Regulated expression of the interferon-beta gene in mice.

A single plasmid regulated expression vector based upon a mifepristone-inducible two plasmid system, termed pBRES, has been constructed and tested in mice using murine interferon-b (mIFNb) as the transgene. The expression of mIFNb in the circulation was followed by measuring the systemic induction of IP-10, a validated biomarker for mIFNb in mice. Long-term, inducible expression of mIFNb was demonstrated following a single intramuscular (i.m.) injection of the pBRES mIFNb plasmid vector into the hind limb of mice. Induction of mIFNb expression was achieved by administration of the small molecule inducer, mifepristone (MFP). Plasmid DNA and mIFNb mRNA levels in the injected muscles correlated with mIFNb expression as monitored by IP-10 over a 3-month time period. Renewable transgene expression was achieved following repeat administration of the plasmid at 3 months following the first plasmid injection. A dose-dependent increase in expression was demonstrated by varying the amount of injected plasmid or the amount of the inducer administered to the mice. Finally, the pBRES plasmid expressing mIFNb under control of the inducer, MFP, was shown to be efficacious in a murine model of experimental allergic encephalomyelitis, supporting the feasibility of gene-based therapeutic approaches for treating diseases such as multiple sclerosis.

Effect of viral dose on neutralizing antibody response and transgene expression after AAV1 vector re-administration in mice.

Neutralizing antibodies (nAB) at the time of administration hamper the effectiveness of adeno-associated virus (AAV) as a clinical DNA delivery system. The present study was designed to investigate if AAV re-administration in muscle tissue is dependent on the nAB titer. Recombinant (r)AAV serotype 1, as a promising candidate for targeting skeletal muscle, was used for gene delivery. C57Bl/6 mice were infected intramuscularly with doses between 1 x 10(9) and 5 x 10(10) virus particles (vp) of AAV1-expressing luciferase (AAV1-luc) or human interferon-beta (AAV1-hIFNbeta). Increasing transgene expression was observed over the first 2 months and anti-AAV1 nAB titers peaked between weeks 4 and 8. Six months after the first administration, 5 x 10(10) vp of AAV1-IFNbeta were re-administered. Following re-administration, nAB titers increased but did not significantly affect transgene expression from the AAV vector that had been administered first. In contrast, hIFNbeta expression originating from the second vector administration was significantly diminished and reflected the nAB titer present at the day of re-administration. The present study extends earlier observations that preexisting nAB affects AAV1 re-administration. The level of nAB is proportional to the virus dose used for the first injection and transgene expression following re-administration is dependent on preexisting nAB titer.

Identification and function probing of an antithrombin IIIß conformation-specific antibody.

UNLABELLED: ESSENTIALS: Antithrombin III (AT)ß binds heparin with higher affinity than ATα. A conformation-specific antibody against ATß, TPP2009, was made to investigate ATß in hemostasis. TPP2009 bound specifically to heparin-ATß and greatly reduced the anticoagulant effect of AT. This antibody was effective in elucidating the importance of ATß in hemostasis. BACKGROUND: Antithrombin III (AT)ß is an isoform of AT that lacks the post-translational carbohydrate modification at Asn135. This isoform binds heparin with greater affinity than ATα, and has been shown to target antithrombotic function to the extracellular vascular endothelial injury site. OBJECTIVES: To characterize a conformation-specific antibody against ATß and begin to investigate the role of ATß in maintaining hemostasis. METHODS: Surface plasmon resonance (SPR), antigen binding and functional assays were conducted to characterize the mode of action of antibodies generated against heparin-bound ATß (ATß*H) by the use of phage display. RESULTS: SPR and binding studies showed that one of the antibodies, TPP2009, bound specifically to ATß*H and glycosaminoglycan-associated ATß on endothelial cells. In diluted prothrombin and activated factor X (FXa)-induced clotting assays, TPP2009 dose-dependently reduced the anticoagulant effect of heparin in non-hemophilic and FVIII-deficient human plasma, with half-maximal effective concentrations (EC50 ) of 10.5 nm and 4.7 nm, respectively. In AT-deficient human plasma, TPP2009 dose-dependently inhibited the effects of exogenously added ATß and heparin. In purified systems with ATß and pentasaccharide, TPP2009 restored > 91% of FXa activity. TPP2009 dose-dependently reversed the effects of heparin in rabbit (EC50 , 25.7 nm) and cynomolgus monkey (EC50 , 21.5 nm) plasma, but not in mouse plasma. TPP2009 was also effective in partially restoring FXa activity in rabbit and cynomolgus monkey plasma treated with FVIII function-neutralizing antibodies. CONCLUSIONS: TPP2009 specifically targets a unique conformational epitope on ATß*H and blocks ATß-mediated anticoagulation. It effectively promotes coagulation in plasma, indicating the importance of ATß in hemostasis.

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.

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.

Growth inhibition of an established A431 xenograft tumor by a full-length anti-EGFR antibody following gene delivery by AAV.

Therapeutic monoclonal antibodies continue to achieve clinical success for the treatment of many different diseases, particularly cancer. However, the production and purification of antibodies continues to be a time and labor-intensive process with considerable technical challenges. Gene-based delivery of antibodies may address this, via direct production within the host that achieves therapeutic levels. In this report, we validate the feasibility that gene-based delivery is a viable approach for efficacious delivery of antibodies in the preclinical and, presumably, clinical setting. We demonstrate high and sustained in vivo expression of the murine antihuman epidermal growth factor receptor antibody 14E1 following intramuscular delivery by adeno-associated virus (AAV) 2/1. Incorporating the Furin/2A technology for monocistronic expression of both heavy and light chains, we achieved sustained serum levels of full-length 14E1 peaking over 1 mg ml(-1) in athymic nude mice. In the A431 xenograft tumor model, 14E1 was capable of significantly inhibiting tumor growth and prolonging survival when AAV was administered prior to tumor challenge. Furthermore, 14E1 demonstrated significant antitumor efficacy against well-established tumors (approximately 400 mm(3)) when AAV was administered up to 20 days after tumor challenge. Here we demonstrate for the first time growth inhibition of a well-established tumor by a full-length antibody following delivery by AAV.

The effect of hypoxia on the uptake, replication and lytic potential of group B adenovirus type 3 (Ad3) and type 11p (Ad11p).

Replicating, tumor selective viruses are being tested as potential treatments for human cancers. Hypoxia is a pathophysiological cancer condition that alters the lytic potential of the replication-competent adenovirus serotype 5 (Ad5) virus by a mechanism independent of receptor levels or internalization rates. We extend these initial studies to examine the potential effects of hypoxia on the group B adenoviruses (Ads), adenovirus type 3 (Ad3) (group B1) and adenovirus type 11p (Ad11p) (group B2). Receptor expression (CD46) is not altered by hypoxia. However, the lytic potential is compromised in a cell-dependent fashion. Consequently, our study suggests that group B replicating Ad-based treatments, like the group C Ad-5-based viruses, will need to be modified in order to effectively treat hypoxic components of human tumors.

Effect of hypoxia on Ad5 infection, transgene expression and replication.

Oxygen deprivation (hypoxia) is a common feature of various human maladies, including cardiovascular diseases and cancer; however, the effect of hypoxia on Ad-based gene therapies has not been described. In this study, we evaluated how hypoxia (1% pO(2)) affects different aspects of Ad-based therapies, including attachment and uptake, transgene expression, and replication, in a series of cancer cell lines and primary normal cells. We found that hypoxia had no significant effect on the expression or function of the Ad5 attachment (Coxsackievirus and Adenovirus Receptor) and internalization (alpha(v) integrins) proteins, nor on the human cytomegalovirus-driven expression of an exogenous gene carried by a replication-incompetent Ad. Viral replication, however, was compromised by hypoxic conditions. Our studies revealed hypoxia-induced reductions in E1A levels that were mediated at the post-transcriptional level. E1A drives cells into the viral replication optimal S phase of the cell cycle; consequently, the combination of reduced E1A protein and hypoxia-induced G1 arrest of cells may be responsible for the lack of efficient viral replication under hypoxic conditions. Consequently, while traditional replication-incompetent Ad-based vectors appear to be viable delivery systems for hypoxia-associated disease indications, our studies suggest that Oncolytic Ads may need additional factors to efficiently treat hypoxic regions of human tumors.

Gene delivery from the E3 region of replicating human adenovirus: evaluation of the ADP region.

Genetically modified replication-selective human adenoviruses are currently undergoing testing in the clinical setting as anticancer agents. Coupling the lytic function of these viruses with virus-mediated transgene delivery represents a powerful extension of this treatment. We have designed a unique system for gene delivery from the replicating virus. It takes advantage of the endogenous gene expression control sequences (promoter, splicing, polyadenylation signals) to efficiently and predictably deliver transgenes from the non-essential E3 transcription unit while still maintaining the expression of the remaining E3 genes in the multi-gene transcription unit. In this article, we engineered restriction enzyme sites into the virus genome selectively to delete the ADP gene and replace it with the therapeutic transgenes CD and TNFalpha. We demonstrate that: (1) transgene expression from this region mirrors the substituted ADP gene; (2) the loss of ADP in these viruses results in infected cells with extended viability and protein synthesis when compared with a wild-type Ad5 infected cell; and (3) expression of surrounding E3 genes can be maintained in such a system. The potential advantages of delivering transgenes from the ADP region of the replicating adenovirus are discussed.

Human cytomegalovirus immediate-early two protein region involved in negative regulation of the major immediate-early promoter.

The immediate-early two (IE2) gene products of human cytomegalovirus negatively regulate gene expression from the major immediate-early promoter in permissive human fibroblasts. A mutational analysis of the IE2 proteins indicated that the carboxyl-terminal region is required for negative regulation. The IE2 proteins that lack amino acid residues 365 to 519, or the carboxyl-terminal amino acids failed to negatively regulate. Most of the amino-terminal portion of the IE2 protein was not required for negative regulation. A possible explanation of the negative effect on downstream expression by the IE2 proteins is discussed.

A cis-acting element in the major immediate-early (IE) promoter of human cytomegalovirus is required for negative regulation by IE2.

The major immediate-early promoter (MIEP) of human cytomegalovirus (CMV) contains a number of different enhancer elements in both repetitive and nonrepetitive sequences that influence the level of downstream transcription. This report describes a cis-acting element in the MIEP that responds to negative regulation by the IE2 gene product. Deletion analysis demonstrated that the cis-acting repressor element is located between the TATA box and the transcription initiation site from -13 to -1. The DNA sequence of the repressor element is 5'-CGTTTAGTGAACC-3'. The sequence is found in both the human and simian CMV MIEPs but not the murine CMV MIEP or in several other enhancer-containing promoters. The repressor element was isolated in a DNA fragment from -13 to +3 and was found to be functional in either orientation. It could be transferred to a heterologous enhancer-containing promoter and was functional when placed between the TATA box and the transcription initiation site. The element did not function when placed downstream of the transcription initiation site. Therefore, the cis-acting repressor element is position dependent. The role of the repressor element and the IE2 gene product in human CMV productive or latent infection is discussed.

The amino-terminal portion of CD1 of the adenovirus E1A proteins is required to induce susceptibility to tumor necrosis factor cytolysis in adenovirus-infected mouse cells.

Previous work by our laboratory and others has shown that mouse cells normally resistant to tumor necrosis factor can be made sensitive to the cytokine by the expression of adenovirus E1A. The E1A gene can be introduced by either infection or transfection, and either of the two major E1A proteins, 289R or 243R, can induce this sensitivity. The E1A proteins are multifunctional and modular, with specific domains associated with specific functions. Here, we report that the CD1 domain of E1A is required to induce susceptibility to tumor necrosis factor cytolysis in adenovirus-infected mouse C3HA fibroblasts. Amino acids C terminal to residue 60 and N terminal to residue 36 are not necessary for this function. This conclusion is based on 51Cr-release assays for cytolysis in cells infected with adenovirus mutants with deletions in various portions of E1A. These E1A mutants are all in an H5dl309 background and therefore they lack the tumor necrosis factor protection function provided by the 14.7-kilodalton (14.7K) protein encoded by region E3. Western blot (immunoblot) analysis indicated that most of the mutant E1A proteins were stable in infected C3HA cells, although with certain large deletions the E1A proteins were unstable. The region between residues 36 and 60 is included within but does not precisely correlate with domains in E1A that have been implicated in nuclear localization, enhancer repression, cellular immortalization, cell transformation in cooperation with ras, induction of cellular DNA synthesis and proliferation, induction of DNA degradation, and binding to the 300K protein and the 105K retinoblastoma protein.

Deletion mutation analysis of the adenovirus type 2 E3-gp19K protein: identification of sequences within the endoplasmic reticulum lumenal domain that are required for class I antigen binding and protection from adenovirus-specific cytotoxic T lymphocytes.

Adenovirus E3-gp19K is a transmembrane glycoprotein, localized in the endoplasmic reticulum (ER), which forms a complex with major histocompatibility complex (MHC) class I antigens and retains them in the ER, thereby preventing cytolysis by cytotoxic T lymphocytes (CTL). The ER lumenal domain of gp19K, residues 1 to 107, is known to be sufficient for binding to class I antigens; the transmembrane and cytoplasmic ER retention domains are located at residues ca. 108 to 127 and 128 to 142, respectively. To identify more precisely which gp19K regions are involved in binding to class I antigens, we constructed 13 in-frame virus deletion mutants (4 to 12 amino acids deleted) in the ER lumenal domain of gp19K, and we analyzed the ability of the mutant proteins to form a complex with class I antigens, retain them in the ER, and prevent cytolysis by adenovirus-specific CTL. All mutant proteins except one (residues 102 to 107 deleted) were defective for these properties, indicating that the ability of gp19K to bind to class I antigens is highly sensitive to mutation. All mutant proteins were stable and were retained in the ER. Sequence comparisons among adenovirus serotypes reveal that the ER lumenal domain of gp19K consists of a variable region (residues 1 to 76) and a conserved region (residues 77 to 98). We show, using the mutant proteins, that the gp19K-specific monoclonal antibody Tw1.3 recognizes a noncontiguous epitope in the variable region and that disruption of the variable region by deletion destroys the epitope. The monoclonal antibody and class I antigen binding results, together with the serotype sequence comparisons, are consistent with the idea that the ER lumenal domain of gp19K has three subdomains that we have termed the ER lumenal variable domain (residues 1 to ca. 77 to 83), the ER lumenal conserved domain (residues ca. 84 to 98), and the ER lumenal spacer domain (residues 99 to 107). We suggest that the ER lumenal variable domain of gp19K has a specific tertiary structure that is important for binding to the polymorphic alpha 1 and alpha 2 domains of class I heavy (alpha) chains. We suggest that the ER lumenal conserved domain of gp19K may interact with some conserved protein, perhaps the highly conserved alpha 3 domain of class I heavy chains. Finally, the ER lumenal spacer domain may allow the ER lumenal variable and conserved domains to extend out from the ER membrane so that they can interact with class I heavy chains.

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.

Sequence and functional analysis of the human adenovirus type 7 E3-gp19K protein from 17 clinical isolates.

The adenovirus (Ad) early region 3 (E3) glycoprotein of 19K (gp19K) binds major histocompatibility (MHC) class I antigens in the endoplasmic reticulum (ER), and the gp19K-class I complex is retained in the ER through an ER retention signal at the C-terminus of gp19K. This retention of class I antigens blocks cytolysis of gp19K-expressing cells by cytotoxic T lymphocytes (CTL). Animal models infected with Ad mutants lacking gp19K support a role for gp19K in counteracting a CTL response. Gp19K binds with different avidities to different class I antigens, and portions of the gp19K sequence are highly variable among Ad serotypes in different subgroups (Ad3, 11, and Ad35 in subgroup B; Ad2 and Ad5 in subgroup C); this raises the possibility that certain human individuals may be more susceptible to productive or persistent infection by particular serotypes of Ad, depending on the haplotype of the individual and the type of Ad. To begin to address this possibility, the gp19K gene from 17 very diverse Ad7 (subgroup B) clinical isolates was amplified by the polymerase chain reaction, and the DNA sequences were determined. The Ad7 gp19K sequence was 98% identical to that of Ad3. Surprisingly, we found complete conservation of the amino acid sequence of gp19K from all but one of the clinical isolates; one isolate had a conservative Ala to Val substitution. Gp19K from Ad7 clinical isolates representing distinct Ad7 genotypes co-immunoprecipitated with class I antigens. Our data indicate that there is very strong evolutionary pressure to maintain the sequence of gp19K in Ad7. The only known function for gp19K from different Ad serotypes is binding to class I antigens. It is interesting to consider, therefore, what selective pressure operates to maintain the sequence of gp19K among serotypes within a subgroup, and yet allows for very significant divergence in the sequence of gp19K among serotypes in different subgroups. The possible role of MHC class I antigens in this selection process is discussed.

Identification and characterization of the human cytomegalovirus immediate-early region 2 gene that stimulates gene expression from an inducible promoter.

The human cytomegalovirus (HCMV) XbaI E cloned DNA fragment of approximately 20 kilobases can complement an adenovirus mutant (dl312) defective in the E1a viral gene product (D. J. Spector and M. J. Tevethia, Virology 151:329-338, 1986). This viral DNA fragment contains three immediate-early (IE) genes between 0.709 and 0.751 map units (M. F. Stinski, D. R. Thomsen, R. M. Stenberg, and L. C. Goldstein, J. Virol. 46:1-14, 1983). Two of the IE genes, IE1 and IE2, were isolated and tested for a role in regulating viral gene expression. Since HCMV early and late promoters require additional characterization, the chloramphenicol acetyl transferase (cat) gene, driven by the adenovirus E2 promoter, was used as an indicator of gene expression. cat expression from this heterologous viral promoter was shown to be stimulated by HCMV at early times after infection. The IE1 gene product did not function independently in activating this promoter. The IE2 gene products could independently stimulate the expression of a plasmid of a plasmid when the cat gene was placed downstream of the inducible E2 promoter (E2CAT). Five proteins of different sizes have been predicted to originate from IE2, depending on mRNA splicing. The protein products specified by the IE2 gene were characterized with an antibody to a synthetic peptide according to the open reading frame of exon 2. Three of the five proteins are encoded by exon 2. Three viral proteins of 82, 54, and 28 kilodaltons (kDa) were detected. The exons contained in the region designated as IE2a have open reading frames that could code for two of the smaller proteins of 27 and 30 kDa. This region, when driven by the HCMV enhancer, could independently stimulate gene expression from E2CAT to a high level. A plasmid with the HCMV enhancer upstream of exons, that could code for the HCMV IE2 proteins of 48 and 51 kDa, as well as 27- and 30-kDa proteins, also stimulated E2CAT expression but at a lower level. The activity of this plasmid was augmented by the IE1 gene product, despite the fact that the latter gene product alone was inactive. It is proposed that the HCMV IE region 2 gene products are involved in the regulation of viral or host cell promoters either independently or in combination with other HCMV IE proteins.

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 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 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.