4 research outputs found
Characterization of a Membrane-Associated Protein Implicated in Visna Virus Binding and Infection
No full textAbstractThe identity of the cellular receptor(s) for visna virus, an ovine lentivirus, is currently unknown; however, previous studies from our laboratory have identified membrane-associated proteins expressed selectively in susceptible cells which bind visna virus. Moreover, a polyclonal antibody (2-23), raised against a 45-kDa visna virus binding protein, bound specifically to the surface of susceptible cells in immunofluorescence assays and significantly reduced binding of visna virus to cells (S. E. Crane et al., 1991, J. Virol., 65, 6137–6143). In this report we extend our studies of this antibody (2-23), showing both that 2-23 significantly reduces visna virus infection of susceptible cells and that 2-23 immunoprecipitates a putative protein complex consisting of a prominent 30-kDa protein, as well as the 45-kDa immunogen, specifically from radiolabeled virus-susceptible sheep cells. Further, we demonstrate that the 30-kDa protein is a membrane-associated proteoglycan substituted with a chondroitin sulfate glycosaminoglycan (GAG) chain(s) and that treatment of susceptible cells with an inhibitor of GAG synthesis significantly reduces visna virus production. Collectively, these data support a role for a proteoglycan in visna virus cell binding and infection
Involvement of a Membrane-Associated Serine/Threonine Kinase Complex in Cellular Binding of Visna Virus
No full textAbstractPrevious studies from our laboratory identified cellular membrane proteins that mediate binding of visna virus to susceptible cells. In the pilot report, antiserum raised to one of these proteins, ∼45 kDa, was shown to both label the surface of susceptible cells and block the binding of visna virus to cell membranes. In a recent study, we reported that the same antiserum, designated 2-23, significantly inhibited infection by visna virus and specifically immunoprecipitated a membrane-associated protein complex from susceptible cells, comprised of a ∼45- kDa protein, as well as a 30-kDa protein. Because the 30-kDa protein was readily detectable in TRANS[35S]-LABELed susceptible cells, we were able to characterize this protein biochemically, as a chondroitin sulfate proteoglycan. In the present study, we sought to characterize the ∼45-kDa protein and examined 2-23 immune complexes for the presence of kinase activity. Our data indicate that although in vitro kinase assays of 2-23 immunoprecipitates specifically result in the phosphorylation of the ∼45-kDa protein as well as a novel ∼56-kDa protein, only the ∼45-kDa protein exhibits inherent serine/threonine kinase activity. In addition, the kinase activity can be isolated in 2-23 immunoprecipitates of membranes prepared from visna virus-susceptible cells. Finally, in an effort to evaluate the biological relevance of our in vitro observations, we examined 2-23 immunoprecipitates of [32P]orthophosphate-labeled visna-susceptible cells and report that the ∼56-kDa protein is phosphorylated constitutively on serine in vivo. Collectively, these data implicate a serine/threonine kinase complex in the binding/infection of visna virus
Visna Virus-Induced Activation of MAPK Is Required for Virus Replication and Correlates with Virus-Induced Neuropathology
No full textCharacterisation of T cell defects in acute myeloid leukaemia
No full textPhDUnderstanding the immune system in patients with cancer and how it interacts with malignant cells is critical for the development of successful immunotherapeutic strategies at a time when novel cancer treatment approaches are required. Acute myeloid leukaemia (AML) results in widespread interaction between the malignant cells and T cells and as such, offers an opportunity to study these interactions. A flow cytometric analysis of T cells in the peripheral blood of patients presenting with AML illustrated that the absolute number of T cells is increased in AML compared with healthy controls. Furthermore, a large population of CD3+56+ cells was identified. These cells are not natural killer T cells but effector T cells that may represent a failing immunosurveillance mechanism. Two technical issues were explored: how to separate T cells from the peripheral blood of newly diagnosed AML patients and the impact of the method of immunomagnetic cell separation on the gene expression profile of healthy T cells. Gene expression profiling was subsequently performed on T cells from AML patients compared with healthy controls. Global differences in transcription were observed suggesting aberrant T cell activation patterns in AML. As differentially regulated genes involved in actin cytoskeletal formation were noted, a functional assessment of the ability of T cells from AML patients to form immunological synapses was performed. This illustrated that although T cells from AML patients can form conjugates with autologous blasts, their ability to form immune synapses and recruit phosphotyrosine signalling molecules to that signalling interface is impaired. Taken together, these findings demonstrate that numerically T cells are plentiful in AML however they are abnormal in terms of the genes they are transcribing and in their interactions with tumour cells. Targeting immunological synapse formation may represent an important means of improving T cell recognition of tumour cells across a range of cancers
