshift mutation in the E4orf3 open reading frame and H5pm4149 carries stop codons in the E1B-55K open reading frame, so they do not express E4orf3 and E1B-55K, respectively (65,66). All viruses were propagated, and titers were determined as fluorescenceforming units (FFU), in HEK293 monolayer cultures (63).…”