Integrin Activation Pma4/26/2021
Cells were then infected with the recombinant parvovirus B19-Luc vector for 1 hour at 37C in a CO 2 incubator.Yoder, Arun Srivastava; 51 integrin as a cellular coreceptor for human parvovirus B19: requirement of functional activation of 1 integrin for viral entry.
Although blood group P antigen has been reported to be the cell surface receptor for parvovirus B19, a number of nonerythroid cells, which express P antigen, are not permissive for parvovirus B19 infection. We have documented that P antigen is necessary for parvovirus B19 binding but not sufficient for virus entry into cells. To test whether parvovirus B19 utilizes a cell surface coreceptor for entry, we used human erythroleukemia cells (K562), which allow parvovirus B19 binding but not entry. We report here that upon treatment with phorbol esters, K562 cells become adherent and permissive for parvovirus B19 entry, which is mediated by 51 integrins, but only in their high-affinity conformation. Mature human red blood cells (RBCs), which express high levels of P antigen, but not 51 integrins, bind parvovirus B19 but do not allow viral entry. In contrast, primary human erythroid progenitor cells express high levels of both P antigen and 51 integrins and allow 1 integrinmediated entry of parvovirus B19. Thus, in a natural course of infection, RBCs are likely exploited for a highly efficient systemic dissemination of parvovirus B19. Similarly, B19p6 promoter-driven expression analyses were carried out with plasmid DNA transfections, 11-13 which do not accurately reflect a natural infection by parvovirus B19. Thus, P antigen alone is insufficient to impart erythroid specificity to parvovirus B19. ![]() Normal donor red blood cells (RBCs) were purchased from the Central Indiana Regional Blood Center, Indianapolis, and used on the day of purchase. Human bone marrowderived highly purified CD71 primary erythroid progenitor cells were purchased from AllCells (Berkeley, CA). Recombinant parvovirus B19-Luc vector stocks were generated and purified by CsCl equilibrium density gradient centrifugation as previously described. Wild-type parvovirus B19-containing sera were a kind gift from Kent Dupuis, Cerus, Concord, CA. Activating and inhibitory monoclonal antibodies against and integrins were purchased from Chemicon (Temecula, CA). To induce an adhesive phenotype in K562 cells pharmacologically, suspension K562 cells were treated with 15 nM or 32 nM phorbol 12-myristate 13-acetate (PMA) for 48 hours, as described previously. After infection cells were either directly transferred to glass coverslips coated with TAK adhesive protein (Sigma) (K562 cells) or either mock treated or treated with 0.05 trypsin plus 0.02 EDTA (ethylenediaminetetraacetic acid) at 37C for 5 minutes (RBCs) and then transferred to the coverslips. ![]() After another washing step, nuclei (K562 cells) were stained using SYTO 16 green fluorescent nucleic acid stain (Molecular Probes). ![]() A mean of 12 to 20 slices per visible field were acquired using a multitrack mode to record red and green fluorescence separately, and overlay images were subsequently generated using the LSM5 software (Zeiss). For RBCs, image acquisition was achieved with red fluorescence and transmission light to visualize cell contourmorphology. In some experiments, cells were either mock treated or preincubated with 10 gmL of activating (N29, 21C8) and inhibitory (P4C10, JB1a) 1 integrin antibodies for 30 minutes at room temperature or 500 gmL GRGDS peptides for 20 minutes at room temperature. For integrin cross-linking, K562 cells were first incubated with 10 gmL of 1 integrin inhibitory antibodies (JB1a) or 500 gmL GRGDS peptides for 20 minutes at room temperature followed by that with 5 gmL of goat antimouse immunoglobulin G (IgG) antibodies for an additional 20 minutes at room temperature.
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