Peter Tomasec

Viral evasion of natural killer cells during human cytomegalovirus infection

Cytotoxic T cells are major players in the immune defence against human cytomegalovirus (HCMV). The virus has, however, developed several mechanisms to escape from this control. In particular, it down-regulates cell surface expression of HLA class I molecules. Because natural killer (NK) cells recognize and eliminate cells that lack HLA class I molecules, HCMV-infected cells could be more susceptible to NK lysis. In this review, we discuss the role played by NK cells in immune defence against HCMV and we describe potential strategies the virus has developed to escape from NK cell-mediated lysis. We focus in particular on a newly described protein, HCMV gpUL40, that induces cell surface expression of HLA-E, a non-classical class I molecule known to regulate NK cell functions.

Use of recombinant herpes simplex virus type 1 vectors for gene transfer into tumour and normal anterior pituitary cells

In this paper we demonstrate the use of recombinant viral vectors derived from herpes simplex virus type 1 (HSV1) to transfer reporter genes in vitro into rat anterior pituitary cells grown in primary cultures and the anterior pituitary tumour cell lines GH3 and AtT20. The three vectors used were, tsK/beta-galactosidase (beta-gal), tsK/CRH and tsK/TIMP, the corresponding transgene products respectively being E. coli beta-gal, pre-procorticotropin releasing hormone (ppCRH), and the chimeric protein TIMP/Thy1 (tissue inhibitor of metalloproteinases (TIMP)/linked to the carboxy terminus of Thy1 which confers the addition of a glycolipid glycosyl-phosphatidylinositol anchor in the ER). Double labelling immunofluorescence experiments to detect reporter proteins and transduced cell types indicated that the three vectors could transfer and express the reporter genes in normal and tumour anterior pituitary cells. Virus infection of pituitary cells was characterised, and it was shown that infection with tsK/beta-gal at multiplicities of infection (MOI)=10, 100% of tumour and non-endocrine anterior pituitary cells expressed beta-gal, whereas 75% endocrine anterior pituitary cells expressed the transgene. Long-term expression studies after infection with tsK/beta-gal indicated that anterior pituitary cells in primary cultures expressed the transgene for significant longer periods than tumour anterior pituitary cells. Growth arrest by serum starvation markedly decreased the frequency of transgene expression in anterior pituitary cells following infection with tsK/beta-gal. Transgenic products expressed from tsK were targeted to their correct intracellular domain in both anterior pituitary cells in primary cultures and in pituitary tumour cell lines. We conclude that transgenes can be delivered into anterior pituitary cells in primary culture and pituitary tumour cell lines using tsK derived HSV1 vectors. The prospect of employing viral vectors to transfer genes into endocrine cells opens up the potential exploration of various molecular aspects of pituitary cell function both in vitro and in vivo, as well as the use of gene transfer into the pituitary for potentially therapeutic applications, such as the treatment of pituitary tumours.

Mitogenic effects and nuclear localisation of procorticotrophin-releasing hormone expressed within stably transfected fibroblast cells (CHO-K1)

To investigate the intracellular localisation and biological activity of procorticotrophin-releasing hormone (proCRH), we have established stably transfected CHO-K1 cells expressing the rat pre-proCRH cDNA. Using immunoblot analysis of cell lysates of transfected CHO-K1 cells, we detected a major CRH immunoreactive band with an apparent molecular weight of approximately 19 kDa. This 19 kDa band could account for full length proCRH molecule which has not undergone post-translational modifications. Metabolic labelling followed by immunoprecipitation, SDS-PAGE and autoradiography indicated that no endoproteolytic processing of proCRH takes place within the transfected CHO-K1 cells. Immunofluorescence staining localises the CRH precursor to both the cytoplasm and to the nucleus in transfected CHO-K1 cells. This result was confirmed using subcellular fractionation techniques on radiolabelled CHO-K1 cells expressing immunoreactive CRH. A major CRH-immunoreactive band of 19 kDa was detected both in the microsomal and secreted fractions, indicating the presence of proCRH within the secretory pathway of these cells. This was also evident in the nuclear fraction, therefore confirming the nuclear localisation of proCRH. Analysis of DNA concentration, cell number and DNA synthesis showed that stably transfected CHO-K1 cells expressing proCRH have a higher proliferation and DNA synthesis rate than wildtype CHO-K1 cells or CHO-K1 cells transfected with pEE14 alone. Our results therefore suggest a mitogenic role for the intact proCRH molecule within CHO-K1 cells. Furthermore, treatment of mouse corticotrophic tumour cells (AtT20/D16-16) with conditioned medium from transfected CHO-K1 cells expressing proCRH, stimulated both DNA synthesis and cell proliferation above basal levels. Our results constitute the first reported direct evidence of a mitogenic role for proCRH acting on a corticotrophic cell population.

Simultaneous detection of amplicon and HSV-1 helper encoded proteins reveals that neurons and astrocytoma cells do express amplicon-borne transgenes in the absence of synthesis of virus immediate early proteins

HSV-1 amplicon vectors were used to express either a cytoplasmic (beta-galactosidase) or a membrane targeted protein (TIMP-Thy1) in primary neuronal cultures, and a human astrocytoma cell line. Whereas some cells became infected by vector particles alone others were simultaneously infected by both vector and helper particles. Our results show that IEHCMV and HSV-1 IE3 promoters are able to direct transgene expression in these cells in the absence of synthesis of helper virus transacting proteins, and stress the need of monitoring expression from both partners of an amplicon population, in order to differentiate transgene expression in cells singly infected with amplicon particles, from those infected by both amplicon and helper particles.

Expression of biologically active procorticotrophin-releasing hormone (proCRH) in stably transfected CHO-K1 cells: characterization of nuclear proCRH.

Corticotrophin‐releasing hormone (CRH) is a 41 amino acid neuropeptide which is cleaved at a pair of dibasic amino acids from a larger precursor molecule (pre‐proCRH) by the action of endopeptidases. In cells possessing a regulated secretory pathway, sorting of proneuropeptides and prohormones occurs within the trans‐Golgi network, where they are finally packaged into secretory vesicles to be released in response to an external stimulus. Such cells also possess a constitutive secretory pathway, and neuropeptides are also translocated into this subcellular compartment. We have recently established stably transfected CHO‐K1 cells expressing the rat pre‐proCRH cDNA, and shown that proCRH was localized within the secretory pathway and the nucleus of transfected cells. Both the cytoplasmic and nuclear species of IR‐CRH displayed an apparent molecular weight of approximately 19 kDa, consistent with the size of the uncleaved CRH precursor molecule.

Generation of a Recombinant Herpes Simplex Virus Type 1 Expressing the Rat Corticotropin- Releasing Hormone Precursor: Endoproteolytic Processing, Intracellular Targeting and Biological Activity

We describe the generation of a recombinant herpes simplex virus type 1 (HSV1) vector, tsK/CRH10, derived from the temperature-sensitive mutant tsK, expressing rat pre-procorticotropin-releasing hormone (ppCRH). In hypothalamic neurons, within the paraventricular and supraoptic nuclei, this neuropeptide precursor is processed to mature CRH (1–41), the key modulator of the hypothalamic-pituitary-adrenal stress response. We used the recombinant HSV1 tsK/CRH10 to study posttranslational processing, intracellular localization and biological activity of proCRH (pCRH) within neuronal, glial and epithelial cell lines. We showed that CRH-like immunoreactivity expressed in neuronal, glial and epithelial cells infected with tsK/CRH10 was biologically active, could be detected intracellularly and was also secreted. Our data also show that within Neuro2a and NG115 cells, the CRH precursor is cleaved to yield a CRH-like immunoreactive fragment of approximately 4.75 kD which could account for mature CRH (1–41). No endoproteolytic processing of the precursor takes place within the astrocytic 1321 NI cell line. Using immunocytochemistry techniques we detected CRH-like immunoreactivity within the endoplasmic reticulum-Golgi region in all cells and within secretory vesicles of Neuro2a and NG115 cells, suggesting correct targeting to the regulated secretory pathway within these cells. Our results demonstrate that the HSV1 recombinant vector expressing the full-length CRH precursor molecule constitutes an excellent delivery system for both cell lines and postmitotic neurons in vitro, which has enabled the study of targeting, endoproteolytic processing and biological activity of this neuropeptide precursor. Furthermore, it can also be used to generate transient transgenesis of the CRH precursor in vivo, to study neuroendocrine-immune interactions within the mammalian central nervous system.

Synaptogenesis and distribution of presynaptic axonal varicosities in low density primary cultures of neocortex: an immunocytochemical study utilizing synaptic vesicle-specific antibodies, and an electrophysiological examination utilizing whole cell recording

SummaryLow-density primary cultures of neocortical neurons were utilized to examine: (i) early interactions of growing neurites with morphological characteristics of axons with other neuronal elements, and (ii) the distribution of presynaptic axonal varicosities closely apposed to MAP-2 immunoreactive, putatively postsynaptic, dendrites. At the light microscopical level axonal varicosites, presumably presynaptic terminals, were identified using immunocytochemistry incorporating antibodies specific for the synaptic vesicle antigens synaptophysin and synapsin. The presence of synaptophysin- and synapsin-immunoreactive swellings along axonal processes was first detected at 5 days post-plating and was also apparent in axons growing in isolation. At 5–7 daysin vitro, immunolabelled axonal varicosities in close apposition to putative postsynaptic dendrites (MAP-2 immunoreactive) dendrites were detected. Electrophysiologically active synaptic contacts can also readily be detected at this stage. After 3 weeksin vitro presynaptic contacts do appear to be distributed heterogeneously along postsynaptic dendrites of many neurons in culture. As the culture matures a higher number of presynaptic profiles can be seen along dendrites, with a centrifugal distribution, e.g. a higher density of presynaptic axonal terminals in close apposition to more distal regions of larger dendrites, putatively considered to be apical dendrites of pyramidal-like neurons. In our cultures, the overall increase in the density and the pattern of distribution of presynaptic axon terminals immunoreactive for synaptic vesicle antigens closely apposed to putative post-synaptic structures mimics the general postnatal increase of synaptic density in the neocortexin vivo. Thus, low density primary cultures of neocortical neurons offer a valuable system to explore and manipulate (i) the molecular and cellular basis of neocortical synaptogenesis, and (ii) the pharmacology of neocortical synaptic transmission.

Generation and characterization of an antiserum reactive with a proteolytic processing site within rat procorticotrophin-releasing hormone

In this paper we report the generation of an antibody specific for the cleavage site within procorticotrophin-releasing hormone (proCRH) at the N-terminus proCRH/CRH (1-41) junction. Using radioimmunoassay techniques were show that the antibody generated (781) cross-reacts specifically with the proCRH (137-150) Tyr fragment, corresponding to the cleavage site within the full length precursor molecule. The anti-cleavage site antibody does not crossreact with the endoproteolytic products originated from the CRH precursor molecule, i.e. CRH (1-41) or proCRH (125-151) or with any of the CRH-immunoreactive fragments tested i.e. CRH (36-41), CRH (1-20) and CRH (30-41). It also shows no cross-reactivity with CRH-related substances from other species, i.e. urotensin I (fish) and sauvagine (frog). The cleavage site antibody (781), recognizes the full length proCRH molecule in Western blotting and in liquid phase radioimmunoassay from transfected CHO-K1 cells expressing the full length pre-proCRH cDNA. Using immunofluorescence and immunoprecipitation techniques followed by SDS-PAGE and autoradiography, we confirm the presence of the intact CRH precursor molecule within the nucleus and the cytoplasm of stably transfected CHO-K1 cells expressing immunoreactive proCRH. The immunofluorescence studies using primary cultures of hypothalamic neurons, show that immunoreactive (IR) proCRH is localized within the perinuclear region and was also seen along the neuronal processes where it accumulates at their tips. Our results, therefore, show that this antibody will be an invaluable tool in the study of intracellular trafficking in relation to the endoproteolytic processing of the CRH precursor molecule.

Co-localisation of autoimmune antibodies specific for double stranded DNA with procorticotrophin-releasing hormone within the nucleus of stably transfected CHO-K1 cells

Human autoantibodies and corticotrophin-releasing hormone (CRH)-specific antibodies have been used in a double-labelling immunofluorescence technique to demonstrate that immunoreactive CRH structures are co-localised with immunostaining produced by double stranded DNA-specific human autoantibodies within the nucleus of cultured ovarian cells of Chinese hamsters (CHO-K1). This co-localisation was confirmed using confocal microscopy. A metabolic labelling technique was used to investigate the role of the cytoskeleton in mediating nuclear translocation of proCRH within stably transfected CHO-K1 cells and showed that microtubule and actin disrupting agents had no effect upon the nuclear translocation of proCRH. These results, therefore, suggest that nuclear translocation of proCRH is not affected by drugs which disrupt the cytoskeleton and, consequently, modify the diameter of the nuclear pores.

Intracellular Retention of the Corticotrophin-releasing Hormone (CRH) Precursor Within COS-7 Cells

We investigated the intracellular localization of CRH in transiently transfected COS-7 cells expressing the full-length rat corticotropin-releasing hormone (CRH) precursor cDNA. CRH synthesized by transfected COS-7 cells is mainly stored intracellularly. In contrast, CHO-K1 cells expressing the same CRH precursor stored and released equal amounts of immunoreactive (IR)-CRH. Ultrastructural analysis revealed that CRH is stored in electron-dense aggregates in the RER of transiently transfected COS-7 cells and does not migrate into the Golgi apparatus. On the basis of the different intracellular localization, storage, and release of CRH in COS-7 and CHO-K1 cells, we hypothesize that the intracellular trafficking of CRH within the constitutive secretory pathway for protein secretion not only depends on its primary amino acid sequence but might also be influenced by intracellular conditions or factors.