Limited but durable changes to cellular gene expression in a model of latent adenovirus infection are reflected in childhood leukemic cell lines.

Mucosal lymphocytes support latent infections of species C adenoviruses. Because infected lymphocytes resist re-infection with adenovirus, we sought to identify changes in cellular gene expression that could inhibit the infectious process. The expression of over 30,000 genes was evaluated by microarray in persistently infected B-and T-lymphocytic cells. BBS9, BNIP3, BTG3, CXADR, SLFN11 and SPARCL1 were the only genes differentially expressed between mock and infected B cells. Most of these genes are associated with oncogenesis or cancer progression. Histone deacetylase and DNA methyltransferase inhibitors released the repression of some of these genes. Cellular and viral gene expression was compared among leukemic cell lines following adenovirus infection. Childhood leukemic B-cell lines resist adenovirus infection and also show reduced expression of CXADR and SPARCL. Thus adenovirus induces limited changes to infected B-cell lines that are similar to changes observed in childhood leukemic cell lines.

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.

Adenovirus DNA in Guthrie cards from children who develop acute lymphoblastic leukaemia (ALL).

BACKGROUND: In search of a proposed viral aetiology of childhood acute lymphoblastic leukaemia (ALL), the common species C adenoviruses were analysed in Guthrie cards. METHODS: Guthrie cards from 243 children who later developed ALL and from 486 matched controls were collected and analysed by nested polymerase chain reaction for the presence of adenovirus DNA. RESULTS: Adenovirus DNA was reliably detected from only two subjects, both of whom developed ALL. CONCLUSION: Adenovirus DNA is detected in Guthrie card samples at too low a frequency to reveal an association between adenovirus and the development of leukaemia.

Latent species C adenoviruses in human tonsil tissues.

Although species C human adenoviruses establish persistent infections, the molecular details of this lifestyle remain poorly understood. We previously reported that adenovirus DNA is found in human mucosal T lymphocytes in a noninfectious form (C. T. Garnett, D. Erdman, W. Xu, and L. R. Gooding, J. Virol. 76:10608-10616, 2002). In this study, human tonsil and adenoid tissues were analyzed to determine the dynamics of infection, the rate of clearance of viral DNA, and the possibility of reactivation of virus from these tissues. The presence of viral DNA peaked at 4 years of age and declined thereafter. The average number of viral genomes declined with the age of the donor. The frequency of virus-bearing cells ranged from 3 x 10(-7) to 3.4 x 10(-4), while the amount of viral DNA per cell varied less, with an average of 280 copies per cell. All species C serotypes were represented in these tissues, although adenovirus type 6 was notably rare. Infectious virus was detected infrequently (13 of 94 of donors tested), even among donors with the highest levels of adenoviral DNA. Adenovirus transcripts were rarely detected in uncultured lymphocytes (2 of 12 donors) but appeared following stimulation and culture (11 of 13 donors). Viral DNA replication could be stimulated in most donor samples by lymphocyte stimulation in culture. New infectious virus was detected in 13 of 15 donors following in vitro stimulation. These data suggest that species C adenoviruses can establish latent infections in mucosal lymphocytes and that stimulation of these cells can cause viral reactivation resulting in RNA transcription, DNA replication, and infectious virus production.

Adenovirus DNA is detected at increased frequency in Guthrie cards from children who develop acute lymphoblastic leukaemia.

Epidemiological evidence suggests that childhood acute lymphoblastic leukaemia (ALL) may be initiated by an in infection in utero. Adenovirus DNA was detected in 13 of 49 neonatal blood spots from ALL patients but only in 3 of 47 controls (P=0.012) suggesting a correlation between prenatal adenovirus infection and the development of ALL.

Three adenovirus E3 proteins cooperate to evade apoptosis by tumor necrosis factor-related apoptosis-inducing ligand receptor-1 and -2.

Adenovirus encodes multiple gene products that regulate proapoptotic cellular responses to viral infection mediated by both the innate and adaptive immune systems. The E3-10.4K and 14.5K gene products are known to modulate the death receptor Fas. In this study, we demonstrate that an additional viral E3 protein, 6.7K, functions in the specific modulation of the two death receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The 6.7K protein is expressed on the cell surface and forms a complex with the 10.4K and 14.5K proteins, and this complex is sufficient to induce down-modulation of TRAIL receptor-1 and -2 from the cell surface and reverse the sensitivity of infected cells to TRAIL-mediated apoptosis. Down-modulation of TRAIL-R2 by the E3 complex is dependent on the cytoplasmic tail of the receptor, but the death domain alone is not sufficient. These results identify a mechanism for viral modulation of TRAIL receptor-mediated apoptosis and suggest the E3 protein complex has evolved to regulate the signaling of selected cytokine receptors.

Immune evasion by adenoviruses.

Adenovirus is a human pathogen that infects mainly respiratory and gastrointestinal epithelia. While the pathology caused by this virus is generally not life threatening in immunocompetent individuals, there is a large literature describing its ability to establish a persistent infection. These persistent infections typically occur in apparently healthy individuals with no outward signs of disease. Such a long term and benign interaction between virus and immune system requires adenoviruses to dampen host antiviral effector mechanisms that would otherwise eliminate the virus and cause immune-mediated pathology to the host. Adenovirus devotes a significant portion of its genome to gene products whose sole function seems to be the modulation of host immune responses. This review focuses on what is currently understood about how these immunomodulatory mechanisms work and how they might play a role in maintaining the virus in a persistent state.

The adenovirus E3-10.4K/14.5K complex mediates loss of cell surface Fas (CD95) and resistance to Fas-induced apoptosis.

Cytotoxic T cells use Fas (CD95), a member of the tumor necrosis factor (TNF) receptor superfamily, to eliminate virus-infected cells by activation of the apoptotic pathway for cell death. The adenovirus E3 region encodes several proteins that modify immune defenses, including TNF-dependent cell death, which may allow this virus to establish a persistent infection. Here we show that, as an early event during infection, the adenovirus E3-10.4K/14.5K complex selectively induces loss of Fas surface expression and blocks Fas-induced apoptosis of virus-infected cells. Loss of surface Fas occurs within the first 4 h postinfection and is not due to decreased production of Fas protein. The decrease in surface Fas is distinct from the 10.4K/14.5K-mediated loss of the epidermal growth factor receptor on the same cells, because intracellular stores of Fas are not affected. Further, 10.4K/14.5K, which was previously shown to protect against TNF cytolysis, does not induce a loss of TNF receptor, indicating that this complex mediates more than one function to block host defense mechanisms. These results suggest yet another mechanism by which adenovirus modulates host cytotoxic responses that may contribute to persistent infection by human adenoviruses.

Generation of cytotoxic T lymphocytes against immunorecessive epitopes after multiple immunizations with adenovirus vectors is dependent on haplotype.

Currently, adenovirus (Ad) is being considered as a vector for the treatment of cystic fibrosis as well as other diseases. However, the cytotoxic T lymphocyte (CTL) response to Ad could limit the effectiveness of such approaches. Since the CTL response to virus infection is often focused on one or a few immunodominant epitopes, one approach to circumvent this response is to create vectors that lack these immunodominant epitopes. The effectiveness of this approach was tested by immunizing mice with human group C adenoviruses. Three mouse strains (C57BL/10SnJ [H-2b], C3HeB/FeJ [H-2k], and BALB/cByJ [H-2d]) were immunized with wild-type Ad or Ad vectors lacking the immunodominant antigen(s), and the CTL responses were measured. In C57BL/10 (B10) mice, a single inoculation intraperitoneally (i.p.) led to the recognition of an immunodominant antigen in E1A. When B10 mice were inoculated multiple times either i.p. or intranasally with wild-type Ad or an Ad vector lacking most of the E1 region, subdominant epitopes outside this region were recognized. In contrast, C3H mice inoculated with wild-type Ad recognized an epitope mapping within E1B. When inoculated twice with Ad vectors lacking both E1A and E1B, no immunorecessive epitopes were recognized. The immune response to Ad in BALB/c mice was more complex. CTLs from BALB/c mice inoculated i.p. with wild-type Ad recognized E1B in the context of the major histocompatibility complex (MHC) class I Dd allele and a region outside E1 associated with the Kd allele. When BALB/c mice were inoculated with E1-deleted Ad vectors, only the immunodominant Kd-restricted epitope was recognized, and Dd-restricted CTLs did not develop. This report indicates that the emergence of CTLs against immunorecessive epitopes following multiple administrations of Ad vectors lacking immunodominant antigens is dependent on haplotype and could present an obstacle to gene therapy in an MHC-diverse human population.

Characterization of factors involved in modulating persistence of transgene expression from recombinant adenovirus in the mouse lung.

One potential limitation of adenovirus (Ad)-based vectors for the gene therapy of cystic fibrosis (CF) and other genetic diseases is the transience of expression observed in most in vivo systems. In this study, the influence of various factors on persistence of transgene expression in the lung was investigated. In the absence of immune pressure, such as in the nude mouse, the genomic structure of the vector was found to be predominant in determining the persistence of expression; Ad vector constructs with an E1-E3+E4ORF6+ backbone encoding beta-galactosidase (beta-Gal) or the cystic fibrosis transmembrane conductance regulator (CFTR) produced declining levels of expression while an Ad/CMV beta Gal vector with an E1-E3+E4+ backbone gave rise to sustained, long-term reporter gene expression. The ability of the latter vector to persist was in turn limited in part by the presence of cytotoxic T lymphocytes (CTLs). Adoptive transfer experiments indicated that CTLs directed against either viral proteins or the beta-Gal reporter gene product were able to reduce expression in nude C57BL/6 mice stably expressing beta-Gal from the E4+ vector. Finally, the specificity and strength of the CTL response elicited by Ad vector was found to vary considerably depending on mouse strain haplotype. These results indicate that persistence of transgene expression in a given system is determined by the interplay between several factors including genomic structure of the vector, host background, and immune response.

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.

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

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

Activation of intracellular proteases is an early event in TNF-induced apoptosis.

The serine protease inhibitor tosyl-argenine methyl ester inhibits TNF-induced apoptosis, suggesting that proteolysis is necessary for this response. To test this hypothesis, we asked whether protein fragmentation occurs during the death of C3HA fibroblasts, a 3T3-like cell that was rendered sensitive to TNF by cycloheximide. Our results show that the binding of fluorescamine, which binds primary amines, was increased in apoptotic cells by approximately 50%. We also found that 10-15% of the protein in apoptotic cells was no longer precipitable by TCA. Evidence for proteolysis was also revealed by SDS-PAGE analysis and from Western blots. We observed fragmentation and/or degradation of lamin B, topoisomerase I, histone H1, protein kinase C beta 1, and cPLA2, indicating that proteolysis during apoptosis is non-specific. We also found evidence of proteolysis in C3HA cells sensitized to TNF by the adenovirus dl758 (which lacks the E3 14.7-kDa resistance gene) suggesting that protease activation is common in TNF-induced apoptosis. In contrast, the adenovirus E3 14.7-kDa resistance gene prevented proteolysis suggesting that this protein acts at, or upstream of the proteases activated in this response. Finally, because tosyl-argenine methyl ester inhibits the release of [3H]arachidonic acid from apoptotic cells, we tested whether proteolysis of cPLA2 is necessary for enzyme activation. Our results failed, however, to reveal a common proteolytic fragment in different cell types, and when tested in vitro the cytosol from apoptotic cells had less cPLA2 activity. It is unlikely, therefore, that proteolysis is necessary for the activation of this enzyme during TNF-induced apoptosis.

Cytolysis of adenovirus-infected murine fibroblasts by IFN-gamma-primed macrophages is TNF- and contact-dependent.

The effect of interferon-gamma (IFN-gamma) priming on macrophages for cytolysis of adenovirus-infected murine fibroblasts was examined using peritoneal macrophages and the RAW264.7 (RAW) murine macrophage cell line. Adenovirus-infected cells were lysed by IFN-gamma-primed RAW macrophages via a TNF- and contact-dependent mechanism under conditions in which little or no soluble TNF was detected in the supernatant of these effectors. TNF involvement in the lytic mechanism of IFN-gamma-primed macrophages is shown by (a) cytolysis of TNF-sensitive LM and adenovirus E1A-expressing cells, (b) protection from cytolysis by the adenovirus E3-14.7K protein and the E3-10.4/14.5K complex of proteins, and (c) inhibition of cytolysis when neutralizing anti-TNF serum is added to cocultures of macrophages and susceptible adenovirus-infected targets. Physical separation of effectors and targets prevents cytolysis, indicating that cell contact is required. Nonetheless, IFN-gamma-primed RAW macrophages are unable to lyse E8 tumor cells, which are killed by fully activated (triggered) macrophages. These findings indicate that IFN-gamma-primed macrophages are cytolytic for TNF-sensitive targets without soluble TNF release, but they lack the full cytolytic capacity of LPS-triggered macrophages.

Regulation of TNF-mediated cell death and inflammation by human adenoviruses.

Human adenoviruses are among a growing number of human, animal, and even plant viruses that encode gene products that interfere with the defense mechanisms of the host. The host mechanisms most often affected by these viral products are the innate and inflammatory responses such as interferon and the proinflammatory cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF). This review discusses examples of viral anti-immune mechanisms from many different viruses and then focuses on the molecular interactions between adenoviruses and TNF and the effect of these interactions on the ensuing pathogenesis of virus infection.

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.

Characterization of mutants within the gene for the adenovirus E3 14.7-kilodalton protein which prevents cytolysis by tumor necrosis factor.

The 14,700-Da protein (14.7K protein) encoded by the E3 region of adenovirus has previously been shown to protect mouse cells from cytolysis by tumor necrosis factor (TNF). Delineating the sequences in the 14.7K protein that are required for this activity may provide insight into the mechanism of protection from TNF by 14.7K as well as the mechanism of TNF cytolysis. In the present study, we examined the ability of 14.7K mutants to protect cells from lysis by TNF. In-frame deletions as well as Cys-to-Ser mutations in the 14.7K gene were generated by site-directed mutagenesis and then built into the genome of a modified adenovirus type 5 (dl7001) that lacks all E3 genes. dl7001, which replicates to the same titers as does adenovirus type 5 in cultured cells, has the largest E3 deletion analyzed to date. 51Cr release was used to assay TNF cytolysis. Our results indicate that most mutations in the 14.7K gene result in a loss of function, suggesting that nearly the entire protein rather than a specific domain functions to prevent TNF cytolysis.