Transcriptional regulation of the hepatitis B virus large surface antigen gene

Hepatitis B virus (HBV) is a hepatotropic virus of highly restricted host range and tissue specificity. Although the mechanisms governing this tropism are not fully understood, it is likely that restrictions occur at multiple steps in the viral life cycle. The liver-specific regulation of HBV gene expression suggests that transcription may be an important factor in the hepatotropism of the virus. An analysis of tissue- or cell- line-specific regulation of the HBV promoters may elucidate the role of transcriptional regulation in the hepatotropism of the virus. The major aim of this project was to characterize the transcriptional regulation of the large surface antigen gene of hepatitis B virus. To achieve this, the regions of the HBV genome involved in the regulation of the expression of the large surface antigen gene were identified using a transient transfection system in mammalian cell lines. The transcriptional activities of the four HBV promoters were compared in the human differentiated hepatoma cell lines Hep3B, PLC/PRF/5, HepG2 and Huh7, a human dedifferentiated hepatoma cell line HepG2.1, and the nonhepatoma cell lines HeLa S3 and NIH 3T3. To determine the relative transcriptional activities of the four HBV promoters, reporter gene plasmids were generated such that the expression of the firefly luciferase gene was under the control of each of the HBV promoters in the context of the complete genome. The nucleocapsid promoter and large surface antigen promoter displayed higher relative activities in the differentiated hepatoma cell lines, indicating that these promoters are preferentially active in these cell lines. A series of large surface antigen promoter deletion plasmids were constructed to identify the important regulatory regions of the large surface antigen promoter. The deletion analysis demonstrated that the region responsible for the high relative activity in differentiated hepatoma cell lines is located between -90 and -76 relative to the transcription initiation site (-*■!) located at map position 2809. This sequence element contains the binding site (GTTAATCATTACT) for the liver-enriched transcription factor hepatocyte nuclear factor I, HNF1. A eukaryotic expression vector containing the HNFI cDNA under the control of the mouse metallothionein I promoter was cotransfected with the HBV promoter constructs in Huh7 and HepG2.1 cells, and the relative levels of activity were determined. The Huh7 cell line was used because it is one of the cell lines in which HBV replication and particle production can occur and may represent the tissue culture system closest to the natural environment for the HBV life cycle, the liver cell. The cloned transcription factor HNFI activated transcription from the large surface antigen promoter, but not from any of the other HBV promoters. Cotransfection experiments using the HNFI cDNA expression vector and large surface antigen promoter deletion constructs demonstrated that this transactivation was mediated through the HNFI binding site located between -90 and -76 in the large surface antigen promoter. A series of deletion mutants of the cDNA in the HNFI expression vector was generated to determine the transcriptional activation domain of the HNFI polypeptide. The major domain of the HNFI polypeptide involved in transcriptional activation of the large surface antigen promoter in the human hepatoma cell line HepG2.1 was mapped to a region rich in glutamine and proline residues (9 of 18 residues). To demonstrate directly that the HNFI polypeptide produced by the expression of the HNFI cDNA could bind the large surface antigen promoter HNFI recognition sequence, and to determine whether a protein present in the differentiated hepatoma cell line Huh7 bound the HNFI element, gel mobility shift analysis was performed. This analysis demonstrated that a protein present in nuclear extracts from Huh7 cells formed a specific complex with the HNFI binding site which had similar migration properties to the complex formed between exogenously expressed HNFI and the HNFI recognition sequence. DNase I footprinting analysis demonstrated the binding of a protein present in the differentiated hepatoma cell line Huh7 to the HNFI recognition sequence in the large surface antigen promoter. DNase I footprinting also showed that purified TATA binding protein binds the TATA box element located between -31 and -23 in the large surface antigen promoter. The analysis of synthetic promoter constructs suggested that the HNFI and TATA box elements were the only elements necessary for maximal activity from the large surface antigen promoter, and analysis of clustered point mutations in the large surface antigen minimal promoter region demonstrated that sequences between the HNFI and TATA box elements were not required for the HNF1- dependent activity of the large surface antigen promoter. These studies suggested that the liver-enriched transcription factor HNFI plays a critical role in the cell-line and tissue-specific regulation of the HBV large surface antigen promoter

Regulation of T cell lymphokine production by killer cell inhibitory receptor recognition of self HLA class I alleles.

The killer cell inhibitory receptors (KIRs) are surface glycoproteins expressed by natural killer (NK) and T cells that specifically recognize defined groups of polymorphic human histocompatibility leukocyte antigen (HLA) class I molecules. Interactions between KIRs on NK or T cells and major histocompatibility complex (MHC) class I molecules on potential target cells inhibit cell-mediated cytotoxicity, presumably by delivering a negative signal preventing lymphocyte activation. In this study we examined whether KIRs also regulate cytokine production induced in response to T cell receptor-dependent T cell activation. CD4+ and CD8+ T cell clones were stimulated by bacterial superantigens in the presence or absence of monoclonal antibodies (mAbs) against the KIR NKB1 or MHC class I molecules, and production of tumor necrosis factor alpha and interferon gamma was evaluated. When bacterial superantigen was presented by an autologous antigen-presenting cell (APC) to a KIR+ T cell clone, cytokine production was always enhanced in the presence of anti-MHC class I mAb. Similarly, anti-KIR mAb also augmented cytokine production, provided that the APC expressed a HLA class I allele recognized by the KIR. These results suggest that recognition of autologous MHC class I molecules by KIR+ T cells provides a regulatory mechanism acting to modulate the potency of their responses to antigenic challenge

"Tolerization" of human T-helper cell clones by chronic exposure to alloantigen

Induction of clonal anergy in T-helper (Th) cells may have a role in regulating immune responses. A model system for studying Th cell tolerization at the clonal level in vitro could be useful for investigating the mechanisms involved. Accordingly, alloreactive helper cells were maintained in culture with interleukin 2 (IL 2) by intermittent stimulation with specific antigen. Regardless of the frequency of antigen stimulation, clones of age less than ca. 35 population doublings (PD) were found to undergo antigen-specific autocrine clonal expansion in the absence of exogenous IL 2. Such young clones (designated as phase I) could therefore not be "tolerized" by frequent exposure to antigen. In contrast, most clones of age greater than ca. 35 PD could be tolerized by frequent exposure to antigen (designated as phase II clones). Their autocrine proliferation was then blocked, although they still recognized antigen specifically as shown by their retained ability to secrete interferon-gamma (IFN-gamma) and granulocyte-macrophage colony stimulating factor (GM-CSF). The mechanism of response failure involved both an inability to upregulate IL 2 receptors in the absence of exogenous IL 2, as well as an inability to secrete IL 2. These defects were not overcome by stimulation with mitogens or calcium ionophore and phorbol esther in place of alloantigen. T-cell receptor, alpha, beta, and gamma-chain gene rearrangements remained identical in phase I and phase II clones. Tolerization of phase II clones could be avoided by increasing the period between antigen exposures. Despite this, whether or not phase II cells were capable of autocrine proliferation, they were found to have acquired the novel function of inducing suppressive activity in fresh lymphocytes. Suppressor-induction was blocked by the broadly reactive MHC class II-specific monoclonal antibody (moAb) TU39, but not by moAb preferentially reacting only with HLA-DR, DQ, or DP. Sequential immunoprecipitation on T-cell clones showed the presence of a putative non-DR, DQ, DP, TU39+ molecule on phase II clones. However, this molecule was also found on phase I clones. The nature of the TU39-blockable suppressor-inducing determinant present on phase II but not on (most) phase I clones thus remains to be clarified. In addition to suppressor-induction activity, phase II clones also acquired lytic potential as measured in a lectin approximation system. Cytotoxic (CTX) potential was also not influenced by the frequency of antigenic stimulation and could be viewed as a constitutive modulation of clonal functio

Helper T cells for cytotoxic T lymphocytes need not be I region restricted.

We investigated the antigenic requirements for restimulation of H-2- restricted cytolytic T lymphocytes (CTL) in vitro to determine whether H-2 I region-restricted helper T cells are required in these responses. In one set of experiments, we studied the in vitro response of (responder x nonresponder)F(1) female T cells to the male antigen H-Y. We chose to examine this response because it has been suggested that the defect in nonresponder strains is a failure of helper T cells to recognize H-Y in association with nonresponder I region determinants. However, we find that nonresponder male stimulator cells are as effective as F(1) male stimulator cells at inducing H-Y-specific CTL responses. This finding calls into question reports that secondary CTL responses to H-Y are dependent upon the activation of H-Y- specific helper T cells restricted to responder type I region determinants. In a second set of experiments, we examined the requirements for restimulation of H-2-restricted T cells specific for minor-histocompatibility antigens from long-term mixed lymphocyte cultures. These cultures were established by repeatedly restimulating cultures of specific T cells with H- 2-matched stimulator cells expressing foreign minor histocompatibility antigens. We found that H-2D-restricted T ceils, including CTL, could be restimulated with cells that were matched with the responding cells at only the D region genes. This response did not appear to result from positive allogeneic effects or from antigen processing and representation by responder type APC that might contaminate the cultures. Thus, we find no evidence for a requirement for I region-restricted helper T cells in these CTL responses. However, helper T cells are required because we find that CTL lines derived by limit-dilution cloning from these long-term MLC are absolutely dependent upon exogenous helper factors for growth. The most simple interpretation of these results is that the helper cells are restricted to H-2 antigens other than I region antigens or to antigens that code outside of the H-2 complex. Finally, we show that factor-dependent CTL lines must recognize their specific antigen to proliferate, even in the presence of exogenous factors. The requirement of activated CTL for antigen to proliferate provides an explanation for how specific CTL can be selectively enriched in MLC by specific antigen stimulation. Furthermore, it is at variance with reports that memory CTL or activated CTL require only interleukin 2 for restimulation

Concomitant induction of the cell surface expression of Ia determinants and accessory cell function by a murine macrophage tumor cell line.

This study demonstrates that an uncharacterized soluble factor produced in concanavalin A-induced rat spleen cell suspensions has the capacity to induce the increased expression of cell surface H-2K and H-2D molecules and the expression of I-region gene products on murine monocyte-macrophage lineage tumors that are not Ia positive in the absence of the factor. In parallel with induction of serologically defined Ia specificities, Ia-induced WEHI-3 macrophage tumor cells are capable of providing accessory cell function in stimulating IL-2 production by T-T hybridomas that are activated in a major histocompatibility complex-restricted, antigen-dependent fashion. The uninduced Ia-negative WEHI-3 tumor cells do not trigger a comparable response in this assay system

Varicellovirus UL 49.5 proteins differentially affect the function of the transporter associated with antigen processing, TAP

Cytotoxic T-lymphocytes play an important role in the protection against viral infections, which they detect through the recognition of virus-derived peptides, presented in the context of MHC class I molecules at the surface of the infected cell. The transporter associated with antigen processing (TAP) plays an essential role in MHC class I–restricted antigen presentation, as TAP imports peptides into the ER, where peptide loading of MHC class I molecules takes place. In this study, the UL49.5 proteins of the varicelloviruses bovine herpesvirus 1 (BHV-1), pseudorabies virus (PRV), and equine herpesvirus 1 and 4 (EHV-1 and EHV-4) are characterized as members of a novel class of viral immune evasion proteins. These UL49.5 proteins interfere with MHC class I antigen presentation by blocking the supply of antigenic peptides through inhibition of TAP. BHV-1, PRV, and EHV-1 recombinant viruses lacking UL49.5 no longer interfere with peptide transport. Combined with the observation that the individually expressed UL49.5 proteins block TAP as well, these data indicate that UL49.5 is the viral factor that is both necessary and sufficient to abolish TAP function during productive infection by these viruses. The mechanisms through which the UL49.5 proteins of BHV-1, PRV, EHV-1, and EHV-4 block TAP exhibit surprising diversity. BHV-1 UL49.5 targets TAP for proteasomal degradation, whereas EHV-1 and EHV-4 UL49.5 interfere with the binding of ATP to TAP. In contrast, TAP stability and ATP recruitment are not affected by PRV UL49.5, although it has the capacity to arrest the peptide transporter in a translocation-incompetent state, a property shared with the BHV-1 and EHV-1 UL49.5. Taken together, these results classify the UL49.5 gene products of BHV-1, PRV, EHV-1, and EHV-4 as members of a novel family of viral immune evasion proteins, inhibiting TAP through a variety of mechanisms