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Introduction: The early transcription unit 3 (E3) of human adenoviruses (HAdVs) encodes several immunoevasins, including the E3/49K protein, which is unique for species D of HAdVs. It is expressed as surface transmembrane protein and shed. E3/49K of HAdV-D64 binds to the protein tyrosine phosphatase surface receptor CD45, thereby modulating activation of T and NK cells., Methods: Considering that E3/49K represents the most polymorphic viral protein among species D HAdVs, we demonstrate here that all tested E3/49K orthologs bind to the immunologically important regulator CD45. Thus, this feature is conserved regardless of the pathological associations of the respective HAdV types., Results: It appeared that modulation of CD45 is a unique property restricted to HAdVs of species D. Moreover, E3/49K treatment inhibited B cell receptor (BCR) signaling and impaired BCR signal phenotypes. The latter were highly comparable to B cells having defects in the expression of CD45, suggesting E3/49K as a potential tool to investigate CD45 specific functions., Conclusion: We identified B cells as new direct target of E3/49K-mediated immune modulation, representing a novel viral immunosubversive mechanism., Competing Interests: Author MB was employed by Navita S.r.l. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Hildenbrand, Cramer, Bertolotti, Kaiser, Kläsener, Nickel, Reth, Heim, Hengel, Burgert and Ruzsics.)

Human adenoviruses (HAdVs) are widespread pathogens that generally cause mild infections in immunocompetent individuals but severe or even fatal diseases in immunocompromised patients. In order to counteract the host immune defenses, HAdVs encode various immunomodulatory proteins in the early transcription unit 3 (E3). The E3/49K protein is a highly glycosylated type I transmembrane protein uniquely expressed by species D HAdVs. Its N-terminal ectodomain sec49K is released by metalloprotease-mediated shedding at the cell surface and binds to the receptor-like protein tyrosine phosphatase CD45, a critical regulator of leukocyte activation and functions. It remained elusive which domains of CD45 and E3/49K are involved in the interaction and whether such an interaction can also occur on the cell surface with membrane-anchored full-length E3/49K. Here, we show that the two extracellular domains R1 and R2 of E3/49K bind to the same site in the domain d3 of CD45. This interaction enforces the dimerization of CD45, causing the inhibition of T cell receptor signaling. Intriguingly, the membrane-anchored E3/49K appears to be designed like a "molecular fishing rod" using an extended disordered region of E3/49K as a "fishing line" to bridge the distance between the plasma membrane of infected cells and the CD45 binding site on T cells to effectively position the domains R1 and R2 as baits for CD45 binding. This design strongly suggests that both secreted sec49K as well as membrane-anchored full-length E3/49K have immunomodulatory functions. The forced dimerization of CD45 may be applied as a therapeutic strategy in chronic inflammatory disorders and cancer. IMPORTANCE The battle between viruses and their hosts is an ongoing arms race. Whereas the host tries to detect and eliminate the virus, the latter counteracts such antiviral measures to replicate and spread. Adenoviruses have evolved various mechanisms to evade the human immune response. The E3/49K protein of species D adenoviruses mediates the inhibition of immune cell function via binding to the protein tyrosine phosphatase CD45. Here, we show that E3/49K triggers the dimerization of CD45 and thereby inhibits its phosphatase activity. Intriguingly, the membrane-anchored E3/49K seems to be designed like a "molecular fishing rod" with the two CD45 binding domains of E3/49K as baits positioned at the end of an extended disordered region reminiscent of a fishing line. The adenoviral strategy to inhibit CD45 activity by forced dimerization may be used for therapeutic intervention in autoimmune diseases or to prevent graft rejection after transplantation., Competing Interests: The authors declare no conflict of interest.

Recombinant adenovirus (rAd) vectors represent one of the most frequently used vehicles for gene transfer applications in vitro and in vivo . rAd genomes are constructed in Escherichia coli where their genomes can be maintained, propagated, and modified in form of circular plasmids or bacterial artificial chromosomes. Although the rescue of rAds from their circular plasmid or bacmid forms is well established, it works with relatively low primary efficiency, preventing this technology for library applications. To overcome this barrier, we tested a novel strategy for the reconstitution of rAds that utilizes the CRISPR/Cas-machinery to cleave the circular rAd genomes in close proximity to their inverted terminal repeats (ITRs) within the producer cells upon transfection. This CRISPR/Cas-mediated in vivo terminal resolution allowed efficient rescue of vectors derived from different human adenovirus (HAdV) species. By this means, it was not only possible to increase the efficiency of virus rescue by about 50-fold, but the presented methodology appeared also remarkably simpler and faster than traditional rAd reconstitution methods., Competing Interests: AR, JF, H-GB, and ZR are co-inventors in patent application EP20198944 by the Albert Ludwig University of Freiburg that describes the use of CRISPR/Cas-mediated rescue of recombinant adenoviruses from circular plasmids., (Copyright © 2022 Riedl, Fischer, Burgert and Ruzsics.)

Persistent viruses cause chronic disease, and threaten the lives of immunosuppressed individuals. Here, we elucidate a mechanism supporting the persistence of human adenovirus (AdV), a virus that can kill immunosuppressed patients. Cell biological analyses, genetics and chemical interference demonstrate that one of five AdV membrane proteins, the E3-19K glycoprotein specifically triggers the unfolded protein response (UPR) sensor IRE1α in the endoplasmic reticulum (ER), but not other UPR sensors, such as protein kinase R-like ER kinase (PERK) and activating transcription factor 6 (ATF6). The E3-19K lumenal domain activates the IRE1α nuclease, which initiates mRNA splicing of X-box binding protein-1 (XBP1). XBP1s binds to the viral E1A-enhancer/promoter sequence, and boosts E1A transcription, E3-19K levels and lytic infection. Inhibition of IRE1α nuclease interrupts the five components feedforward loop, E1A, E3-19K, IRE1α, XBP1s, E1A enhancer/promoter. This loop sustains persistent infection in the presence of the immune activator interferon, and lytic infection in the absence of interferon.

The E3 transcription unit of human species C adenoviruses (Ads) encodes immunomodulatory proteins that mediate direct protection of infected cells. Recently, we described a novel immunomodulatory function for E3/49K, an E3 protein uniquely expressed by species D Ads. E3/49K of Ad19a/Ad64, a serotype that causes epidemic keratokonjunctivitis, is synthesized as a highly glycosylated type I transmembrane protein that is subsequently cleaved, resulting in secretion of its large ectodomain (sec49K). sec49K binds to CD45 on leukocytes, impairing activation and functions of natural killer cells and T cells. E3/49K is localized in the Golgi/trans-Golgi network (TGN), in the early endosomes, and on the plasma membrane, yet the cellular compartment where E3/49K is cleaved and the protease involved remained elusive. Here we show that TGN-localized E3/49K comprises both newly synthesized and recycled molecules. Full-length E3/49K was not detected in late endosomes/lysosomes, but the C-terminal fragment accumulated in this compartment at late times of infection. Inhibitor studies showed that cleavage occurs in a post-TGN compartment and that lysosomotropic agents enhance secretion. Interestingly, the cytoplasmic tail of E3/49K contains two potential sorting motifs, YXXΦ (where Φ represents a bulky hydrophobic amino acid) and LL, that are important for binding the clathrin adaptor proteins AP-1 and AP-2in vitro Surprisingly, mutating the LL motif, either alone or together with YXXΦ, did not prevent proteolytic processing but increased cell surface expression and secretion. Upon brefeldin A treatment, cell surface expression was rapidly lost, even for mutants lacking all known endocytosis motifs. Together with immunofluorescence data, we propose a model for intracellular E3/49K transport whereby cleavage takes place on the cell surface by matrix metalloproteases., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The E3 transcription unit of human adenoviruses (Ads) encodes immunomodulatory proteins. Interestingly, the size and composition of the E3 region differs considerably among Ad species, suggesting that distinct sets of immunomodulatory E3 proteins may influence their interaction with the human host and the disease pattern. However, to date, only common immune evasion functions of species C E3 proteins have been described. Here we report on the immunomodulatory activity of a species D-specific E3 protein, E3/49K. Unlike all other E3 proteins that act on infected cells, E3/49K seems to target uninfected cells. Initially synthesized as an 80- to 100-kDa type I transmembrane protein, E3/49K is subsequently cleaved, with the large ectodomain (sec49K) secreted. We found that purified sec49K exhibits specific binding to lymphoid cell lines and all primary leukocytes, but not to fibroblasts or epithelial cells. Consistent with this binding profile and the molecular mass, the sec49K receptor was identified as the cell surface protein tyrosine phosphatase CD45. Antibody-blocking studies suggested that sec49K binds to the membrane proximal domains present in all CD45 isoforms. Functional studies showed that sec49K can suppress the activation and cytotoxicity of natural killer cells as well as the activation, signaling, and cytokine production of T cells. Thus, we have discovered an adenovirus protein that is actively secreted and describe immunomodulatory activities of an E3 protein uniquely expressed by a single Ad species.

The human adenovirus E3/19K protein is a type I transmembrane glycoprotein of the endoplasmic reticulum (ER) that abrogates cell surface transport of major histocompatibility complex class I (MHC-I) and MHC-I-related chain A and B (MICA/B) molecules. Previous data suggested that E3/19K comprises two functional modules: a luminal domain for interaction with MHC-I and MICA/B molecules and a dilysine motif in the cytoplasmic tail that confers retrieval from the Golgi apparatus back to the ER. This study was prompted by the unexpected phenotype of an E3/19K molecule that was largely retained intracellularly despite having a mutated ER retrieval motif. To identify additional structural determinants responsible for ER localization, chimeric molecules were generated containing the luminal E3/19K domain and the cytoplasmic and/or transmembrane domain (TMD) of the cell surface protein MHC-I K(d). These chimeras were analyzed for transport, cell surface expression, and impact on MHC-I and MICA/B downregulation. As with the retrieval mutant, replacement of the cytoplasmic tail of E3/19K allowed only limited transport of the chimera to the cell surface. Efficient cell surface expression was achieved only by additionally replacing the TMD of E3/19K with that of MHC-I, suggesting that the E3/19K TMD may confer static ER retention. This was verified by ER retention of an MHC-I K(d) molecule with the TMD replaced by that of E3/19K. Thus, we have identified the E3/19K TMD as a novel functional element that mediates static ER retention, thereby increasing the concentration of E3/19K in the ER. Remarkably, the ER retrieval signal alone, without the E3/19K TMD, did not mediate efficient HLA downregulation, even in the context of infection. This suggests that the TMD is required together with the ER retrieval function to ensure efficient ER localization and transport inhibition of MHC-I and MICA/B molecules.

The adenovirus (Ad) early transcription unit 3 (E3) encodes multiple immunosubversive functions that are presumed to facilitate the establishment and persistence of infection. Indeed, the capacity of E3/19K to inhibit transport of HLA class I (HLA-I) to the cell surface, thereby preventing peptide presentation to CD8(+) T cells, has long been recognized as a paradigm for viral immune evasion. However, HLA-I downregulation has the potential to render Ad-infected cells vulnerable to natural killer (NK) cell recognition. Furthermore, expression of the immediate-early Ad gene E1A is associated with efficient induction of ligands for the key NK cell-activating receptor NKG2D. Here we show that while infection with wild-type Ad enhances synthesis of the NKG2D ligands, major histocompatibility complex class I chain-related proteins A and B (MICA and MICB), their expression on the cell surface is actively suppressed. Both MICA and MICB are retained within the endoplasmic reticulum as immature endoglycosidase H-sensitive forms. By analyzing a range of cell lines and viruses carrying mutated versions of the E3 gene region, E3/19K was identified as the gene responsible for this activity. The structural requirements within E3/19K necessary to sequester MICA/B and HLA-I are similar. In functional assays, deletion of E3/19K rendered Ad-infected cells more sensitive to NK cell recognition. We report the first NK evasion function in the Adenoviridae and describe a novel function for E3/19K. Thus, E3/19K has a dual function: inhibition of T-cell recognition and NK cell activation.

Human enteric adenoviruses propagate poorly in conventional human cell lines used to grow other adenovirus serotypes. As human enteric adenoviruses have a defect in counteracting the cellular interferon (IFN) response in cell culture, to aid in growth of the virus, a 293-based cell line defective in its ability to respond to IFN was constructed. This cell line (293-SV5/V) constitutively expresses V-protein of the paramyxovirus Simian virus 5, which degrades the signal transducer and activator of transcription 1 (STAT1) and thereby prevents the STAT1-mediated IFN response. Analysis of human enteric adenovirus type 40 (HAdV-40)-infected 293-SV5/V cells compared with parental 293 cells shows that the recombinant line allows more rapid production of virus and results in higher titres. These results suggest that the defect in HAdV-40 in counteracting the IFN response can be overcome at least partially through the use of 293-SV5/V cell lines.

Until recently, adenovirus (Ad)-mediated gene therapy was almost exclusively based on human Ad type 5 (Ad5). Preexisting immunity and the limited, coxsackievirus and adenovirus receptor-dependent tropism of Ad5 stimulated attempts to exploit the natural diversity in tropism of the other 50 known human Ad serotypes. Aiming in particular at immunotherapy and vaccination, we have screened representative serotypes from different Ad species for their ability to infect dendritic cells. Ad19a, an Ad from species D, was selected for development as a new vector for vaccination and cancer gene therapy. To clone and manipulate its genome, we have developed a novel methodology, coined "exposon mutagenesis," that allows the rapid and precise introduction of virtually any genetic alteration (deletions, point mutations, or insertions) into recombinant Ad bacterial artificial chromosomes. The versatility of the system was exemplified by deleting the E3 region of Ad19a, by specifically knocking out expression of a species-specific E3 gene, E3/49K, and by reinserting E3/49K into an E3 null Ad19a mutant. The technology requires only limited sequence information and is applicable to other Ad species. Therefore, it should be extremely valuable for the analysis of gene functions from any Ad species. In addition, a basic, replication-defective E1- and E3-deleted Ad19a vector expressing GFP (Ad19aGFP) was generated. This new vector based on species D Ads exhibits a very promising tropism for lymphoid and muscle cells and shows great potential as an alternative vector for transduction of cell types that are resistant to or only poorly transduced by conventional Ad5-based vectors.

Until recently, adenovirus-based gene therapy has been almost exclusively based on human adenovirus serotype 5 (Ad5). The aim of this study was to systematically compare the efficiency of transduction of primary muscle cells from various species by two adenoviral vectors from subgroups C and D. Transduction of a panel of myoblasts demonstrated a striking specificity of an Ad19a-based replication-defective E1-deleted vector (Ad19aEGFP) for human cells, whereas the Ad5-based vector had high affinity for nonhuman primate myoblasts. Transgene expression correlated well with cell-associated vector genomes. Up to 6.59% of the initially applied Ad19aEGFP vector particles were taken up by human myoblasts, as compared with 0.1% of the corresponding Ad5 vector. Remarkably, Ad19aEGFP but not Ad5EGFP efficiently transduced differentiated human myotubes, an in vitro model for skeletal muscle transduction. Uptake of Ad19aEGFP vector particles in human myotubes was 12-fold more efficient than that of Ad5EGFP. Moreover, both vectors demonstrated an early block at the level of vector uptake in mouse myoblasts and rat L6 cells. Investigation of the underlying mechanism for binding and uptake of the two vectors by human myoblasts showed high susceptibility for Ad19a to neuraminidase and wheat germ agglutinin (WGA) lectin, whereas Ad5-mediated transduction was dependent on binding to the coxsackie-adenovirus receptor (CAR) and sensitive to soluble RGD peptide and heparin. Our study offers insights into species-dependent factors that determine Ad tropism and, moreover, provides a basis for application of the novel Ad19a-based vector for gene transfer into human skeletal muscle.

The adenovirus (Ad) early transcription unit E3 encodes immunosubversive functions. The E3 transmembrane proteins 10.4 and 14.5 form a complex that down-regulates the epidermal growth factor receptor and apoptosis receptors from the cell surface by diverting them to endosomes/lysosomes for degradation. The latter process protects infected cells from ligand-induced apoptosis. The mechanism by which 10.4-14.5 mediate re-routing remains elusive. We examined the role of putative YXX Phi and dileucine (LL) transport motifs within Ad2 10.4-14.5 for target protein modulation. By generating stable E3 transfectants expressing 10.4-14.5 proteins with alanine substitutions in these motifs, we show that 3 of the 5 motifs are essential for functional activity. Whereas tyrosine 74 in 14.5 appears to be important for efficient 10.4-14.5 interaction, the 122YXX Phi motif in 14.5 and the dileucine motif Leu 87-Leu88 in 10.4 constitute genuine transport motifs: disruption of either motif abolished binding to the cellular adaptor proteins AP-1 and AP-2, as shown by surface plasmon resonance spectroscopy, and caused missorting, dramatically altering cell surface appearance and the intracellular location of viral proteins. Fluorescence-activated cell sorter analysis and immunofluorescence data provide evidence that Tyr122 in 14.5 is essential for rapid endocytosis of the 10.4-14.5 complex, whereas the 10.4LL motif acts down-stream and protects 10.4-14.5 from extensive degradation by rerouting it into a recycling pathway. Infection of primary cells with adenoviruses carrying the relevant point mutations confirmed the crucial role of these transport motifs for down-regulation of Fas, TRAIL-R1, TRAIL-R2, and epidermal growth factor receptor. Thus, two distinct transport motifs present in two proteins synergize for efficient target removal and immune evasion.

A protein of unknown physiological function, called amyloid precursor-like protein 2 (APLP2), forms an association with the murine class I molecule K(d) that is up-regulated by the presence of the adenoviral protein E3/19K. We have extended these findings to show that APLP2 and E3/19K associate preferentially with folded K(d) and not with the open form. APLP2 was detectable at the cell surface, but its surface expression was not up-regulated by the concurrent expression of K(d). Experimental down-regulation of APLP2 expression caused a consistent increase in the surface expression of K(d), indicating that APLP2 normally reduces K(d) surface expression. These data suggest a role for APLP2 in controlling the maturation of major histocompatibility complex class I molecules.

The early transcription unit 3 (E3) of adenoviruses (Ads) encodes immunomodulatory functions. We previously described a novel gene of 49K within the E3 region of Ad19a, an Ad of subgenus D that is similar to Ad8 and Ad37 causes epidemic keratoconjunctivitis (EKC). Interestingly, 49K was reported not to be present in Ad9 and Ad17, other subgenus D Ads not causing EKC. Therefore, we investigated whether 49K is selectively expressed in EKC-causing Ads. Using specific DNA probes, we detect 49K-homologous genes in all subgenus D Ads tested. Moreover, 49K-specific antibodies recognize a high molecular weight protein in cells infected with all subgenus D serotypes irrespective of their ability to cause EKC. Sequencing of several 49K genes reveals a high homology without a distinct feature recognizable for those of EKC-associated Ad strains. Thus, E3/49K is a subgenus D specific E3 protein whose expression does not correlate with the EKC-causing phenotype and thus may rather be implicated in illnesses commonly caused by this subgenus. Interestingly, the 49K sequences of Ad19a and Ad37 are identical. To estimate the extent of the sequence identity between these two viruses, we initially sequenced the right ITR and the hexon. This analysis revealed that the right ITR of Ad19a is identical to Ad37, while the hexon sequence is Ad19p-like. This suggested that the region of identity is much larger and that Ad19a arose by recombination of Ad37 with an Ad19p-like Ad. Further sequencing mapped the crossover within the DNA binding protein. Thus, Ad19a contains a large sequence block ( approximately 13 kb), from the 100K gene to the right ITR, identical to Ad37. The implications of these findings in light of the temporal appearance of the EKC-causing Ad strains are discussed.

The early transcription unit 3 (E3) of human adenoviruses (Ads) encodes proteins with various immunomodulatory functions. Ads from different subgenera differ considerably in their E3 coding capacity, suggesting that distinct sets of immunomodulatory E3 proteins may influence the disease pattern associated with different Ad subgenera. Interestingly, the E3 region of Ads classified in subgenus D, which are often isolated from AIDS patients and have the propensity to cause eye infections, contains a unique gene, named E3/49K, that may encode a protein with a calculated molecular weight of 48,984 that might be implicated in diseases caused by this subgenus. The 49K sequence predicts a highly glycosylated type I transmembrane protein with a short cytoplasmic tail containing two motifs, YXXPhi and LL, potentially involved in targeting the protein to endosomal or lysosomal compartments. Remarkably, the 49K protein is predicted to contain an unusual immunoglobulin-like fold. Here we have characterized the E3/49K protein of Ad type 19a, an Ad of subgenus D which causes epidemic keratoconjunctivitis. E3/49K was synthesized as an 80- to 100-kDa protein, which is unusually large for an E3 protein. In contrast to another early protein, E3/19K, the expression of E3/49K started early but continued throughout the infection cycle. Analysis of the 49K glycosylation revealed that the majority of 49K molecules contained only 12 of the predicted 14 N-glycans. Furthermore, we provide evidence that 49K is O-glycosylated. At steady state, E3/49K was localized in the Golgi-trans-Golgi network and in early endosomes. Interestingly, the 49K protein has a rather short half-life and seems to be proteolytically cleaved. A processing pattern similar to that in the early stages of infection is seen in transfected cells, constitutively expressing 49K in the absence of other Ad proteins. Together, our data provide the first biochemical and cell biological characterization of an unique E3 protein of subgenus D Ads.

Murine cytomegalovirus (MCMV) interferes with the MHC class I pathway of antigen presentation. The type I transmembrane glycoprotein gp40, encoded by the gene m152, retains major histocompatibility complex (MHC) class I complexes in the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC)/cis-Golgi. These MHC class I complexes are stable, show an extended half-life and do not exchange beta(2)-microglobulin, whereas gp40 reaches an endosomal/lysosomal compartment where it subsequently is degraded. The analysis of regions within the viral protein that are essential for MHC class I retention revealed that a secreted form of gp40, lacking the cytoplasmic tail and the transmembrane region, still has the capacity to retain MHC class I complexes. Continuous expression of gp40 is not required for MHC class I retention. Our data indicate that the retention of MHC class I complexes in the ERGIC/cis-Golgi is triggered by gp40 and does not require the further presence of the viral protein.

Amyloid precursor-like protein 2 (APLP2) is a member of a protein family related to the amyloid precursor protein, which is implicated in Alzheimer's disease. Little is known about the physiological function of this protein family. The adenovirus E3/19K protein binds to major histocompatibility complex (MHC) class I antigens in the endoplasmic reticulum, thereby preventing their transport to the cell surface. In cells coexpressing E3/19K and the MHC K(d) molecule, K(d) is associated with E3/19K and two cellular protein species with masses of 100 and 110 kDa, termed p100/110. Interestingly, p100/110 are released from the complex upon the addition of K(d)-binding peptides, suggesting a role for these proteins in peptide transfer to MHC molecules. Here we demonstrate by microsequencing, reactivity with APLP2-specific antibodies, and comparison of biochemical parameters that p100/110 is identical to human APLP2. We further show that the APLP2/K(d) association does not require the physical presence of E3/19K. Thus, APLP2 exhibits an intrinsic affinity for the MHC K(d) molecule. Similar to the binding of MHC molecules to the transporter associated with antigen processing, complex formation between APLP2 and K(d) strictly depends upon the presence of beta(2)-microglobulin. Conditions that prolong the residency of K(d) in the endoplasmic reticulum lead to a profound increase of the association and a drastic reduction of APLP2 transport. Therefore, this unexpected interplay between these unrelated molecules may have implications for both MHC antigen and APLP2 function.

Mouse cytomegalovirus (MCMV) early gene expression interferes with the major histocompatibility complex class I (MHC class I) pathway of antigen presentation. Here we identify a 48 kDa type I transmembrane glycoprotein encoded by the MCMV early gene m06, which tightly binds to properly folded beta2-microglobulin (beta2m)-associated MHC class I molecules in the endoplasmic reticulum (ER). This association is mediated by the lumenal/transmembrane part of the protein. gp48-MHC class I complexes are transported out of the ER, pass the Golgi, but instead of being expressed on the cell surface, they are redirected to the endocytic route and rapidly degraded in a Lamp-1(+) compartment. As a result, m06-expressing cells are impaired in presenting antigenic peptides to CD8(+) T cells. The cytoplasmic tail of gp48 contains two di-leucine motifs. Mutation of the membrane-proximal di-leucine motif of gp48 restored surface expression of MHC class I, while mutation of the distal one had no effect. The results establish a novel viral mechanism for downregulation of MHC class I molecules by directly binding surface-destined MHC complexes and exploiting the cellular di-leucine sorting machinery for lysosomal degradation.

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.

The early transcription unit 3 (E3) of human adenoviruses encodes proteins which appear to subvert host defense mechanisms. For example, the E3/19K protein inhibits the transport of major histocompatibility complex (MHC) class I molecules to the cell surface and thereby prevents cell lysis by cytotoxic T cells. Tumor necrosis factor alpha (TNF) stimulates expression of MHC molecules on the cell surface of normal cells but not of E3(+) cells, rather, a further reduction of MHC expression is evident. This was attributed to the increased expression of E3/19K upon TNF treatment, an effect also observed for other E3 proteins. We investigated the mechanism of the TNF-mediated up-regulation of E3 products. We show that TNF stimulates expression of a luciferase reporter gene driven by the E3 promoter. Mutation of individual transcription factor binding sites within the E3 promoter reveals the importance of the NF-kappaB binding site kappa2 for TNF inducibility. Electrophoretic mobility shift assays using antibodies directed against various members of the NF-kappaB family demonstrate that stimulation by TNF is mediated by the p50-p65 NF-kappaB complex. TNF inducibility does not depend on coexpression of E1A and can be observed during infection. Interestingly, the E3 promoter seems to be the only early promoter responsive to TNF and the only adenovirus promoter containing an NF-kappaB site. The implications of this regulatory mechanism for the adenovirus life cycle and its pathogenesis are discussed.

Early region 3 (E3) of human adenoviruses (Ads) codes for proteins that appear to control viral interactions with the host. For example, the most abundant E3 protein, E3/19K, inhibits the transport of newly synthesized class I major histocompatibility molecules to the cell surface, thereby interfering with antigen presentation. So far, the E3 regions of Ad subgroups A, B, C, and F have been characterized. We have cloned the E3A region of Ad type 19a (Ad19a), which belongs to the largest subgroup, D, and causes epidemic keratoconjunctivitis in humans. The sequence reveals five open reading frames (ORFs) with the potential to encode the Ad19 equivalent of pVIII, as well as proteins 12.2K, 16.2K, and 18.6K. The last ORF predicts a novel 49K protein which has no counterpart in other subgroups. Both the sequence and the overall organization of the E3 region from Ad19a shows a closer relationship to group B than to group C Ads. The 18.6K ORF represents the Ad19 homolog of the Ad2 E3/19K protein. By using 293 cells stably transfected with the Adl9a E3A region, we showed by immunoprecipitation, pulse-chase experiments, and fluorescence-activated cell sorter analysis that the Ad19 E3/19K protein binds to and prevents the transport of major histocompatibility complex molecules to the cell surface. The similar but distinct functional activity of the Ad19 E3/19K protein, combined with the new sequence which differs from those of subgroup B and C proteins, allows a more precise definition of amino acids essential for HLA binding.

We have recently shown that the accumulation of diverse viral and cellular membrane proteins in the ER activates the higher eukaryotic transcription factor NF-kappaB. This defined a novel ER-nuclear signal transduction pathway, which is distinct from the previously described unfolded protein response (UPR). The well characterized UPR pathway is activated by the presence of un- or malfolded proteins in the ER. In contrast, the ER stress signal which activates the NF-kappaB pathway is not known. Here we used the adenovirus early region protein E3/19K as a model to investigate the nature of the NF-kappaB-activating signal emitted by the ER. E3/19K resides in the endoplasmic reticulum where it binds to MHC class I molecules, thereby preventing their transport to the cell surface. It is maintained in the ER by a retention signal sequence in its carboxy terminus, which causes the protein to be continuously retrieved to the ER from post-ER compartments. Mutation of this sequence allows E3/19K to reach the cell surface. We show here that expression of E3/19K potently activates a functional NF-kappaB transcription factor. The activated NF-kappaB complexes contained p50/p65 and p50/c-rel heterodimers. E3/19K interaction with MHC class I was not important for NF-kappaB activation since mutant proteins which no longer bind MHC molecules remained fully capable of inducing NF-kappaB. However, activation of both NF-kappaB DNA binding and kappaB-dependent transactivation relied on E3/19K ER retention: mutants, which were expressed on the cell surface, could no longer activate the transcription factor. This identifies the NF-kappaB-activating signal as the accumulation of proteins in the ER membrane, a condition we have termed "ER overload." We show that ER overload-mediated NF-kappaB activation but not TNF-stimulated NF-kappaB induction can be inhibited by the intracellular Ca2+ chelator TMB-8. Moreover, treatment of cells with two inhibitors of the ER-resident Ca(2+) -dependent ATPase, thapsigargin and cyclopiazonic acid, which causes a rapid release of Ca2+ from the ER, strongly activated NF-kappaB. We therefore propose that ER overload activates NF-kappaB by causing Ca2+ release from the ER. Because NF-kappaB plays a key role in mounting an immune response, ER overload caused by viral proteins may constitute a simple antiviral response with broad specificity.

The E3/19K protein of human adenovirus type 2 is a resident transmembrane glycoprotein of the endoplasmic reticulum. Its capacity to associate with class I histocompatibility (MHC) antigens abrogates cell surface expression and the antigen presentation function of MHC antigens. At present, it is unclear exactly which structure of the E3/19K protein mediates binding to MHC molecules. Apart from a stretch of approximately 20 conserved amino acids in front of the transmembrane segment, E3/19K molecules from different adenovirus subgroups (B and C) share little homology. Remarkably, the majority of cysteines are conserved. In this report, we examined the importance of cysteine residues for the structure and function of E3/19K. We show that E3/19K contains intramolecular disulfide bonds. By using site-directed mutagenesis, individual cysteines were replaced by serines and mutant proteins were stably expressed in 293 cells. On the basis of the differential binding of monoclonal antibody Tw1.3 and cyanogen bromide cleavage experiments, a structural model of E3/19K is proposed, in which Cys-11 and Cys-28 as well as Cys-22 and Cys-83 are linked by disulfide bonds. Both disulfide bonds (all four cysteines) are absolutely critical for the interaction with human MHC antigens. This was demonstrated by three criteria: loss of E3/19K coprecipitation, lack of transport inhibition, and normal cell surface expression of MHC molecules. Mutation of the three other cysteines had no effect. This indicates that a conformational determinant based on two disulfide bonds is crucial for the function of the E3/19K molecule, namely, to bind and to inhibit transport of MHC antigens.

Adenoviruses of subgroup C can establish persistent infections in human beings. The exact site of persistence has not been established, but lymphoid tissues are certainly one reservoir. Experimental evidence suggests that early transcription unit 3 (E3) of the virus is involved in this phenomenon. In particular, the most abundant protein of this region, the E3/19K protein, seems to fulfill an important role in viral escape from the immune response. We previously demonstrated that in nonlymphoid cells E3/19K interferes with the antigen presentation function of class I major histocompatibility complex (MHC) antigens by inhibiting their transport to the cell surface. However, the function of the E3 products in lymphoid cells was not investigated. To examine this, the T-lymphoma cell line Jurkat was transfected with a DNA fragment comprising the entire E3 region of adenovirus type 2. We show here that E3/19K is expressed in the absence of the viral transactivator E1A with a rate of biosynthesis similar to that in nonlymphoid 293 cells. Furthermore, inhibition of transport and down-regulation of MHC antigens was comparable in both cell lines. In contrast, various T-cell molecules containing immunoglobulin-like domains showed a normal expression pattern in the transfected cells. A detailed analysis of the interaction between E3/19K and the MHC class I antigens of Jurkat (HLA-A3 and HLA-B35) revealed a differential sensitivity for down-regulation by E3/19K. The data demonstrate that E3/19K exerts its function also in lymphoid cells without affecting other lymphoid cell surface molecules. The implications for persistence of adenovirus in lymphoid cells in vivo are discussed.

Assembly of histocompatibility class I antigens (MHC) with beta 2-microglobulin (beta 2m) and peptide takes place in the rough endoplasmic reticulum (ER). At present, it is unclear why peptides generated in the cytosol or ER by proteolysis are not further degraded. Also, it is an open question whether assembly and/or peptide binding is self-instructive or is promoted by additional molecules, for example, chaperones. We previously demonstrated that the adenovirus glycoprotein E3/19K binds to human and some mouse MHC molecules in the ER, interfering with their transport to the cell surface. Here we show that immunoprecipitates from human cells that express transfected E3/19K and murine MHC Kd molecules not only contain MHC heavy chain, beta 2m and E3/19K but also two additional proteins with apparent molecular weights of 100 kDa and 110 kDa. Biochemical characterization of these proteins, designated p100 and p110, demonstrates that they are transmembrane glycoproteins with a similar if not identical protein backbone. Consistent with a role as chaperones, we find that glucose starvation induces complex formation between p100/110 and MHC-E3/19K. Most interestingly, p100 and p110 are displaced from the complex by addition of Kd-specific peptides. Therefore, p100 and p110 might be chaperones that promote correct folding of MHC antigens and/or peptide binding to MHC.

Human adenovirus (Ad) can cause persistent infections in humans. Early region 3 (E3) of the virus appears to be implicated in this phenomenon. This transcription unit encodes proteins that interfere in various ways with host cell functions, including (i) cell-surface expression of histocompatibility class I antigens (HLA), (ii) cell-surface expression of the epidermal growth factor receptor (EGF-R), and (iii) the biological activity of tumor necrosis factor alpha (TNF-alpha). We transfected the human cell line 293 with the entire E3 region of Ad2 and investigated the influence of the cytokines TNF-alpha and interferon gamma (IFN-gamma) on cell-surface expression of HLA class I and the EGF-R. Whereas IFN-gamma treatment induced expression of HLA to some extent but not that of the EGF-R, TNF-alpha treatment augmented the reduction of these cell-surface molecules. Subsequent studies on the mechanism of this effect showed a TNF-alpha-dependent upregulation of E3 protein (E3/19K) and mRNA. The significance of this phenomenon was confirmed in infection experiments. A dramatic increase in the amount of E3/19K, even after short induction with low doses of TNF-alpha could be demonstrated. The study provides evidence for an interaction between the immune system and Ad in which the virus takes advantage of an immune mediator to escape immunosurveillance of the host.

We have previously expressed in transgenic mice a chimeric H-2Kd/Kk protein called C31, which contains the extracellular alpha 1 domain of Kd, whereas the rest of the molecule is of Kk origin. This molecule functions as a restriction element for alloreactive and influenza A-specific cytotoxic T lymphocytes (CTL) but is only weakly expressed at the cell surface of splenocytes. Here, we show that the low cell surface expression is the result of slow intracellular transport and processing of the C31 protein. A set of hybrid molecules between Kd and Kk were used to localize the regions in major histocompatibility complex (MHC) molecules that are important for their intracellular transport and to further localize the structures responsible for binding to the adenovirus 2 E3/19K protein. This protein appears to be an important mediator of adenovirus persistence. It acts by binding to the immaturely glycosylated forms of MHC class I proteins in the endoplasmic reticulum (ER), preventing their passage to the cell surface and thereby reducing the recognition of infected cells by virus-specific T cells. We find the surprising result that intracellular transport and E3/19K binding are controlled primarily by the first half of the second domain of Kd, thus localizing these phenomena to the five polymorphic residues in this region of the Kd protein. This result implies that the E3/19K protein may act by inhibiting peptide binding or by disrupting the oligomerization of MHC class I molecules required for transport out of the ER. Alternatively, the E3/19K protein may inhibit the function of a positively acting transport molecule necessary for cell surface expression of MHC class I molecules.

Clonal deletion plays a major part in the maintenance of natural self-tolerance in both normal and transgenic mice. Self antigens that are expressed in the thymus result in the physical elimination of autoreactive thymocytes at a particular stage in their development. For example, the majority V beta 6- and V beta 8.1-bearing T cells that recognize the minor lymphocyte-stimulating antigen, Mls-1a (ref. 10) , are clonally deleted in the thymuses of normal mice and transgenic mice expressing Mls-1a (refs 2, 3, 9). In contrast, a very different mechanism of tolerance involving the functional inactivation, but not elimination, of autoreactive cells, termed clonal inactivation or clonal anergy, has been implicated in some experimentally manipulated systems of tolerance. To test further the mechanisms involved in self-tolerance, we have generated transgenic mice expressing a V beta 8.1 beta chain on greater than 95% of peripheral T cells and have tested tolerance to Mls-1a in these mice. Surprisingly, a significant fraction of the CD4+ peripheral cells that survived deletion were non-responsive in vitro to any stimulus tested. Naturally occurring tolerance to a self antigen expressed in the thymus can thus be mediated by clonal anergy, as well as by clonal deletion.

Tolerance to IE molecules leads to deletion of V beta 17a-bearing T cells. Both, the CD4+ as well as the CD8+ T cell subsets are affected. A large percentage of CD4+ V beta 17a+ T cell hybrids recognize IE molecules. We now have investigated the reactivity for IE antigens of CD8+ V beta 17a+ T cell hybrids. Using a transfection approach, we have introduced the murine CD8 molecule into different V beta 17a+ T cell hybrids. Furthermore, the CD8 cDNA was transfected into the BW5147 alpha-beta- fusion partner. This allowed us to generate a large number of V beta 17a+ T cell hybrids by fusion with the appropriate T cells. Only 6% of T cell hybrids were stimulated to produce IL-2 upon incubation with IE+ cells. However, in those, the CD8 molecule seemed not to contribute to the IE reactivity of the hybrid, since mAbs against the CD8 molecule failed to inhibit their reactivity. This low percentage of V beta 17a+ CD8+ IE-reactive T cell hybrids contrasts with the strong reduction of CD8+ V beta 17a+ T cells in IE+ mice, strongly suggesting that elimination of such cells in the thymus occurs when they are coexpressing CD4 and CD8. This view was confirmed by the occasional expression of CD4 in some hybrids in which case IE reactivity was detected. Furthermore, we demonstrated the functional integrity of the introduced CD8 molecule by: (a) reconstitution of the IL-2 response in a class I-restricted TNP-specific T cell hybrid; and (b) by generation of alloreactive class I-restricted T cell hybrids using the new CD8+ fusion cell line. This CD8+ fusion partner, BWLyt2-4, should prove useful to study antigen processing and antigen presentation requirements of class I-restricted T cells.

The E3/19K protein of human adenovirus type 2 binds to HLA class I antigens and blocks their terminal glycosylation and cell surface expression. The nature of this interaction is non-covalent and involves neither disulfide bridges between the two molecules nor their carbohydrates. The murine H-2 Kd antigen associates with the E3/19K protein in a similar fashion to human HLA antigens whereas the allelic product H-2 Kk does not. Hybrid genes between the Kd and Kk alleles were constructed and their products were expressed in embryonic kidney cells together with the E3/19K protein. This allowed us to identify the alpha 1 and alpha 2 domains as the essential structures of the histocompatibility antigens for binding the viral protein. Interestingly, these domains are also crucial for T cell recognition. The implications for the evolution of adenoviruses and their ability to cause persistent infections are discussed.

Hybrid genes were constructed for the localization of allodeterminants on murine class I antigens recognized by antibodies and cytolytic T lymphocytes. By using deletion subclones of the H-2Kd and H-2Kk genes, homologous regions were exchanged between the two alleles. The altered genes were introduced and expressed in mouse fibroblast and fibrosarcoma cells. Cells expressing hybrid antigens were analyzed with 29 monoclonal anti-H-2Kd and anti-H-2Kk antibodies and with 150 short-term alloreactive cytolytic T-cell clones. When only the first or only the second amino-terminal domain was exchanged, most T cells and 60% of the antibodies lost their reactivity to the H-2K antigen. No T-cell clone was directed against the third extracellular domain, whereas three antibodies could bind to this domain. This implies that nearly all determinants essential for a cytolytic T-cell response or for antibody binding lie on the two external domains and are conformational structures generated by the interaction of these two domains.

The E3 19,000-dalton protein termed "E3/19K" of adenovirus type 2 binds to human class I histocompatibility antigens (HLA antigens). Human 293.12 cultured cells that express a cloned gene for the E3/19K protein show reduced levels of HLA antigens on the cell surface compared to parental 293 cells. We have transfected these cell lines with plasmid DNA containing the murine histocompatibility H-2Kd allele to demonstrate that this antigen binds also to the E3/19K protein. The resulting association prevents the H-2Kd antigen from being terminally glycosylated and inhibits its cell-surface expression. Two murine cytolytic T-lymphocyte clones specific for HLA antigens and restricted by the H-2Kd antigen lyse the human 293Kd cells. In the presence of the E3/19K protein, a dramatically reduced cell surface density of both HLA and H-2Kd antigens was shown. This decreased amount of cell-surface HLA/H-2Kd antigens correlated with a reduction in susceptibility to lysis of the target cells. In particular, the cell-surface level of the H-2Kd antigen, which is the restricting element, was crucial for efficient lysis. Thus, the E3/19K protein of adenovirus type 2 indirectly reduces the cellular immune recognition in the in vitro system. This might be the mechanism involved in latent and persistent infections caused by adenoviruses in vivo.

We have determined the DNA sequence of the H-2Kk gene of the mouse major histocompatibility complex (MHC). Comparison on the nucleotide and protein level of three H-2K alleles (Kk, Kb and Kd) reveals a high degree of homology, in particular between the Kb and Kk alleles. Differences between the two latter antigens are almost exclusively confined to the alpha 1 and alpha 2 domains. At nine positions in the extracellular part of the molecules we have found allele-specific amino acids. Interestingly, 78% of these residues are either polar or carry hydroxyl-groups. This makes it likely that they are exposed on the surface of the molecules and might then be part of antigenic determinants. We have also identified potentially allele-specific nucleotide sequences of the K genes which might be used as specific DNA probes.