ИСТИНА

HIV can spread as a cell-free virus or via intercellular contactcalled virological synapse (VS). Cell-to-cell transmission is anefficient mechanism that help virus to escape from neutralizingantibodies and to increase multiplicity of infection, but has beendifficult to quantify. To accurately measure the level of HIV cell-to-cell infection, earlier we have developed replication-dependentreporter vectors inLuc and inGFP. However, this methodrequired transient co-transfection of T cells with the packaging,reporter, and Env expressing vectors making it costly. The goalof this study is to generate human T cell lines Jurkat and CEMthat would stably express indicated vectors and, ones mixed withtarget Raji/CD4 cells, will enable measuring VS formation andcell-to-cell infection using flow cytometry. The VS will beassessed by formation of red-blue conjugates between RFP+ pro-ducer cells and BFP+ target cells. For this purpose, we intro-duced BFP expression cassette into AAVS1 locus of Raji/CD4target cells using zinc finger nuclease (ZFN). The RFP expressioncassette was combined to inGFP/Luc construct in order to labelproducer cells and co-select the silent inGFP/Luc reporter, whichbecomes active only in target cells after completing one cycle ofHIV-1 replication. HIV-1 packaging construct was modified byadding CD5HA2 marker (Zotova A et al. (2019) Sci Rep 9,3132) at the C-terminus of Nef for isolation by FACS sorting.To avoid cell cycle arrest, we generated and tested Vpr(-) andNef(-) packaging constructs and found that the first one is suit-able for stable transgenesis. Among the three gene delivery strate-gies exploiting AAVS1-directed ZFN or CRISPR-Cas9 knock-in,FRT recombination and transposition, we selected transgenesiswith Sleeping beauty as the most efficient. In summary, we pre-pared a genetic platform to create a cellular system for HIV-1cell-to-cell transmission evaluation. This work was supported byRFBR grant 18-29-07052.