A protein serologically and functionally related to the group C E3 14,700-kilodalton protein is found in multiple adenovirus serotypes.

A 14.7-kilodalton protein (14.7K protein) encoded by the E3 region of group C adenoviruses has been shown to protect virus-infected fibroblasts from lysis by tumor necrosis factor (TNF) (L.R. Gooding, L.W. Elmore, A.E. Tollefson, H.A. Brady, and W.S.M. Wold, Cell 53:341-346, 1988). In this study we show that adenoviruses of other groups are also protected from TNF-induced cytolysis. Representative serotypes of groups A, B, D, and E produce a protein analogous to the 14.7K protein found in human group C adenoviruses. Deletion of this protein in group C viruses permits virus infection to induce cellular susceptibility to TNF killing. As with group C adenoviruses, cells infected with wild-type adenoviruses of other serotypes are not killed by TNF and are protected from lysis induced by TNF plus cycloheximide. However, cells are susceptible to TNF-induced lysis when infected with adenovirus type 4 mutants from which the 14.7K gene has been deleted. Although all known adenovirus serotypes infect epithelial cells, adenoviruses cause several diseases with various degrees of pathogenesis. Our findings suggest that the 14.7K protein provides a function required for the in vivo cytotoxicity of many adenoviruses independent of the site of infection or degree of pathogenesis.     

Resistance of human cells to the adenovirus E3 effect on class I MHC antigen expression. Implications for antiviral immunity

Group C human adenovirus (Ad) serotypes (e.g., Ad2 and Ad5) cause persistent infections in man. One proposed mechanisms to explain human adenovirus persistence is an ineffective CTL response due to reduced cell surface expression of class I MHC Ag on virally infected cells, an effect mediated by the 19-kDa glycoprotein encoded by Ad early region 3 (E3). In the present study, the generality of this phenomenon was tested by analyzing E3 19-kDa glycoprotein down-regulation of cell surface class 1 MHC Ag on a variety of human cell types. With the exception of the Ad5 early region 1 (E1) transformed cell line, 293, Ad2/5 infection of fibroblastic, epithelial, and lymphoid cells did not cause major decreases in surface class I Ag until the terminal stages of infection when cell death is imminent. Furthermore, newly synthesized class I Ag continued to be surface expressed on most cell types at times when infected cells contained large amounts of Ad E3 19-kDa glycoprotein. These data indicate that most types of human cells are resistant to the E3 19-kDa glycoprotein effect, suggesting that virus-specific CTL recognition and lysis of most Ad2/5-infected human cells should not be limited by E3 19-kDa-mediated reduction in class I MHC Ag expression.     

Cutting edge: adenovirus E19 has two mechanisms for affecting class I MHC expression

Viral strategies for immune evasion include inhibition of various steps in the class I MHC assembly pathway. Here, we demonstrate that adenovirus produces one gene product with a dual function in this regard. It is well established that adenovirus E19 binds class I molecules and retains them in the endoplasmic reticulum (ER). However, E19 also delays the expression of class I alleles to which it cannot tightly bind. Here, we show that E19 binds TAP and acts as a tapasin inhibitor, preventing class I/TAP association. DeltaE19, an E19 mutant lacking the ER-retention signal, delays maturation of class I molecules, indicating that E19's inhibition of class I/TAP interaction is sufficient to delay class I expression. These data identify tapasin inhibition as a novel mechanism of viral immune evasion and suggest that, through this secondary mechanism, adenovirus can affect Ag presentation by MHC alleles that it can only weakly affect by direct retention.     

Mouse anti-adenovirus cytotoxic T lymphocytes. Inhibition of lysis by E3 gp19K but not E3 14.7K

Early region E3 of adenovirus (Ad) appears to encode proteins involved in the interaction of the virus with the host immune system. The E3 region 19-kDa glycoprotein (gp19K) binds to class I MHC Ag in the endoplasmic reticulum and inhibits their transport to the cell surface; it has been proposed that this protects virus infected cells from lysis by CTL. We have found that the E3 14.7-kDa protein (14.7K) inhibits lysis of infected cells by TNF, and here we show that it also protects cells from lysis by lymphotoxin, which has been implicated as a mediator of CTL lysis. We have developed a method for producing CTL specific for human Ad2 and Ad5 in mice, in order to test directly which of the genes in the E3 region protect infected cells from lysis by virus specific CTL. The presence of the E3 region inhibits both the induction of Ad-specific CTL in culture and the lysis of infected target cells by these CTL. The inhibition varies between different mouse strains, with almost complete inhibition in C57BL/10 (H-2b) mice, partial inhibition with BALB/c (H-2d) and little or no inhibition with C3H (H-2k); results were similar for Ad2 and Ad5. By using a panel of E3 deletion mutants, inhibition of target cell lysis by Ad5 specific CTL was mapped exclusively to the gp19K gene. The 14.7K gene had no effect on CTL lysis despite its ability to protect cells against lysis by lymphotoxin. gp19K was synthesized abundantly in mouse cells by mutants retaining the gp19K gene; some mutant forms of the protein were synthesized but were nonfunctional. These data support the hypothesis that gp19K can protect Ad infected cells against lysis by virus specific CTL.     

Characterization of a major histocompatibility complex class I antigen-binding glycoprotein from adenovirus type 35, a type associated with immunocompromised hosts

Adenovirus type 35 (Ad35) is a group B adenovirus that has been isolated primarily from patients with acquired immunodeficiency syndrome and other immunodeficiency disorders. We have studied the interaction of this unique adenovirus with the immune system by analyzing Ad35 early viral proteins in infected HeLa cells. We have identified a 29,000-Mr Ad35 early glycoprotein, E29, which associates with class I antigens of the major histocompatibility complex (MHC) in the endoplasmic reticulum. Ad35 E29 is analogous to the group C Ad2 early glycoprotein E3-19K (E19), which has been shown to interfere with the expression of class I antigens on the cell surface (H. Burgert and S. Kvist, Cell 41:987-997, 1985). In contrast to the Ad2 glycoprotein, Ad35 E29 was synthesized in much smaller amounts, was more extensively glycosylated, and did not cross-react with polyclonal antibody against the Ad2 protein. As a control, a class I antigen-binding glycoprotein from another group B adenovirus, Ad7, was also characterized and was found to have properties similar to those of Ad35 E29. Therefore, the differences in the glycosylation and quantity of class I antigen-binding glycoproteins between Ad35 and Ad2 are group related. Inhibition of the expression of MHC class I antigens, which are needed for cytotoxic-T-lymphocyte recognition of virus-infected cells, appears to play a vital role in the adenovirus life cycle in vivo. Our data indicate that this function has been conserved despite significant differences in the MHC class I antigen-binding glycoprotein and in the pathogenicity between serotypes.     

Role of the adenovirus E3-19k conserved region in binding major histocompatibility complex class I molecules.

The adenovirus early region 3 glycoprotein E3-19k binds to and down regulates major histocompatibility complex (MHC) class I molecules in infected cells. We previously identified a 20-amino-acid conserved region in E3-19k by comparison of protein sequences from four different adenovirus serotypes. The roles of the E3-19k C-terminal and adjacent conserved regions in the interaction with MHC class I molecules have been examined. A functional class I-binding glycoprotein was expressed from the cloned E3 18.5-kDa open reading frame of adenovirus type 35. Truncations and single-amino-acid mutations in the adenovirus type 35 glycoprotein were created by site-directed in vitro mutagenesis and tested for the ability to associate with MHC class I molecules. Deletion of most of the transmembrane domain and cytoplasmic tail did not affect binding to class I molecules. However, removal of an additional 11 amino acids eliminated binding and changed the conformation of the adjacent conserved region. Separate mutations of residues Asp-107 and Met-110, within the conserved region, severely reduced or eliminated binding. These data indicate that the E3-19k conserved region plays a crucial role in binding to MHC class I molecules.     

Adenovirus E3-19K proteins of different serotypes and subgroups have similar, yet distinct, immunomodulatory functions toward major histocompatibility class I molecules

Our understanding of the mechanism by which the E3-19K protein from adenovirus (Ad) targets major histocompatibility complex (MHC) class I molecules for retention in the endoplasmic reticulum is derived largely from studies of Ad serotype 2 (subgroup C). It is not well understood to what extent observations on the Ad2 E3-19K/MHC I association can be generalized to E3-19K proteins of other serotypes and subgroups. The low levels of amino acid sequence homology between E3-19K proteins suggest that these proteins are likely to manifest distinct MHC I binding properties. This information is important as the E3-19K/MHC I interaction is thought to play a critical role in enabling Ads to cause persistent infections. Here, we characterized interaction between E3-19K proteins of serotypes 7 and 35 (subgroup B), 5 (subgroup C), 37 (subgroup D), and 4 (subgroup E) and a panel of HLA-A, -B, and -C molecules using native gel, surface plasmon resonance (SPR), and flow cytometry. Results show that all E3-19K proteins exhibited allele specificity toward HLA-A and -B molecules; this was less evident for Ad37 E3-19K. The allele specificity for HLA-A molecules was remarkably similar for different serotypes of subgroup B as well as subgroup C. Interestingly, all E3-19K proteins characterized also exhibited MHC I locus specificity. Importantly, we show that Lys(91) in the conserved region of Ad2 E3-19K targets the C terminus of the α2-helix (MHC residue 177) on MHC class I molecules. From our data, we propose a model of interaction between E3-19K and MHC class I molecules.     

IFN increases class I MHC antigen expression on adenovirus-infected human cells without inducing resistance to natural killer cell killing.

It has been previously shown that unstimulated NK cells cannot preferentially lyse adenovirus serotypes 2 and 5-infected human cells. In this study, the ability of IFN to promote the selective NK cell-mediated lysis of adenovirus-infected human cells was determined. The relationship between target cell susceptibility to NK cell-mediated killing and class I Ag expression was also analyzed through the use of adenovirus serotype 2 and 5 mutants that do not make the adenovirus early region 3 19-kDa class I binding protein. IFN induced the selective lysis of adenovirus serotype 2 and 5-infected human cells by activating NK cells (IFN-alpha) and protecting uninfected, but not adenovirus-infected cells, from NK cell-mediated lysis (IFN-gamma). IFN-gamma increased the expression of class I Ag on the surface of cells infected with the adenovirus early region 3 deletion mutants, dl327 or dl801, to a level equal to or greater than that expressed on uninfected cells. Despite the increased expression of class I Ag, IFN-gamma could not protect these adenovirus-infected cells from NK cell-mediated lysis. Thus, dl327 or dl801 infection prevented IFN-gamma's induction of cytolytic resistance to NK cell-mediated killing but left IFN-gamma's induction of class I Ag intact. Surface class I Ag levels were substantially higher on IFN-gamma-treated, dl327-, and dl801-infected cells in comparison to cells infected with wild type adenovirus serotype 5. Again, higher target cell levels of class I Ag did not correlate with increased resistance to NK cell-mediated lysis because there was equivalent NK cell-mediated killing of IFN-gamma-treated adenovirus serotype 5-, dl327-, or dl801-infected cells. Thus, IFN-gamma only protects uninfected cells from NK cell-mediated killing, irrespective of target class I Ag levels, and thereby concentrates NK lytic activity on just adenovirus-infected cells. These data demonstrate that IFN-gamma's ability to protect target cells from NK cell-mediated cytolysis is unrelated to IFN-gamma's induction of surface class I MHC Ag.     

Processing and presentation of murine cytomegalovirus pORFm164-derived peptide in fibroblasts in the face of all viral immunosubversive early gene functions

CD8 T cells are the principal effector cells in the resolution of acute murine cytomegalovirus (mCMV) infection in host organs. This undoubted antiviral and protective in vivo function of CD8 T cells appeared to be inconsistent with immunosubversive strategies of the virus effected by early (E)-phase genes m04, m06, and m152. The so-called immune evasion proteins gp34, gp48, and gp37/40, respectively, were found to interfere with peptide presentation at different steps in the major histocompatibility complex (MHC) class I pathway of antigen processing and presentation in fibroblasts. Accordingly, they were proposed to prevent recognition and lysis of infected fibroblasts by cytolytic T lymphocytes (CTL) during the E phase of viral gene expression. We document here that the previously identified MHC class I D(d)-restricted antigenic peptide (257)AGPPRYSRI(265) encoded by gene m164 is processed as well as presented for recognition by m164-specific CTL during the E and late phases of viral replication in the very same cells in which the immunosubversive viral proteins are effectual in preventing the presentation of processed immediate-early 1 (m123-exon 4) peptide (168)YPHFMPTNL(176). Thus, while immunosubversion is a reality, these mechanisms are apparently not as efficient as the term immune evasion implies. The pORFm164-derived peptide is the first noted peptide that constitutively escapes the immunosubversive viral functions. The most important consequence is that even the concerted action of all immunosubversive E-phase proteins eventually fails to prevent immune recognition in the E phase. The bottom-line message is that there exists no immune evasion of mCMV in fibroblasts.     

Cytotoxic T-Lymphocyte Target Proteins and Their Major Histocompatibility Complex Class I Restriction in Response to Adenovirus Vectors Delivered to Mouse Liver

The activation of cytotoxic T lymphocytes (CTLs) to cells infected with adenovirus vectors contributes to problems of inflammation and transient gene expression that attend their use in gene therapy. The goal of this study was to identify in a murine model of liver gene therapy the proteins that provide targets to CTLs and to characterize the major histocompatibility complex (MHC) class I restricting elements. Mice of different MHC haplotypes were infected with an E1-deleted adenovirus expressing human alkaline phosphatase (ALP) or β-galactosidase as a reporter protein, and splenocytes were harvested for in vitro CTL assays to aid in the characterization of CTL epitopes. A library of vaccinia viruses was created to express individual viral open reading frames, as well as the ALP and lacZ transgenes. The MHC haplotype had a dramatic impact on the distribution of CTL targets: in C57BL/6 mice, the hexon protein presented by both H-2K^b and H2D^b was dominant, and in C3H mice, H-2D^k-restricted presentation of ALP was dominant. Adoptive transfer of CTLs specific for various adenovirus proteins or transgene products into either Rag-I or C3H-scid mice infected previously with an E1-deleted adenovirus verified the in vivo relevance of the adenovirus-specific CTL targets identified in vitro. The results of these experiments illustrate the impact of lr gene control on the response to gene therapy with adenovirus vectors and suggest that the efficacy of therapy with adenovirus vectors may exhibit considerable heterogeneity when applied in human populations.

A prerequisite for successful human gene therapy is the development of efficient and safe transfer technologies. Recombinant adenovirus vectors have several features which make them attractive vehicles for gene delivery. They transduce a wide variety of cell types, do not require host cell proliferation for gene transfer, and are able to transduce cells in vivo (6, 17, 19, 20, 25). The adenovirus genome is comprised of early and late genes; expression of the former leads to the activation of a cascade which culminates in the formation of new virions (9). First-generation recombinant adenoviruses used in gene therapy have been rendered replication defective by deletion of the E1A and E1B genes.Enthusiasm for the use of recombinant adenoviruses with deletions of the E1 genes in gene therapy has been diminished by the observation that deletion of E1 sequences is insufficient to completely ablate expression of other early and late viral genes. Previous studies revealed that the block in replication achieved by deleting E1 can be overcome in vitro with high multiplicities of infection (MOIs) or through cellular factors with E1-like function (10, 24). Expression of viral genes in infected-host cells leads to direct toxicity or to the stimulation of adenovirus-specific, major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTLs) which can contribute to the loss of transgene expression (31, 33, 34). The antigenic potential of reporter proteins as well as therapeutic proteins in models of gene replacement therapy is another potential problem. A few recent reports have highlighted the role of the transgene product in inducing destructive cellular immune responses (28, 32).The capsid proteins of adenovirus vectors stimulate CD4⁺ T helper cells which recognize antigenic peptides in association with MHC class II determinants. Both T_H1 and T_H2 subsets are activated, the former contributing to the CTL response by augmenting the stimulation of CD8⁺ T cells as well as by increasing expression of MHC class I on the target cell via gamma interferon (36).The antigenic targets recognized by MHC class I-restricted CTLs have been extensively studied for a variety of viruses including influenza virus, vesicular stomatitis virus, and lymphocytic choriomeningitis virus. Epitopes within internal regulatory proteins, as well as integral membrane proteins, have been identified as targets for CTL-mediated destruction of virus-infected cells (1, 2, 29, 37, 38). For adenovirus, however, little is known about the antigen specificity of the CTL response. Studies with replication-competent, wild-type adenovirus revealed E1A encoded within the E1 locus as a strong immunodominant antigen (15, 18). These data, however, are not relevant to the use of adenoviruses in gene therapy when E1A and E1B had been deleted.In this study, a library of recombinant vaccinia viruses expressing viral regulatory and structural genes as well as two reporter genes was used to precisely identify the targets within E1-deleted recombinant adenoviruses which elicit CD8⁺ T-cell responses. Experiments were performed in four strains of inbred mice, three of which had different mouse H-2 haplotypes. This study revealed that the level of CTL response to adenovirus antigens or the transgene product is dependent on the MHC haplotype of the host.     

An adenovirus type 2 glycoprotein blocks cell surface expression of human histocompatibility class I antigens

The adenovirus type 2 encoded protein E3/19K binds to human histocompatibility class I antigens (HLA). This association occurs both in adenovirus-infected cells and in cells that have been transfected with the gene encoding the E3/19K protein. The formation of the HLA-E3/19K complex prevents the HLA antigens from being correctly processed by inhibiting their terminal glycosylation. This effect is specific for HLA antigens and does not generally involve the glycosyltransferases. Furthermore, the HLA-E3/19K association dramatically reduces the cell surface expression of the HLA antigens. This reduced level of antigens might influence the cytotoxic T cell response. Therefore, our results show a possible molecular mechanism whereby adenoviruses, and perhaps other viruses, delay or escape the cellular immune system of the host.     

The E3/19K protein of adenovirus type 2 binds to the domains of histocompatibility antigens required for CTL recognition

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.     

Cowpox virus evades CTL recognition and inhibits the intracellular transport of MHC class I molecules

Orthopoxviruses evade host immune responses by using a number of strategies, including decoy chemokine receptors, regulation of apoptosis, and evasion of complement-mediated lysis. Different from other poxviral subfamilies, however, orthopoxviruses are not known to evade recognition by CTL. In fact, vaccinia virus (VV) is used as a vaccine against smallpox and a vector for eliciting strong T cell responses to foreign Ags. and both human and mouse T cells are readily stimulated by VV-infected APC in vitro. Surprisingly, however, CD8(+) T cells of mice infected with cowpox virus (CPV) or VV recognized APC infected with VV but not APC infected with CPV. Likewise, CD8(+) T cells from vaccinated human subjects could not be activated by CPV-infected targets and CPV prevented the recognition of VV-infected APC upon coinfection. Because CD8(+) T cells recognize viral peptides presented by MHC class I (MHC I), we examined surface expression, total levels, and intracellular maturation of MHC I in CPV- and VV-infected human and mouse cells. Although total levels of MHC I were unchanged, CPV reduced surface levels and inhibited the intracellular transport of MHC I early during infection. CPV did not prevent peptide loading of MHC I but completely inhibited MHC I exit from the endoplasmic reticulum. Because this inhibition was independent of viral replication, we conclude that an early gene product of CPV abrogates MHC I trafficking, thus rendering CPV-infected cells "invisible" to T cells. The absence of this immune evasion mechanism in VV likely limits virulence without compromising immunogenicity.     

Retrovirus-induced changes in major histocompatibility complex antigen expression influence susceptibility to lysis by cytotoxic T lymphocytes

Retrovirus infection of murine fibroblasts was found to alter the expression of major histocompatibility complex (MHC) antigens. Fibroblasts infected with Moloney murine leukemia virus (M-MuLV) exhibited up to a 10-fold increase in cell surface expression of all three class I MHC antigens. Increases in MHC expression resulted in the increased susceptibility of M-MuLV-infected cells to lysis by allospecific cytotoxic T lymphocytes (CTL). M-MuLV appears to exert its effect at the genomic level, because mRNA specific for class I antigens, as well as beta 2-microglobulin, show a fourfold increase. Fibroblasts infected with the Moloney sarcoma virus (MSV):M-MuLV complex show no increase in MHC antigen expression or class I mRNA synthesis, suggesting that co-infection with MSV inhibits M-MuLV enhancement of MHC gene expression. Quantitative differences in class I antigen expression on virus-infected cells were also found to influence the susceptibility of infected cells to lysis by H-2-restricted, virus-specific CTL. Differential lysis of infected cells expressing varied levels of class I antigens by M-MuLV-specific bulk CTL populations and CTL clones suggests that individual clones may have different quantitative requirements for class I antigen expression. The MSV inhibition of MHC expression could be reversed by interferon-gamma. Treatment of MSV:M-MuLV-infected fibroblasts with interferon-gamma increased their susceptibility to lysis by both allogeneic and syngeneic CTL. The data suggest that interferon-gamma may function in the host's immune response to viral infections by enhancing MHC antigen expression, thereby increasing the susceptibility of virus-infected cells to lysis by H-2-restricted, virus-specific CTL.