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W S Wold

Publications and source records attributed to W S Wold.

At least 19 recordsLinked to original sources
PubMed2001

Inhibition of TRAIL-induced apoptosis and forced internalization of TRAIL receptor 1 by adenovirus proteins.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis through two receptors, TRAIL-R1 (also known as death receptor 4) and TRAIL-R2 (also known as death receptor 5), that are members of the TNF receptor superfamily of death domain-containing receptors. We show that human adenovirus type 5 encodes three proteins, named RID (previously named E3-10.4K/14.5K), E3-14.7K, and E1B-19K, that independently inhibit TRAIL-induced apoptosis of infected human cells. This conclusion was derived from studies using wild-type adenovirus, adenovirus replication-competent mutants that lack one or more of the RID, E3-14.7K, and E1B-19K genes, and adenovirus E1-minus replication-defective vectors that express all E3 genes, RID plus E3-14.7K only, RID only, or E3-14.7K only. RID inhibits TRAIL-induced apoptosis when cells are sensitized to TRAIL either by adenovirus infection or treatment with cycloheximide. RID induces the internalization of TRAIL-R1 from the cell surface, as shown by flow cytometry and indirect immunofluorescence for TRAIL-R1. TRAIL-R1 was internalized in distinct vesicles which are very likely to be endosomes and lysosomes. TRAIL-R1 is degraded, as indicated by the disappearance of the TRAIL-R1 immunofluorescence signal. Degradation was inhibited by bafilomycin A1, a drug that prevents acidification of vesicles and the sorting of receptors from late endosomes to lysosomes, implying that degradation occurs in lysosomes. RID was also shown previously to internalize and degrade another death domain receptor, Fas, and to prevent apoptosis through Fas and the TNF receptor. RID was shown previously to force the internalization and degradation of the epidermal growth factor receptor. E1B-19K was shown previously to block apoptosis through Fas, and both E1B-19K and E3-14.7K were found to prevent apoptosis through the TNF receptor. These findings suggest that the receptors for TRAIL, Fas ligand, and TNF play a role in limiting virus infections. The ability of adenovirus to inhibit killing through these receptors may prolong acute and persistent infections.

Adenoviridae↗
PubMed2001

Tissue-specific, tumor-selective, replication-competent adenovirus vector for cancer gene therapy.

We have previously described two replication-competent adenovirus vectors, named KD1 and KD3, for potential use in cancer gene therapy. KD1 and KD3 have two small deletions in the E1A gene that restrict efficient replication of these vectors to human cancer cell lines. These vectors also have increased capacity to lyse cells and spread from cell to cell because they overexpress the adenovirus death protein, an adenovirus protein required for efficient cell lysis and release of adenovirus from the cell. We now describe a new vector, named KD1-SPB, which is the KD1 vector with the E4 promoter replaced by the promoter for surfactant protein B (SPB). SPB promoter activity is restricted in the adult to type II alveolar epithelial cells and bronchial epithelial cells. Because KD1-SPB has the E1A mutations, it should replicate within and destroy only alveolar and bronchial cancer cells. We show that KD1-SPB replicates, lyses cells, and spreads from cell to cell as well as does KD1 in H441 cells, a human cancer cell line where the SPB promoter is active. KD1-SPB replicates, lyses cells, and spreads only poorly in Hep3B liver cancer cells. Replication was determined by expression of the E4ORF3 protein, viral DNA accumulation, fiber synthesis, and virus yield. Cell lysis and vector spread were measured by lactate dehydrogenase release and a "vector spread" assay. In addition to Hep3B cells, KD1-SPB also did not express E4ORF3 in HT29.14S (colon), HeLa (cervix), KB (nasopharynx), or LNCaP (prostate) cancer cell lines, in which the SPB promoter is not expected to be active. Following injection into H441 or Hep3B tumors growing in nude mice, KD1-SPB caused a three- to fourfold suppression of growth of H441 tumors, similar to that seen with KD1. KD1-SPB had only a minimal effect on the growth of Hep3B tumors, whereas KD1 again caused a three- to fourfold suppression. These results establish that the adenovirus E4 promoter can be replaced by a tissue-specific promoter in a replication-competent vector. The vector has three engineered safety features: the tissue-specific promoter, the mutations in E1A that preclude efficient replication in nondividing cells, and a deletion of the E3 genes which shield the virus from attack by the immune system. KD1-SPB may have use in treating human lung cancers in which the SPB promoter is active.

Adenovirus E1A Proteins↗
PubMed2001

Functional analysis of the leukemia protein ELL: evidence for a role in the regulation of cell growth and survival.

The ELL gene encodes an RNA polymerase II transcription factor that frequently undergoes translocation with the MLL gene in acute human myeloid leukemia. Here, we report that ELL can regulate cell proliferation and survival. In order to better understand the physiological role of the ELL protein, we have developed an ELL-inducible cell line. Cells expressing ELL were uniformly inhibited for growth by a loss of the G(1) population and an increase in the G(2)/M population. This decrease in cell growth is followed by the condensation of chromosomal DNA, activation of caspase 3, poly(ADP ribose) polymerase cleavage, and an increase in sub-G(1) population, which are all indicators of the process of programmed cell death. In support of the role of ELL in induction of cell death, expression of an ELL antisense RNA or addition of the caspase inhibitor ZVAD-fmk results in a reversal of ELL-mediated death. We have also demonstrated that the C-terminal domain of ELL, which is conserved among the ELL family of proteins that we have cloned (ELL, ELL2, and ELL3), is required for ELL's activity in the regulation of cell growth. These novel results indicate that ELL can regulate cell growth and survival and may explain how ELL translocations result in the development of human malignancies.

Amino Acid Chloromethyl Ketones↗
PubMed2000

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.

Adenovirus E3 Proteins↗
PubMed2000

Tumor-specific, replication-competent adenovirus vectors overexpressing the adenovirus death protein.

We have constructed two novel adenovirus (Ad) replication-competent vectors, named KD1 and KD3, that may have use in anticancer therapy. The vectors have two key features. First, they markedly overexpress the Ad death protein (ADP), an Ad nuclear membrane glycoprotein required at late stages of infection for efficient cell lysis and release of Ad from cells. Overexpression of ADP was achieved by deleting the E3 region and reinserting the adp gene. Because ADP is overexpressed, KD1 and KD3 are expected to spread more rapidly and effectively through tumors. Second, KD1 and KD3 have two E1A mutations (from the mutant dl1101/1107) that prevent efficient replication in nondividing cells but allow replication in dividing cancer cells. These E1A mutations preclude binding of E1A proteins to p300 and pRB. As a result, the virus should not be able to drive cells from G(0) to S phase and therefore should not be able to replicate in normal tissues. We show that KD1 and KD3 do not replicate well in quiescent HEL-299 cells or in primary human bronchial epithelial cells, small airway epithelial cells, or endothelial cells; however, they replicate well in proliferating HEL-299 cells and human A549 lung carcinoma cells. In cultured A549 cells, KD1 and KD3 lyse cells and spread from cell to cell more rapidly than their control virus, dl1101/1107, or wild-type Ad. They are also more efficient than dl1101/1107 or wild-type Ad in complementing the spread from cell to cell of an E1(-) E3(-) replication-defective vector expressing beta-galactosidase. A549 cells form rapidly growing solid tumors when injected into the hind flanks of immunodeficient nude mice; however, when A549 cells were infected with 10(-4) PFU of KD3/cell prior to injection into mice, tumor formation was nearly completely suppressed. When established A549 tumors in nude mice were examined, tumors injected with buffer grew 13.3-fold over 5 weeks, tumors injected with dl1101/1107 grew 8-fold, and tumors injected with KD1 or KD3 grew 2.6-fold. Hep 3B tumors injected with buffer grew 12-fold over 3.5 weeks, whereas tumors injected with KD1 or KD3 grew 4-fold. We conclude that KD1 and KD3 show promise as anticancer therapeutics.

Adenovirus E3 Proteins↗
PubMed1999

Immune responses to adenoviruses: viral evasion mechanisms and their implications for the clinic.

Adenoviruses encode proteins that block responses to interferons, intrinsic cellular apoptosis, killing by CD8(+) cytotoxic T lymphocytes and killing by the death ligands TNF, Fas ligand and TRAIL. The viral proteins are believed to prolong acute and persistent adenovirus infections. The proteins may prove useful in protecting adenovirus gene therapy vectors and transplanted cells from the immune system.

Adenoviridae↗
PubMed1998

Lung-specific expression of adenovirus E3-14.7K in transgenic mice attenuates adenoviral vector-mediated lung inflammation and enhances transgene expression.

Herein, we report that the adenovirus E3-14.7K protein inhibits the inflammatory response to adenovirus in transgenic mice in which the E3-14.7K gene was selectively expressed in the respiratory epithelium, using the human surfactant protein C (SP-C) promoter. E3-14.7K mRNA and protein were detected specifically in the lungs of SPC/E3-14.7K transgenic mice. Responses of the transgenic mice to Av1Luc1, an E1-E3-deleted Ad vector encoding the luciferase reporter gene, were examined, including vector transgene expression and lung inflammation. In wild-type mice, luciferase activity declined rapidly and was lost 14 days following Av1Luc1 administration. The loss of luciferase activity was associated with pulmonary infiltration by macrophages and lymphocytes. In heterozygous SPC/E3-14.7K mice, luciferase activity was increased by 7 days compared with control littermates, and pulmonary infiltration by macrophages was decreased. In homozygous (+/+) SPC/E3-14.7K mice, luciferase activity was increased 7, 14, and 21 days following administration compared with wild-type mice, and lung inflammation was markedly reduced. After Av1Luc1 administration, PCNA staining of bronchiolar and alveolar respiratory epithelial cells was decreased in SPC/E3-14.7K transgenic mice, indicating decreased epithelial cell proliferation, a finding consistent with the observed reduction in inflammation. CD4 and CD8 lymphocyte populations were only mildly altered, while humoral responses to adenoviral vectors were unchanged in the SPC/E3-14.7K mice. The E3-14.7K protein expressed selectively in respiratory epithelial cells suppresses Ad-induced pulmonary epithelial cell cytotoxicity and lung inflammation in vivo and prolongs reporter gene expression.

Adenoviridae↗
PubMed1998

Forced degradation of Fas inhibits apoptosis in adenovirus-infected cells.

DNA viruses have evolved elaborate mechanisms to overcome host antiviral defences. In adenovirus-infected cells, programmed cell death (apoptosis) induced by the cytokine tumour necrosis factor (TNF) is inhibited by several adenovirus-encoded proteins. Occupation of the cell-surface receptor Fas, a member of the TNF-receptor superfamily that is expressed on most cell types, triggers apoptosis of that cell. Here we show that the adenovirus RID (for receptor internalization and degradation) protein complex, which is an inhibitor of TNF-induced apoptosis, mediates internalization of cell-surface Fas and its destruction inside lysosomes within the cell. Fas has not previously been shown to be internalized and then degraded. RID also mediates internalization of the receptor for epidermal growth factor, but it does not affect the transferrin receptor or class I antigens of the major histocompatibility complex. Removal of Fas from the surface of adenovirus-infected cells expressing RID may allow infected cells to resist Fas-mediated cell death and thus promote their survival.

Adenoviridae↗
PubMed1998

Inhibition of tumor necrosis factor and interferon triggered responses by DNA viruses.

DNA viruses use elegant mechanisms to overcome the antiviral responses mediated by tumor necrosis factor (TNF), the TNF receptor family member Fas and the interferons. TNF, which is secreted by activated monocytes and lymphocytes, induces apoptosis as well as expression of genes involved in the inflammatory and immune responses. Depending on the DNA virus and the viral proteins, the following mechanisms to prevent TNF receptor- and Fas-induced apoptosis are used: (1) absorption of extracellular TNF by secreted homologs of the TNF receptor; (2) degradation of Fas; (3) inhibition of the assembly of FADD and Caspase 8 with TNFR1 and Fas; (4) direct inhibition of proapoptotic caspase enzymatic activity; and (5) inhibition of the proapoptotic members of the Bcl-2 family. Interferons induce expression of multiple antiviral genes. DNA viruses encode proteins that function in different ways to block interferon-induced transcription as well as the activity of enzymes that block viral protein synthesis. These antiviral proteins prolong acute and persistent infections.

Animals↗
PubMed1997

Adenovirus E3-10.4K/14.5K protein complex inhibits tumor necrosis factor-induced translocation of cytosolic phospholipase A2 to membranes.

We have reported that three adenovirus (Ad) proteins, named E3-10.4K/14.5K, E3-14.7K, and E1B-19K, independently inhibit tumor necrosis factor (TNF)-induced apoptosis in Ad-infected cells. E3-10.4K/14.5K and E3-14.7K also inhibit TNF-induced release of arachidonic acid (AA). TNF-induced apoptosis and AA release are thought to require TNF-activation of the 85-kDa cytosolic phospholipase A2 (cPLA2). cPLA2 normally exists in a latent form in the cytosol; it is activated by phosphorylation by mitogen-activated protein kinase, and in the presence of agents that mobilize intracellular Ca2+, cPLA2 translocates to membranes where it cleaves AA from membrane phospholipids. We now report that TNF induces translocation of cPLA2 from the cytosol to membranes in Ad-infected human A549 cells and that E3-10.4K/14.5K but not E3-14.7K or E1B-19K is required to inhibit TNF-induced translocation of cPLA2. Ad infection also inhibited TNF-induced release of AA. Under the same conditions, Ad infection did not inhibit TNF-induced phosphorylation of cPLA2 or TNF activation of NFkappaB. Ad infection also inhibited cPLA2 translocation in response to the Ca2+ ionophore A23187 and to cycloheximide, but this inhibition did not require E3-10.4K/14.5K. Ad infection did not inhibit cPLA2 translocation in response to interleukin-1beta or platelet-derived growth factor. We propose that E3-10.4K/14.5K inhibits TNF-induced AA release and apoptosis by directly or indirectly inhibiting TNF-induced translocation of cPLA2 from the cytosol to membranes. AA formed by cPLA2 can be metabolized to prostaglandins, leukotrienes, and lipoxyns, molecules that amplify inflammation. E3-10.4K/14.5K probably functions in Ad infections to inhibit both TNF-induced apoptosis and inflammation.

Adenovirus E3 Proteins↗
PubMed1996

The E3-11.6-kDa adenovirus death protein (ADP) is required for efficient cell death: characterization of cells infected with adp mutants.

We have reported that an 11,600-Da nuclear membrane glycoprotein named adenovirus death protein (ADP), encoded by the E3 region, is required for the efficient death (lysis) of adenovirus (Ad)-infected cells. We postulated that ADP mediates the release of virions from cells at the conclusion of replication. Here we provide further characterization of cells infected by adp+ and adp- Ads. Using virus mutants with deletions in the individual E3 genes, we show that only mutants that lack ADP have small plaques that are slow to develop. Mutants in the adp gene replicated as well as wild-type Ad, but the cells lysed much more slowly. Cell lysis and viability were determined by plaque size, cell morphology, trypan blue exclusion, the release of lactate dehydrogenase, and the MTT assay for mitochondrial activity. ADP is required for efficient lysis of human A549, KB, 293, and MCF-7 cells. A549 cells infected with adp+ Ads began to die at 2-3 days postinfection and were dead by 6 days. With adp mutants, > 80% of cells remained viable for 5-6 days; when the medium was changed, > 80% of cells were viable after 7 days and 10-20% after 14 days. When the MTT assay was used, there was an increase in mitochondrial activity, suggesting that Ad infection stimulates respiratory metabolism. Nearly all nuclei from wild-type Adinfected cells lacked DAPI-stained DNA by 7 days, whereas with an adp mutant nearly all nuclei stained brightly after 15 days. Nuclei from adp mutant-infected cells were extremely swollen and full of virus, and appeared to have an intact nuclear membrane. Cells infected with wild-type Ad had many vacuoles and perhaps a disrupted nuclear membrane; they did not display features typical of apoptosis.

Adenovirus E3 Proteins↗
PubMed1996

Induction of susceptibility to tumor necrosis factor by E1A is dependent on binding to either p300 or p105-Rb and induction of DNA synthesis.

The introduction of the adenovirus early region 1A (E1A) gene products into normal cells sensitizes these cells to the cytotoxic effects of tumor necrosis factor (TNF). Previous studies have shown that the region of E1A responsible for susceptibility is CR1, a conserved region within E1A which binds the cellular proteins p300 and p105-Rb at nonoverlapping sites. Binding of these and other cellular proteins by E1A results in the induction of E1A-associated activities such as transformation, immortalization, DNA synthesis, and apoptosis. To investigate the mechanism by which E1A induces susceptibility to TNF, the NIH 3T3 mouse fibroblast cell line was infected with viruses containing mutations within E1A which abrogate binding of some or all of the cellular proteins to E1A. The results show that TNF susceptibility is induced by E1A binding to either p300 or p105-Rb. E1A mutants that bind neither p300 nor p105-Rb do not induce susceptibility to TNF. Experiments with stable cell lines created by transfection with either wild-type or mutant E1A lead to these same conclusions. In addition, a correlation between induction of DNA synthesis and induction of TNF sensitivity is seen. Only viruses which induce DNA synthesis can induce TNF sensitivity. Those viruses which do not induce DNA synthesis also do not induce TNF sensitivity. These data suggest that the mechanisms underlying induction of susceptibility to TNF by E1A are intimately connected to E1A's capacity to override cell cycle controls.

3T3 Cells↗
PubMed1996

The adenovirus death protein (E3-11.6K) is required at very late stages of infection for efficient cell lysis and release of adenovirus from infected cells.

Adenovirus (Ad) infection is concluded by assembly of virions in the cell nucleus followed by lysis of cells by an unknown mechanism. We have described an Ad nuclear membrane glycoprotein of 11,600 kDa (E3-11.6K) which is encoded by the E3 transcription unit and which is synthesized in small amounts from the E3 promoter at early stages of infection but in large amounts from the major late promoter at very late stages of infection. We now report that E3-11.6K is required for the efficient lysis (death) of Ad-infected cells, and we propose that the function of E3-11.6K is to mediate the release of Ad progeny from infected cells. We have renamed E3-11.6K the Ad death protein (ADP). Virus mutants that lack ADP replicated as well as adp+ Ad, but the cells lysed more slowly, virus release from the cell was retarded, and the plaques were small and developed slowly. Cells infected with adp+ viruses began to lyse at 2 or 3 days postinfection (p.i.) and were completely lysed by 5 or 6 days p.i. In contrast, cells infected with adp mutants did not begin significant lysis until 5 or 6 days p.i. Cell lysis and viability were determined by plaque size, extracellular virus, cell morphology, release of lactate dehydrogenase, trypan blue exclusion, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay for mitochondrial activity, RNA degradation, and DNA degradation as determined by agarose gel electrophoresis and the terminal deoxynucleotidyltransferase end labeling assay. Protein synthesis was almost nonexistent at 3 days p.i. in cells infected with adp+ Ads, but it was still increasing in cells infected with adp mutants. Host cell protein synthesis was undetectable at 1 day p.i. in cells infected with adp+ Ads or adp mutants. Cells infected with adp mutants showed Ad cytopathic effect at 1 or 2 days p.i. in that they rounded up and detached, but the cells remained metabolically active and intact for >5 days p.i. When examined by electron microscopy, the nuclei were extremely swollen and full of virus, and the nuclear membrane appeared to be intact. ADP is unrelated in sequence to other known cell death-promoting proteins.

Adenovirus E3 Proteins↗
PubMed1996

The role of human adenovirus early region 3 proteins (gp19K, 10.4K, 14.5K, and 14.7K) in a murine pneumonia model.

Products of human adenovirus (Ad) early region 3 (E3) inhibit both specific (cytotoxic T lymphocytes [CTLs]) and innate (tumor necrosis factor alpha [TNF-alpha]) immune responses in vitro. The E3 gp19K protein prevents CTL recognition of Ad-infected fibroblasts by sequestering major histocompatibility complex class I proteins in the endoplasmic reticulum. E3 proteins 10.4K, 14.5K, and 14.7K function to protect infected cells from TNF-alpha cytolysis. To address the in vivo functions of these proteins, Ad mutants that lack the E3 genes encoding these proteins were inoculated intranasally into C57BL/10SnJ (H-2b) mice. Mutants that lack the gp19K gene failed to alter CTL generation or to affect Ad-induced pulmonary infiltrates. Since gamma interferon (IFN-gamma) is capable of overcoming gp19K suppression of CTL lysis in vitro, mice were depleted of IFN-gamma and inoculated with gp19K mutants. Even when IFN-gamma was depleted, gp19K was incapable of altering pulmonary lesions. These resuls are not in accord with the function of gp19K in vitro and suggest that gp19K does not affect immune recognition in vivo during an acute virus infection, yet they do not exclude the possibility that gp19K blocks immune recognition of Ad during a persistent infection. In contrast, when mice were inoculated with Ad mutants that lack the TNF resistance genes (14.7K and either 10.4K or 14.5K), there was a marked increase in alveolar infiltration and no change in the amounts of perivascular/peribronchiolar infiltration compared with wild-type-Ad-induced pathology. These findings demonstrate the importance of TNF susceptibility and TNF by-products for recruiting inflammatory cells into the lungs during Ad infections.

Adenoviridae Infections↗
PubMed1996

The adenovirus E3-14.7K protein and the E3-10.4K/14.5K complex of proteins, which independently inhibit tumor necrosis factor (TNF)-induced apoptosis, also independently inhibit TNF-induced release of arachidonic acid.

Tumor necrosis factor (TNF) is an inflammatory cytokine that inhibits the replication of many viruses in cultured cells. We have reported that adenovirus (Ad) infection of TNF-resistant mouse cells renders them susceptible to lysis by TNF and that two sets of proteins encoded by the E3 transcription unit block TNF cytolysis. The E3 protein sets are named E3-14.7K (14,700 kDa) and E3-10.4K/14.5K (a complex of two proteins of 10,400 and 14,500 kDa). TNF activation of the 85-kDa cytosolic phospholipase A2 (cPLA2) is thought to be essential for TNF cytolysis (i.e.,TNF-induced apoptosis). Here we provide evidence that cPLA2 is important in the response of Ad-infected cells to TNF and that the mechanism by which E3-14.7K and E3-10.4K/14.5K inhibit TNF cytolysis is by inhibiting TNF activation of cPLA2. cPLA2 cleaves arachidonic acid (AA) specifically from membrane phospholipids; therefore, cPLA2 activity was measured by the release of 3H-AA from cells prelabeled with 3H-AA. Uninfected cells or cells infected with wild-type Ad were not lysed and did not release 3H-AA in response to TNF. In contrast, TNF treatment induced cytolysis and 3H-AA release in uninfected cells sensitized to TNF by treatment with cycloheximide and also in infected cells sensitized to TNF by expression of E1A. In C127 cells, in which either E3-14.7K or E3-10.4K/14.5K inhibits TNF cytolysis, either set of proteins inhibited TNF-induced release of 3H-AA. In C3HA cells, in which E3-14.7K but not E3-10.4K/14.5K prevents TNF cytolysis, E3-14.7K but not E3-10.4K/14.5K prevented TNF-induced release of 3H-AA. When five virus mutants with lesions in E3-14.7K were examined, there was a perfect correlation between a mutant's ability to inhibit both TNF-induced cytolysis and release of 3H-AA. E3-14.7K expressed in two stably transfected C127 cell lines prevented both TNF-cycloheximide-induced cytolysis and release of 3H-AA. The E3 proteins also prevented TNF-induced cytolysis and release of 3H-AA in mouse L929 cells, which are spontaneously sensitive to TNF. TNF cytolysis was blocked by dexamethasone, an inhibitor of PLA2 activity, and by nordihydroquaiaretic acid, which inhibits the metabolism of AA to the leukotrienes. Indomethacin, which blocks the formation of prostaglandins from AA, did not inhibit TNF cytolysis. The leukotrienes and prostaglandins are amplifiers of the inflammatory response. We propose that E3-14.7K and E3-10.4K/14.5K function independently in Ad infection to inhibit both cytolysis and inflammation induced by TNF.

Adenoviridae Infections↗
PubMed1995

The E3-20.5K membrane protein of subgroup B human adenoviruses contains O-linked and complex N-linked oligosaccharides.

Subgroup B adenoviruses (Ad3, -7, -11, -35) contain two open reading frames (ORFs) in the early E3 transcription unit that are not present in subgroup C adenoviruses (Ad2, Ad5). The product of one of these ORFs, a 20,500-kDa (20.5K) protein, was shown previously to be expressed as two diffuse 22K and 36K bands on SDS-PAGE; the 22K appeared to be the precursor to the 36K species. As judged by its predicted sequence, 20.5K is a type I membrane glycoprotein with two potential sites for N-glycosylation and a transmembrane domain near its COOH-terminus. Here we show that when Ad3- or Ad7-infected cells were radiolabeled in the presence of tunicamycin, which prevents the addition of N-linked oligosaccharides, both the 22K and the 36K forms of 20.5K showed increased mobility in SDS-PAGE, indicating that both forms contain N-linked sugars. Both the 22K and the 36K forms were sensitive to digestion by endoglycosidase F and N-glycanase, again indicating that they both contain N-linked sugars. Only the 22K species was sensitive to endoglycosidase H, indicating that it contains high-mannose-type oligosaccharides and that the 36K species contains complex-type carbohydrates. The 36K form was sensitive to neuraminidase, indicating that its sugars contain terminal sialic acid. When digested with N-glycanase and neuraminidase, the 36K form was sensitive to O-glycanase, indicating that the 36K form has O-linked oligosaccharides. The 22K form was labeled with [3H]mannose and the 36K form was labeled with [3H]glucosamine and to a much lesser extent by [3H]mannose. Altogether these results indicate that the 20.5K protein is cotranslationally modified with N-linked high-mannose oligosaccharides, then the protein moves into the Golgi and trans-Golgi network where it acquires O-linked and complex N-linked oligosaccharides.

Adenovirus E3 Proteins↗
PubMed1995

A 20,500-Dalton protein is coded by region E3 of subgroup B but not subgroup C human adenoviruses.

There is an open reading frame (ORF) between ATG2089 and TGA2656 in the early E3 transcription unit of subgroup B adenovirus 3 (Ad3) that could encode a protein of 20,500 MW (20.5K). This ORF also exists in Ad7, Ad11, and Ad35 (subgroup B). An antipeptide antiserum made in rabbits against the predicted Ad3/7 20.5K protein immunoprecipitated two diffuse bands with apparent molecular weights of about 22K and 36K from Ad3- or Ad7-infected cells. These bands were also detected in immunoblots. These bands were not seen in mock-infected cells or cells infected with Ad3 or Ad7 mutants that delete the gene for 20.5K. In vitro transcription and translation of the 20.5K gene yielded a protein of about 18K, suggesting that this may be the primary translation product and that the 22K and 36K forms of 20.5K arise by post-translation processing. Pulse/chase experiments suggest that the half-life of the 22K form is short, and that this form is further modified to the 36K species. In accord with these results and as judged by its predicted sequence, 20.5K appears to be a membrane glycoprotein with a potential N-terminal signal sequence, a second hydrophobic putative transmembrane domain, and two potential Asn-linked glycosylation sites. The 20.5K protein was synthesized throughout the course of the infection. Ad3 and Ad7 mutants lacking 20.5K grew as well as wild-type Ad3 and Ad7, indicating that, in common with subgroup C E3 proteins, the 20.5K protein is dispensable for virus replication in cultured cells. The 20.5K gene is totally absent from the E3 region of Ad2 and Ad5 (subgroup C). The gene is also absent or highly diverged in the E3 region of Ad12 (subgroup A) and Ad40 (subgroup F). Given that E3 genes may counteract host defenses, the 20.5K protein may contribute to the unique pathogenic properties of subgroup B human adenoviruses.

Adenovirus E3 Proteins↗