The spread of retroviruses between cells is estimated to be 2-3 orders of magnitude more efficient when cells can physically interact with each other 1,2 . The underlying mechanism is largely unknown, but transfer is believed to occur through large-surface interfaces, called virological or infectious synapses 3-6 . Here, we report the direct visualization of cell-to-cell transmission of retroviruses in living cells. Our results reveal a mechanism of virus transport from infected to non-infected cells, involving thin filopodial bridges. These filopodia originate from non-infected cells and interact, through their tips, with infected cells. A strong association of the viral envelope glycoprotein (Env) in an infected cell with the receptor molecules in a target cell generates a stable bridge. Viruses then move along the outer surface of the filopodial bridge toward the target cell. Our data suggest that retroviruses spread by exploiting an inherent ability of filopodia to transport ligands from cell to cell.To study the spread of retroviruses between living cells, we used the murine leukemia virus (MLV) as a model. MLV was fluorescently labelled in infected cells by expressing a CFPfusion with the capsid protein Gag (MLV Gag-CFP), as well as an envelope protein (Env) carrying a YFP-insertion (MLV Env-YFP) 7,8 . Infected cells were then cocultured with noninfected target cells expressing a CFP fusion with the MLV receptor mCAT1 (mCAT1-CFP) 8 . Infected cells were readily identified by the presence of retroviral particles, observed as punctae displaying both YFP and CFP fluorescence (Fig. 1A). Receptor-expressing target cells were characterized by homogeneous CFP fluorescence at the plasma membrane. Strikingly, essentially all virus particles moving from infected to target cells migrated along thin, elongated filopodia ( Fig. 1A and see Supplementary Information, Movie 1). The particles moved unidirectionally at an average rate of 0.7 μm min −1 (n = 117) and required, on average, approximately 18 min to move from one cell to the other (Fig. 1B, C). Identical observations were made when target cells were labelled with mCAT1-YFP (see Supplementary Information, Movie 2).Filopodial bridges were only observed between infected and non-infected cells. They averaged 5.8 μm in length (n = 59) and were long lived ( Fig. 1D, E; observed up to the maximum imaging time of 4 h). In contrast, normal filopodia of target cells that did not connect with an infected cell were significantly shorter (average length, 2.37 μm; n = 60) and highly dynamic, rapidly undergoing cycles of growth and retraction (Fig. 1D, E). To visualize moving viral particles at higher resolution, cells were cocultured on a coverslip with a lettered grid, and a filopodial bridge actively transporting virus was observed using a fluorescence microscope. The sample was then fixed and the same area visualized in the scanning electron microscope. The correlated images revealed that viral particles of a size of approximately 100 nm moved on the outer surfac...

Viruses have often been observed in association with the dense microvilli of polarized epithelia as well as the filopodia of nonpolarized cells, yet whether interactions with these structures contribute to infection has remained unknown. Here we show that virus binding to filopodia induces a rapid and highly ordered lateral movement, “surfing” toward the cell body before cell entry. Virus cell surfing along filopodia is mediated by the underlying actin cytoskeleton and depends on functional myosin II. Any disruption of virus cell surfing significantly reduces viral infection. Our results reveal another example of viruses hijacking host machineries for efficient infection by using the inherent ability of filopodia to transport ligands to the cell body.

Retroviral assembly and budding is driven by the Gag polyprotein and requires the host‐derived vacuolar protein sorting (vps) machinery. With the exception of human immunodeficiency virus (HIV)‐infected macrophages, current models predict that the vps machinery is recruited by Gag to viral budding sites at the cell surface. However, here we demonstrate that HIV Gag and murine leukemia virus (MLV) Gag also drive assembly intracellularly in cell types including 293 and HeLa cells, previously believed to exclusively support budding from the plasma membrane. Using live confocal microscopy in conjunction with electron microscopy of cells generating fluorescently labeled virions or virus‐like particles, we observed that these retroviruses utilize late endosomal membranes/multivesicular bodies as assembly sites, implying an endosome‐based pathway for viral egress. These data suggest that retroviruses can interact with the vps sorting machinery in a more traditional sense, directly linked to the mechanism by which cellular proteins are sorted into multivesicular endosomes.

2Viral infections spread based on the ability of viruses to overcome multiple barriers and move from cell to cell, tissue to tissue, and person to person and even across species. While there are fundamental differences between these types of transmissions, it has emerged that the ability of viruses to utilize and manipulate cell-cell contact contributes to the success of viral infections. Central to the excitement in the field of virus cell-to-cell transmission is the idea that cell-to-cell spread is more than the sum of the processes of virus release and entry. This implies that virus release and entry are efficiently coordinated to sites of cell-cell contact, resulting in a process that is distinct from its individual components. In this review, we will present support for this model, illustrate the ability of viruses to utilize and manipulate cell adhesion molecules, and discuss the mechanism and driving forces of directional spreading. An understanding of viral cell-to-cell spreading will enhance our ability to intervene in the efficient spreading of viral infections.

Cells use a variety of intercellular structures, including gap junctions and synapses, for cell-cell communication. Here, we present recent advances in the understanding of thin membrane bridges that function in cell-cell signaling and intercellular transport. Cytonemes or filopodial bridges connect neighboring cells via mechanisms of adhesion, which enable ligand-receptor-mediated transfer of surface-associated cargoes from cell to cell. By contrast, tunneling nanotubes establish tubular conduits between cells that provide for the exchange of both cell-surface molecules and cytoplasmic content. We propose models for the biogenesis of both types of membrane bridges and describe how viruses use these structures for the purpose of cell-to-cell spread.