Analysis of the Maize Polyubiquitin-1 Promoter Heat Shock Elements and Generation of Promoter Variants with Modified Expression Characteristics

The maize polyubiquitin-1 (Ubi-1) promoter is one of a few select promoters used to express foreign genes in monocots, such that recombinant proteins can be produced at commercially viable levels. Modifying the activity, specificity and responsiveness of such promoters provides a means to achieve desired levels and patterns of expression of genes encoding target products. Ubi-1 is constitutively expressed but is further induced by heat shock. The promoter contains two overlapping sequences with similarity to defined heat shock elements and we show that these sequences are also present upstream of the Ubi-1 homologue isolated from teosinte. Both the maize and teosinte promoters can mediate a heat shock response in transgenic maize. We have dissected the overlapping maize Ubi-1 promoter heat shock elements and demonstrate that the 3' element is required to mediate a heat shock response. The Ubi-1 promoter is particularly active in tissues consisting of rapidly dividing cells, and within the seed it is strongly biased towards driving expression in the embryo. However, replacement of the heat shock elements with a trimer of a basic domain/leucine zipper factor binding site of a pea lectin promoter shifts the balance in seed expression towards the endosperm. The Ubi-1 variants described here differ in their overall activity in the seed, but they all show potential for driving high levels of heterologous gene expression in maize.     

Immunogenetics of the NKG2D ligand gene family

NKG2D ligands (NKG2DLs) are a group of major histocompatibility complex (MHC) class I-like molecules, the expression of which is induced by cellular stresses such as infection, tumorigenesis, heat shock, tissue damage, and DNA damage. They act as a molecular danger signal alerting the immune system for infected or neoplastic cells. Mammals have two families of NKG2DL genes: the MHC-encoded MIC gene family and the ULBP gene family encoded outside the MHC region in most mammals. Rodents such as mice and rats lack the MIC family of ligands. Interestingly, some mammals have NKG2DL-like molecules named MILL that are phylogenetically related to MIC, but do not function as NKG2DLs. In this paper, we review our current knowledge of the MIC, ULBP, and MILL gene families in representative mammalian species and discuss the origin and evolution of the NKG2DL gene family.     

Two Novel Human Cytomegalovirus NK Cell Evasion Functions Target MICA for Lysosomal Degradation

NKG2D plays a major role in controlling immune responses through the regulation of natural killer (NK) cells, αβ and γδ T-cell function. This activating receptor recognizes eight distinct ligands (the MHC Class I polypeptide-related sequences (MIC) A andB, and UL16-binding proteins (ULBP)1–6) induced by cellular stress to promote recognition cells perturbed by malignant transformation or microbial infection. Studies into human cytomegalovirus (HCMV) have aided both the identification and characterization of NKG2D ligands (NKG2DLs). HCMV immediate early (IE) gene up regulates NKGDLs, and we now describe the differential activation of ULBP2 and MICA/B by IE1 and IE2 respectively. Despite activation by IE functions, HCMV effectively suppressed cell surface expression of NKGDLs through both the early and late phases of infection. The immune evasion functions UL16, UL142, and microRNA(miR)-UL112 are known to target NKG2DLs. While infection with a UL16 deletion mutant caused the expected increase in MICB and ULBP2 cell surface expression, deletion of UL142 did not have a similar impact on its target, MICA. We therefore performed a systematic screen of the viral genome to search of addition functions that targeted MICA. US18 and US20 were identified as novel NK cell evasion functions capable of acting independently to promote MICA degradation by lysosomal degradation. The most dramatic effect on MICA expression was achieved when US18 and US20 acted in concert. US18 and US20 are the first members of the US12 gene family to have been assigned a function. The US12 family has 10 members encoded sequentially through US12–US21; a genetic arrangement, which is suggestive of an ‘accordion’ expansion of an ancestral gene in response to a selective pressure. This expansion must have be an ancient event as the whole family is conserved across simian cytomegaloviruses from old world monkeys. The evolutionary benefit bestowed by the combinatorial effect of US18 and US20 on MICA may have contributed to sustaining the US12 gene family.     

The heat shock protein HSP70 promotes mouse NK cell activity against tumors that express inducible NKG2D ligands

The stress-inducible heat shock protein (HSP) 70 is known to function as an endogenous danger signal that can increase the immunogenicity of tumors and induce CTL responses. We show in this study that HSP70 also activates mouse NK cells that recognize stress-inducible NKG2D ligands on tumor cells. Tumor size and the rate of metastases derived from HSP70-overexpressing human melanoma cells were found to be reduced in T and B cell-deficient SCID mice, but not in SCID/beige mice that lack additionally functional NK cells. In the SCID mice with HSP70-overexpressing tumors, NK cells were activated so that they killed ex vivo tumor cells that expressed NKG2D ligands. In the tumors, the MHC class I chain-related (MIC) A and B molecules were found to be expressed. Interestingly, a counter selection was observed against the expression of MICA/B in HSP70-overexpressing tumors compared with control tumors in SCID, but not in SCID/beige mice, suggesting a functional relevance of MICA/B expression. The melanoma cells were found to release exosomes. HSP70-positive exosomes from the HSP70-overexpressing cells, in contrast to HSP70-negative exosomes from the control cells, were able to activate mouse NK cells in vitro to kill YAC-1 cells, which express NKG2D ligands constitutively, or the human melanoma cells, in which MICA/B expression was induced. Thus, HSP70 and inducible NKG2D ligands synergistically promote the activation of mouse NK cells resulting in a reduced tumor growth and suppression of metastatic disease.     

Varicella-zoster virus IE62 protein utilizes the human mediator complex in promoter activation

The varicella-zoster virus (VZV) major transactivator, IE62, is involved in the expression of all kinetic classes of VZV genes and can also activate cellular promoters, promoters from heterologous viruses, and artificial promoters containing only TATA elements. A key component of the mechanism of IE62 transactivation is an acidic activation domain comprising the N-terminal 86 amino acids of IE62. However, the cellular target of this N-terminal acidic activation is unknown. In the work presented here, we show that the IE62 activation domain targets the human Mediator complex via the Med25 (ARC92) subunit and that this interaction appears to be fundamental for transactivation by the IE62 activation domain. In contrast, the Med23 subunit (Sur2/TRAP150beta/DRIP130/CRSP130) of the Mediator complex is not essential for IE62-mediated activation. Further, the IE62 activation domain appears to selectively interact with a form of the Mediator complex lacking CDK8. Chromatin immunoprecipitation experiments showed that IE62 stimulates recruitment of Mediator to an IE62-responsive model promoter. Finally, immunofluorescence microscopy of VZV-infected cells demonstrated intranuclear translocation of the Mediator complex to viral replication compartments. These studies suggest that Mediator is an essential component for efficient VZV gene expression.     

Repression of gene expression upon infection of cells with herpes simplex virus type 1 mutants impaired for immediate-early protein synthesis.

Herpes simplex virus type 1 (HSV-1) mutants defective in immediate-early (IE) gene expression do not readily enter productive replication after infection of tissue culture cells. Instead, their genomes are retained in a quiescent, nonreplicating state in which the production of viral gene products cannot be detected. To investigate the block to virus replication, we used the HSV-1 triple mutant in1820K, which, under appropriate conditions, is effectively devoid of the transactivators VP16 (a virion protein), ICP0, and ICP4 (both IE proteins). Promoters for the HSV-1 IE ICP0 gene or the human cytomegalovirus (HCMV) major IE gene, cloned upstream of the Escherichia coli lacZ coding sequences, were introduced into the in1820K genome. The regulation of these promoters and of the endogenous HSV-1 IE promoters was investigated upon conversion of the virus to a quiescent state. Within 24 h of infection, the ICP0 promoter became much less sensitive to transactivation by VP16 whereas the same element, when used to transform Vero cells, retained its responsiveness. The HCMV IE promoter, which is not activated by VP16, also became less sensitive to the HCMV functional homolog of VP16. Both elements remained available for transactivation by HSV-1 IE proteins at 24 h postinfection, showing that the in1820K genome was not irreversibly inactivated. The promoters controlling the HSV-1 ICP4, ICP22, and ICP27 genes also became essentially unresponsive to transactivation by VP16. The ICP0 promoter was induced when hexamethylene bisacetamide was added to cultures at the time of infection, but the response to this agent was also lost by 24 h after infection. Therefore, promoter elements within the HSV-1 genome are actively repressed in the absence of IE gene expression, and repression is not restricted specifically to HSV-1 IE promoters.     

Expression of the RAE-1 family of stimulatory NK-cell ligands requires activation of the PI3K pathway during viral infection and transformation

Natural killer (NK) cells are lymphocytes that play a major role in the elimination of virally-infected cells and tumor cells. NK cells recognize and target abnormal cells through activation of stimulatory receptors such as NKG2D. NKG2D ligands are self-proteins, which are absent or expressed at low levels on healthy cells but are induced upon cellular stress, transformation, or viral infection. The exact molecular mechanisms driving expression of these ligands remain poorly understood. Here we show that murine cytomegalovirus (MCMV) infection activates the phosphatidylinositol-3-kinase (PI3K) pathway and that this activation is required for the induction of the RAE-1 family of mouse NKG2D ligands. Among the multiple PI3K catalytic subunits, inhibition of the p110α catalytic subunit blocks this induction. Similarly, inhibition of p110α PI3K reduces cell surface expression of RAE-1 on transformed cells. Many viruses manipulate the PI3K pathway, and tumors frequently mutate the p110α oncogene. Thus, our findings suggest that dysregulation of the PI3K pathway is an important signal to induce expression of RAE-1, and this may represent a commonality among various types of cellular stresses that result in the induction of NKG2D ligands.     

Systemic NKG2D down-regulation impairs NK and CD8 T cell responses in vivo

The immunoreceptor NKG2D stimulates activation of cytotoxic lymphocytes upon engagement with MHC class I-related NKG2D ligands of which at least some are expressed inducibly upon exposure to carcinogens, cell stress, or viruses. In this study, we investigated consequences of a persistent NKG2D ligand expression in vivo by using transgenic mice expressing MHC class I chain-related protein A (MICA) under control of the H2-K(b) promoter. Although MICA functions as a potent activating ligand of mouse NKG2D, H2-K(b)-MICA mice appear healthy without aberrations in lymphocyte subsets. However, NKG2D-mediated cytotoxicity of H2-K(b)-MICA NK cells is severely impaired in vitro and in vivo. This deficiency concurs with a pronounced down-regulation of surface NKG2D that is also seen on activated CD8 T cells. As a consequence, H2-K(b)-MICA mice fail to reject MICA-expressing tumors and to mount normal CD8 T cell responses upon Listeria infection emphasizing the importance of NKG2D in immunity against tumors and intracellular infectious agents.     

The human cytomegalovirus 80-kilodalton but not the 72-kilodalton immediate-early protein transactivates heterologous promoters in a TATA box-dependent mechanism and interacts directly with TFIID.

We have asked how the human cytomegalovirus major immediate-early 1 (IE1) and 2 (IE2) proteins act to transactivate heterologous cellular and viral promoters. Here we show that transactivation of the human immunodeficiency virus long terminal repeat and the 70,000-molecular-weight heat shock protein (hsp70) promoter by IE1 is TATA box independent and that the IE1 protein does not interact directly with the TATA box-binding factor TFIID. Conversely, transactivation of these promoters by IE2 is TATA box dependent and a direct interaction between IE2 and TFIID occurs, suggesting that IE2 transactivation is mediated through interaction with TFIID.     

The 89,000-Mr murine cytomegalovirus immediate-early protein activates gene transcription

To study trans-activation of gene expression by murine cytomegalovirus (MCMV) immediate-early (IE) proteins, the IE coding region 1 (ie1), which encodes the 89,000-Mr IE phosphoprotein (pp89), was stably introduced into L cells. A cell line was selected and characterized that efficiently expressed the authentic viral protein. The pp89 that was constitutively expressed in L cells stimulated the expression of transfected recombinant constructs containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of viral promoters. The regulatory function of the ie1 product was confirmed by transient expression assays in which MCMV IE genes were cotransfected into L cells together with recombinant constructs of the CAT gene. For CAT activation by the ie1 product, a promoter region was required, but there was no preferential activation of a herpes simplex virus type 1 delayed-early promoter. All plasmid constructs that contained the intact coding sequences for pp89 induced gene expression in trans. The MCMV enhancer region was not essential for the expression of a functional IE gene product, and testing of the cis-regulatory activity of the MCMV enhancer revealed a low activity in L cells. Another region transcribed at IE times of infection, IE coding region 2, was unable to induce CAT expression and also did not augment the functional activity of ie1 after cotransfection.