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Abstract

Transmission of HIV-1 after particle assembly and budding from the membrane of infected T cells can occur by two different modes: cell-free infection, where particles are released into the extracellular space and traffic to susceptible cells by diffusion, and cell-associated transmission by direct cell-cell contacts via so-called virological synapses (VS). In this dissertation, I established a novel approach to visualize HIV-1 cell-to-cell transmission using pulse-chase labeling to separately identify viral transfer and productive infection. To this end, I successfully established and characterized a fully replication-competent labeled HIV-1 derivative, HIV-1iSNAPf(opt). I could demonstrate that this derivative exhibited near wild-type levels of infectivity and remarkably stable integration of SNAPf into the group-specific antigen (Gag), as evidenced by the detection of SNAPf-tag expression by flow cytometry and Western blot after prolonged passaging in A3.01 T cells. In addition, the effect of codon optimization within sfGFP and SNAPf upon insertion into Gag was investigated, based on the previous observation that an increased level of CpG dinucleotides leads to RNA degradation by the Zinc-finger Antiviral Protein (ZAP). No notable enhancement in replication kinetics or infectivity was observed for the codon-optimized derivatives, when a time course was conducted in infected A3.01 T cells and infectivity assays were performed in reporter cell lines. Furthermore, I developed a real-time method for the detection of productive cell-to-cell transmission. This was accomplished by labeling Gag.SNAPf in the donor cell population with a cell-permeable benzylguanine (BG)-conjugated SNAP dye, followed by continuous observation of Gag.SNAPf expression in contacted target cells, in presence of a novel, highly fluorogenic dye “SNAP23”. Moreover, a semi-automated analytical pipeline, developed in collaboration with ZEISS Arivis, was utilized to quantitatively analyze the factors influencing the dynamics of VS formation and to establish a correlation between cell-to-cell transfer and Gag.SNAPf expression in contacted target cells. The results of the quantitative analyses indicated that a single contact was sufficient to induce new Gag expression in the target cell, regardless of the duration of the contact. Furthermore, new Gag.SNAPf expression was observed in target cells as early as 30 minutes after VS formation, indicating the potential for direct translation of incoming genomic RNA. In conclusion, pulse-chase labeling of HIV-1 infection and cell-to-cell transmission offers a versatile tool for studying the various stages of the HIV-1 replication cycle. In the context of VS, this is the first instance in which distinct contact events have been directly correlated with novel Gag.SNAPf(opt) expression in target cells.