Adenovirus death protein (ADP) is required for lytic infection of human lymphocytes.

The adenovirus death protein (ADP) is expressed at late times during a lytic infection of species C adenoviruses. ADP promotes the release of progeny virus by accelerating the lysis and death of the host cell. Since some human lymphocytes survive while maintaining a persistent infection with species C adenovirus, we compared ADP expression in these cells with ADP expression in lymphocytes that proceed with a lytic infection. Levels of ADP were low in KE37 and BJAB cells, which support a persistent infection. In contrast, levels of ADP mRNA and protein were higher in Jurkat cells, which proceed with a lytic infection. Epithelial cells infected with an ADP-overexpressing virus died more quickly than epithelial cells infected with an ADP-deleted virus. However, KE37, and BJAB cells remained viable after infection with the ADP-overexpressing virus. Although the levels of ADP mRNA increased in KE37 and BJAB cells infected with the ADP-overexpressing virus, the fraction of cells with detectable ADP was unchanged, suggesting that the control of ADP expression differs between epithelial and lymphocytic cells. When infected with an ADP-deleted adenovirus, Jurkat cells survived and maintained viral DNA for greater than 1 month. These findings are consistent with the notion that the level of ADP expression determines whether lymphocytic cells proceed with a lytic or a persistent adenovirus infection.

The role of cyclophosphamide in enhancing antitumor efficacy of an adenovirus oncolytic vector in subcutaneous Syrian hamster tumors.

We have previously reported that intratumoral injection of VRX-007--an Ad5 (a species C adenovirus)-based vector overexpressing adenovirus death protein--can suppress the growth of subcutaneous HaK (hamster renal cancer) tumors. VRX-007 replication and tumor growth inhibition are enhanced when the hamsters are immunosuppressed by a high dose of cyclophosphamide (CP), an immunosuppressive and chemotherapeutic agent. Here, we report that continuous immunosuppression with CP was not required for increased oncolytic activity of VRX-007 because short-term dosing or continuous dosing with the drug yielded similar antitumor results. Prolonged viral replication was found only in animals on continuous CP treatment. We used 007-Luc, a replication-competent, luciferase-expressing vector similar to VRX-007, to investigate the replication of the vector over time. Tumor growth inhibition was similar in hamsters given CP treatment either 1 week before or 1 week after 007-Luc injection, which suggests that CP exerts its antitumor efficacy independently of vector therapy. 007-Luc did not spread far from the inoculation site, even in immunosuppressed, CP-treated animals. Our results indicate that the enhanced effectiveness that is produced by the combination of VRX-007 and CP therapies is due to their two independent mechanisms and that they do not have to be given simultaneously for the improved outcome.

INGN 007, an oncolytic adenovirus vector, replicates in Syrian hamsters but not mice: comparison of biodistribution studies.

Preclinical biodistribution studies with INGN 007, an oncolytic adenovirus (Ad) vector, supporting an early stage clinical trial were conducted in Syrian hamsters, which are permissive for Ad replication, and mice, which are a standard model for assessing toxicity and biodistribution of replication-defective (RD) Ad vectors. Vector dissemination and pharmacokinetics following intravenous administration were examined by real-time PCR in nine tissues and blood at five time points spanning 1 year. Select organs were also examined for the presence of infectious vector/virus. INGN 007 (VRX-007), wild-type Ad5 and AdCMVpA (an RD vector) were compared in the hamster model, whereas only INGN 007 was examined in mice. DNA of all vectors was widely disseminated early after injection, but decayed rapidly in most organs. In the hamster model, DNA of INGN 007 and Ad5 was more abundant than that of the RD vector AdCMVpA at early times after injection, but similar levels were seen later. An increased level of INGN 007 and Ad5 DNA but not AdCMVpA DNA in certain organs early after injection, and the presence of infectious INGN 007 and Ad5 in lung and liver samples at early times after injection, strongly suggests that replication of INGN 007 and Ad5 occurred in several Syrian hamster organs. There was no evidence of INGN 007 replication in mice. In addition to providing important information about INGN 007, the results underscore the utility of the Syrian hamster as a permissive immunocompetent model for Ad5 pathogenesis and oncolytic Ad vectors.

An acute toxicology study with INGN 007, an oncolytic adenovirus vector, in mice and permissive Syrian hamsters; comparisons with wild-type Ad5 and a replication-defective adenovirus vector.

Oncolytic (replication-competent) adenoviruses as anticancer agents provide new, promising tools to fight cancer. In support of a Phase I clinical trial, here we report safety data with INGN 007 (VRX-007), an oncolytic adenovirus with increased anti-tumor efficacy due to overexpression of the adenovirus-encoded ADP protein. Wild-type adenovirus type 5 (Ad5) and a replication-defective version of Ad5 were also studied as controls. A parallel study investigating the biodistribution of these viruses is described elsewhere in this issue. The toxicology experiments were conducted in two species, the Syrian hamster, which is permissive for INGN 007 and Ad5 replication and the poorly permissive mouse. The studies demonstrated that the safety profile of INGN 007 is similar to Ad5. Both viruses caused transient liver damage upon intravenous injection that resolved by 28 days post-infection. The No-Observable-Adverse-Effect-Level (NOAEL) for INGN 007 in hamsters was 3 x 10(10) viral particles per kg. In hamsters, the replication-defective vector caused less toxicity, indicating that replication of Ad vectors in the host is an important factor in pathogenesis. With mice, INGN 007 and Ad5 caused toxicity comparable to the replication-defective adenovirus vector. Partially based on these results, the FDA granted permission to enter into a Phase I clinical trial with INGN 007.

Oncolytic adenovirus that overproduces ADP and replicates selectively in tumors due to hTERT promoter-regulated E4 gene expression.

We have constructed a novel oncolytic adenovirus (Ad) vector, named VRX-011, in which the replication of the vector is targeted to cancer cells by the replacement of the wild-type Ad E4 promoter with the human telomerase reverse transcriptase (hTERT) promoter. Genes in the Ad E4 transcription unit are essential for Ad replication; therefore, VRX-011 will grow efficiently only in cells in which the hTERT promoter is active, that is, in a wide range of cancer and immortalized cells but not in most somatic cells. Consistent with these expectations, VRX-011 replicated efficiently in all cancer cell lines examined, while its growth was restricted in various primary and normal cells. VRX-011 overexpresses ADP (also known as E3-11.6K), an Ad protein required for efficient cell lysis and release of virions from cells at late stages of infection. This overexpression enhances cell-to-cell spread and could significantly increase antitumor efficacy. In a xenograft model in nude mice, both intratumoral and intravenous administration of VRX-011 effectively suppressed the growth of subcutaneous Hep3B human liver tumors. Also, intravenous delivery of VRX-011 greatly reduced the number and size of A549 human lung cancer cell nodules in a disseminated lung tumor model in nude mice. Importantly, tail vein administration of different doses of VRX-011 in C57BL/6 mice showed minimal liver toxicity. Considering its broad range of lytic replication in cancer cells, its attenuated phenotype in primary cells, its efficacy in suppressing xenografts, and its low toxicity in mouse liver, VRX-011 is a promising candidate for further evaluation as an anticancer therapeutic.

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.

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.

GP73, a novel Golgi-localized protein upregulated by viral infection.

We report the isolation and characterization of GP73, a novel 73kDa human Golgi protein. The GP73 cDNA was cloned by differential screening of a cDNA library derived from the liver of a patient with adult giant-cell hepatitis (GCH), a rare form of hepatitis with presumed viral etiology. In vitro transcription-translation studies indicate that GP73 is an integral membrane protein, and immunolocalization experiments using epitope-tagged GP73 demonstrate that the protein is localized to the Golgi apparatus. Northern blot analysis of RNA from multiple human tissues reveals a single GP73 mRNA transcript with a size of approximately 3.0kb. Immunohistochemical studies using rabbit polyclonal antisera directed against recombinant GP73 demonstrate that the protein is preferentially expressed by epithelial cells in many human tissues. In normal livers, GP73 is consistently present in biliary epithelial cells, whereas hepatocytes show little or no signal. In contrast, livers of patients with GCH display strong GP73 immunoreactivity in multinucleated hepatocytes. GP73 mRNA and protein are expressed in highly differentiated HepG2 hepatoma cells after infection with adenovirus in vitro. We conclude that GP73 represents a novel, epithelial cell-specific integral membrane Golgi protein that can be upregulated in response to viral infection.

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.

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.

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.

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.

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.

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.

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.

The adenovirus E3 10.4K and 14.5K proteins, which function to prevent cytolysis by tumor necrosis factor and to down-regulate the epidermal growth factor receptor, are localized in the plasma membrane.

The adenovirus type 2 and 5 E3 10,400- and 14,500-molecular-weight (10.4K and 14.5K) proteins are both required to protect some cell lines from lysis by tumor necrosis factor and to down-regulate the epidermal growth factor receptor. We have shown previously that both 10.4K and 14.5K are integral membrane proteins and that 14.5K is phosphorylated and O glycosylated. The 10.4K protein coimmunoprecipitates with 14.5K, indicating that the two proteins function as a complex. Here we show, using immunofluorescence and two different cell surface-labeling techniques, that both proteins are localized in the plasma membrane. In addition, we show that trafficking of each protein to the plasma membrane depends on concomitant expression of the other protein. Finally, neither protein could be immunoprecipitated from conditioned media, indicating that neither is secreted. Taken together, these results suggest that the plasma membrane is the site at which 10.4K and 14.5K function to inhibit cytolysis by tumor necrosis factor and to down-regulate the epidermal growth factor receptor.

Adenovirus proteins that subvert host defenses.

Adenovirus encodes numerous products that counteract host defenses. A virus-encoded RNA, VA RNA1, prevents interferon-mediated shut-off of protein synthesis. Other protein products inhibit interferon-induced gene transcription, prevent cell killing by cytotoxic T cells or block apoptosis, and three sets of proteins independently block the cytolysis and inflammation induced by tumor necrosis factor. Studies of these factors are providing insights into viral pathogenesis.

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 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.