Assembly of the Murine Leukemia Virus Is Directed towards Sites of Cell–Cell Contact

Applying 4D imaging, this study investigates the mechanism by which cell-cell contact enhances retrovirus spreading and demonstrates that viral budding is highly polarized towards sites of cell-cell contact.We have investigated the underlying mechanism by which direct cell–cell contact enhances the efficiency of cell-to-cell transmission of retroviruses. Applying 4D imaging to a model retrovirus, the murine leukemia virus, we directly monitor and quantify sequential assembly, release, and transmission events for individual viral particles as they happen in living cells. We demonstrate that de novo assembly is highly polarized towards zones of cell–cell contact. Viruses assembled approximately 10-fold more frequently at zones of cell contact with no change in assembly kinetics. Gag proteins were drawn to adhesive zones formed by viral Env glycoprotein and its cognate receptor to promote virus assembly at cell–cell contact. This process was dependent on the cytoplasmic tail of viral Env. Env lacking the cytoplasmic tail while still allowing for contact formation, failed to direct virus assembly towards contact sites. Our data describe a novel role for the viral Env glycoprotein in establishing cell–cell adhesion and polarization of assembly prior to becoming a fusion protein to allow virus entry into cells.Author Summary: Retroviruses such as the human immunodeficiency virus are known to spread much more efficiently under conditions of direct cell–cell contact as compared to cell-free conditions. How cell–cell contact stimulates virus spreading is poorly understood. In this study, we apply four-dimensional imaging (3D space over time) of a model retrovirus to directly monitor and quantify the events of assembly, release, and transmission of individual viral particles in real time in living cells. Our work reveals that after contacts are established between virus-producing cells and uninfected target cells, the majority of virus particle assembly is initiated at sites of cell–cell contact. The ability of the virus to direct assembly of its particles towards sites of cell–cell contact is dependent on the presence of the cytoplasmic tail of the viral envelope glycoprotein. When this cytoplasmic tail was deleted, virus assembly at cell–cell contacts was no longer enhanced. This study contributes to an emerging model in which several steps of the viral life cycle are efficiently coordinated at sites of cell–cell contact, thereby promoting the spreading of viral infection to neighboring cells.