Ad-infected Cells Research Articles

IFN-γ secretion is polarized at effector immunological synapses (IS) in vivo. (87.52)

Abstract In vitro cytokine secretion by T cells is polarized. For example, IFN-γ secretion is focused towards the immunological synapse (IS), while secretion of TNFα is multi-directional. Whether polarized cytokine secretion occurs in vivo is unknown. We studied the intracellular distribution of IFN-γ within T cells establishing IS with infected astrocytes in vivo. Astrocytes were infected with an adenovirus (Ads) expressing Thymidine Kinase (TK), a marker for infected cells. 30 days later we induced a systemic immune response against Ads, and 14 days after the immunization animals were sacrificed and brains studied by immunohistochemistry and confocal microscopy. Brain sections were stained for TCR, LFA-1, TK and IFN-γ. Confocal analysis revealed that IFN-γ is polarized in T cells establishing close anatomical contacts with Ad infected cells. Polarization was found in cells with or without polarized LFA-1 or TCR. This demonstrates that in vivo IFN-γ is selectively polarized towards the APC, and further suggests that maturation of the IS is not necessary for IFN-γ polarization. In consequence, polarized IFN-γ secretion may precede the maturation of immunological synapses. This work was funded by grants from NINDS, NIH.

Adeno-associated virus induces apoptosis during coinfection with adenovirus

Adeno-associated virus (AAV) is a nonpathogenic parvovirus that efficiently replicates in the presence of adenovirus (Ad). Exogenous expression of the AAV replication proteins induces caspase-dependent apoptosis, but determining if AAV infection causes apoptosis during viral infection is complicated by Ad-mediated programmed cell death. To eliminate Ad-induced cytolysis, we used an E3 adenoviral death protein (ADP) mutant, pm534. AAV and pm534-coinfected cells exhibited increased cell killing compared to pm534 alone. Relative to cells infected with Ad alone, AAV and wild-type Ad-infected cells displayed decreased ADP expression, increased cytolysis until the third day of the infection, and decreased cytolysis thereafter. Biochemical and morphological characteristics of apoptosis were observed during coinfections with AAV and pm534 or Ad, including a moderate degree of caspase activation that was not present during infections with pm534 or Ad alone. AAV coinfection also increased extracellular pH. These studies suggest that AAV induces caspase-dependent and caspase-independent apoptosis.

Cancer‐specific targeting of an adenovirus‐delivered herpes simplex virus thymidine kinase suicide gene using translational control

Two technical hurdles, gene delivery and target specificity, have hindered the development of effective cancer gene therapies. In order to circumvent the problem of tumor specificity, the suicide gene, HSV-1 thymidine kinase (HSV-Tk), was modified with a complex 5' upstream-untranslated region (5'-UTR) that limits efficient translation to cells expressing high levels of the translation initiation factor, eIF4E. Since previous studies have shown that most tumor cells express elevated levels of eIF4E, tumor-specific gene delivery was optimized by incorporation of the 5'-UTR-modified suicide gene (HSV-UTk) into an adenovirus vector (Ad-CMV-UTk). The efficacy of this novel approach of targeting suicide gene expression and limiting cytotoxicity by means of translational restriction was tested in vitro with the use of the human breast cancer cell lines (MCF-7, MDA-MB435, and ZR-75-1). As controls, normal MCF10A, HMEC, and HMSC cell lines that express relatively low levels of eIF4E were used. Real-time reverse-transcription polymerase chain reaction (RT-PCR) was used to quantify HSV-Tk mRNA for cells infected with Ad-CMV-UTk as well as with Ad-CMV-Tk (a control adenovirus in which HSV-Tk is not regulated at the level of translation). Translation of HSV-Tk in the Ad-infected cells was measured by Western blot analysis. In addition, cytotoxicity was determined following treatment with the pro-drug ganciclovir (GCV) using an MTT viability assay. Finally, microPET imaging was used to assess cancer cell-specific expression of HSV-Tk and expression in normal tissues in vivo after intraperitoneal injection of Ad-CMV-Tk or Ad-CMV-UTk. These data collectively showed enhanced cancer cell-specific gene expression and reduced normal tissue gene expression for the Ad-HSV-UTk compared to the Ad-CMV-Tk, leading to increased cancer cell-enhanced GCV cytotoxicity. These results indicate that translational targeting of suicide gene expression in tumor cells in vitro and in vivo is effective and may provide a platform for enhanced cancer gene therapy specificity.

Adenovirus E3-6.7K protein is required in conjunction with the E3-RID protein complex for the internalization and degradation of TRAIL receptor 2.

Adenoviruses (Ads) encode several proteins within the early region 3 (E3) transcription unit that help protect infected cells from elimination by the immune system. Among these immunomodulatory proteins, the receptor internalization and degradation (RID) protein complex, which is composed of the RIDalpha (formerly E3-10.4K) and RIDbeta (formerly E3-14.5K) subunits, stimulates the internalization and degradation of certain members of the tumor necrosis factor (TNF) receptor superfamily, thus blocking apoptosis initiated by Fas and TNF-related apoptosis-inducing ligand (TRAIL). The experiments reported here show that TRAIL receptor 2 (TR2) is cleared from the cell surface in Ad-infected cells. Virus mutants containing deletions that span E3 were used to show that the RID and E3-6.7K proteins are both necessary for the internalization and degradation of TR2, whereas only the RID protein is required for TRAIL receptor 1 downregulation. In addition, replication-defective Ad vectors that express individual E3 proteins were used to establish that the RID and E3-6.7K proteins are sufficient to clear TR2. These data demonstrate that E3-6.7K is an important component of the antiapoptosis arsenal encoded by the E3 transcription unit of subgroup C Ads.

Protein arginine methylation during lytic adenovirus infection.

Arginine methylation of proteins affects major processes in the cell, including transcriptional regulation, mRNA metabolism, signal transduction and protein sorting. Arginine methylation of Ad (adenovirus) E1B 55-kDa-associated protein E1B-AP5 was recently described by us [Kzhyshkowska, Schutt, Liss, Kremmer, Stauber, Wolf and Dobner (2001) Biochem. J. 358, 305-314]. In this first example of protein arginine methylation analysis in Ad-infected cells, we investigated methylation of the E1B-AP5 and the viral L4-100 kDa protein. We demonstrate that E1B-AP5 methylation is enhanced during the course of infection in a cell-type-specific manner. We also show that L4-100 kDa is efficiently methylated in Ad-infected cells. L4-100 kDa formed complex with methyltransferase in vivo during productive infection, and can be methylated by HRMT1L2 (human protein arginine methyltransferase 1) in vitro. Comparative analysis of E1B-AP5 and L4-100 kDa protein methylation in Ad-infected HeLa, MCF-7 and H1299 cells revealed that the profile of protein arginine methylation correlates with the efficiency of Ad proteins production. Our results suggest that protein arginine methylation is an important host-cell function required for efficient Ad replication.

Strain-specific rate of shutdown of CMV enhancer activity in murine liver confirmed by use of persistent [E1 −, E2b −] adenoviral vectors

The systemic delivery of [E1 −] adenoviral (Ad) vectors encoding a transgene results in efficient viral uptake and abundant transgene expression in the liver. However, [E1 −]Ad vector persistence is transient due to cytotoxic T lymphocyte (CTL)-mediated loss of the Ad-infected cells. Our laboratory has previously demonstrated that additional modifications to the [E1 −]Ad vector genome, by deletion of the Ad E2b genes, significantly decreased virus-genome-derived gene expression and simultaneously improved the long-term performance of the resultant [E1 −, E2b −]Ad vector. In this study, we confirmed that [E1 −, E2b −]Ad vector genomes could persist equally well in C57Bl/6 or Balb/c mouse hepatocytes. Despite vector genome persistence, we observed a strain-dependent variability in the duration of CMV enhancer/promoter-driven transgene expression in the liver. While Balb/c mice rapidly shut down [E1 −, E2b −]Ad-derived transgene expression, C57Bl/6 mice allowed for prolonged transgene expression. This occurred even when both strains were crossed into a severe combined immune-deficient background, demonstrating that host adaptive immune responses are not responsible for the phenomenon. Furthermore, differential methylation of the CMV enhancer/promoter was also not demonstrated in either strain of mouse, eliminating this mechanism as causative. Thus, alternative mechanisms for this phenomenon are discussed.

Input Virion Proteins: Cryptic Targets of Antivector Immune Responses in Preimmunized Subjects

Input Virion Proteins: Cryptic Targets of Antivector Immune Responses in Preimmunized Subjects

Spatial and temporal organization of adeno-associated virus DNA replication in live cells.

Upon cell entry, the genomes of herpes simplex virus type 1 (HSV-1) and adenovirus (Ad) associate with distinct nuclear structures termed ND10 or promyelocytic leukemia (PML) nuclear bodies (NBs). PML NB morphology is altered or disrupted by specific viral proteins as replication proceeds. We examined whether adeno-associated virus (AAV) replication compartments also associate with PML NBs, and whether modification or disruption of these by HSV-1 or Ad, both of which are helper viruses for AAV, is necessary at all. Furthermore, to add a fourth dimension to our present view of AAV replication, we established an assay that allows visualization of AAV replication in live cells. A recombinant AAV containing 40 lac repressor binding sites between the AAV inverted terminal repeats was constructed. AAV Rep protein and helper virus-mediated replication of this recombinant AAV genome was visualized by binding of enhanced yellow fluorescent protein-lac repressor fusion protein to double-stranded AAV replication intermediates. We demonstrate in live cells that AAV DNA replication occurs in compartments which colocalize with AAV Rep. Early after infection, the replication compartments were small and varied in numbers from 2 to more than 40 per cell nucleus. Within 4 to 8 h, individual small replication compartments expanded and fused to larger structures which filled out much of the cell nucleus. We also show that AAV replication compartments can associate with modified PML NBs in Ad-infected cells. In wild-type HSV-1-infected cells, AAV replication compartments and PML NBs did not coexist, presumably because PML was completely disrupted by the HSV-1 ICP0 protein. However, alteration or disruption of PML appears not to be a prerequisite for AAV replication, as the formation of replication compartments was normal when the ICP0 mutants HSV-1 dl1403 and HSV-1 FXE, which do not affect PML NBs, were used as the helper viruses; under these conditions, AAV replication compartments did not associate with PML NBs.

Ternary complex formation between DNA-adenovirus core protein VII and TAF-Iβ/SET, an acidic molecular chaperone

The adenovirus (Ad) genome complexed with viral core proteins designated Ad core is the template for transcription of early genes and the first round of replication in Ad-infected cells. A cellular protein designated template-activating factor-I (TAF-I) is found to be involved in remodeling of the Ad core in vitro. Here we found that TAF-I interacts with the Ad DNA through core protein VII in infected cells in early phases of infection. In vitro binding assays using recombinant proteins showed that TAF-I forms ternary complexes with DNA–protein VII complexes.

Distinct domains in the adenovirus E3 RIDalpha protein are required for degradation of Fas and the epidermal growth factor receptor.

Adenovirus (Ad) types 2 and 5 encode at least five proteins within the E3 transcription unit that help the virus evade the immune system. Two such proteins, RIDalpha (formerly E3-10.4K) and RIDbeta (formerly E3-14.5K), form the RID (receptor internalization and degradation) complex (formerly E3-10.4K/14.5K). RID mediates clearance from the cell surface and lysosomal degradation of a number of important members in the tumor necrosis factor receptor (TNFR) superfamily and the receptor tyrosine kinase receptor family. Affected receptors include Fas, TRAIL (TNF-related apoptosis-inducing ligand) receptor 1 (TR1), TR2, and epidermal growth factor receptor (EGFR). Degradation of Fas and TRAIL receptors protects Ad-infected cells from apoptosis. To investigate the mechanism of action of RIDalpha, 14 mutant RIDalpha proteins, each containing a three- to five-amino-acid deletion, were constructed and then expressed from the E3 region of a replication-competent recombinant Ad in the same context as wild-type RIDalpha. Each mutant protein was characterized with regard to five physical properties associated with wild-type RIDalpha, namely, protein stability, proteolytic cleavage, insertion into the membrane, complex formation with RIDbeta, and transport to the cell surface. Additionally, the mutant proteins were tested for their ability to mediate internalization and degradation of EGFR and Fas and to protect cells from Fas-mediated apoptosis. The majority of mutant RIDalpha proteins (8 out of 14) were physically similar to wild-type RIDalpha. With regard to functional characteristics, the cytoplasmic domain of RIDalpha is largely unimportant for receptor internalization and degradation and the extracellular domain of RIDalpha is important for down-regulation of EGFR but not Fas.

Gene transduction efficiency and maturation status in mouse bone marrow-derived dendritic cells infected with conventional or RGD fiber-mutant adenovirus vectors.

Since dendritic cells (DCs) play a critical role in establishing antigen-specific adaptive immune responses, in the past several years, therapeutic strategies using genetically modified DCs against cancer and infectious diseases have attracted increasing attention. In the present study, we demonstrated that RGD fiber-mutant adenovirus vector (AdRGD) exhibited markedly superior gene transduction efficiency in mouse bone marrow-derived DCs (mBM-DCs) compared to conventional adenovirus vector (Ad). Likewise, this vector exhibited superior major histocompatibility complex class I-restricted presentation of antigen derived from the delivered gene in mBM-DCs. In order to investigate the effect of Ad-infection on the DC-differentiation process (maturation), we used three types of AdRGD and three conventional Ad to transduce mBM-DCs. These vectors carried either no transgene, LacZ gene, or gp100 gene. Infection by any of the Ad vectors enhanced the expression of MHC class II molecules in mBM-DCs. CD80, CD86, and CD40 expression and IL-12 production were more efficient in AdRGD-infected mBM-DCs than in conventional Ad-infected cells. Contrary to our expectations, endocytotic activity of mBM-DCs decreased only slightly upon Ad-infection, whereas antigen uptake by lipopolysaccharide (LPS)-driven mature mBM-DCs was significantly impaired. However, our reverse transcription-polymerase chain reaction analysis revealed that Ad-infection resulted in the upregulation of the chemokine receptor CCR7 and downregulation of CCR6 in mBM-DCs and LPS-stimulated cells. We, therefore, concluded that Ad-infection directly influenced DC-maturation, although the effects were milder than under LPS-stimulation. In addition, this change in the immunologic properties of DCs resulted primarily from an increase in the number of Ad-particles capable of invading the cells rather than from the expression of foreign genes. AdRGD-infection caused greater induction of maturation than conventional Ad-infection, irrespective of the type of transgene inserted.

Genetic modification of adenovirus 5 tropism by a novel class of ligands based on a three-helix bundle scaffold derived from staphylococcal protein A.

The use of adenovirus (Ad) as an efficient and versatile vector for in vivo tumor therapy requires the modulation of its cellular tropism. We previously developed a method to genetically alter the tropism of Ad5 fibers by replacing the fiber knob domain by an extrinsic trimerization motif and a new cellular ligand. However, fibers carrying complex ligands such as single-chain antibody fragments did not assemble into functional pentons in vitro in the presence of penton base, and failed to be rescued into infectious virions because of their inability to fold correctly within the cytoplasm of Ad-infected cells. Here we show that the coding sequence for a disulfide bond-independent three-helix bundle scaffold Z, derived from domain B of Staphylococcal protein A and capable of binding to the Fc portion of immunoglobulin (Ig) G1, could be incorporated into modified knobless Ad fiber gene constructs with seven shaft repeats. These fiber gene constructs could be rescued into viable virions that were demonstrated to enter 293 cells engineered for IgG Fc surface expression but not unmodified 293 cells, via a mechanism that could be specifically blocked with soluble Fc target protein. However, the tropism modified viruses showed a slightly impaired cellular entry and a lower infectivity than wildtype (WT) virus. In addition, we generated recombinant fibers containing an IgA binding Affibody ligand, derived from combinatorial specificity-engineering of the Z domain scaffold. Such fiber constructs also showed the expected target specific binding, indicating that the affibody protein class is ideally suited for genetic engineering of Ad tropism.

Downregulation of CXCR4 gene expression in primary human endothelial cells following infection with E1(-)E4(+) adenovirus gene transfer vectors.

Infection of human endothelial cells with first-generation E1(-)E4(+) adenovirus (Ad) vectors leads to prolonged cell survival and changes in the cell phenotype to a more quiescent stage. Based on the concept that the CXCR4, the receptor for the endothelial chemoattractant stromal-derived factor-&alpha (SDF-alpha), is constitutively expressed by quiescent, resting endothelial cells, the present study analyzes the effect of Ad vector infection on CXCR4 expression and SDF-alpha responses of human umbilical vein endothelial cells (HUVEC). CXCR4 transcripts were markedly downregulated in E1(-)E4(+) Ad-infected cells 48 h following infection, but not in uninfected control cells or when the cells were infected with an E1(-)E4(-) Ad vector. Analysis of surface CXCR4 expression by flow cytometry demonstrated marked reduction of the CXCR4 receptor on cells infected with E1(-)E4(+) Ad compared to uninfected control cells or E1(-)E4(-) Ad-infected cells. Infection of other cell types which express CXCR4, such as dendritic cells and myeloma cells, did not exhibit CXCR4 receptor downregulation following infection with E1(-)E4(+) Ad. Consistent with the observed downregulation of CXCR4 mRNA and surface protein, infection of the endothelial cells with an E1(-)E4(+) Ad rendered the cells unresponsive to the chemoattractant SDF-alpha compared to naive or E1(-)E4(-) Ad-infected cells. Together, the data suggest that first-generation Ad vectors, likely the E4 region, modify the ability of endothelial cells to respond to at least one important chemoattractant.

Different effects of dominant negative mutants of desmocollin and desmoglein on the cell-cell adhesion of keratinocytes.

Desmosomes contain two types of cadherin: desmocollin (Dsc) and desmoglein (Dsg). In this study, we examined the different roles that Dsc and Dsg play in the formation of desmosomes, by using dominant-negative mutants. We constructed recombinant adenoviruses (Ad) containing truncated mutants of E-cadherin, desmocollin 3a, and desmoglein 3 lacking a large part of their extracellular domains (EcaddeltaEC, Dsc3adeltaEC, Dsg3deltaEC), using the Cre-loxP Ad system to circumvent the problem of the toxicity of the mutants to virus-producing cells. When Dsc3adeltaEC Ad-infected HaCaT cells were cultured with high levels of calcium, E-cadherin and beta-catenin, which are marker molecules for the adherens junction, disappeared from the cell-cell contact sites, and cell-cell adhesion was disrupted. This also occurred in the cells infected with EcaddeltaEC Ad. With Dsg3deltaEC Ad infection, keratin insertion at the cell-cell contact sites was inhibited and desmoplakin, a marker of desmosomes, was stained in perinuclear dots while the adherens junctions remained intact. Dsc3adeltaEC Ad inhibited the induction of adherens junctions and the subsequent formation of desmosomes with the calcium shift, while Dsg3deltaEC Ad only inhibited the formation of desmosomes. To further determine whether Dsc3adeltaEC directly affected adherens junctions, mouse fibroblast L cells transfected with E-cadherin (LEC5) were infected with these mutant Ads. Both Dsc3adeltaEC and EcaddeltaEC inhibited the cell-cell adhesion of LEC5 cells, as determined by the cell aggregation assay, while Dsg3deltaEC did not. These results indicate that the dominant negative effects of Dsg3deltaEC were restricted to desmosomes, while those of Dsc3adeltaEC were observed in both desmosomes and adherens junctions. Furthermore, the cytoplasmic domain of Dsc3adeltaEC coprecipitated both plakoglobin and beta-catenin in HaCaT cells. In addition, beta-catenin was found to bind the endogenous Dsc in HaCaT cells. These findings lead us to speculate that Dsc interacts with components of the adherens junctions through beta-catenin, and plays a role in nucleating desmosomes after the adherens junctions have been established.

A helper virus-free packaging system for recombinant adeno-associated virus vectors.

Adeno-associated virus (AAV) is a human parvovirus that is currently receiving widespread attention for its potential use as a gene therapy vector. Construction of the recombinant AAV vector (rAAV) involves replacing most of the viral genome with a transgene of interest and then packaging this recombinant genome into an infectious virion. Most current protocols for generating rAAV entail the co-transfection of a vector plasmid and a packaging plasmid that expresses the viral replication and structural genes onto adenovirus (Ad) infected cells growing in culture. Limitations of this procedure include (1) contamination of rAAV with the Ad helper virus, (2) low yields of rAAV and (3) production of replication-competent AAV. In this report we describe new helper plasmids (pSH3 and pSH5) that eliminate the Ad co-infection requirement. The helper plasmids express the AAV rep and cap genes and the Ad E2A, VAI and E4 genes. When the helper plasmids are co-transfected onto human 293 cells with a vector plasmid in the absence of Ad infection, the rAAV vector yield is up to 80-fold greater than those obtained with the pAAV/Ad packaging plasmid. Moreover, replication competent AAV in the rAAV preparations is less than 0.00125%. The major advantages of this system are (1) the absence of infectious adenovirus and (2) the use of only two plasmids, which enhances transfection efficiencies and hence vector production. We believe that this two-plasmid transfection system will allow for more widespread use of the AAV vector system because of its simplicity and high yields. This system will be especially useful for preclinical analyses of multiple rAAV vectors.

Organization of Splicing Factors in Adenovirus-Infected Cells Reflects Changes in Gene Expression during the Early to Late Phase Transition

The spatial distribution of splicing factors is temporally regulated during adenovirus (ad) infection. Here we focus on two splicing factor distribution patterns characteristic of ad-infected cells. During the intermediate phase splicing factors surround sites of viral DNA accumulation in regions of high transcriptional activity. This distribution appears as a series of interconnected rings when viewed by microscopy. We refer to cells with this staining pattern as “ring cells.” We have previously shown that at late times after infection, splicing factors are present in discrete structures identified as enlarged interchromatin granules (IGs) that also contain spliced viral RNA. We refer to cells with this pattern as “cluster cells.” We determined which steps in viral gene expression occurred in ring and cluster cells. We found that transcription and some splicing of viral late genes had occurred in ring cells. Late RNA was present at transcription sites in ring cells. Cluster cells contained spliced viral late RNA in nuclear IGs and in the cytoplasm. The presence of cluster cells in the infected culture was well correlated with the export of viral RNA to the cytoplasm. Cluster cells had synthesized late proteins. Our data show that the dynamic localization of splicing factors reflects changes in gene expression activity of the infected cell as it switches over to late gene expression.

E1B 55-kilodalton-associated protein: a cellular protein with RNA-binding activity implicated in nucleocytoplasmic transport of adenovirus and cellular mRNAs.

The adenovirus type 5 (Ad5) early 1B 55-kDa protein (E1B-55kDa) is a multifunctional phosphoprotein that regulates viral DNA replication and nucleocytoplasmic RNA transport in lytically infected cells. In addition, E1B-55kDa provides functions required for complete oncogenic transformation of rodent cells in cooperation with the E1A proteins. Using the far-Western technique, we have isolated human genes encoding E1B-55kDa-associated proteins (E1B-APs). The E1B-AP5 gene encodes a novel nuclear RNA-binding protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) family that is highly related to hnRNP-U/SAF-A. Immunoprecipitation experiments indicate that two distinct segments in the 55-kDa polypeptide which partly overlap regions responsible for p53 binding are required for complex formation with E1B-AP5 in Ad-infected cells and that this protein interaction is modulated by the adenovirus E4orf6 protein. Expression of E1B-AP5 efficiently interferes with Ad5 E1A/E1B-mediated transformation of primary rat cells. Furthermore, stable expression of E1B-AP5 in Ad-infected cells overcomes the E1B-dependent inhibition of cytoplasmic host mRNA accumulation. These data suggest that E1B-AP5 might play a role in RNA transport and that this function is modulated by E1B-55kDa in Ad-infected cells.

The adenovirus E3/10.4K-14.5K proteins down-modulate the apoptosis receptor Fas/Apo-1 by inducing its internalization.

Adenoviruses (Ads) have evolved multiple mechanisms to evade the host immune response. Several of the immunomodulatory Ad proteins are encoded in early transcription unit 3 (E3). The E3/19K protein interferes with antigen presentation and T cell recognition, whereas the E3/10.4K, 14.5K, and 14.7K proteins can protect cells from tumor necrosis factor alpha-mediated lysis. Here, we describe an additional activity of E3 proteins. Transfectants expressing all E3 proteins of Ad2 exhibit a profound reduction of the apoptosis receptor CD95 (Fas, APO-1) on the cell surface. In contrast, cells expressing only the E3A region have normal Fas levels. Thus, one of the E3B proteins (10.4K, 14.5K, or 14.7K) seems to be responsible for this effect. To identify the E3B products involved, each individual E3B ORF was selectively disrupted. Examination of stable cell lines containing the mutated E3 regions showed that Fas expression is restored when either the 10.4K or the 14.5K ORF is disrupted, whereas mutation of the 14.7K ORF does not rescue Fas expression. Loss of Fas on the cell surface is accompanied by a similar decrease of total Fas levels. However, in the presence of lysosomotropic agents Fas accumulates in endosomal/lysosomal vesicles, indicating that 10.4K-14.5K induce internalization and degradation of Fas. Down-regulation of Fas but not CD40 is also observed during infection and as a consequence, Ad-infected cells are protected from Fas-mediated apoptosis. Thus, the Fas system is implicated in Ad pathogenesis.

Adenovirus-mediated gene transfer of fibroblast growth factor-1: angiogenesis and tumorigenicity in nude mice.

Gene transfer of angiogenic growth factors with replication-deficient recombinant adenovirus (Ad) vectors may provide a new approach to the treatment of ischemic diseases. To determine if Ad-infected cells could stimulate angiogenesis in vivo and to assess the tumorigenicity of cells infected with these vectors, NIH3T3 fibroblasts infected with Ad vectors coding for human acidic fibroblast growth factor (aFGF-1) were used in angiogenic and tumorigenic assays. Infected cells induced a strong angiogenic response in vivo, while cells infected with control virus did not. Stable 3T3 transfectants expressing the FGF-1 gene were also highly angiogenic and exhibited growth in soft agar, while Ad-infected cells did not. Ad-infected cells grew transiently in nude mice, whereas 3T3 transfectants formed large tumors which grew exponentially. Extrapolation of cell dose-response curves showed that a minimum of 1.5 x 10(4) infected cells were required for transient tumor cell growth in vivo. Ad-infected cells cultured in vitro for 30 days lost their invasive phenotype and the ability for transient cell growth in nude mice. Thus, phenotypic changes induced by Ad-mediated gene transfer of FGF-1 are transient both in vitro and in vivo, suggesting that these Ad vectors do not have tumorigenic potential. Stimulation of angiogenesis by Ad-infected cells may be useful for the evaluation of anti-angiogenic and anti-tumor agents.

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.