Wolfgang Hübner, Ping Chen, Matthew Spinelli, Armando del Portillo, Yuxin Liu and Benjamin Chen

Division of Infectious Diseases, Department of Medicine, Department of Pharmacology and Systems Therapeutics, Immunology Institute

Mount Sinai School of Medicine,

New York, USA. Wolfgang.Huebner@mssm.edu

HIV can be efficiently transferred from T cell-to-T cell through adhesive structures called virological synapses (VS).  This means of viral spread may be central to our understanding of HIV transmission and viral dissemination within the host.  While cell free HIV-1 is capable of productive infections, VS mediated transfer is quantitatively more efficient.  During contact of an infected and uninfected T cell, VS are formed in an actin-, Env- and CD4-dependent manner.  We developed a replication competent HIV-1 molecular clone that carries a Gag-internal green fluorescent protein (iGFP).  This novel GFP fusion approach permits us to track the expression and localization of Gag in a cell and to monitor the state of viral assembly.  The infected cells and the progeny viruses are strongly labelled with GFP permitting us to follow VS-mediated viral transfer.  At the VS strong accumulations of Gag are often observed forming button like shapes in the donor cell at the site of cell-cell contact.  FRET analysis show intense Gag oligomerization at this site indicating virus assembly.  Dynamic live imaging of the VS reveals that HIV-expressing cells are polarized and make long-lived, env-dependent contacts with target cells. Four-dimensional, time-lapse confocal imaging shows a temporal progression of synapse formation and particle transfer in which budding from the donor cell is highly coordinated with endocytosis by the target cell. The observed transfer is massive and rapid, occuring within minutes. Transfered particles show very dynamic mobility in the target cells.  The high efficiency of transfer into primary human CD4+ T cells, the primary target of HIV in vivo, suggests the VS may be important in dissemination of HIV-1.