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Human T-lymphotropic virus type 1 (HTLV-1) is a human pathogenic retrovirus that infects mainly CD4+ T-lymphocytes and causes adult T-cell leukemia or an inflammatory disorder called HTLV-1-associated myelopathy/tropical spastic paraparesis. Being very inefficient in infecting cells via isolated virions, HTLV-1 has evolved several clever strategies to transmit from cell to cell which mechanisms are not fully understood. We performed a novel method of GeCKO library screening in HTLV-1 infection assay to determine cellular factors (including not completely discovered viral receptors) involved in HTLV-1 cell-to-cell transmission and replication. GeCKO library is designed for loss-of-function screens followed by deep sequencing analysis of 20-nt gRNA targets. Each target sequence recovered by PCR from the integrated lentiviral DNA matches the open reading frames of the genes and suggests knockout of the corresponding gene. First, we generated CEM and Raji/CD4 cell lines with GeCKO knockout library, and evaluated applicability of cell libraries to identify the BF4 monoclonal antibody that specifically stained viral biofilms on the surface of the HTLV-1 infected T cells, but its target antigen was unknown. Afterwards, we assessed the level of gene representation in library cells by next-generation sequencing (NGS) analysis. Finally, we performed infection assay by mixing library cells with chronically HTLV-1 infected MT2 cells. HTLV-1 resistant library cells were analyzed by NGS in order to reveal factors, which mediate resistance to HTLV-1. Among them we found CD82, KCTD5 (potassium channel tetramerization domain containing 5), and KPNA1 (karyopherin subunit alpha 1). HIV-1 uses KPNA1 to translocate its genome into the nucleus. Now we are investigating the role of KPNA-1 in HTLV-1 pathogenesis. We believe that the results obtained with GeCKO library screening will broaden our knowledge about molecular mechanisms of HTLV-1 cell-to-cell transmission.