A nonessential glycoprotein is coded by early region E3 of adenovirus type 7

Ad7 was shown to induce polypeptides of 66,000 daltons (66K), 28K, and 16K during early stages of infection. The 28K could be labeled with [2-³H]mannose and it bound to concanavalin A-agarose, indicating that it is a glycoprotein. Neither the glycoprotein nor the 16K polypeptide were induced by a viable early region E3 deletion mutant or an E3 insertion mutant, indicating that the polypeptides are coded by early region E3, and that they are nonessential for growth of Ad7 on cultured KB cells. The Ad7 glycoprotein was compared to the Ad2 E3-coded glycoprotein in terms of size and oligosaccharide linkage. The Ad7 glycoprotein has an apparent molecular weight of 28K versus 25K for the Ad2 glycoprotein, as estimated by SDS-PAGE. The Ad7 glycoprotein and the purified 25K Ad2 glycoprotein were digested with endo-β-acetylglucosaminidase H (endo H) to determine the type of oligosaccharide linkages as well as the apparent molecular weights of the polypeptide chains. Endo H reduced the apparent molecular weights of the Ad7 and Ad2 glycopolypeptides from 28K and 25K, to 17.5K and 19K, respectively. This establishes that both proteins have Asn-linked oligosaccharides of the high-mannose type, and that the Ad7 polypeptide chain is slightly smaller than that of Ad2, 17.5K versus 19K. Thus, the E3s of both Ad7 (group B) and Ad2 (group C) encode glycoproteins that are similar in apparent molecular weight, that have high-mannose oligosaccharides, and that are nonessential for virus growth on cultured KB cells.     

Identification and characterization of a 30K protein (Ad4E3-30K) encoded by the E3 region of human adenovirus type 4

Human adenovirus type 4 (Ad4), the sole member of subgroup E, contains an open reading frame in the E3 region predicted to encode a unique 30-kDa protein (named Ad4E3-30K). Ad4E3-30K is predicted to be an integral membrane protein containing an N-terminal signal sequence, a lumenal domain, a transmembrane domain near the C-terminus, and a 37-amino-acid cytoplasmic tail. To determine whether this protein is expressed, rabbit polyclonal antisera were raised against 30K-containing fusion proteins expressed in bacteria. A 30K protein was detected by immunoprecipitation from cell-free translation products and from Ad4-infected A549 cells radiolabeled in the presence of tunicamycin. The protein was detected at only low levels in infected cells. It was not synthesized by a mutant with a large E3 deletion that includes the Ad4E3-30K gene. This mutant grows as well as wild-type Ad4 in culture. Features of Ad4E3-30K were characterized in different transient expression systems. The protein underwent glycosylation by addition of approximately six asparagine-linked oligosaccharides. These glycans were sensitive to endoglycosidase H, indicating that they were either high-mannose or hybrid types, but not complex types, and that the protein did not pass through the Golgi apparatus. Immunofluorescence staining of transfected cells revealed that Ad4E3-30K was localized primarily in the endoplasmic reticulum and nuclear envelope.     

The E3-11.6K protein of adenovirus is an Asn-glycosylated integral membrane protein that localizes to the nuclear membrane.

The 11,600 MW (101 amino acids; 11.6K) protein of adenovirus 2 (Ad2) is a protein of unknown function which is synthesized in low amounts during early stages of infection but in very high amounts at late stages. The 11.6K protein migrates as three major groupings of diffuse bands of ca. 14K, 21K, and 31K on SDS-PAGE, indicating that 11.6K undergoes post-translational modification. We show here that 11.6K is Asn-glycosylated with complex (endo H-resistant) oligosaccharides and that 11.6K is an integral membrane protein. Immunofluorescence indicated that 11.6K initially is associated with the endoplasmic reticulum and Golgi apparatus and that it ultimately localizes to the nuclear membrane. The 11.6K protein is predicted to have a single signal-anchor sequence at residues 41-62 and only one potential Asn-linked glycosylation site at residue 14; thus, 11.6K must be oriented in the membranes with its NH2-terminus in the lumen and its COOH-terminus in the cytoplasm. The signal-anchor and glycosylation features of 11.6K are preserved in Ad2 and Ad5 (group C), and in Ad3 and Ad7 (group B), but the sequence of 11.6K is more diverged among these serotypes than is the sequence of most other adenovirus proteins.     

The adenovirus E3-14.5K protein which is required for prevention of TNF cytolysis and for down-regulation of the EGF receptor contains phosphoserine.

The E3-14.5K and E3-10.4K proteins form a complex and function to down-regulate the epidermal growth factor receptor and to prevent tumor necrosis factor cytolysis in adenovirus-infected cells. Both 14.5K and 10.4K are cytoplasmic membrane proteins with a Ccyt orientation in the membrane. We show here that 14.5K is phosphorylated on serine residues in cells infected by adenoviruses that synthesize both 14.5K and 10.4K. 14.5K is phosphorylated on both serine and threonine in cells infected by a mutant that does not synthesize 10.4K; thus, the presence or absence of 10.4K affects the phosphorylation of 14.5K. Phosphotyrosine was not detected. 14.5K is also phosphorylated when translated in vitro in a rabbit reticulocyte extract. Both in vivo and in vitro, at least one of the phosphorylation sites is near the C-terminus, in the cytoplasmic domain of 14.5K. This C-terminal region of 14.5K is the most conserved among Ad5, Ad2, Ad3, and Ad7, and it is essential for 14.5K to prevent tumor necrosis factor cytolysis.     

Overexpression of adenovirus E3-11.6K protein induces cell killing by both caspase-dependent and caspase-independent mechanisms

Recent studies have shown enhanced antitumor efficacy with adenoviruses that either lack E1B-19K or overexpress E3-11.6K (also known as adenoviral death protein). E1B-19K is a well-characterized anti-apoptotic protein, and viruses with E1B-19K deletions show increased cytopathicity. However, the mechanism of cell killing by E3-11.6K, which plays an important role in killing infected cells and virion release, is not well characterized. To understand the mechanism of cell killing following E3-11.6K overexpression, we constructed a recombinant adenovirus, Ad-ME, by introducing viral major late promoter upstream of the E3-11.6K sequence. Similar to the E1B-19K-deleted virus, E1B/19K-, Ad-ME induced cell death to a greater extent than the wild-type virus. Cell shrinkage, membrane blebbing, activation of caspases 3 and 9, cleavage of poly(ADP-ribose)polymerase (PARP), DNA degradation, and ratio of ADP to ATP in Ad-ME-infected cells indicated that apoptosis contributes to cell death following E3-11.6K overexpression. However, the levels of activation of caspases 3 and 9 were lower in cells infected with Ad-ME compared to those infected with E1B/19K-. Furthermore, cell killing by Ad-ME was not effectively inhibited by Z-VAD-FMK, a general caspase inhibitor. Taken together, our results suggest both caspase-dependent and caspase-independent mechanisms of cell killing due to overexpression of E3-11.6K.     

The E3-14.5K integral membrane protein of adenovirus that is required for down-regulation of the EGF receptor and for prevention of TNF cytolysis is O-glycosylated but not N-glycosylated.

The adenovirus E3-14.5K protein is a cytoplasmic integral membrane protein that functions in concert with the E3-10.4K protein to down-regulate the epidermal growth factor receptor and to prevent tumor necrosis factor cytolysis in adenovirus-infected cells. The 14.5K protein migrates as multiple bands in SDS-PAGE, indicating that it undergoes post-translational modification. The 14.5K protein is known to be phosphorylated on serine. We show here that 14.5K can be metabolically labeled with [3H]glucosamine, that the label is labile to alkali, and that the SDS-PAGE band pattern is simplified in a cell line that is defective in O-glycosylation. Thus, 14.5K is O-glycosylated, probably at a single site in the NH2-terminal lumenal domain. The protein was not metabolically labeled with [3H]mannose, and its SDS-PAGE band pattern was not affected by tunicamycin treatment in vivo or endo F treatment in vitro; thus, 14.5K is not N-glycosylated. There was no evidence that the 10.4K protein is glycosylated, and the 10.4K protein was not required for glycosylation of 14.5K. Virtually all 14.5K molecules appear to contain the core disaccharide Gal beta 1-3GalNAc alpha 1-Ser/Thr which is commonly found on mucin-type O-glycoproteins, and neuraminidase digestion experiments indicated that this disaccharide contains terminal sialic acid.     

The early proteins of the nondefective Ad2-SV40 hybrid viruses: the 19K glycoprotein is coded by Ad2 early region 3

To determine which proteins are coded by early region 3 of Ad2, we compared Ad2-infected HeLa cells to cells infected with a series of nondefective Ad2-SV40 hybrid viruses (Ad2⁺ND) with progressively larger deletions of region 3. Both Ad2- and Ad2⁺ND-infected cells synthesized early proteins with apparent molecular weights of 72K, 16.5K, and 11K on sodium dodecyl sulfate- polyacrylamide gels. Ad2-infected cells contained, in addition, a 19K and a 17K glycoprotein and a 14K protein. None of the Ad2+ND-infected cells synthesized the 14K protein. Ad2⁺ND-infected cells also did not synthesize the 19K and 17K proteins, but cells infected with Ad2⁺ND₄, the ND with the smallest deletion in region 3, synthesized an 18K glycoprotein which had several methionine-containing peptides in common with the 19K and 17K proteins. We have, therefore, placed the genes for the 19K, 17K, and 14K proteins in early region 3. Two previous studies had assigned the 19K protein to early region 4 when in vitro translation of mRNA selected with restriction fragments from region 4 yielded a protein with an apparent molecular weight of 19K. However, our search of the literature revealed that the translation system used in those studies does not glycosylate proteins. Therefore, the 19K protein made in vitro was not glycosylated and could only have been the same protein as the 19K protein from infected cells if the glycosylated and nonglycosylated forms of the protein had the same molecular weight. By treating Ad2-infected cells with glucosamine or deoxyglucose we have determined that the nonglycosylated form of the 19K protein is a 15.5K protein.     

Structural analysis of the adenovirus type 2 E3/19K protein using mutagenesis and a panel of conformation-sensitive monoclonal antibodies

The E3/19K protein of human adenovirus type 2 (Ad2) was the first viral protein shown to interfere with antigen presentation. This 25 kDa transmembrane glycoprotein binds to major histocompatibility complex (MHC) class I molecules in the endoplasmic reticulum (ER), thereby preventing transport of newly synthesized peptide-MHC complexes to the cell surface and consequently T cell recognition. Recent data suggest that E3/19K also sequesters MHC class I like ligands intracellularly to suppress natural killer (NK) cell recognition. While the mechanism of ER retention is well understood, the structure of E3/19K remains elusive. To further dissect the structural and antigenic topography of E3/19K we carried out site-directed mutagenesis and raised monoclonal antibodies (mAbs) against a recombinant version of Ad2 E3/19K comprising the lumenal domain followed by a C-terminal histidine tag. Using peptide scanning, the epitopes of three mAbs were mapped to different regions of the lumenal domain, comprising amino acids 3-13, 15-21 and 41-45, respectively. Interestingly, mAb 3F4 reacted only weakly with wild-type E3/19K, but showed drastically increased binding to mutant E3/19K molecules, e.g. those with disrupted disulfide bonds, suggesting that 3F4 can sense unfolding of the protein. MAb 10A2 binds to an epitope apparently buried within E3/19K while that of 3A9 is exposed. Secondary structure prediction suggests that the lumenal domain contains six beta-strands and an alpha-helix adjacent to the transmembrane domain. Interestingly, all mAbs bind to non-structured loops. Using a large panel of E3/19K mutants the structural alterations of the mutations were determined. With this knowledge the panel of mAbs will be valuable tools to further dissect structure/function relationships of E3/19K regarding down regulation of MHC class I and MHC class I like molecules and its effect on both T cell and NK cell recognition.     

Actin-independent exclusion of CD95 by PI3K/AKT signalling: implications for apoptosis

The immune system eliminates infected or transformed cells through the activation of the death receptor CD95. CD95 engagement drives the recruitment of the adaptor protein Fas-associated death domain protein (FADD), which in turn aggregates and activates initiator caspases-8 and -10. The CD95-mediated apoptotic signal relies on the capacity to form the CD95/FADD/caspases complex termed the death-inducing signalling complex (DISC). Cells are classified according to the magnitude of DISC formation as either type I (efficient DISC formation) or type II (inefficient). CD95 localised to lipid rafts in type I cells, whereas the death receptor was excluded from these domains in type II cells. Here, we show that inhibition of both PI3K class IA and serine-threonine kinase Akt in type II cells promoted the redistribution of CD95 into lipid rafts, DISC formation and the initiation of the apoptotic signal. Strikingly, these molecular events took place independently of CD95L and the actin cytoskeleton. Overall, these findings highlight that the oncogenic PI3K/Akt signalling pathway participates in maintaining cells in a type II phenotype by excluding CD95 from lipid rafts.     

The adenovirus early region 4 open reading frame 6/7 protein regulates the DNA binding activity of the cellular transcription factor, E2F, through a direct complex

Nuclear factor E2F is a cellular protein that binds to the adenovirus E2 promoter and E1A enhancer regions and to the cellular c-myc P2 promoter region. The DNA binding activity of E2F, detected in vitro using nuclear extracts prepared from HeLa cells, is increased by adenovirus infection (termed E2F induction). We demonstrate here that a 19.5-kD protein, encoded by adenovirus early region 4 (E4) open reading frame (ORF) 6/7, is primarily responsible for the induction of E2F DNA binding activity to the E2 promoter region. Viral mutants that contain frame-shift mutations in E4 ORF 6/7 failed to induce E2F binding activity; a virus that carries an E4 ORF 6/7 cDNA in place of the E4-coding sequences induced E2F efficiently. Using gel mobility shift assays, we demonstrate that the E4 ORF 6/7 product induces the binding of E2F to the E2 promoter via a direct complex. The addition of a peptide-specific antiserum, directed against the E4 ORF 6/7 protein, to an in vitro E2F-binding reaction resulted in the formation of a DNA-protein complex with reduced gel mobility compared to the normal, adenovirus-induced E2F-E2 promoter complex. The formation of the E2F-E2 promoter-antibody complex was blocked by the addition of the cognate peptide used to generate the antiserum but not by a nonspecific peptide. Nuclear extracts prepared from adenovirus-infected HeLa cells were cleared of E2F binding activity using the ORF 6/7 peptide-specific serum, but not the preimmune serum, suggesting that E2F and the E4 ORF 6/7 product form a protein-protein complex in solution. The adenovirus E1A proteins are not absolutely required for the induction of E2F binding activity because the infection of HeLa cells with an E1A mutant, dl312, at high multiplicity resulted in E2F induction. Under these conditions of infection, the E4 ORF 6/7 product was synthesized. E2F binding activity was induced, but inefficiently, in cells infected with E4 ORF 6/7 mutants, indicating that an additional pathway may lead to E2F induction.     

The nucleotide sequence of adenovirus type 11 early 3 region: comparison of genome type Ad11p and Ad11a.

The early 3 region (E3) of two strains (genome type Ad11p and Ad11a) of human adenovirus serotype 11, causing persistent urinary and acute respiratory illnesses, respectively, has been identified and partially sequenced. The sequenced E3 regions of Ad11p and Ad11a were 1980 and 1966 bp long and encoded three complete ORFs, 18.5, 20.3, 20.6k within the Ad11p genome and 18.5, 20.3, 20.2k within the Ad11a genome. The sequence analysis of the 18.5k gene product demonstrated that a transmembrane domain and a cytoplasmic domain of Ad11p, Ad11a, and Ad35 was identical. Ad11p and Ad35 were homologous in the signal sequence. There was one amino acid mismatch between Ad11p and Ad11a, represented by an alanine instead of a proline. The endoplasmic reticulum lumenal domain, which binds to class I MHC, was relatively conserved between Ad11p and Ad11a with the exception of Glu80 and Glu104 in Ad11p, which were replaced by Gln80 and Lys104 in Ad11a. Within the 20.2k protein of Ad11a, the amino acid sequence Thr-Thr-Ser-His was deleted from a position immediately upstream the transmembrane region of the Ad11p 20.6k protein. The 9.0k E3 open reading frame (ORF) of Ad3 was deleted in the genomes of Ad11p and Ad11a. It is noteworthy that Ad11p and Ad35 which both cause persistent infection of the urinary tract display a remarkable similarity in several ORFs of the E3 region.     

Repression of the immunoglobulin heavy chain 3′ enhancer by helix-loop-helix protein Id3 via a functionally important E47/E12 binding site: implications for developmental control of enhancer function

The activity of the immunoglobulin 3′ enhancer is restricted to the late stages of B lymphoid development. Here we further examine the molecular basis for the temporally restricted activity of the B-lymphoid IgH 3′ enhancer. We demonstrate that a binding site (E5 site) for the E47 and/or E12 proteins is functionally important for enhancer activity. The multimerized E5 site acts as a B cell-specific enhancer and, when assayed in COS cells, can be transactivated by E47/E12 proteins. This transactivation in COS cells, as well as the activity of the full length 3′ enhancer in plasma cells, can be repressed by overexpression of the dominant negative nuclear regulator Id3. When examining the tissue distribution of Id3 in murine cell lines, we find that Id3 is expressed throughout the pre-B and B cell stages, but is down-regulated at the plasma cell stage. Thus, Id3 may contribute to the temporal regulation of the IgH 3′ enhancer.     

The mouse Mid1 gene: implications for the pathogenesis of Opitz syndrome and the evolution of the mammalian pseudoautosomal region

We have recently reported isolation of the gene responsible for X- linked Opitz G/BBB syndrome, a defect of midline development. MID1 is located on the distal short arm of the human X chromosome (Xp22. 3) and encodes a novel member of the B box family of zinc finger proteins. We have now cloned the murine homolog of MID1 and performed preliminary expression studies during development. Mid1 expression in undifferentiated cells in the central nervous, gastrointestinal and urogenital systems suggests that abnormal cell proliferation may underlie the defect in midline development characteristic of Opitz syndrome. We have also found that Mid1 is located within the mouse pseudoautosomal region (PAR) in Mus musculus , while it seems to be X- specific in Mus spretus. Therefore, Mid1 is likely to be a recent acquisition of the M. musculus PAR. Genetic and FISH analyses also demonstrated a high frequency of unequal crossovers in the murine PAR, creating spontaneous deletion/duplication events involving Mid1. These data provide evidence for the first time that genetic instability of the PAR may affect functionally important genes. In addition, we show that MID1 is the first example of a gene subject to X-inactivation in man while escaping it in mouse. These data contribute to a better understanding of the molecular content and evolution of the rodent PAR.